Advanced Drug Delivery Reviews 65 (2013) 1496–1532

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Advanced Drug Delivery Reviews

journal homepage: www.elsevier.com/locate/addr

Microfluidic and lab-on-a-chip preparation routes for organic

nanoparticles and vesicular systems for nanomedicine applications☆
Lorenzo Capretto a,⁎,1, Dario Carugo b,c,1, Stefania Mazzitelli d, Claudio Nastruzzi d, Xunli Zhang b,e
a
Department of Pharmaceutics, UCL School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX, United Kingdom
b
Bioengineering Sciences Research Group, Faculty of Engineering and the Environment, University of Southampton, Southampton SO17 1BJ, United Kingdom
c
Electro-Mechanical Engineering Group, Faculty of Engineering and the Environment, University of Southampton, Southampton SO17 1BJ, United Kingdom
d
Department of Life Sciences and Biotechnology, University of Ferrara, Via F. Mortara 17/19, 44121 Ferrara, Italy
e
Institute for Life Sciences, University of Southampton, Southampton, SO17 1BJ, United Kingdom

a r t i c l e i n f o a b s t r a c t

Article history: In recent years, advancements in the fields of microfluidic and lab-on-a-chip technologies have provided unique
Accepted 1 August 2013 opportunities for the implementation of nanomaterial production processes owing to the miniaturisation of the
Available online 8 August 2013 fluidic environment. It has been demonstrated that microfluidic reactors offer a range of advantages compared to
conventional batch reactors, including improved controllability and uniformity of nanomaterial characteristics. In
Keywords:
addition, the fast mixing achieved within microchannels, and the predictability of the laminar flow conditions,
Microfluidic
Lab-on-a-chip
can be leveraged to investigate the nanomaterial formation dynamics. In this article recent developments in
Nanomedicine the field of microfluidic production of nanomaterials for drug delivery applications are reviewed. The features
Nanomaterial that make microfluidic reactors a suitable technological platform are discussed in terms of controllability of
Polymeric micelles nanomaterials production. An overview of the various strategies developed for the production of organic
Liposomes nanoparticles and colloidal assemblies is presented, focusing on those nanomaterials that could have an impact
Polymersomes on nanomedicine field such as drug nanoparticles, polymeric micelles, liposomes, polymersomes, polyplexes and
hybrid nanoparticles. The effect of microfluidic environment on nanomaterials formation dynamics, as well as the
use of microdevices as tools for nanomaterial investigation is also discussed.
© 2013 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1497
2. Overview of synthetic approaches for organic nanoparticles and vesicular systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1498
2.1. Methods of preparation for vesicular systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1498
2.1.1. Mechanism of vesicle formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1498
2.1.2. Classical production techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1499
2.2. Methods of preparation for organic nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1499
2.2.1. Top-down approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1499
2.2.2. Bottom-up approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1499
2.2.3. Nanoprecipitation mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1499
3. Microfluidic synthesis of nanomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1500
3.1. The microfluidic environment and mixing principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1500
3.1.1. Reynolds number and diffusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1500
3.1.2. Mixing in microfluidic devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1501
3.2. Features of microfluidic reactors for the production of nanomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1501
3.2.1. Micromixing and reactor design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1502
3.2.2. Temperature control and heat transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1503
3.2.3. Kinetic control and investigation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1503
3.2.4. On-chip analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1504
3.2.5. High throughput production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1504

☆ This review is part of the Advanced Drug Delivery Reviews theme issue on "Design production and characterization of drug delivery systems by Lab-on-a-Chip technology".
⁎ Corresponding author. Tel.: +44 207 753 5847.
E-mail address: l.capretto@soton.ac.uk (L. Capretto).
1
The authors have equally contributed to the work.

0169-409X/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.addr.2013.08.002
 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532 1497

3.3. Materials used in the fabrication of microfluidic chip for the production of nanomaterials . . . . . . . . . . . . . . . . . . . . . . . . 1505
4. Microfluidic production of nanoparticle and vesicular systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1505
4.1. Organic nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1505
4.2. Nanomaterial from self-assembly of amphiphilic molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1505
4.2.1. Lipid vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1506
4.2.2. Niosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1511
4.2.3. Polymersomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1511
4.2.4. Polymeric micelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1515
4.2.5. Polymer nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1517
4.2.6. Hybrid nanoparticles and complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1517
4.2.7. Nanomaterial produced through complexation mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1518
5. Microfluidic-based manipulation, immobilisation and in situ analysis of nanoparticles and vesicular systems . . . . . . . . . . . . . . . . . . 1519
5.1. Nanoparticles and vesicular systems manipulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1520
5.2. Nanoparticles and vesicular systems immobilisation and in situ analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1521
6. Effect of microfluidic environment on dynamics of nanomaterial formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1521
6.1. Computational fluid dynamic (CFD) models for indirect investigation of the nanomaterial formation dynamics . . . . . . . . . . . . . . 1522
6.2. Methods for direct investigation of nanomaterial formation dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1522
6.3. Effect of shear stress on nanomaterial characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1523
7. Assessing the biological performance of nanomaterials produced by microfluidic technology: in-vitro and in-vivo tests . . . . . . . . . . . . . 1523
8. Conclusion and outlooks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1525
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1527

1. Introduction
Over the past two decades, research in the field of nanoparticles
(NPs), that are particles with dimension in the range of about a few
tens to a few hundreds of nanometres, has experienced a tremendous
growth. The importance of NPs has been demonstrated and NPs have
been used in a wide range of applications including microelectronics,
chemistry, energy, life sciences and pharmaceutics [1–4]. Until recently,
nanotechnology has experienced an intense research on inorganic NPs,
which is reflected in the plethora of synthetic methods available for
such NPs and established applications in various fields such as medical
imaging, catalysis and electronics [5,6]. In comparison, only few studies
have addressed the production of organic NPs. This is surprising,
considering the high potential of organic NPs in the fields of
pharmaceutics and nanomedicine [7,8], food technology [9] as well
as chemo- and bio-sensing [10,11]. In the next sections, we will
mainly focus on the preparation of nanosized dosage forms, which
represent the most widely, explored area in the field of organic
nanotechnology.
Many different types of nanosized dosage forms have been explored
for their possible use in drug delivery and pharmaceutics. These mainly
include nanoemulsions [12–14], organic NPs [15,16], macromolecules
such as dendrimers [17], and supramolecular aggregates such as micelles
[18], liposomes [19] and polyplexes [20]. The reason for the use of
nanosized dosage forms in pharmaceutics stems from the beneficial effect
that a reduction in size, down to the nanoscale, has on bioavailability and
targetability of a pharmaceutical active [21,22]. In this review emphasis is
placed on those nanosized dosage forms that have stimulated interest in
the design of microfluidic reactors for their preparation. These include
both polymer and non-polymer based “hard” organic nanoparticles
(i.e. kinetically stable after preparation) and “soft” vesicular systems
(i.e. in dynamic equilibrium after preparation) including liposomes,
niosomes, polymersomes, and micelles. It should be noted that
“frozen” core micelles can be included within the category of “hard”
nanoparticles, given the associated slow rate of unimer exchange [23].
Similar to their inorganic counterparts, organic NPs display size-
dependent characteristics. However, the characteristics of organic NPs
result from both (i) different electronic properties of the molecules at
the NP surface [24] and (ii) a largely increased surface-to-volume ratio
(SVR) [25,26], rather than depending on the quantum confinement
effect as in the case of metal NPs [27].
It is well known that the bioavailability of an active formulated as NP
suspension can be greatly increased by synergistically enhancing both
thermodynamic solubility and dissolution rate. For small particles, the
NP apparent solubility (Sapp) is increased due to the high curvature of
the particle at the surface according to Kelvin's equation [28]
Sapp 2γV
¼ e RTr ð1Þ
So
where S0 is the equilibrium solubility for the bulk material, γ is the
interfacial free energy, V is the molar volume, R is the gas constant, T
is the absolute temperature, and r is the particle's radius. An increase
in Sapp also results in increased dissolution rate dM=dt , according to
the Noyes Whitney equation [29] (2)
dM  
¼ kA Sapp −C ðt Þ ð2Þ
dt
where A is the solid surface area, C(t) is the solute concentration at time
t, and k is the constant of the dissolution process. In addition, it should
be noted that A increases with decreasing the particle dimension,
which synergistically contributes to the increased dissolution rate.
Furthermore, an additional increase of solubility can derive from an
increase in γ due to the production of nanoparticle constituted of
thermodynamically unstable polymorphs or amorphous phase [3,30].
In addition to enhancing the solubility of poorly soluble drugs, the
possibility to realise a time controlled release [31], and a passive (through
EPR effect [32]) or active targeted delivery [33], has also contributed to
drive the research in the field of nanomedicine. Finally, in view of the
almost infinite range of chemical substances from which nanoparticles
can be produced, their immense potential in different areas of future
applications appears evident.
Despite significant advancements in the field that have been
achieved so far, challenges still remain in synthesising and
formulating organic compound-based nanostructured materials,
while the synthesis of inorganic nanomaterials has been extensively
studied over decades with optimised control over both particle
shape and size being reported [34–36]. Consequently, special
preparation techniques are required in order to disperse solid
organic materials into water, maintain the dispersion for a certain
period of time and functionalise organic NPs.
Microfluidics is a new and emerging science and technology field
dealing with miniaturised systems that process, or manipulate, small
volumes of fluids (10− 9 to 10− 18 L), by means of channels with
dimensions ranging from tens to hundreds of micrometres [37].
1498 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532
Microfluidic and lab-on-a-chip technologies rely on the concept of
“shrinking” traditional bench chemical systems down to the size of
a few centimetres squares. The main feature of such systems is the
miniaturisation of the fluidic environment, with a channel width of
about 100 μm (about the diameter of a human hair), where chemicals
are brought together using a variety of pumping techniques for
synthesis, separation or analysis purposes.
Recent years have seen the implementation of microfluidic-based
and lab-on-chip technologies as tools in pharmaceutical research. For
instance, lab-on-a-chip devices have been used to support complex
chemical processes [38,39], to screen these processes [40–42], to study
cell-formulation interactions [43] and the mechanisms of action of
drug devices combination products [44], and to produce microsized
particles or droplets with potential applications in drug delivery [45]
and cell encapsulation [46–49].
Relevant to the formulation of organic nanomaterial, a series of
features make microfluidic and lab-on-a-chip devices appealing
technological platforms not only to produce particles with a well-
controlled size distribution, but also to investigate the nanomaterial
formation process by integration with a number of measurement
systems into the microreactor [35].
However, despite their potential, at present few reports have
comprehensively discussed microfluidic techniques for the formation
of organic nanomaterials. Hence, this review aims to discuss the
advantages of microfluidic and lab-on-a-chip technology for the
development and understanding of organic nanomaterial production,
and its current or potential application in drug discovery and drug
delivery.
In this article, the most recent research and developments about
the production of organic nanomaterials for drug delivery applications
using microfluidic technology are reviewed, with the aim of elucidating
the importance of developing novel strategies for achieving improved
nanoparticle formation. The fluid dynamic characteristics of the
microfluidic environment and the advantageous features of the
microfluidic devices are discussed in the context of organic nanomaterial
formation, followed by a summary of the various strategies developed for
the microfluidic and lab-on-a-chip production of nanoparticles and
vesicular systems. Subsequently the use of microdevices in the
manipulation and analysis of nanomaterials are discussed providing
examples on how microfluidic devices could be employed as useful
tools in the investigation of nanomaterial formation processes. The
biological performance of nanomaterials produced bymicrofluidic
technology is also reviewed. Further, a perspective for future research
is provided highlighting the issues that still hinder the widespread
success of microfluidic devices for nanomaterial production.
2. Overview of synthetic approaches for organic nanoparticles and
vesicular systems
One of the major challenges within nanotechnology field has
been the development of synthetic methods for nanosized dosage
forms production. This interest is mostly driven by the need for
developing methodologies capable of producing nanomaterials
with tuneable characteristics (i.e. size, physico-chemical properties
and concentration) and with high homogeneity, in order to possibly
reduce post-production steps while obtaining high standard products.
The following paragraphs provide an overview of the synthetic
approaches developed for either organic NPs or vesicular-like
systems in rapid dynamic equilibrium, together with their possible
mechanisms of formation.
2.1. Methods of preparation for vesicular systems
Surfactants and amphiphilic block copolymers at low concentration
levels usually self-assemble forming micelles, with physical and
architectural properties depending on the relative hydrophobicity and
on the relative composition of hydrophobic moieties (or tectons)
and hydrophilic moieties [50]. Conversely, lipid molecules generally
form lamellar bilayered structures and ― in dilute solutions ― they
arrange into vesicles, referred to as liposomes [51]. Vesicles formed
by self-assembly of polymers and polypeptides are instead referred
to as polymersomes [52] and peptosomes [53], respectively. In the
present paragraph classical methods of vesicular systems production
are described, together with essential information about their
mechanisms of formation and related governing parameters.
2.1.1. Mechanism of vesicle formation
The process of vesicular systems formation can be categorised
as a two-step self-assembly [50]. In this process, the amphiphile
forms a bilayer in the first step, which subsequently closes to form
a vesicle in the second step. The size of the hydrophobic moiety
relative to the hydrophilic moiety determines the shape of the
self-assembled amphiphilic structure, by influencing the curvature of
the hydrophobic–hydrophilic interface. The latter can be expressed in
terms of mean curvature (H) and Gaussian curvature (KG), which are
related to the surfactant packing parameter (V am =a  L) by the following
equation [54]:
V am K  L3
¼1þHLþ G ð3Þ
aL 3
where Vam is the amphiphile hydrophobic volume, a is the interfacial
area, and L is the chain length normal to the interface. With this respect,
packing parameters of ~0.33, 0.5 and 1 correspond to the formation of
spherical, cylindrical and bilayered structures, respectively [50]. Based
on these purely geometrical considerations, it thus appears evident
that it is possible to actively modulate the architecture of the obtained
structures by controlling the governing parameters (i.e. hydrophilic–
hydrophobic block ratio, in case of block copolymer structures).
However, a more comprehensive analysis of vesicular systems
formation must take into account the relation between structures
architecture and free energy. With this respect, in the case of
amphiphilic molecules, the free energy depends on both the
interfacial energy of the hydrophobic–hydrophilic interface and
the loss of entropy associated with the process of surfactants or
polymer chains aggregation into microdomains [50]. Notably,
when the interfacial energy is large and the entropy loss is relatively
small, the thermodynamics of the process is dominated by the
minimisation of the interfacial area. In this specific scenario,
hydrophobic and hydrophilic phases are highly segregated and this
state is usually referred to as superstrong segregation limit (SSSL)
[55]. Polymers (or tectons) characterised by low conformational
entropy are generally stiff molecules, having low internal degrees
of freedom. In this case, amphiphiles will form structures which
minimise the interfacial area per unit volume, corresponding to
planar interfaces.
The formation of vesicular systems relies on the principle that
tectons energetically prefer a parallel molecular spatial arrangement
[50]. Based on this consideration, sheet-like micelles should represent
the optimal architecture. However, at low tecton concentration levels
when the sheet-like structures are too large, the energy loss due to
surface tension effects forces the structure to close forming a vesicular
system [51]. This process gives rise to a line energy depending on the
line tension (γL); and the bending of the bilayer during closure requires
an additional energy depending on the bilayer bending modulus (κ).
The balance between line tension and bending energy determines the
minimal vesicular size [56]. Notably, vesicle formation is generally
favoured when the bending elasticity of the bilayer is low, and the
surface tension is high.
However, commonly employed lipids and polymeric structures are
characterised by a low molecular solubility and thus the architectural
properties of the formed vesicles do not depend predominately on
 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532
exchange and equilibrium processes, but they are mainly governed
by the preparation conditions. For this reason, these vesicular
systems are usually referred to as ‘non-equilibrium vesicles’ [50].
Thus, in this case, the vesicle size depends less significantly on
thermodynamic factors but mostly on non-equilibrium aspects of
the formation process. Notably, the shape of the obtained vesicle is
the one associated with the minimisation of the bending energy for
a given difference in the number of amphiphiles in the inner and
outer monolayers, which depend on the production technique [51].
Consequently, vesicle diameter and shape can be controlled by
relying on different production methods [57].
2.1.2. Classical production techniques
In conventional vesicle preparation routes, the contact between
the aqueous phase and amphiphiles can be achieved by (i) phase
inversion and (ii) organic solvent-free methods [57]. In phase
inversion approaches, the amphiphile is initially dissolved in an
organic solvent. Subsequently, the solvent is exchanged by water,
via evaporation or dialysis [58,59]. These methods rely on the
variation of the packing parameters and, thus, the achievement of
an optimal surface area per molecule, as mentioned previously.
However, while effective control over vesicle size and architecture
can be achieved with these techniques, the formed membrane
structure contains residual organic solvent, which can potentially
compromise the functioning of bioactive molecules encapsulated
within the bilayer for biomedical purposes.
On the other hand, in solvent-free approaches, the amphiphile is
generally deposited onto a substrate and subsequently hydrated
(referred to as hydration method). This is usually performed in the
presence of an external energy source (i.e. mechanical or electrical)
[60]. The formation of vesicles from polymeric or lipid films upon
application of alternating electric field is generally referred to as
electroformation, and it has been observed to result in the formation
of micrometre-sized vesicles.
However, batch methods of production are generally associated
with high vesicle size polydispersity, requiring post-processing steps
(i.e. extrusion or sonication) to improve size uniformity. Further details
about these methods and their translation into microscale systems will
be provided in the next sections.
2.2. Methods of preparation for organic nanoparticles
Methods of preparation of organic NPs are typically categorised in two
groups: (i) top-down and (ii) bottom-up approaches. In the following
paragraphs the methodologies developed for the two approaches are
briefly discussed. Subsequently, the mechanism of nanoprecipitation
exploited in the bottom-up approaches is described more in detail.
2.2.1. Top-down approaches
Top-down approaches rely on reduction of size of macroscopic
material down to nanometre scale. Various procedures have been
developed with mechanical milling being the most obvious. However,
although milling processes have been widely used in organic NP
formation [5], they are generally considered unsuitable for pharma-
ceutical actives. This applies particularly to the production of NPs with
narrow distribution and small size, because mechanical energy in the
form of shearing and impact forces which is applied during particle
milling, make it difficult to reduce the size of the particles without
simultaneously inducing particle agglomeration [3]. In addition,
abrasion during the milling process can cause contamination of the
end product which is difficult to separate, especially in active compound
formulations, while it may be difficult to produce nanosized organic
particles consistently when using solid-particle milling [61]. A similar
approach is represented by the “high-pressure homogenization”
technique [62,63], where cavitation forces are exploited for the
reduction of particle size.
1499
Lithographic methods such as “Particle replication In Non-wetting
Templates” (PRINT) [64,65] and “Step and Flash Imprint Lithography”
(S-FIL) [66], have also been conveniently employed for the production
of organic NPs with effective control over NP size and shape. PRINT
utilises a non-wetting perfluoropolyether (PFPE) as a mould to create
monodisperse NPs with various shapes in mild conditions that
allow for the use of a range of different active substances including
those more subjected to degradation. Similarly, S-FIL relies on a
nanoimprinting process to produce NPs but differs from PRINT for the
method of release of NPs after preparation, which is realised by the
solubilisation of a sacrificial layer.
2.2.2. Bottom-up approaches
Bottom-up approaches involve the conversion of molecular matter
dispersed in liquid or gas phase into NPs by a condensation process.
They are far more utilised in research setting since they require less
expensive equipment and smaller amount of reagents, and often grant
better control over NP characteristics in terms of composition and
size. The bottom-up approaches can be further classified in emulsion-
based and nanoprecipitation methods. In the emulsion-based methods
the emulsion generally works as intermediate step to confine the
organic phase in small droplets, which act as a template for the organic
NPs. In a typical setting, the organic compound is firstly dissolved in an
organic solvent. This solution is then emulsified with controlling the
droplet size by homogenisation method. The NP production is achieved
by separating the solvent via evaporation [67] or diffusion process
[68,69].
Instead, nanoprecipitation procedures rely on the creation of a
supersaturated solution, by means of temperature change [70] or
solvent shifting [71], which ultimately leads to the formation of a
precipitate [72]. The mechanism and theory at the basis of the
nanoprecipitation approaches will be discussed in the following section.
Based on this theory, it is clear that crucial for the preparation of organic
NP is the creation of a rapid and spatially uniform supersaturation. In
order to improve mixing performance, various modifications to the
basic methodology of nanoprecipitation have been developed. NPs
have been produced using bath reaction vessel where the solutions
were mixed by means of magnetic stirring [73] or ultrasound [74].
Moving from conventional bulk systems, membrane systems have
also been used [75] while Johnson and Prud'homme [9–11] have
developed mesoscale continuous-flow confined impinging jets (CIJ)
and multiple inlets vortex mixers (MIVM). With the latter they have
further demonstrated that a fast mixing is crucial in order to achieve
a kinetically controlled nanoprecipitation, and consequently a fine
control over the size and polydispersity of NPs [12,13].
2.2.3. Nanoprecipitation mechanism
The framework of classical crystallization theory provides a useful
model to describe the formation of NPs via bottom-up approaches,
and for understanding the role that this process exerts in controlling
the characteristics of the produced NPs [3,76]. Starting from a
multicomponent, single phase system, the precipitation (or phase
separation) occurs at the onset of supersaturation when the system is
perturbed by modifying a boundary condition such as temperature or
concentration, such that the phase separation becomes favourable to
reduce system free energy.
Fig. 1 shows a simplified representation of the nanoprecipitation
process. After a system perturbation, the solute concentration
increases above the saturation limit (i.e. solubility) and reaches a
critical concentration at which the precipitation process is triggered.
NP formation model involves an initial nucleation phase in which
seed particles, referred to as nuclei, precipitate spontaneously and
a subsequent growth phase in which the initial seeds capture the
remaining dissolved solute.
The nucleation phase occurs when the concentration of the solute
reaches a critical nucleation concentration at which a shower of nuclei
1500 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532
Fig. 1. Schematic representation of solute concentration trend during the nanoprecipitation
process (adapted with permission from [3]. Copyright 2001; John Wiley and Sons).
is formed. At this stage, the nuclei form by condensation of molecules
until they reach a critical size at which they are stable against
dissolution. The nucleation phase proceeds until the concentration of
the solute falls below the critical nucleation threshold, where new
nuclei cannot be any longer formed [25]. However, this concentration
is still sufficient to allow the growth of the nuclei already formed. The
growth phase proceeds until the concentration of the still-dissolved
material has fallen to the equilibrium concentration (i.e. bulk solubility).
A precise control over the nucleation phase is often difficult to achieve
therefore, heterogeneous nanoprecipitation, by means of preformed
seed crystals [77] or additives [78] can be used.
To obtain NP batches with narrow size distribution it is important to
tune the process in order to ensure that the nucleation occurs in a short
period compared to the time required for the growth phase. In the
nucleation phase, nucleation and growth occur concurrently therefore,
the relative kinetics of the two processes greatly affects the quality of
the produced NPs. In this regard, it should be noted that the nucleation
process is far more dependent on the supersaturation level than on the
growth process; hence, high supersaturation is beneficial to favour the
nucleation over the growth [25].
In addition, since nucleation and growth rates depend on
the supersaturation level, in order to obtain a monodisperse batch,
a homogenous environment is required in terms of temperature
and species concentration during both nucleation and growth phases
(i.e. temperature variations affect the solubility of the species and thus
the supersaturation level) [79]. Batch synthesis of NPs is typically
carried out in stirred flasks, where stirring is conventionally used to
rapidly mix reactants and keep growing particles in suspension.
Generally, during the synthesis process, the controlled addition of
secondary reactants is required. In such cases, the addition rate and
mixing speed often determine the presence or absence of secondary
particle nucleation, homogeneity and state of aggregation of the final
mixture. As mentioned earlier, the homogeneity of concentration and
temperature is also crucial for monodisperse particle size distributions
to be obtained, due to the sensitivity of colloidal nucleation and
morphology to the local temperature and composition [3]. However,
macroscale synthesis often grants poor control over the spatial and
temporal distribution of the governing physical and chemical
parameters (i.e. temperature and species' concentration) [80], as
well as over species additions and mixing rates. These two factors
lead to non-homogenous rate of nucleation and growth, resulting
in polydisperse NP output.
Furthermore, batch synthesis tends to suffer from poor repro-
ducibility of size and quality of the NPs from batch to batch. In addition,
the relatively large amount of material used and the time-consuming
processes further complicate screening and optimisation of the
synthesis conditions and therefore the scale-up to batch-scale stirred
flask synthesis for larger production [4].
3. Microfluidic synthesis of nanomaterials
Size, shape and crystal-structure characteristics strongly affect
NPs' physical and chemical properties. Therefore, it is not surprising
that in the production of NPs a superior control over the process
parameters, in order to produce material of required features, is
desirable [35,36,81,82]. In this respect, microfluidic reactors offer a
series of potential advantages for the production of NPs since they enable
accurate control and manipulation of fluids and fluid interfaces. In
microfluidic devices, reactions are carried out within small reaction
channels, with diameter between a few tens and a few hundreds of
microns. The small dimension, and the resulting large SVR, enables
rapid and uniform heat and mass transfer that can dramatically improve
NP yield and size distribution while reducing undesirable by-product
formation. In addition, the possibility of solvent recycling, and integrated
separation techniques, are likely to provide cost effective and
environment friendly technology for the production of NPs [34].
3.1. The microfluidic environment and mixing principles
In this section, the fundamental theory of fluid and mass transfer,
and the implication of using microfluidic devices for mixing are firstly
discussed. In principle, fluid flow in microfluidic environments is
governed by the same laws that apply to the flow at the macroscale.
However, the miniaturisation confers additional characteristics that
can be exploited in order to perform processes not otherwise possible
at the macroscale. Notably, microfluidic devices are not simply a
miniaturised version of their macroscale counterparts. This is because
many physical characteristics, including SVR and diffusion-based mass
transfer, do not scale linearly from macro- to micro-domains. Another
peculiarity of microfluidic systems is represented by the omnipresence
of laminar flow, due to the predominant role exerted by viscous forces.
These factors thus become significant at the microscale, and their effects
should be considered while designing and developing lab-on-a-chip
devices.
In other words, a microfluidic mixer is not simply a miniaturised
version of a macroscale mixing device; it should be designed in such a
way to leverage the physical characteristics of mass and fluid transfer
in a micro-confined domain.
3.1.1. Reynolds number and diffusion
Fluid flow is generally categorised into two regimes: laminar and
turbulent. In laminar regime the fluid flows in parallel layers with no
cross currents perpendicular to the main flow direction, whereas
turbulent flow is generally characterised by the formation of vortices
and flow fluctuations in space and time. From a physical perspective,
the two regimes differ in terms of the relative importance of viscous
forces (i.e. the friction imparted by the channel walls) and inertial forces
(i.e. fluid momentum) which is measured by the Reynolds number (Re)
[83]:
ρuDh uDh
Re ¼ ¼ ð4Þ
μ v
where ρ and μ are the fluid density and dynamic viscosity, respectively;
ν is the fluid kinematic viscosity; u is the mean fluid velocity and Dh is
the hydraulic diameter of the channel. The latter is a characteristic
number which depends on the cross-sectional geometry of the channel,
and is given by [83]
4A
Dh ¼ ð5Þ
P wet
 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532
where A and Pwet are channel cross-sectional area and wetted perimeter,
respectively.
At low Re, viscous effects overcome inertial effects and a fully
laminar flow occurs. In this situation, fluid streams flow parallel to
each other and the velocity at any location within the fluid stream
is time invariant when boundary conditions are stationary. Therefore
mass transfer by advection can occur only in the direction of the fluid
flow, and mixing between streams is dominated by molecular
diffusion [84]. Conversely, at the high Re associated with turbulent
regime the flow is dominated by inertial effects and exhibits motion
that is random in both space and time. In this situation there are
advective mass transport phenomena in all spatial directions [85].
A transitional flow regime usually occurs between laminar
and turbulent regimes. The exact transitional Reynolds number is a
function of many parameters, including channel geometry and
surface roughness. The transitional Re is generally in the range
1500–2500 for the large majority of fluid dynamic problems [86].
In microfluidic systems, Re is typically lower than 100 and the flow
is thus regarded as laminar. This characteristic of fluid flow has a
direct effect on mixing within microfluidic devices.
Notably, in a fluidic domain where the flow is laminar, mass transfer
is dominated by passive molecular diffusion and advection. Diffusion is
defined as the mechanism of molecule transport from a domain of
higher concentration to a domain of lower concentration by Brownian
motion, resulting in a gradual mixing of material. Diffusion-based
mass transfer is described mathematically using Fick's laws [87]
dφ
j ¼ −D
dx
where φ is the species concentration, x is a spatial coordinate, and D is
the diffusion coefficient. For spherical particles, D can be derived from
the Stokes–Einstein equation [88]:
kT
D¼
6πμr
where k is Boltzmann's constant; T is the absolute temperature; r is
the radius of the particles (or molecules) and μ is the dynamic viscosity
of the fluid medium. The diffusion coefficient is usually expressed
in m2s−1 (or cm2s−1), and for a small molecule in water at room
temperature it assumes the typical value of 10−9 m2s−1 [89].
Diffusion is a non-linear process in which the time t required for a
species to diffuse scales quadratically with the distance x covered.
Thus a simple, one-dimensional diffusion process can be modelled
using the following equation [90]:
2
x ¼ 2D  t
where t is the average diffusion time over the distance x. In a
microfluidic channel, x usually represents the stream width of the
fluid to be mixed along the microfluidic channel. On a microscale,
the diffusion distance can be extremely small, especially if fluid
streams are hydrodynamically focused. From the moment that t
scales with the square power of x (see Eq. (8)), a decrease in
diffusion distance has the effect of dramatically reducing the time
required for complete mixing to be achieved. Therefore, diffusion
becomes a viable method to transport particles and mix chemical
species in microfluidic systems.
3.1.2. Mixing in microfluidic devices
At the macroscale, mixing is generally achieved by generation of
turbulent flow, making it possible to segregate fluid in small domains,
thereby leading to an increase of the contact surface and a decrease of
the mixing path. As discussed in the previous paragraph, due to the
low Re, mixing in microfluidic devices is achieved by taking advantage
1501
of the small length scale, which dramatically increases the effect of
diffusion and advection, realising efficient and fast mixing [91].
Micromixer architecture is generally designed in such a way
to decrease the mixing path and increase the contact surface area.
Micromixers are frequently classified as being passive or active, depending
on the mechanism exploited to generate mixing at the microscale.
Active micromixers use external energy sources, as well as fluid
pumping energy, in order to generate time-dependent perturbations
of the flow field and accelerate the mixing process [92]. Based on
the type of external force employed, micromixers can be categorised
as pressure field-driven [93], acoustic (ultrasonic)-driven [94],
temperature-induced [95] or magneto-hydrodynamic [96]. Active
micromixers have generally higher mixing efficiency compared to
passive micromixers [97]. However, the need for system integration
with peripheral devices (i.e. actuators for external power source)
and the laborious and expensive fabrication processes, have limited
the implementation of such devices in practical applications. In
addition, the use of exogenous energy sources (i.e. in the form of
ultrasonic waves) may result in the generation of high temperature
gradients which can potentially damage the bio-actives. For this reason,
active mixers are not a popular choice when applying microfluidics to
pharmaceutical, chemical and biological applications [97].
Passive mixers instead rely entirely on fluid pumping energy, and
make use of specific channel architectures to restructure the fluid flow
in such a way to reduce the diffusion length and maximize the contact
surface area. They were the first microfluidic devices reported due
to the associated cost-effectiveness and convenient fabrication
routes compared to active micromixers. Furthermore, they can be
ð6Þ
easily integrated with more complex lab-on-a-chip devices. The
reduction of mixing time can be achieved by different means, such
as splitting the fluid stream by means of serial or parallel lamination
[98,99], hydrodynamically focusing fluid streams [100], introducing
bubbles of gas (slug) or liquid (droplet) into the flow [101,102], or
enhancing chaotic advection using ribs and groves designed on the
channel walls [103,104]. An extensive overview about the different
ð7Þ
types and designs of micromixers has been provided elsewhere
[105].
3.2. Features of microfluidic reactors for the production of nanomaterials
A series of features make microfluidic reactors particularly appealing
in the production and investigation of nanomaterials as compared with
conventional macroscale reactors. Most of these features derive from
the unique characteristics of the flow in microchannels and in general
of the microfluidic environment, such as the omnipresence of the
highly predictable laminar flow and a large SVR. The implications and
ð8Þ advantages of these characteristics on nanomaterial production are
described in the following paragraphs. Briefly, these characteristics
include:
• Efficient and controllable mixing under continuous flow conditions
resulting in a homogeneous reaction environment.
• Improved and efficient temperature control and heat transfer.
• In situ monitoring of the progress of NP formation through residence
time based resolution.
• Temporal control of reactions by adding reagent at precise time
intervals during the reaction process.
• Control over characteristics of NP by controlling the kinetics of the
process.
• High-throughput screening of various formulations by on-line
variation of the process parameters.
• Opportunity to integrate post-synthesis processes and measurement
systems on a single technology platform.
• Possible scale-up of the process by increasing the number of the
microreactors.
1502 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532
The aforementioned set of characteristics reveals the significant
potential of microfluidics to transform current classical batch technology
for the production of NPs into continuous microfluidic process. Different
research groups have worked towards this aim and achieved promising
advancements, but clearly, this science is still in its infancy and
much remains to be done in order to demonstrate the superiority
of microfluidic process over the conventional batch process.
3.2.1. Micromixing and reactor design
The traditional batch methods lack precise control over the
mixing and supersaturation level, leading to uncontrolled nucleation
and growth processes, resulting in poor control over final particle
characteristics [106]. A rapid heat/mass transfer can greatly improve
the controllability of these characteristics that in turn determine
the physico-chemical properties of the produced nanomaterials.
In this respect, microscale reactors can provide homogeneous, fast
(in the order of fraction to hundreds of milliseconds) and reproducible
mixing conditions, thus potentially representing a method to obtain
nanomaterials with excellent dimensional characteristics.
Microscale mixers/reactors are characterised by a larger SVR,
offering the possibility to enhance mass and heat transfer as compared
to conventional bulk mixing systems [91,107]. This leads to a massively
reduced mixing time that becomes comparable with the nucleation and
growth kinetics, opening the possibility to exert a control on them [4].
With respect to the mixing approach and device characteristics,
microreactors for nanomaterial production can be grouped in two
categories: continuous flow and segmented flow microreactors.
3.2.1.1. Continuous flow microreactors. Continuous flow microreactors,
compared to segmented flow reactors, are generally characterised by
higher productivity and the possibility to continuously vary the reactant
composition along the reaction channel [34]. In this way, it is possible to
realise multi-step processes by connecting in series different reactors
[108,109]. Additionally, due to the simplicity of the flow pattern, process
scaling-up can be achieved by simply increasing the flow rate [110].
Continuous flow microreactors can be further categorised in three
main sub-groups depending on the architecture of the microchannel
network. These comprise capillary tube, coaxial flow and micromixer.
Capillary tube microreactors represent the simplest device
configuration. They are constituted of silica [111], steel [112] or
polymer [113] capillary tubes with channel lumen dimension in
the order of microns. They have been generally applied for the
production of metal nanomaterials, where an improved NP output
was realised through a fast and accurate temperature control. Their ease
of manufacture and operation, together with the possibility to use
robust materials able to withstand the high temperature requirements,
has attracted the attention on capillary device for the production of
metal and semiconductor nanocrystals. However, capillary devices
suffer from the risk of lumen blockage, adhesion of chemicals to the
channel surface, and relatively high product polydispersity [114].
In order to address these issues, coaxial flow microreactors have
been proposed [109,114–116]. The ensheathing flow prevents direct
contact of the reaction mixture with the channel walls thus minimising
clogging and adhesion to channel walls. In addition, the ensheathing
provides a way to minimise the polydispersity of the formed NP by
constraining the flow containing the precipitating species at the centre
of the channel, where a more homogeneous fluid velocity field subsists.
This in turn results in more homogeneous residence time distribution
(RTD) of the growing NPs within the microreactor; hence, different
growing NPs experience a similar growth process duration, with
beneficial effect on nanoparticle polydispersity. The implication of RTD
on microreactor-based nanoparticle synthesis is discussed later in this
section (see paragraph 3.2.1.2).
A broad RTD also characterises laminar flow micromixers. However,
the possibility to integrate on-chip characterisation systems and realise
tailored channel architectures, together with the potential for tuning
mixing performance, have attracted attention on the use of micromixers
for nanoparticle production. Different types of micromixers have been
conveniently applied.
A basic “Y-shaped” channel configuration was used to produce
nanoparticle of hydrocortisone, a poorly water soluble drug [117].
In this study, the authors demonstrated the production of nano-
sized dispersions, and the possibility to control the mean particle size in
the range of 80–450 nm by modifying the experimental parameters and
design of microreactors. Using a similar setup, in a subsequent research
[117], the same group employed artificial neural networks to identify
relationships between variables affecting drug nanoprecipitation,
demonstrating the critical role of antisolvent flow rate. A “T” type
micromixer has been also employed to prepare nanocrystals of
barium sulphate and boehmite [110]. The authors, leveraging the
impingement of the two streams, enhanced the mixing of the reactants,
and exerted a control over nanoprecipitation output modifying the
velocity of the impinging streams. The simple “T” and “Y” configurations
have also been integrated with on-line measurement systems to
investigate the fundamental reaction processes [118], and to provide
information for an on-line algorithm-based control of the process
parameters, which can drive the system towards a desired goal
[119].
Wilde et al. [120] introduced a radial interdigitate microreactor
based on the principle of parallel lamination for the production of
thiol functionalised gold NPs. The design was constituted of a 16 inputs
radial interdigitate mixer, which represented a modification of the
classical straight design for parallel lamination micromixer. The size
standard deviations of the NPs produced with this micromixer ranged
between 0.6 and 0.9 nm, in comparison with a range between 1.3 and
2.1 nm for the bulk synthesis. Results also indicated a control over the
final mean size diameter by controlling the flow rate and, in turn, the
residence time and the growth phase. Notably, the device was able to
mix the reactants with high volumetric throughput up to 4500 μL/min.
Parallel lamination micromixers were also used for the production of
CdS NPs [121,122]. Based on the measurement of the adsorption
spectra, it was observed that the NPs produced in the microfluidic
device were more homogeneous in terms of size, than those produced
with conventional bulk methods. Moreover, a tendency toward the
NP uniformity with the increase of the flow rate ratio was reported.
Increasing the flow rate reduced the likelihood of nanoparticle
coalescence since the NPs were extracted from the reaction volume
more quickly.
The use of a split and recombine (SAR) micromixer has been
presented for the synthesis of metal NPs [123–125], demonstrating
that the mixing conditions depend strongly on flow rate and local
geometry. However, due to the complicated 3D architecture, the
fabrication of this type of mixers might be non-trivial.
In contrast, a simple planar flow focusing architecture does not
require long fabrication process, and provides the possibility to control
the dimension of the produced nanomaterials by simply varying the
volumetric flow rates of the three inlet streams. The control over
the dimensional characteristics is particularly important in the case of
nanomaterials for drug delivery application, since the dimension
strongly influences the spatial and temporal release of the actives.
Therefore, planar flow focusing architectures have been extensively
applied for the production of different drug delivery carriers such as
liposomes [126], solid lipid NPs (SLN) [127] and polymeric micelles
[128]. The various microfluidic reactors employed for the production
of nanomaterial for drug delivery application are discussed more in
detail in paragraph 4.
When high molecular weight hydrophobic species (i.e., N45 kDa),
such as poly-lactic-glycolic acid polymer (PLGA) are nanoprecipitated
using planar focusing devices fabricated in PDMS, they tend to
aggregate on the channel inner surface, potentially resulting in
clogging of the device. The aggregation is caused by the adsorption
of the hydrophobic species on the hydrophobic walls of the device,
 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532
resulting in substantially reduced robustness of operation and,
eventually, irreversible device failure. A 3D focusing, in both the
horizontal and the vertical dimensions, has been proposed to
overcome this issue [129]. The 3D focusing device did not require
complicated fabrication process since it was composed of a
monolithic single layer characterised by three sequential inlets for
vertical focusing, followed by a conventional cross-junction. However,
since the vertical focusing was achieved through two additional
streams of solvent, it caused a dilution of the initial polymeric stream
resulting in a decrease of the attained supersaturation level.
A complete reactor system has been fabricated by integrating
different units with the mixing device [130]. It integrated pumps,
mixers, valves, micro-heaters and temperature controllers. The authors
reported a significant reduction of the reaction time for the formation of
hexagonal gold nanoparticles that passed from 30 min to 5 min for
traditional bulk and microfluidic syntheses, respectively.
3.2.1.2. Segmented flow microreactors. Alongside the laminar flow
microfluidic reactors, various authors presented alternative approaches
to the chemical synthesis of NPs based on segmented flow microreactors.
These microreactors can be further categorised as slug flow (gas–liquid)
or multiphase flow (liquid–liquid) microfluidic reactors [131,132].
In chemical synthesis of NPs, an important parameter which strongly
affects the monodispersity of the produced samples is represented by the
residence time distribution, which reflects the mean time that each
particle spends inside the reactor. In laminar flow microfluidic reactors
the parabolic flow profile (fluid moving slower near the channel wall
than in the centre) and the associated axial dispersion, cause a variation
in residence time, which in turn leads to a wider size distribution of
the produced NPs [133]. This problem with laminar flow reactors can
be overcome with using segmented flow microreactors, which lead to
a better control of nanoparticle size distribution. This is because the
droplets (liquid–liquid) or slugs (gas–liquid) can work as a microsized
reactor that flows along the channel with a time that is determined
only by the flow rate. Mixing in these microdevices is achieved leveraging
the microstreaming created within the slug or droplet while it is flowing
along the straight and winding channels.
It has been experimentally demonstrated that segmentation is
beneficial in controlling the size distribution of the synthesised NPs.
Kahn et al. [133] studied the effect of the reactor design on the synthesis
of silica NPs. Two reactor designs were compared: (i) single phase
laminar flow reactor (LFR) and (ii) segmented (gas–liquid) flow reactor
(SFR). The size of the particles produced with LFR was found to be
correlated with the residence time. Mean particle sizes increased with
residence time in the reactor, as expected, because of the larger growth
time available for the particles. Notably, the LFR produced wider particle
size distribution at higher linear velocity due to the steeper velocity
profile, which caused higher axial dispersion of the growing colloidal
particles as they flowed through the reactor. In agreement with the
theory stated above, they found that the use of a SFR could minimise
the axial dispersion effect, and lead to a narrower size distribution of
the produced NPs. Additional advantage of slug-flow reactors is the
simple separation of the gas from the final reaction mixture. Therefore,
they do not require post processing purification steps. However, careful
operation and low throughput are usually required to achieve the
stability of the multiphase gas-flow pattern [110].
Shestopalov et al. [132] reported the use of a droplet-flow (liquid–
liquid) reactor, where the multiphase flow was generated from two
immiscible fluids rather than from a gas–liquid flow. They further
demonstrated the utility of this droplet-based microfluidic method
to perform a multi-step synthesis of CdS/CdSe core–shell particles.
Slug-flow reactors present the problem of the physical contact of
the particle with the wall of the microchannel that could result in
cross contamination and channel clogging. In contrast, microfluidic
droplet-based reactors represent a possible solution to overcome this
problem, since the reaction droplets are not in physical contact with
1503
the wall, but are rather surrounded by the carrier fluid. An example of
droplet-based microreactor was presented for the synthesis of CdSe
nanocrystals in droplets flowing in a perfluorinated carrier fluid. The
droplets were formed by utilising a flow-focusing nanojet structure
with a steep increase in channel height [131].
The possibility to create stable droplet-flow not only provided a
way to control the residence time but also provided the opportunity
for running multiphase processes. Wang et al. [134] developed a
method for the formation of TiO2 NPs using a microreactor that created
a stable interface between two insoluble liquids. The two insoluble
liquid systems used in this study were: 1-hexanol/formamide and
cyclohexane/water. The interface between the two phases represented
the site of the hydrolysis reaction between titanium tetraisopropoxide
(TTIP) and water that led to the production of the titania NPs. Droplet-
based microreactors can also be used to implement multi-step synthesis
[102] by adding additional reagent to droplets through a droplets fusion
process. However, the velocity of the flows must be carefully adjusted to
permit the fusion of the droplets limiting the process versatility [132].
3.2.2. Temperature control and heat transfer
Temperature plays a critical role in controlling solubility, super-
saturation level and process kinetics during nanomaterial formation;
therefore, efficient temperature control can improve nanomaterial
synthesis. Typically, microfluidic devices contain channels with
characteristic dimension in the range 10–400 μm. This small dimension
results in increased SVR (in the range 10,000–50,000m2·m−3), compared
with 100–2000 m2·m−3 of their macroscale counterparts [135].
Typical microfluidic devices exhibit high thermal transfer
efficiencies, and this allows performing exothermic and/or high
temperature reactions in an efficient and controllable (isothermal)
manner [136,137]. Microfluidic environments have been shown to
provide an efficient temperature control in continuous flow reactors
chemical synthesis [135,138].
The large SVR also offers the possibility to accelerate heat exchange
for nanoparticle synthesis. In this respect, it has been shown that only
0.4 s were needed for a 200 μm diameter channel to heat a liquid from
20 to 300 °C [139]. Nakamura et al. reported the use of capillary tubing
in hot oil bath for the production of quantum dots. Efficient and
fine temperature control of the microreactor environment was
beneficial for controlling particle diameter and achieving reproducible
preparation of CdSe nanocrystals [139,140]. By varying the temperature
and the flow rate ratio, NPs with different size were produced. In
addition, the author reported a high reproducibility of particle size
distribution that was considered to be caused by increased accuracy
in temperature control. A method has also been reported that
utilises a three-step process in order to form CdSe–ZnS composite
NPs. The method involves immersion of capillaries in different
heated oil bathes, combined with a microfluidic mixer intermediate
step [141].
3.2.3. Kinetic control and investigation
In addition to providing efficient control over the dimensional
characteristics of the nanomaterials produced, microreactors also
offer the possibility to investigate and control the fundamental
reaction processes of the NP formation. Microfluidic devices provide
a platform for in situ monitoring of the temporal evolution of NP
formation through the ability to spatially resolve nucleation and
growth phases in the reactor during synthesis [142]. Investigation
of the mechanisms of nanocrystal nucleation and growth is critical
to understand how to control nanostructure synthesis on a
production scale. Different techniques such as spatially resolved
photoluminescence imaging and spectroscopy [118], and small
angle X-ray scattering (SAXS) [143] were used to investigate the
kinetics and mechanisms of nanoparticle nucleation and growth
during synthesis in a microfluidic reaction channel, demonstrating
1504 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532
the capability of microreactors as investigation tools on the
fundamentals of nanoprecipitation.
Nanomaterials synthesis in microfluidic devices takes also
advantage of the unique ability of microfluidic channels to operate
at continuous flow regime, allowing for spatial and temporal control
of reactions by adding reagents at precise time intervals during the
reaction process. Such a feature endows microfluidic reactors with
the ability to carry out pre– and post-treatments and multi-step
syntheses in the same reactor.
Shestopalov et al. [132] reported a droplet-based microfluidic
method for performing multi-step synthesis of quantum dots with
millisecond time-control. The system is constituted of two separate
and independent reaction zones that allow for the control of two
different reactions or stages of the same reaction. For the production
of CdS quantum dots, a mixture of CdCl2 and mercaptopropionic acid
(MPA), a solution of NaOH and a solution of Na2S were infused into
the left, centre and right inlets of the device, respectively. Droplets
were formed when these aqueous streams were infused in the oil
stream. The reaction was allowed to take place for 75 ms and then
quenched with a stream containing MPA. A comparison of the results
obtained for the reactions with and without the quenching step
indicated a decrease in monodispersity when the quenching step
was eliminated. They were also able to produce CdS/CdSe core shell
NPs infusing Na2Se instead of MPA, from the quench channel. A system
for growth of shell coatings with tuneable thickness and without
complications due to secondary nucleation has been also presented by
Jensen et al. [144], for coating colloidal silica core particles with titania
layers through controlled hydrolysis of titanium tetraethoxide (TEOT).
By controlling flow rates, reaction times and quenching procedure
Kumar et al. [36] were able to prepare Co NPs with three different
crystal structures. Results from this study seem to indicate that the
difference in flow rate ratio and residence time influences the
nucleation phase in a critical way, leading to the production of NPs
with different crystalline structures. This study suggests the possible
application of microfluidic device to obtain NPs with different
structure by controlling the process parameters, hence the kinetics of
reaction. It is well known that synthesis of NPs via metal salt reduction
is often kinetically ― rather than thermodynamically ― controlled,
leading to the possibility of generating particle in metastable phases.
Control of the crystal structure is one of the key issues in nanoparticle
synthesis since physical and chemical characteristics also depend
directly on crystal structure [145].
3.2.4. On-chip analysis
Miniaturised and integrated on-chip analysis of nanomaterial can
play an important role in high throughput characterisation of various
drug delivery formulations, accelerating the understanding of the effect
and the optimisation of the process parameters. On-chip detection
devices are situated further downstream the nanoprecipitation zone to
allow in situ measurements. Positioning the detection unit at different
positions along the channel also enables the collection of spatial and
temporal information about the reaction process. Unfortunately, few
methods exist for on-line size characterisation of the particles in the
channel. In traditional nanoparticle sizing, Dynamic Light Scattering
(DLS) is a powerful tool for in situ size analysis of nanometre structures.
In addition, DLS is ideally suited to be incorporated into microfluidic
devices because it can be adapted to measure very small fluid samples.
Chastek and colleagues reported a microfluidic chip coupled with a
DLS apparatus through fibre optic probes that provided a quantitative
measurement of micelles as small as 10nm in diameter [146,147]. Kuiper
et al. [148] described the use of an apparatus in which confocal
correlation spectroscopy (CCS) was employed for sizing both fluorescent
and non-fluorescent NPs on-chip. CCS compared to other sizing
techniques, such as light scattering, has the ability to measure dilute
samples in small volumes. With such a device, they measured particles
ranging in diameter from 11 to 300 nm.
The potential of on-line characterisation to offer a fast and effective
way to optimise reactions based on real-time readout of particle
dimensions is obvious, however, very little research has been conducted
on on-chip characterisation of NPs, and the available information does
not demonstrate comparable results with traditional off-chip methods.
In particular, both on-chip DLS and CCS provide the opportunity for
on-line measurement of nanoparticle size as small as 10 nm; however,
this limits their application when NPs with size comparable with the
lower detectability limit are produced. In this respect, 100 nm limit is
considered as the larger optimal size for NPs intended for cancer
therapy applications [149].
Notably, DeMello's group proposed an improved system constituted
by a microfluidic reactor to carry out the synthesis, and an on-line
spectrometer to monitor the emission spectra of the emergent
particles [119]. The data collected were fed into a control algorithm
that automatically updated, without any human intervention, the
reaction conditions (flow rates and temperature) driving the system
to the desired goal.
3.2.5. High throughput production
The use of microreactors for industrial-scale production of
nanomaterials requires extensive reactor parallelization, since each
individual unit provides only a small volume dedicated to reaction.
Scaling up microfluidic device size signifies a marked increase of
the volumetric flow rate of the liquid that is processed through the
microdevice, according to the following equation [34]
Q ¼ U·A ð9Þ
where Q is the volumetric flow rate, U is the average fluid velocity,
and A is the cumulative cross-sectional area of the microchannels.
From the moment that a significant increase in fluid average velocity
generally results in undesired increase of the pressure drop across
the microchannel, a scale-up strategy usually involves increasing
the cumulative flow through the cross-section of microchannels,
by increasing the number of microreactors. Generally, there are
three levels of scale-up:
• Increasing the number of channels arraying identical channels in the
same lamina.
• Increasing the number of layers adding multi-layers with channel
arrays.
• Increasing the number of devices using identical devices connected in
parallel.
In order to achieve an efficient scale up of NP production in
microfluidic channel, the same flow rate must be conveyed into all the
arrayed microchannels. In fact, as explained in the previous section,
the flow rate across the channel is important in controlling the reaction
condition such as heat and mass transfer, and species residence time
[150]. The use of pumps for each channel can meet the requirement of
uniform distribution of the flow but is not practical from the standpoint
of the cost. A more reasonable strategy consisted of distributing the flow
from a common reservoir through the microchannels to a common
product reservoir. However, the distribution of flow rate uniformly in
each channel is not an easy task, and so far, only few authors have
conducted investigation in this direction.
Amador et al. [150] studied, using a method based on electrical
resistance networks, the flow distribution in arrayed microchannel for
two different manifold structures called “consecutive” and “bifurcation”.
They found that in the absence of channel dimensional variations
the bifurcation structure always produced flow equipartitioning as long
as the length of the straight channel after each channel bend was
sufficient for a symmetrical velocity profile to develop. Commenge et al.
[151] analysed the influence of the geometrical dimensions of the
reactor microstructure on the velocity distribution between channels,
and optimised the flow distribution in a multichannel system with
 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532
a consecutive distribution. The results obtained were used to design
geometries resulting in uniform velocity distributions between the
channels for single-phase flow.
3.3. Materials used in the fabrication of microfluidic chip for the production
of nanomaterials
The fabrication techniques of microfluidic reactors are derived
from microelectronic technology, where silicon is extensively used
[136,152,153]. The use of monocrystalline silicon is justified because
of its ready availability, the possible integration with electronic
circuits and its physicochemical characteristics that are compatible
with a large number of applications.
Besides silicon, glass has also found application in microreactors
fabrication. Glass is a particularly favourable substrate material for
microfluidic reactors because it has good optical properties, efficient
heat dissipation and a high resistance to mechanical and chemical stress
[154]. The most commonly used glass types in microfluidics are
borosilicate, quartz and crown white glass (because of their excellent
optical properties) and soda-lime glass. In addition, their low cost
meets the economic requirement of disposable devices.
However, the main disadvantage in using glass as a substrate is its
amorphous structure. This can generate non-parallel walls when it is
processed using an isotropic HF wet etching technique. Because the
etching process occurs on the exposed glass surface, as the channel
etches deeper the side walls are also etched leading to low aspect
ratio channel geometries. To obtain a very deep channel, a dry etching
technique such as deep reactive ion etching (DRIE) is used, but this
requires expensive instrumentation to be performed [152].
The long cycle times and sophisticated equipment associated with
silicon and glass microfabrication require more laborious and less
accessible fabrication methods. Polymers have assumed the leading
role as substrate materials for microfluidic devices in recent years
[155], of which poly(dimethylsiloxane) (PDMS) has become the
preferred material for the construction of microfluidic devices because
it can be easily moulded and patterned into channels, it can reproduce
micrometre-sized features with high fidelity, it is optically transparent
and it has low permeability to water. However, the major disadvantage
of PDMS in organic nanoparticles synthesis is its organic solvent
resistance; it can swell in contact with organic solvents, such as aliphatic
and aromatic hydrocarbons, and even dissolve in amine and strong
acids (e.g. sulphuric and trifluoroacetic acid) [156]. Alternative polymer
materials (i.e. modified PDMS, acrylates, PEEK and COC) have recently
been considered in a rapid prototyping technique to fabricate more
solvent-resistant microfluidic reactors [157–159].
Procedures of microfluidic devices fabrication are not addressed
here, since they fall out of the scope of this chapter. The reader is
therefore directed to a number of excellent, recent review articles and
books on this specific subject [152,155,160,161].
4. Microfluidic production of nanoparticle and vesicular systems
In the past decade, there has been a tremendous interest in the
development of microfluidic methods for chemical synthesis of inorganic
nanoparticles for the production of nanocrystalline semiconductors with
tuneable size and uniform distribution of size [35]. However, there has
been much less research on microfluidic methods for the production of
organic nanoparticles and colloidal systems.
The reason for the investigation of such type of materials mainly
stems from their possible use as innovative pharmaceutical formulations
able to increase, in different ways, the efficacy of poorly soluble drugs. In
the following section the various strategies applied for the microfluidic-
based production of nanomaterials and vesicular systems for drug
delivery applications will be reviewed. For the sake of clarity, we have
presented the various strategies dividing them in terms of type of
nanomaterial produced.
1505
4.1. Organic nanoparticles
A summary of the reviewed studies reporting microfluidic-based
production of organic nanoparticles is provided in Table 1, together
with the type of nanoparticles produced and the microfluidic strategy
adopted.
The group of Yao reported a novel method of generation of solid lipid
nanoparticles (SLN) using a flow focusing strategy [127,162]. Solid lipid
nanoparticles are a drug delivery system constituted of biodegradable
physiological lipids or lipid substance, and represent an alternative
drug delivery system to liposomes and polymeric nanoparticles [163].
The microfluidic method presented involves the precipitation of
nanoparticles from a solution of lipid in water-miscible solvent when
it is brought in contact with two lateral aqueous streams. Using this
strategy, the authors were able to produce SLN, with an almost spherical
shape, in a range of mean diameters between 100 and 200nm by simply
varying the flow rate ratio of the two phases.
Ali and colleagues [117] recently reported a microfluidic strategy
based on and Y-shaped micromixer for the preparation of hydrocortisone
nanosuspension through a nanoprecipitation technique. A relatively
stable aqueous hydrocortisone nanosuspension was obtained, with
nanoparticle dimension being controlled by flow rate and design of
microfluidic reactor. A similar technique was adopted by Su et al. [164]
to produce 2,2-dipyridylamine (DPA) nanoparticle. Interestingly they
used a commercial T-junction micromixer connected to 0.5mm ID tubing
as microreactors instead of a more conventional planar microfluidic
device. Genot et al. [165] proposed the use of a simple 3D microfluidic
device in order to solve the problem of fouling and clogging within the
main channel due to the deposition of the precipitating species. They
constructed a simple PDMS Y-junction device in which they later inserted
a glass microcapillary positioned into the intersection between the
channels. Without any particular laborious microfabrication process
they were able to produce a stable 3D focused stream and estimated
mixing efficiency and residence time for the production of rubrene
nanoparticles as model organic nanomaterial.
Finally, Dev et al. [166] reported the use of an unusual mixer for the
solvent-free production of crystalline meloxicam nanoparticles. The
procedure involved the mixing of an alkaline solution of the drug with
a citric acid solution within a rotating tube processor (RTP) [167]. The
RTP consisted of a 6 × 30 cm (diameter × length) aluminium tube which
rotated at a speed between 100 and 2000 rpm. The two solutions were
fed at one end of the tube. When the solution came in contact with the
inner wall of the tube, it spread on its surface and the rotating movement
intensified the mixing process. Authors reported that the fast mixing
kinetics within the RTP caused the formation of thermodynamically
unstable semicrystalline nanoparticles which slowly turned to needle-
like particles after 2 h from preparation.
4.2. Nanomaterial from self-assembly of amphiphilic molecules
The synthesis of drug carriers, such as liposomes [168], niosomes
[169] and polymeric micelles (PMs) [1,128,170–172], produced
through the self-assembly of amphiphilic molecules (phospholipids,
Table 1
Summary of the studies investigating microfluidic-based production of organic
nanoparticles.
Nanoparticles type Microfluidic strategy References
Solid lipid nanoparticles (SLN) Planar flow focusing mixer Zhang et al. [127],
Yun et al. [162]
Hydrocortisone nanosuspension Y-shaped mixer Ali et al. [117]
2,2-dipyridylamine (DPA) T-shaped mixer; multiphase Su et al. [164]
Rubrene 3D flow focusing mixer Genot et al. [165]
Meloxicam Rotating tube processor (RTP) Dev et al. [166]
1506 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532
non-ionic surfactants and block-copolymers, respectively) has also
been investigated. The aggregation of amphiphilic molecules
to form nanomaterial slightly differs from traditional nucleation
and crystal growth that govern the nanoprecipitation of non-
amphiphilic molecules (i.e. inorganic material and hydrocortisone)
[173]. In the nanoprecipitation of non-amphiphilic molecules, the
size of the nanoparticles is predominately controlled by the level of
supersaturation of the solute to be precipitated, and by the duration
of the precipitation process. These two parameters can be finely
adjusted in microfluidic devices by varying the relative flow rate
ratio of the solvent and non-solvent streams and the residence
time distribution respectively, as demonstrated by other researchers
[117,132,144]. In contrast, the size of nanoparticles obtained from
amphiphilic molecules is dictated by the magnitude of the steric
and electrostatic interaction between the amphiphilic molecules,
which is controlled by the polarity of the surrounding solvent
environment (i.e. solvent vs. non-solvent ratio) [173–176]. As the
mixing between solvent and non-solvent proceeds, the polarity of
the environment surrounding the amphiphilic molecules increases
(i.e. increase in water content), and forces the closure of the growing
assembly to minimise the exposure of the hydrophobic portion to
the aqueous environment. As a result, an insertion barrier is created
that prevents new amphiphilic molecules from entering inside the
closed assembly, resulting in kinetically quenching of the growth
phase [173–175].
For example, HFF-based reactors provide the possibility of tuning the
mixing time by changing the width of the focused stream. This gives
opportunity to control the rate of solvent shift, and exert a control over
the length of the aggregation time before the formation of kinetically
quenched nanoparticles. This ultimately results in control over the
assembly process, resulting in tuneable characteristics of the produced
nanomaterial. It is important to note that when drug and amphiphilic
molecules are co-precipitated the two mechanisms of aggregation, for
amphiphilic and non-amphiphilic molecules, act in concert to determine
the nanoparticle characteristics [1].
A summary of the reviewed studies reporting microfluidic-based
production of NPs formed by self-assembly of amphiphilic molecules
is reported in Table 2.
4.2.1. Lipid vesicles
The bulk hydration of lipids in aqueous buffer generally yields large,
polydisperse and multilamellar lipid vesicles (i.e. constituted of multiple
lipid bilayers concentrically organised as an onion-like structure). This
method and other traditional bulk production methods, such as freeze–
thaw cycling, film hydration and reversed phase evaporation, are often
Table 2
Summary of the studies investigating microfluidic-based production of nanomaterials from self-assembly of amphiphilic molecules.
Self-assembly type Microfluidic strategy
Liposome Electroformation
Liposome Hydration
Liposome Extrusion
Liposome Planar flow focusing mixer
Liposome 3D flow focusing mixer
Liposome Ultrasonic mixing
Liposome Pulsed jetting
Liposome Transient membrane ejection
Liposome Double emulsion template
Liposome Droplet emulsion transfer
Lipid-stabilised microbubbles Planar flow focusing mixer
Niosomes Planar flow focusing mixer
Polymersomes Emulsion templates
Polymersomes Mixing-based
Polymer-stabilised microbubbles Emulsion templates
associated with heterogeneous and poorly-controlled chemical and
mechanical conditions during lipid vesicles formation, and result in
non-uniform vesicles in both size and lamellarity.
On the other hand, a range of different methods have been
implemented for the production, manipulation and analysis of lipid
vesicles ― such as liposomes ― within microscale environments
[177,178]. In recent years, considerable attention has been devoted
particularly to the implementation of microfluidic-based strategies for
the production of lipid vesicles, with the aim of overcoming the above
mentioned limitations associated with conventional batch systems.
In this regard, it is possible to distinguish between methodologies
which mostly rely on the scaling-down of analogous macroscale
techniques and methodologies which have been designed specifically
for application at the microscale. In the present paragraph, an overview
about recent advancements in the field of microscale production of lipid
vesicles is provided.
4.2.1.1. Electroformation. Electroformation has been widely-adopted for
producing lipid unilamellar vesicles [179]. The process has been firstly
described in 1986 by Angelova and Dimitrov [60] and involves the
application of an electric field, typically alternating, across a lipid film
which has been previously deposited on the surface of an electrode
and immersed in an aqueous solution. In this way, lipids interact with
the aqueous solution and the applied electric field by peeling off the
surface of the electrode in layers and self-assembling into giant vesicles.
It is believed that the electric field generates fluctuations in the lipid
bilayers which are responsible for the film detachment [179].
This method has been successfully translated from the macroscale to
the microscale, firstly by Kuribayashi et al. [180,181]. In this works,
authors produced various types of giant liposomes encapsulating nano/
micro-functional materials (i.e. fluorescent polystyrene beads) within
squared cross-section microfluidic channels. Channels were sandwiched
between glass layers which were previously coated with indium tin
oxide (ITO) electrodes. An electric field was applied across the electrode,
initiating the vesicle formation process. The method was compared with
other techniques (i.e. gentle hydration) showing increased number of
giant liposomes and reduced enclosing of smaller liposomes within larger
ones [181]. However, liposomes obtained with this technique were
polydisperse, implying that further technological advancements were
required to improve control over vesicles dimensional characteristics.
In order to address this issue, a development of this set-up was later
presented [182], which differentiated from systems previously available
given that the lipid film was micro-patterned onto the electrode surface.
This was achieved by deposition of a phospholipid solution onto a film
of Parylene, which was previously patterned by photolithography and
References
Kuribayashi et al. [180–183], Le Berre et al. [184], Diguet et al. [185]
Lin et al. [189,190], Osaki et al. [192]
Dittrich et al. [193]
Jahn et al. [126,195,199], Wi et al. [196], Zook et al. [197]
Kennedy et al. [200], Paphal et al. [202]
Huang et al. [203], Yamashita et al. [204]
Funakoshi et al. [209], Stachoviak et al. [211,213], Richmond et al. [212]
Ota et al. [214], Kurakazu et al. [215]
Shum et al. [210], Tan et al. [217], Teh et al. [218], Davies et al. [221]
Matosevic et al. [228]
Hettiarachchi et al. [205,207], Talu et al. [206]
Lo et al. [169]
Shum et al. [230,231], Duncanson et al. [229], Seth et al. [232],
Kim et al. [233,235,236,239], Perro et al. [234], Thiele et al. [238]
Thiele et al. [241], Brown et al. [242], Thiermann et al. [243]
Lee et al. [240]
 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532 1507

etched with O2 plasma. Thus, by removing the Parylene film, authors
obtained a micro-patterned lipid film. With this technique, it has
been demonstrated that different liposome shapes could be obtained
depending on the spatial arrangement of the patterns. Notably,
densely-spaced patterns resulted in the production of balloon-like
liposomes, while sparsely-spaced patterns resulted in the production
of hemi-spherical dome-shaped liposomes.
An analogous microfluidic-based device was employed to produce
solvent-free lipid membranes by electroformation [183]. For this
purpose, a stack of lipid-coated Parylene masks was aligned onto a
micro-patterned Cr/Au aperture array. Circular lipid patterns aligned
with the micro-apertures were created by removing successive layers of
Parylene. A second electrode was then placed above the micropatterned
surface and an electric potential was applied across these plates, resulting
in the formation of solvent-free lipid membranes. This approach allowed
overcoming limitations associated with the difficulty of removing
solvents from the formed lipid vesicles, which can potentially limit their
application as in-vitro models of biological cells. Notably, the correct
functioning of membrane proteins may be significantly compromised
by the presence of residual organic solvent within the lipid bilayer.
Significant steps towards increased control over liposome size were
further demonstrated by Le Berre et al. [184] (Fig. 2a–b) and Diguet
et al. [185] by combining surface micro-patterning and electroformation.
In these works the authors deposited lipid films onto a silicone (Si)
substrate having controlled surface properties (Fig. 2a). By changing the
chemical and physical properties of the Si surface, it was demonstrated
that the properties of the final vesicles (i.e. size and size distribution)
could be finely controlled (Fig. 2b). It should be highlighted that these
techniques were capable of producing monodisperse liposomes, thus
representing a significant improvement over standard electroformation
methods.
To conclude, it must be mentioned that the effect of the applied
electric field on the integrity of membrane proteins or other bio-active
molecules has not yet been fully investigated [177], hence representing
a subject of future studies, although incorporation of functional proteins
within lipid vesicles membranes produced by electroformation has
been already reported [186,187].
4.2.1.2. Hydration. Hydration is another widely-adopted method to
produce lipid vesicles, in the form of either multilamellar and unilamellar
giant liposomes [179,188]. In this method, an osmotic pressure gradient
drives water from an aqueous buffered solution into a dried lipid film,
causing the lipid layers to separate [179]. Furthermore, by imposing a
fluid shear stress over the lipid-coated surface results in lipid film rupture
and self-assembly into liposomes [177]. However, due to the poor
control over film peeling, vesicles obtained with this method are generally
polydisperse and multilamellar. Additionally, the performance is
extremely sensitive to the physical properties of the fluid medium,
including pH and temperature. In an attempt to overcome these
limitations, hydration has been performed within microfluidic-based
devices where individual physical variables can be controlled more
precisely. The translation of this method in a microscale system has
been firstly described by Lin et al. [189,190] (Fig. 2c). Authors fabricated
a squared cross-section microchannel within a PDMS layer by soft
lithography technique, which was then bonded to a glass layer. Two
cavities were created for buffer pumping and lipid film hydration,
respectively. Lipid microtubes (1–10 μm in diameter) and vesicles were
formed by flowing a buffered aqueous solution over the lipid film
(Fig. 2c). With this technique it was possible to control the type of
obtained lipid structures by regulating the governing physical parameters
(i.e. fluid flow rate). However, the formed liposomes were neither
unilamellar nor monodisperse.
In order to achieve better control over vesicles size, a technique
combining micropatterning and gentle hydration was later developed
by Osaki et al. [191]. Authors performed selective lipid patterning by
electrospray deposition, following a protocol previously described
by the same group [192]. The patterned substrate was fabricated
by standard photolithography and wet-etching methods. Hydration of
the lipid film resulted in formation of giant liposomes uniform in size,
which diameter could be controlled by regulating the size of the
patterned features. Lipid vesicles produced with this method were
arrayed directly on the substrate, which makes this technique suitable
for on-time in situ bio-chemical analyses.
However, one drawback of hydration methods is the need for lipid
film deposition prior to each process iteration (i.e. vesicle production
step), which limits its potential application in large-scale vesicles
production systems due to the laborious and non-continuous nature
of the procedure [177].
4.2.1.3. Extrusion. An alternative method to produce unilamellar
liposomes with controlled size is represented by extrusion [179]. In
this technique, vesicle dispersion is forced through a membrane with
pores of controlled size, during which lipid membrane rupture and
resealing occur. Vesicles are generally extruded multiple times, until
the desired size is achieved. Notably, the latter can be controlled by
regulating the size of the pores. This technique has been performed in
a microfluidic device by Dittrich et al. [193], with further modifications
with respect to standard macroscale approaches. In this study, a 200 nm
thick micropatterned membrane of silicon nitride was fabricated and
sandwiched between two PDMS layers with channels for fluid delivery

Fig. 2. Microfluidic-based production of lipid vesicles. (a) Schematic of a microfluidic device employed to form liposomes by electroformation. Phase-contrast microscope images of
liposomes obtained by using this device and collected by gravity are reported in (b), for either ITO (left) or Si (right) substrate (reprinted with permission from [184]. Copyright 2008
American Chemical Society). (c) Microscope image illustrating the formation of a single lipid microtube in a PDMS microchannel, by hydration method upon application of a fluid flow
over a thin lipid film (reprinted from [190], Copyright 2006, with permission from Elsevier). (d) Schematic of the process of liposomes formation by self-assembly in a microfluidic
HFF device. Contours of isopropanol/water ratio are reported, illustrating diffusion-governed mass transfer along the microchannel. Furthermore, a three-dimensional contour map of
the fluorescence intensity of a membrane intercalating agent (DiIC18) at the focusing region is illustrated in (e), providing information on the spatial evolution of liposomes formation
(reprinted with permission from [126], Copyright 2004 American Chemical Society).
1508 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532
and release. 3 μm diameter apertures were created by photolithography
technique on the silicone membrane, which was later coated with
an ~ 50 μm multilamellar lipid film. By applying a fluid flow either in
the top or in the bottom channels, the lipid layer was extruded through
the apertures and micrometre-sized lipid vesicles and tubes were
formed. Importantly, the dimensional properties of the obtained
vesicles and tubes were observed to depend on both the size of the
apertures and the pressure difference between the top and bottom
channels. A strong pressure difference favoured the formation of lipid
tubes, while a low pressure difference resulted in the formation of
vesicles. Despite the formed vesicles were polydisperse, a considerable
advantage of this technique was that vesicles could potentially be
generated with high flow speed, making it potentially suitable for
large-scale production applications. However, set-up implementation
(i.e. membrane coating) is non-trivial [177], which poses some
limitations to its widespread usage. Furthermore, the lack of isolation
between the solution to be encapsulated and that in which the
produced vesicles are stored, likely results in poor encapsulation
efficiency [194]. These limitations may represent the subject of future
investigations.
4.2.1.4. Hydrodynamic flow focusing. As mentioned earlier, the most
popular way of producing lipid vesicles in a microfluidic system is
represented by hydrodynamic flow focusing (HFF) [195]. In this
method, a central flow stream of a lipid-containing alcohol solution is
intersected and sheathed by two lateral streams of aqueous solution
(Fig. 2d–e). The lipid stream is hydrodynamically focused into a narrow
sheet, which size can be varied by regulating the volumetric flow rate
ratio between central and lateral streams. Either lateral (i.e. two-
dimensional) or co-axial (i.e. three-dimensional) focusing of the central
stream have been demonstrated [105].
It is believed that, with this technique, the formation of liposomes is
governed mainly by the diffusive mass transfer of species at the miscible
liquids interface. Notably, the alcohol diffuses into the aqueous solution,
until it reaches a critical concentration (i.e. below the solubility limit of
lipids) which triggers the formation of lipid vesicles by self-assembly,
leading to the formation of small, monodisperse and unilamellar
vesicles. It is technically complex to obtain experimental evidence of
the dynamics of vesicle formation in situ, however, it is believed that
the reduction of lipid solubility associated with alcohol and water
diffusion across the two streams causes thermodynamic instabilities at
the edges of bilayer phospholipid fragments (BPF), which bend and
close upon themselves forming the vesicles [195].
The main advantages of this technique include: (i) the viscous-
dominated flow regime (i.e. Re ≪ 1) allowing to finely control the
interfacial region between the fluid streams; (ii) the small scale which
makes the diffusive mass transfer an effective mixing method; (iii) the
possibility to precisely regulate the boundary conditions, which permits
to adjust on-demand the governing physical parameters (i.e. residence
time of solutes); (iv) the continuous-flow format, which can be exploited
for large-scale production of lipid vesicles; and (v) no further post-
processing steps to homogenise vesicles size are generally required.
The first work to have demonstrated production of liposomes using
this method was reported by Jahn and coworkers [126] (Fig. 2d), in
which a trapezoidal cross-section microfluidic device with an angle of
90° between the central and lateral inlet channels (Fig. 2d–e) was
employed to produce monodisperse liposomes in the size range
100–250 nm. Importantly, it was demonstrated that by varying the flow
rate ratio (i.e. ratio between the flow rate in the side inlet channels and
the flow rate in the central inlet channel) it was possible to control the
mean size and the size distribution of the obtained liposomes. Notably,
higher flow rate ratio was associated with lower vesicle size, which
was attributed to the increased shear stress exerted on the liposomes
during self-assembly. Furthermore, continuous-flow encapsulation of
high concentration of a fluorescent probe (carboxy-fluorescein, CF) was
demonstrated with this technique.
A development of this device was later presented [168], which was
characterised by a multiple inlet channel configuration allowing for
water-soluble substances to be encapsulated to the immediate vicinity
of the alcohol stream where the formation of liposomes occurred, thus
reducing sample consumption. Furthermore, channels were deeper and
with a higher aspect ratio, resulting in a more homogeneous velocity
profile across the channel height, reducing surface effects in close
proximity to the top and bottom walls of the channels. In this study,
authors investigated the effect of total flow rate and flow rate ratio on
liposome characteristics. Contrary to previous studies [126], it was
reported that by varying the total flow rate, at a constant flow rate
ratio, had no significant impact on liposome size and size distribution,
which suggested that the absolute magnitude of the shear forces between
the streams had no significant impact on liposomes dimensional
properties. Furthermore, an inverse relationship between flow rate ratio
and liposome mean diameter was determined, which was coherent
with previous studies and was attributed to changes in the dynamics of
species mass transfer across the streams, rather than to the effect of
shear forces at the interface between streams as postulated in previous
studies [126].
In a further study by Jahn et al. [176] the effect of device design
on liposome characteristics was investigated. For this purpose,
different intersection angles between the lateral and central
inlet channels (i.e. 45° and 90°) and different channel cross-sectional
dimensions were considered. Results revealed that microfluidic device
size and geometry did not affect significantly the properties of the
produced vesicles; however, they may have distinct and important
technological implications, since larger devices may be easier to fabricate
and operate, and are generally associated with higher throughput
performance compared with smaller devices (i.e. due to higher flow
rates achievable). These studies clearly showed that nanometre-sized
liposomes with a very narrow size distribution could be produced using
continuous-flow microfluidics and opened the way for potential
applications for on-demand liposome-mediated delivery of point-of-
care personalised therapeutics.
Towards this goal, Wi et al. [196] recently developed a HFF
microdevice for producing liposomes, which were used as delivery
vehicles of genes into a porcine kidney cell line. Liposomes were produced
using PDMS microchannels, fabricated by soft photolithography
technique. At the outlet, a 45° channel intersection was designed
to separate the smaller liposomes (diameter at 25 °C:~200–300 nm) ―
which diffused laterally ― from the larger liposomes and lipid molecules
(diameter at 25 °C: ~400–600 nm). The effect of processing temperature
and flow rate ratio on liposomes size distribution and zeta potential was
evaluated in this study. Liposomes size was observed to be virtually
invariant with changing the flow rate ratio in the range 2–7, while
temperature had a significant effect on liposomes size. With this respect,
liposomes produced at the higher temperatures (i.e. 60 °C) were smaller
than liposomes produced at environmental temperature (i.e. 25 °C). This
was attributed to the reduced interfacial tension between lipid and buffer
streams at the higher temperatures, which favoured species mixing
between the two streams. Furthermore, zeta potential was quantified as
an index of colloidal stability and it was found that temperature had no
significant effect on it, while increasing the flow rate ratio resulted in a
reduction of zeta potential, likely due to the reduced vesicles size [196].
It must be noted that one drawback of HFF devices produced using
deformable PDMS is the reduced control over the hydrodynamic field
due to undesired changes in channels cross-sectional area, particularly
at the higher flow rates required for large scale production [169].
Further investigations about the role played by the operating
temperature on the properties of liposomes formed by HFF have been
performed by Zook et al. [197], by using a microfluidic device fabricated
by ion etching of Si layers. Authors demonstrated that a kinetic theory
could be applied to describe lipid vesicles formation [198], which
provided a suitable hypothesis to explain ― at least qualitatively ― the
results obtained by various liposome formulation strategies currently
 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532
employed. According to this theory, the liposome size mainly depended
on the effect that the operating temperature exerted on the line tension
and the elasticity modulus of the lipid bilayer. In particular, it has been
postulated that, at temperatures lower than the gel-to-liquid crystal
phase transition, the lipid membrane is stiffer and the planar bilayer
discs continue to grow until they reach an energetically favourable
size at which they bend and form spherical liposomes. It was further
demonstrated that, at temperatures not too far from the phase transition,
lower flow rate ratios resulted in the formation of large lipid aggregates
within the focusing region, while at the higher flow rate ratios it was
possible to form medium-sized liposomes (i.e. radius b 50 μm). Results
from this study suggested that considerable attention must be paid to
the transition temperature when producing liposomes using microfluidic
systems [198]. However, further investigations are required in order to
get an all-encompassing understanding of the dynamics of lipid vesicles
formation, and the role played by the local physical properties of the
micro-environment on liposome characteristics.
In order to facilitate the understanding of liposome formation
mechanisms within HFF microdevices, a recent study by Jahn et al.
[199] reported a relevant technological advancement allowing for
instantaneous immobilisation of the focused stream by rapid freezing
with liquid propane, and subsequent cryo-ultramicrotomy and cryo-
SEM imaging of formed liposomes and disc-like intermediate lipid
structures. Interestingly, randomly distributed clusters characterised
by high number densities of spherical and flattened lipid vesicles
were observed, which formation was attributed to the occurrence of
hydrodynamic instabilities between liquids of different dynamic
viscosity. Using this method, the characteristics of lipid vesicles could
be investigated without altering the physical properties of the
molecules of interest. Notably, this technique could be extended to the
production of other nanoparticles, including polymeric micelles and
organic nanoparticles.
While studies mentioned above mostly relied on a two-dimensional
focusing of the lipid stream; co-axial or three-dimensional focusing has
been recently reported, in which the central stream is sheathed in
a cylindrical core at the centre of the channel. This approach allows
overcoming the hydrodynamic effects resulting from the interaction
between the central stream and channel walls, and it is also associated
with a more uniform velocity profile in the focused stream region.
Recently, in a study from Kennedy et al. [200] the interplay between
fluid dynamics and mass transfer within a PDMS-based co-axial
flow focusing microdevice has been thoroughly investigated (Fig. 3a),
together with its effect on the properties of the produced liposomes.
In this device, a lipid solution was focused at the centre of a
125 μm × 125 μm squared cross-section channel [201]. Authors
performed CFD simulations and confocal microscopy acquisitions
to characterise species advection–diffusion along the channel, and
computational results showed agreement with experiments (Fig. 3b)
regarding the shape of the concentration distribution along the
microchannel. Liposomes in the size range 100–300 nm were produced
with this technique and the size of the produced vesicles was observed
to increase with increasing the ionic strength, which was attributed
to competition for water solvation of lipids by salts. Furthermore,
increasing the concentration of phospholipids in the core solution
resulted in the production of smaller liposomes, which was coherent
with previous studies [197]. It must be noted that liposomes produced
with this technique were larger than the ones produced in previous
studies, at comparable sheath-to-core ratios. This is likely due to
differences in microchannel size and architecture, and in the molecular
weight of the lipids employed.
In a recent study, Phapal et al. [202] further investigated the
mechanisms of liposomes formation within 3D HFF microfluidic devices.
For this purpose, an easily reproducible technique was developed
for fabricating microchannels within a PDMS block. With using this
technique, authors demonstrated that at Peclet numbers, Pe≫1, the
size of the formed liposomes depended significantly on the nature of
1509
micro-convective mixing, rather than on diffusive mixing only
(conforming to Pe≪1). With this respect, two hydrodynamic regimes
were identified, one characterised by hydrodynamic instabilities induced
by viscous stratification (at low Re), leading to gentle mixing and lager
liposomes. In the second regime, mixing between the streams did not
occur within the microchannels, but streams mixed as droplets formed
at the outlet when collected in a reservoir. This vigorous mixing led to
the formation of smaller liposomes. Further experimental and theoretical
investigations may be required to support this fascinating hypothesis. In
this respect, the technique recently developed by Jahn et al. [199] may
be employed to further support these experimental findings.
It is worth mentioning that ultrasonic waves have been employed
to enhance species mass transfer within microfluidic environments
[92,94], and few studies have been reported regarding the combined
use of microfluidics and ultrasound (US) for liposomes production. For
example, in a study by Huang et al. [203] a HFF microfluidic device has
been immersed in an ultrasonic chamber and statistically-significant
reduction of mean liposomes size by sonication was demonstrated.
This could be attributed to the increased mixing, resulting from
the generation of cavitational events within the acoustic chamber. In a
study by Yamashita et al. [204] ultrasound exposure of a previously-
formed liposome solution was observed to markedly reduce vesicle size
distribution if performed in a microscale domain (i.e. microcapillary),
rather than in a macroscale system. These preliminary experimental
findings suggest that US may be employed as an external physical
stimulus to trigger or control liposomes formation, thus opening the
way for innovative technological advancements in this research field.
Towards this, a more pervasive understanding of the mechanisms
governing liposomes formation and US–liposome bilayer interaction is
required.
To conclude, it is worth mentioning that the use of microfluidic-
based HFF has been also reported for the production of lipid stabilised
microbubbles which are used as ultrasound contrast agents or delivery
vehicles of bioactive molecules (i.e. anticancer drugs) for US-enhanced
targeted drug delivery [205–207]. Notably, the performance of lipid-
stabilised microbubbles exposed to ultrasonic waves, and associated
biological effects have been recently investigated by using microfluidic
devices [208].
4.2.1.5. Pulsed jetting and transient membrane ejection. The formation of
liposomes by pulsed jetting has been firstly reported by Funakoshi
et al. [209]. In this method vesicles are formed by mechanical
deformation of a planar lipid bilayer established between two aqueous
layers (i.e. two water droplets surrounded by an organic solvent).
Membrane deformation is generated by the action of a finely-
controlled pulsed liquid jet against the lipid bilayer (Fig. 3c). Micro-
nozzles or micro-pipettes are generally employed for this purpose.
The main advantage of this method is that materials contained in the
fluid jet are directly encapsulated in the formed vesicles, with high
efficiency (Fig. 3d–e) [177]. For example, the encapsulation of Jurkat
cells and chromosomes [209] (Fig. 3d–e), nanoparticles and membrane
proteins [211], and signalling lipids [212] have been achieved. With this
technique, mostly unilamellar, monodisperse giant vesicles have
been produced; however, under certain operating conditions, smaller
satellite vesicles can be formed. The average vesicle diameter of both
giant and satellite vesicles has been observed to increase with
increasing the dispense time [209].
In a study by Stachoviak et al. [211], a microfluidic-based pulsed-
jetting system was developed for increased control over the boundary
conditions and the micro-environment. Interestingly, high-speed video
microscopy was employed to reveal the dynamics of fluid–membrane
interaction and vesicle formation. Monodisperse giant vesicles (diameter:
~200μm) were obtained in this study. Furthermore, it was suggested that
a combination of low-volume, high-velocity pulses from a small nozzle is
required for reducing the size of the formed vesicles.
1510 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532

Fig. 3. Microfluidic-based production of lipid vesicles. (a) Schematic depiction of a microfluidic system for liposomes formation based on a three-dimensional HFF. Concentration contours
of a fluorescent solution reconstructed from confocal microscopy acquisitions illustrate device focusing performance (b) (reprinted with permission from [200]. Copyright 2012, American
Institute of Physics). (c) Formation of lipid vesicles by pulsed jetting method. With this technique, cells can be efficiently encapsulated within the vesicle, as demonstrated from
microscope acquisitions (d–e) (reprinted with permission from [209]. Copyright 2007 American Chemical Society). (f) Mechanism of lipid vesicle formation by W/O/W double emulsion
templates. (g) Images of the formation of phospholipid-stabilised W/O/W double emulsions in a glass microcapillary device (top). Optical micrograph of the collected double emulsions.
Drops are characterised by an aqueous core surrounded by a solvent shell containing phospholipid (bottom) (reprinted with permission from [210]. Copyright 2008 American
Chemical Society).

A development of this apparatus was later presented [212,213],
in which an inkjet nozzle was employed to generated fluid jets with
finely regulated characteristics. With this method, control over vesicle
membrane composition, asymmetry and content was demonstrated;
thus making it suitable for highly efficient formation of in-vitro models
of biological cells and high-throughput encapsulation of bio-active
molecules for drug delivery applications.
However, despite the aforementioned advantages, the formation of
liposomes by pulsed jetting represents a complex technological challenge,
requiring advanced engineering tools and skills, and application-specific
optimisation of the experimental protocols depending on the physical
properties of the fluids and the micro-environment [177]. Furthermore,
it has not been clarified yet if the high shear stress levels associated with
fluid jetting may potentially compromise the integrity of encapsulated
bio-molecules or cells [177], thus opening the way for further
investigations in the field.
An analogous technique, referred to as transient membrane ejection
[177], has been developed by Ota et al. [214] for continuous generation
of monodisperse unilamellar vesicles. In this study, a lipid film was
formed in a T-junction, by sequentially infusing water, oil, and water
into the PDMS-based microfluidic device. The cross flow at the junction
acted on the lipid membrane, which was continuously thinned, sheared,
and squeezed. This resulted in the membrane releasing multiple vesicles,
which self-assembled encapsulating water droplets with uniform size.
Notably, fluid displacement within the microchannels was generated by
laser-controlled expansion of a gas bubble in the aqueous solution.
Liposomes produced with this technique were monodisperse (mean
diameter: ~16.5 μm), cell-sized and unilamellar. Furthermore, efficient
encapsulation of bioactive molecules, such as membrane proteins and
gene-expression systems, has been demonstrated.
A development of this method was later presented by Kurakazu et al.
[215], where pneumatically actuated valves were employed to drive the
fluids within the microfluidic channels, resulting in an automated lab-
on-a-chip platform to be potentially employed for the production of
unilamellar and monodisperse vesicles.
However, as for pulsed jetting techniques, this method of vesicle
production required laborious experimental steps to be implemented,
limiting its widespread usability.
4.2.1.6. Double emulsion templates. Water in oil in water (W/O/W)
double emulsification has been employed as a highly-efficient and
convenient route for producing monodisperse giant unilamellar vesicles
[177]. Briefly, W/O/W templates are converted into liposomes by
solvent extraction. Generally, evaporation of the oil phase is performed
by addition of an organic solvent, forcing the lipid monolayers located
at the water–oil interfaces into a lipid bilayer membrane, which
arrange on the double emulsion templates, thereby forming lipid
vesicles (Fig. 3f). A study by Shum et al. [210] firstly demonstrated
microfluidic-based production of liposomes from double emulsion
templates (Fig. 3g). A glass microcapillary device combining a co-flow
and a flow focusing geometry was employed to produce phospholipid-
stabilised W/O/W double emulsions [216] (Fig. 3g), with a droplet
generation frequency of approximately 500 Hz. With this method, the
dimensional properties of the shell of the double emulsions could be
controlled by regulating the flow rates of each fluid phase and capillary
diameter. Droplets produced with this method were uniform in size,
making them suitable templates for obtaining monodisperse vesicles.
Furthermore, authors implemented experimental strategies to improve
the stability of fragile phospholipid vesicles during solvent extraction,
which would be otherwise non-trivial to produce.
A similar method was earlier described by Tan and coworkers
[217] to produce lipid vesicles with associated high encapsulation
efficiency. In this method, the aqueous phase containing the
species to be encapsulated was emulsified in the lipid phase within
a PDMS microfluidic channel, to produce stable lipid emulsions.
The lipid phase consisted of phospholipids dissolved in oleic acid.
The lipid solution emulsion was then injected into a mixture of water
and organic solvent, causing the phospholipids to disassociate from
liquid lipids and arrange around the emulsions into lipid vesicles.
Using this method, nano-sized proteins, 4.12 μm diameter fluorescent
beads and fluorescently labelled HeLa cells have been encapsulated
inside the formed phospholipid vesicles.
More recently, Teh et al. [218] described the production of
monodisperse 20–110 μm diameter lipid vesicles by means of
double emulsion templates. An HFF device was fabricated in
PDMS, for producing emulsion droplets uniform in size. Precise
control over droplets size was achieved by regulation of the flow
 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532
rate ratio. Additionally, authors performed selective hydrophilic
treatment of the PDMS channels surface by coating with PVA,
following an approach described in previous studies [219,220].
With this respect, formation of water-in-oil droplets occurred
within untreated hydrophobic regions, while formation of oil-in-
water droplets occurred within treated hydrophilic regions.
Compared to other hydrophilic surface treatments, the method
developed here did not require laborious mask alignment steps or
the use of hazardous chemicals. With the developed microfluidic
device, authors demonstrated continuous-flow encapsulation of
plasmids and long-term expression of GFP within the formed
liposomes. However, no quantification of the encapsulation
efficiency has been provided. It must be noted that liposomes
produced with this platform had longer stability (N 3 months)
compared to studies previously reported [217].
Three-dimensional HFF was instead employed in a recent study
by Davies et al. [221] for generating double emulsion templates. In
this work, authors developed a novel surface patterning technique
using corona discharge. In this way, wettability of PDMS surfaces
could be spatially controlled, making this technique suitable for
selective surface treatment. Combining surface functionalisation
with axisymmetric flow focusing, the developed microfluidic device
was capable of generating double emulsions in water and oleic acid,
which were then used as templates for liposome formation. Importantly,
surfactants were not required to lower the interfacial tension and create
the conditions for emulsification [222], making the fluidic environment
safe for lipid bilayers membrane formation. Authors further investigated
the effect of the fluidic conditions (i.e. Capillary number) on droplets
characteristics and identified the optimal flow regime for the formation
of single–core–shell templates, suitable as precursors of liposomes.
Liposomes produced with this technique were structurally integer,
as supported by fluorescence recovery after photobleaching (FRAP)
experiments. This study demonstrated the potential of combining
different techniques to achieve formation of unilamellar vesicles to
be employed for a range of potential applications; opening the way
for new advancements in this research field.
4.2.1.7. Droplet emulsion transfer. In this method, water-in-oil emulsions
stabilised by a lipid monolayer are initially produced [223]. Subsequently,
droplets are transferred to an aqueous medium and a second lipid
layer is deposited around the droplet while it crosses the oil–water
interface. In this way, the lipid bilayer is assembled one leaflet at a
time [223]. Importantly, the isolation between vesicle content and
the outer aqueous phase allows for efficient encapsulation. However,
when performed in macroscale batch systems, it results in the
formation of generally polydisperse vesicles.
In order to overcome this limitation, production of droplet
emulsions could be accomplished in a microfluidic-based system,
in order to improve control over droplet size and size distribution
[224–227]. In a study by Matosevic et al. [228], the production of
liposomes by droplet emulsion transfer was performed fully within
a Y-shaped microfluidic device. Lipid-stabilised droplets were
generated and conveyed from one inlet channel, while an aqueous
solution was delivered into the other inlet channel. In this way, after
channels merged together, a lipid-stabilised oil–water interface was
formed in close proximity to the droplets flow. A triangular post was
fabricated in the centre of the channel, which diverted the droplets into
the aqueous phase and forced them to cross the oil–water interface. In
this way, completely on-chip formation of the lipid bilayer was achieved.
Furthermore, authors demonstrated continuous flow encapsulation of
fluorescent probe molecules having different size. However, droplets
displacement stage caused a significant percentage of the droplets to be
disrupted and a gentler alternative may be evaluated in future studies.
A limitation of droplet emulsion transfer methods is the difficulty to
obtain oil-free lipid bilayers, which may limit the functional behaviour
1511
of the produced lipid vesicles and thus limit the range of potential
applications.
4.2.2. Niosomes
A synthetic, non-ionic, counterpart of liposomes is represented by
membrane vesicles formed by self-assembly of non-ionic surfactants,
the so-called niosomes. Hydrophilic molecules can be encapsulated
within their aqueous core, while hydrophobic molecules can be
partitioned into the membrane bilayer. For this reason, they are
generally employed as carriers of bioactive molecules (i.e. anticancer
or anti-inflammatory drugs) and contrast agents for clinical imaging,
and their surface can be functionalised for targeted drug delivery
applications. Conventional bulk methods for producing niosomes
suffer from the poor control over the chemical and mechanical
environment, and thus generally result in large and polydisperse
vesicles. For this reason, post-processing steps (i.e. extrusion or
sonication) are required to reduce and homogenise vesicles size.
With this respect, microfluidic-based production methods can be
employed to achieve a more precise control over the physical and
chemical properties of the fluidic environment. However, to the
best of our knowledge, only one study by Lo et al. [169] has been
reported, demonstrating the production of niosomes in a microfluidic
system. A microfluidic HFF device has been employed for this purpose,
following a previous design reported by Jahn et al. [126] (Fig. 2d), in
which a similar device configuration was employed for producing
liposomes.
In this study, a surfactant mixture (consisting of a sorbitan ester,
cholesterol and dicetyl phosphate) in isopropyl alcohol was injected in
the central stream, while an aqueous buffer was injected into the two
side streams. Either silicon metal or PDMS-glass hybrid microdevices
were employed. Authors investigated the effect of the fluidic conditions,
chemical structure of the surfactant and device materials on the
characteristics of the formed niosomes. It was demonstrated that
increasing the flow rate ratio resulted in a reduction of vesicle
mean diameter, due to the reduced species mixing time. This finding
was coherent with previous studies on liposome formation by HFF
[126,176]. On the contrary, wider channel geometry led to the
formation of larger niosomes, due to the increase in species mixing
time. With respect to the surfactant used, sorbitan ester with the
shorter and saturated alkyl chain assembled into the smallest
vesicles. Additionally, both silicon metal and hybrid glass-PDMS
microdevices were capable of producing niosomes by HFF. However,
as mentioned previously, attention must be paid on the effect that PDMS
distensibility can have on the hydrodynamic focusing performance. By
comparing niosomes produced by bulk method with those produced
by the microfluidic method, it emerged that HFF led to the formation of
smaller vesicles with a narrower size distribution.
Results from this study demonstrated the potential of microfluidics
to produce synthetic membrane vesicles by self-assembly of non-ionic
surfactants. Future investigations should focus on the encapsulation of
molecules within the formed vesicles, thus opening the way for a robust
microfluidic-based alternative to conventional bulk techniques.
4.2.3. Polymersomes
Polymersomes are synthetic vesicles constituted of a bilayer of
amphiphilic molecules (i.e. block copolymers) enclosing an inner
liquid compartment (i.e. aqueous solution). Given the tuneable
permeability of polymersome membrane and the higher mechanical
stability compared to phospholipid vesicles [229], they have been
employed as carriers for drugs, enzymes, proteins, and genetic
material for controlled release applications [230].
Analogous to liposomes production, conventional macroscale
methods for polymersomes synthesis include hydration and
electroformation [231]. However, vesicles produced with these
techniques are generally large in size, polydisperse and poorly
efficient in encapsulating bioactive molecules.
1512 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532

On the other hand, a range of different microfluidic-based devices
have been reported for the production of monodisperse polymersomes.
Here, they have been classified into two main categories, (i) double
emulsion templates and (ii) mixing-based techniques.
4.2.3.1. Double emulsion templates. W/O/W double emulsion templates
have been widely employed to form polymersomes. This method
closely resembles the one used to produce liposomes, and relies on
the evaporation of the solvent of a double emulsion stabilised by a
copolymer [229]. As the solvent evaporates, the polymer self-
assembles into a vesicular structure using the W/O/W emulsion as
a template. Thus, monodisperse templates lead to the formation of
monodisperse polymersomes.
A microfluidic strategy to form polymersomes has been developed
by Shum et al. [231] (Fig. 4a–b). In this study, a glass capillary device
based on a two steps co-flow and flow-focusing architecture [216]
was used to form W/O/W emulsions stabilised by PEG(5000)-b-
PLA(5000) (Fig. 4a). Monodisperse vesicles were obtained by off-
chip solvent evaporation and were characterised by a mean diameter
of approximately 24.5 μm. Furthermore, authors demonstrated
highly-efficient encapsulation of hydrophilic probes (FITC-Dextran)
and the possibility of tuning on-demand the properties of the vesicle
wall (i.e. thickness and permeability) by changing the block ratio of
the unimer. Notably, release of the encapsulated probe could be
triggered by osmotic shock, thus suggesting a simple mechanism for
controlled release of bioactive molecules from vesicles. Furthermore,
this microdevice architecture has been employed in a range of studies
to produce polymersomes with different characteristics. For example,
Seth et al. [232] produced monodisperse polymersomes encapsulating
magnetic nanoparticles, with potential application in MRI detection
and magnetic hyperthermia. Kim et al. [233] instead reported the
formation of polymersomes with a hydrogel core. For this purpose,
the formed polymersomes were irradiated with UV causing cross-
linking of the prepolymers encapsulated within the vesicle. The
hydrogel network was demonstrated (i) to facilitate sustained
release of encapsulated actives, (ii) to provide structural support to
the bilayer membrane preventing its deformation under exposure
to external forces, and (iii) to increase vesicle stability.
With using the same microfluidic device, Shum et al. [230] reported
the formation of multi-compartmental polymersomes, which could be
potentially used to carry multiple actives in a single vesicle (Fig. 4c–d).
Importantly, isolation between different compartments potentially
prevents the cross-contamination between incompatible actives and
could be also exploited to encapsulate reactants and subsequently
trigger the reaction by external physico-chemical stimulation (reaction
on-demand) [230]. In this technique, during solvent evaporation the
copolymers at the inner water–oil (W/O) and at the oil–outer water
(O/W) interfaces are attracted towards each other and form the
membranes. As a consequence of this, adhesion between neighbouring
inner droplets occurs and multi-compartmental vesicles are formed
[229] (Fig. 4c). Authors demonstrated control over the number of
inner droplets within the double emulsion by varying the flow rate of

Fig. 4. Microfluidic-based production of polymersomes. (a) Schematic of a glass microcapillary device for generating W/O/W double emulsions. The injection tube and the collection tube
are tapered from glass capillary tubes having an outer diameter of 1 mm, and an inner diameter of 0.58 mm. Final diameters correspond to 10–50 μm (injection tube) and 40–100 μm
(collection tube). Bright field microscope images of formed double emulsion drops following the evaporation of organic solvent are reported in (b). The emulsion drop consists of an
aqueous core surrounded by a diblock copolymer shell, which is subjected to thinning during solvent evaporation (i.e. from left to right). Images are taken at time intervals of 1 h (reprinted
with permission from [231]. Copyright 2008 American Chemical Society). (c) Schematic illustrating the process of formation of multi-compartmental polymersomes from double emulsion
droplets with multiple inner droplets. A photograph of the operating glass microcapillary device is reported in (d), together with a microscope image of dewetted double emulsion drops
with two inner droplets (adapted with permission from [230]. Copyright 2011; John Wiley and Sons).
 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532
the three phases independently. By slightly modifying the previous
microdevice design, and introducing two separate channels within
the injection capillary, authors demonstrated highly-efficient co-
encapsulation of FITC-Dextran and PEG, thus opening the way for the
use of this technique to co-encapsulate a range of bioactive agents for
therapeutic applications. The production of multi-compartmental
emulsions using a microfluidic-based device was also demonstrated
in a study by Perro et al. [234]. A “glue-free” microdevice consisting
of fused silica capillaries and a T-junction was developed for this
purpose, based on co-flow architecture. Authors thoroughly investigated
the effect of fluids flow rate, surface tension and dynamic viscosity on
the size of the internal and external droplets. Based on the experimental
findings, a predictive model was developed to facilitate the selection of
the optimal combination of operating conditions for a given application.
Multi-compartmental poly(dimethylsiloxane)-graft-poly(ethyleneoxide)
and poly(butadiene)-block-poly(ethyleneoxide) vesicles were obtained
with this technique, and the encapsulation of either hydrophilic or
hydrophobic molecules could be achieved.
However, the achievement of accurate loading of multiple and distinct
components, and their controlled sequential release without cross-
contamination, is still an important challenge towards the application of
multi-compartmental vesicles in drug delivery. In order to address this
issue, Kim et al. [235] developed a microfluidic-based strategy to produce
polymersomes-in-polymersomes. In this technique, single-compartment
polymersomes were produced with using the glass capillary device
mentioned above (first step) [216,231]. The formed polymersomes
were collected in a reservoir and subsequently re-injected as the
inner aqueous phase in another glass capillary microdevice, leading
to the formation of polymersomes-in-polymersomes (second step).
By varying the inner diameter of the collection tube between the
first and the second step, the number of encapsulated polymersomes
could be controlled. Importantly, authors demonstrated programmable
release of encapsulated molecules, in which the temporal evolution of
the release process (i.e. simultaneous or sequential release) could
be controlled by actively changing the stability of the vesicle layer
by means of hydrophobic homopolymers. Notably, higher order
polymersomes-in-polymersomes structures could be achieved, by
means of additional re-injection steps, leading to the encapsulation
of multiple actives. This may open new and innovative perspectives
in the field of controlled release of bioactive molecules.
However, despite polymersome stability being higher compared
to that of lipid vesicles, the integrity of their membrane can be
compromised when exposed to external mechanical or chemical stimuli,
and undesired release of the load may occur in this situation. This can
seriously limit the range of their potential applications. In order to
increase their stability and lower the permeability of vesicles membrane,
a novel strategy has been reported recently by Kim et al. [236] based on a
two-step emulsification method achieved by modifying the design of the
single-capillary device. In this way, water-in-oil-in-water-in-oil-in-water
(W/O/W/O/W) quadruple emulsions were obtained and employed
to form polymersomes-in-polymersomes. Interestingly, the inner
vesicles were characterised by protrusions which extended through
the membrane bilayer of the outer vesicles, leading to the formation
of double bilayers by budding process. Authors demonstrated that
polymersomes having double bilayers were more stable and less
permeable compared to polymersomes which membrane was
composed of a single bilayer.
Another challenge in the field of microfluidic-based production of
polymersomes ― and vesicular systems in general ― is the capability
of scaling-up the production process, which is a necessary prerequisite
towards the practical applicability of the developed microdevices.
With this respect, the use of devices fabricated from PDMS by soft
lithography allows for a significant reduction of the fabrication costs
and time, while preserving accuracy and flexibility [237]. In a study by
Thiele et al. [238] a PDMS microdevice was developed for production
of double emulsion templates. The microdevice consisted of a multi-
1513
step flow-focusing cross-junction geometry, where a diblock copolymer
was dissolved in the organic solvent stream at the first junction,
another organic solvent miscible with the copolymer-loaded solvent
was injected at the second junction, and an aqueous solution was
injected at the third junction. This configuration minimised the
precipitation of the diblock on the channel walls, which can affect the
wettability of the PDMS surface and thus potentially impede emulsion
formation.
Another attempt towards high-throughput formation of poly-
mersomes was recently performed by Kim et al. [239] who demonstrated
the development of a microfluidic device to achieve enhanced production
of W/O/W emulsions used as templates for polymeric vesicles (Fig. 5a–c).
The microdevice relied on the parallelisation of the glass capillary
configuration described in previous studies [210,216] (Fig. 5a). Droplets
formation was performed over a wide range of volumetric flow rates,
enabling high-throughput and stable generation of polymersomes
(i.e. production rate was ~4 times greater than that of single-capillary
devices). Additionally, encapsulation of hydrophilic molecules in the
vesicle aqueous core was performed. However, the size distribution of
the produced vesicles was wider compared to the one of vesicles
obtained by single-capillary devices, suggesting that more accurate
control over the operating conditions and refinement of the device
architecture would be required.
To conclude, it is noteworthy to mention that emulsion templates
have also been employed to produce monodisperse nanoparticle-
shelled bubbles with potential application in the field of US-triggered
drug delivery. For example, Lee et al. [240] produced air-in-oil-in-
water (A/O/W) templates by using the glass-capillary microfluidic
system previously described [216]. Monodisperse and highly uniform
A/O/W compound bubbles were generated at a typical frequency of
several thousand hertz. PVA in the outer aqueous phase was employed
to prevent the coalescence of the generated compound bubbles,
thus leading to the formation of stable nanomaterials for long-term
applications.
4.2.3.2. Mixing-based techniques. Recent studies have demonstrated that
a hydrodynamically well-controlled nucleation and growth process
can be exploited to control polymersomes size and size distribution.
In a study by Thiele et al. [241], a PDMS-based HFF device consisting
of perpendicularly crossed microchannels was developed to focus an
ethanolic block copolymer solution in a stream of water. Polymersomes
nucleated at the periphery of the focused stream, where the ethanol
concentration decreased to a critical level, below which the copolymer
became insoluble. Authors investigated the effect of flow rate ratio
on polymersome characteristics. While at lower flow rate ratios
polymersomes size was almost proportional to the flow rate ratio,
implying good control over vesicles size distribution, a non-linear
relation between polymersome size and flow rate ratio was instead
determined at the higher flow rate ratios. This may imply the
existence of different vesicle growth mechanisms, which merit further
investigations. Overall, with this technique, the mean diameter of
polymersomes could be controlled over a range from 40 nm to 2 μm,
with narrow size distributions and high reproducibility.
In a study by Brown et al. [242], a PDMS-based HFF microdevice was
developed for producing polymersomes from the pH-sensitive block
copolymer poly(2-(methacryloyloxy)ethyl phosphorylcholine)-poly(2-
(diisoprophylamino)ethyl methacrylate) (PMPC-b-PDPA). At the lower
pH (i.e. pH b 6) the polymer chain is completely dissolved in water,
while above a critical pH value (i.e. pH N 7.2) it becomes insoluble and
self-assembles forming stable polymersomes. Thus, by exploiting the
diffusion-governed species mass transfer between fluid streams in
a HFF microdevice, one can finely regulate the local pH levels within
the microenvironment and induce self-assembly of the copolymer.
Authors thoroughly investigated the combined effect of flow rate
ratio and pH of the aqueous buffer solution (lateral streams) on the pH
of the block copolymer solution (central stream) and the dimensional
1514 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532

Fig. 5. Microfluidic-based production of polymersomes. (a) Schematic depiction (left) and photograph (right) of a microfluidic device based on the parallelisation of glass microcapillary
devices, for high-throughput production of polymersomes from W/O/W emulsion templates. Formation of polydisperse emulsion droplets from a widening jet (a, top) and monodisperse
emulsion droplets from a narrowing jet (a, bottom) has been demonstrated with this microdevice. Confocal microscope image of monodisperse polymersomes obtained with this
microfluidic system is reported in (b) (taken from [239], with kind permission from Springer Science and Business Media). (c) Schematic illustration of polymersomes composed of a
bilayer of PEG-b-PLA and PNIPAM-b-PLGA diblock copolymers, where gold nanoparticles are doped into the hydrophobic part of the bilayer. (d–e) Microscope images showing distinct
release behaviours for polymersomes composed of a bilayer of PEG-b-PLA and PNIPAM-b-PLGA at 40 °C (e), and polymersomes having a gold nanoparticle-doped bilayer irradiated
with lasers at room temperature. The white arrow in (f) indicates the rupturing edge (taken with permission from [244]. Copyright 2012; John Wiley and Sons).

characteristics of the produced vesicles. Furthermore, continuous
flow encapsulation of proteins (bovine serum albumin, BSA) was
demonstrated, with encapsulation efficiency comparable to conventional
macroscale production methods. Despite it has lower encapsulation
efficiency compared to other microfluidic-based methods (i.e. W/O/W
emulsion templates), this technique has the advantage of not requiring
organic solvents, which may be retained by the vesicle membrane after
production and affect its performance.
The use of commercially-available micromixers to form poly-
mersomes was recently reported in a study by Thiermann et al.
[243]. Microdevices included stainless steel caterpillar (CPMM),
superfocus (SFIMM) and interdigital slit (SIMM) mixers. Control
over polymersome size and size distribution was achieved with
using these devices, and differences between mixer architecture
resulted in a different performance (i.e. size of the obtained vesicles).
In addition, due to the similar mixing times, CPMM and SIMM displayed
an analogous dependence of vesicles mean diameter on the fluidic
conditions. SFIMM system instead displayed a more significant
variation of the size of the produced vesicles with varying the flow
regime, and vesicles were generally smaller compared with those
obtained with CPMM and SIMM. This study demonstrated that
easy-to-use and commercially-available micromixers could be
employed to form polymersomes with a relatively narrow size
distribution, opening the way for the implementation of microfluidic-
based continuous-flow techniques for the large-scale production of
polymersomes.
4.2.3.3. Methods of actives release from polymersomes. A range of studies
have been devoted to the production of stimuli-sensitive polymersomes
and the development of protocols to achieve programmable and
controlled release of actives encapsulated within the vesicles [245,246].
However, only few studies have been devoted to the investigation of
actives controlled release from stimuli-sensitive polymersomes produced
using microfluidic-based methods.
With this respect, a recent study by Amstad et al. [244]
demonstrated microfluidic-based formation of polymersomes which
membrane bilayer consisted of a mixture of inert and thermosensitive
amphiphiles (Fig. 5e–f) Furthermore, photothermal gold nanoparticles
were encapsulated in the hydrophobic portion of the shell. In this
way, the release of actives from the vesicle could be induced upon
temperature increase above the lower critical solution temperature
(LCST) of the thermosensitive amphiphiles (Fig. 5e), or upon laser
illumination causing nanoparticle heating (Fig. 5f). Either reversible or
irreversible membrane poration was demonstrated with this technique,
suggesting the possibility to achieve control over the kinetics of the
release process.
A study by Martino et al. [247] instead demonstrated the expression
of a membrane-related bacterial protein (MreB) within polymersomes
produced by single-capillary microfluidic technology [231], and the
subsequent triggered release of the protein by osmotic shock. For
this purpose, polymersomes were transferred into an ipo-osmotic
environment causing an osmotic pressure difference between the
inner core of the vesicle and the outer fluid, and thus a net flux of
 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532
fluid directed into the vesicle. For this reason, the membrane bilayer
was subjected to significant strain levels, causing the formation of
transmembrane pores through which the actives could be released
by diffusion. The same release method was adopted in a previous
study to investigate controlled release of fluorescent probes from
within the vesicle [231].
Mechanically-induced rupture of polymersomes bilayer membrane
was demonstrated by Kim et al. [235], by forcing the vesicles to flow
through an orifice with diameter smaller than the vesicle diameter.
With this technique, authors demonstrated sequential release of
molecules from polymersomes-in-polymersomes structures. Notably,
by accurate selection of the operating conditions, one can achieve
reversible or irreversible membrane permeabilisation. In the same
study, controlled release from polymersomes was also demonstrated
by means of a chemical route, simply transferring the vesicle within a
non-aqueous fluidic environment which caused the dissociation of the
lipid bilayer.
4.2.4. Polymeric micelles
While the production of lipid-based nanoparticles and vesicles
in microscale devices represents a relatively established field of
investigation, only a limited number of studies have been reported
regarding the formation of polymeric micelles or copolymer nanoparticles
(i.e. frozen core polymeric micelles) by using microfluidic-based
technology. The large majority of these studies rely on diffusion-
governed mixing processes to induce the self-assembly of block
copolymers. A summary of the reviewed studies reporting microfluidic-
based production of polymeric micelles is reported in Table 3.
A study by Karnink and Gu [128] reported a microfluidic-based
production of PLGA–PEG block copolymer nanoparticles (or PMs) by
enhanced diffusion process in a PDMS-based HFF microdevice. According
to the mechanism of self-assembly of block copolymer nanoparticles
[173,248], the results showed a decrease in nanoparticle size and an
increase in homogeneity with a decrease in mixing time. Notably, the
nanoparticles showed a decrease in size as the relative flow rate of the
polymer stream decreased and no break point was detected, even
when the mixing time was considerably lower than the aggregation
time to produce kinetically frozen polymeric micelles. The authors
hypothesised that the results might be related to the time scale of self-
assembly of the nanoparticles.
Microfluidic-based formation of PMs, constituted of Pluronic® tri-
block copolymer, was also reported by Capretto et al. [170,249]
(Fig. 6a–b). For this purpose, a glass HFF continuous-flow microreactor
was fabricated by pholithography/wet etching procedure [250]. The
flow focusing configuration enabled controllable and fast mixing
process, which assisted the formation of PMs through nanoprecipitation
governed by solvent exchange. Authors investigated the effect of
(i) process operational parameters, (ii) concentration of polymer,
and (iii) microchannel architecture, on the size and size distribution
of the produced PMs. For fixed microchannel architecture, PM size
and polydispersity were observed to decrease with decreasing the
Table 3
Summary of the studies investigating microfluidic-based production of polymeric micelles. PLGA–PEG: poly(lactic-co-glycolic acid)–polyoxyethylene glycol block
copolymer. Poloxamer: Polyoxyethylene–polyoxypropylene block copolymer.
Self-assembly type
PLGA–PEG
Poloxamer
Poloxamer + mithramycin
Poloxamer + β-carotene
Poloxamer + Dexamethasone + ascorbyl-palmitate
Polybenzimidazole
PLGA–PEG
1515
flow rate ratio. This was in agreement with previous studies [128]
and it was attributed to the reduced mixing time required to achieve
complete mixing by diffusion, at the lower flow rate ratios. By
changing microreactor architecture, it was found that PM mean
diameter and polydispersity decreased with reducing the microchannel
dimension. This was further attributed to the improved mixing
efficiency within the microfluidic channels. Additionally, authors
observed that by carefully changing the flow rate ratio, two distinct
microreactors were capable of producing PMs with similar dimensional
properties; however, at a fixed width of the focused stream the smaller
microchannels led to the formation of larger and more polydisperse
PMs. These experimental findings thus demonstrated that microchannel
dimensions, flow rate ratio and fluid physical properties contributed in
concert to influence the nanoprecipitation process. In order to obtain
further insights into the complex interplay between the process
governing parameters, computational fluid dynamics (CFD) simulations
were performed (Fig. 6a). Numerical results revealed the relation
between PM characteristics and the dynamic variations in fluid viscosity
and diffusivity, which affected the mass transfer between solvent and
non-solvent streams. Notably, by comparison with conventional batch
methods, this technique provided better control over PM size and size
distribution (Fig. 6b).
Using a similar microfluidic strategy, the same group reported the
encapsulation of the DNA-binding drug, mithramycin, in polymeric
micelles (PM-MTH) based on Pluronic® block copolymers [171]
(Fig. 6c). The effect of a range of experimental parameters on PM-MTH
characteristics was systematically investigated, by means of a changing-
one-separate-factor-a-time (COST) approach, followed by a more
statistically sound Design of Experiment (DoE) approach. The factors
considered in the present study included: (i) size of the microchannels,
(ii) total flow rate, (iii) flow rate ratio between polymeric and aqueous
solutions, (iv) concentration of polymer, and (v) concentration of drug.
Results showed that empty PMs produced by microfluidic reactor
had a mean radius of 52–61 nm, and that the flow rate ratio positively
correlated with PM size. Furthermore, different polymer initial
concentrations (in the range 7.5–5.0 mM) resulted in empty PMs with
a similar dependence on the flow rate ratio; however, slightly larger
PMs were formed at higher polymer concentration (i.e. 15.0 mM). This
was attributed to the increase of the polymeric solution viscosity,
which resulted in a slowing down of the mixing process. In contrast,
no significant change in PM polydispersity was observed at different
polymer concentrations. The effect of MTH encapsulation on PM
characteristics was further investigated. In this regard, different
concentrations of MTH (in the rage 10.0–55.0 μM) were considered
(Fig. 6d). Due to MTH molecules amphiphilic properties, MTH
intercalated within the polymer chains into the core/shell interface,
leading to larger micelles compared to the empty counterparts.
However, the effect of a non-precipitating amphiphilic molecule
on PM precipitation process is still far from being fully understood,
and it may represent the subject of future investigations. Relevant
to application, it was shown that MTH preferentially partitioned in
the micellar phase.
Microfluidic strategy References
Planar flow focusing mixer Karnink and Gu et al. [128]
Planar flow focusing mixer Capretto et al. [170,251]
Planar flow focusing mixer Capretto et al. [171]
Planar flow focusing mixer Capretto et al. [1]
Planar flow focusing mixer Capretto et al. [172]
Planar flow focusing mixer Hasani-Sadrabadi et al. [252]
3D flow focusing mixer Rhee et al. [129]
1516 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532

Fig. 6. Microfluidic-based production of polymeric micelles and nanoparticles. (a) CFD simulation of species mass transfer in a HFF glass microfluidic device employed to produce block
copolymer micelles. Computational mass fraction contours are reported at three longitudinal positions along the microfluidic device, corresponding to the channels junction, middle
and end of the main mixing channel. PMs size distribution at a polymer concentration of 7.5 × 10−3 M is reported in (b), where the performance of batch reactor is compared with the
one of different HFF microfluidic devices having channel cross-sectional area of 5684 μm2 (Microreactor #1), 1959 μm2 (Microreactor #2) and 844 μm2 (Microreactor #3) (reprinted
from [170], Copyright 2011, with permission from Elsevier). (c) Schematic representation of the preparation procedure for mythramycin encapsulating polymeric micelles by HFF. The
central stream of Pluronic® F127 unimers and mithramycin dissolved in dimethyl sulfoxide is focused by two lateral streams of water. The effect of preparation procedure (i.e. batch
vs. microfluidics) and mithramycin concentration on micelles mean size is reported in (d) (republished with permission of DOVE Medical Press, from [171], Copyright 2012; permission
conveyed through Copyright Clearance Center, Inc.). (e) Atomic Force Microscopy (AFM) images of either empty or β-carotene encapsulating block copolymer micelles produced by HFF.
Size distribution of hybrid NPs (dashed line, solid circles) and empty PMs (dashed line, open circles) at MR = 1.41 is reported (f). The grey solid-line curve represents the sum of the values
of the calculated hybrid NPs and unfilled PMs, while the open squares correspond to the experimental data (reprinted with permission from [1]). (g–i) 3D HFF microfluidic device designed
for the production of polymeric NPs. Device consisted of three sequential inlets for vertical focusing, and a separate inlet for lateral focusing (g). CFD simulations were performed to
optimise device functioning and achieve the desired focusing performance (h). TEM images of produced NPs are shown in (i), at different polymer molecular weight and concentration
(scale bar: 100 nm) (reprinted with permission from [129]. Copyright 2011; John Wiley and Sons).

The same group investigated the production of organic NPs by
HFF, where core–shell structured hybrid NPs containing synthetic
β-carotene were encapsulated by amphiphilic block copolymer
[1] (Fig. 6e–f). A glass microfluidic device, with minor adaptations
with respect to previously mentioned studies [170], was employed
for this purpose to reduce drug deposition on channel walls. NPs size
and the kinetics of the nanoprecipitation process were investigated
under a range of different fluidic conditions, β-carotene and polymer
concentrations, and β-carotene/polymer molecular ratios (MRs).
Authors demonstrated that competitive reactions were responsible for
the formation of two distinct types of NP, either with or without loading
organic β-carotene (Fig. 6f). The corresponding peak area of NPs size
distribution provided a quantitative indication of the amount of each
type of NP, as a function of β-carotene/polymer MR. Furthermore, it
has been demonstrated that increasing β-carotene/polymer MR
resulted in higher mean particle diameter and wider size distribution.
In order to reveal the effect of the complex interplay between molecular
and fluid dynamic phenomena on the nanoprecipitation process, CFD
simulations were performed and integrated with an in-house
developed code that modelled the effect of NP formation on the
kinetics of NP diffusion and advection. Based on the numerical
simulations, a new parameter (nanoprecipitation mismatching)
representing the mismatching between the aggregations of the two
precipitant species was introduced. The variation of this parameter
has been shown to affect the relative amount of “empty” (i.e. unloaded)
PMs and drug loaded stabilised nanoparticles. Understanding the
implication of this parameter on the nanoprecipitation dynamics may
help the selection of stabiliser type, concentration, and process
parameters to obtain stable NPs with high percentage of loading while
reducing the formation of “empty” (i.e. unloaded) PMs. To conclude,
results from this study could be potentially translated to investigate a
variety of similar drug/polymer combinations.
The same group reported co-encapsulation of dexamethasone
(Dex) and ascorbyl-palmitate (AP) in polymeric micelles based on
Pluronic® block-copolymers [251], by using the microfluidic-based
HFF configuration previously mentioned [170]. The effect of the
amount of AP and flow rate ratio on the characteristics of the formed
PMs was investigated. It was shown that increasing the flow rate
ratio from 0.03 to 0.13, micelle mean diameter increased from
~ 207 nm to ~ 1484 nm, which was coherent with results from
previous studies [128,170]. CFD simulations were implemented to
further investigate the effect of the fluidic conditions (i.e. fluid
viscosity, shape of the focused stream) on species mass transfer.
Importantly, as for previous investigations, the microfluidic approach
resulted in PMs with superior dimensional quality compared to those
produced by batch reactor. Furthermore, by increasing the concentration
of AP (i.e. from 0.125 mM to 1.000 mM) resulted in a severe increase of
PM mean diameter (i.e. from 6 nm to 207 nm). A non-linear dependence
of PM size on AP concentration was identified, which was attributed to
the existence of a maximum micellar solubilisation capacity above
 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532
which the excess of drug cannot be solubilised in PM core. The drug in
excess is thus exposed to a solvent which is prone to precipitate in the
form of crystalline suspension causing the observed increase in PM size.
In addition to the aforementioned applications of polymeric
nanoparticles as carriers of bioactive molecules, microfluidic-
based production of NPs for fuel cell technology has been recently
demonstrated by Hasani-Sadrabadi et al. [252]. In this study, an
HFF microdevice was employed to achieve control over the size
and size distribution of organic NPs based on polybenzimidazole.
As for the formation of lipid vesicles, the vast majority of the HFF
microfluidic devices rely on a two-dimensional focusing of the
central fluid stream. However, production of PLGA–PEG and PLGA
polymeric nanoparticles by 3D hydrodynamic flow focusing has
been recently reported by Rhee et al. [129] (Fig. 6g–i). The main
advantage of three-dimensional focusing is the hydrodynamically-
induced isolation of the precipitating polymer from the channel
inner walls, in both the horizontal and the vertical dimensions,
thereby minimising the occurrence of fouling and channel clogging.
However, the fabrication of 3D HFF devices is generally non-trivial
and suffers from limited reproducibility. In this study, a simple design
for a 3D HFF device was described, consisting of three sequential inlets
for vertical focusing and a further inlet for lateral focusing (Fig. 6g).
Authors fabricated a 2D HFF device for direct comparison between the
two techniques. Results showed that NPs produced by 3D HFF had
smaller sizes and improved monodispersity compared to 2D HFF or
bulk production. The monodispersity of NPs produced by 3D HFF
enabled to finely tune NPs size, by varying the polymer block size and
concentration. Mathematical and numerical simulations, together
with confocal microscopy acquisitions (Fig. 6i), were performed in
order to optimise device design and identify the optimal operating
conditions. This simple design may open the way for an easy-to-
fabricate continuous flow microfluidic device, to be potentially
employed for large-scale production of polymeric NPs and capable
of overcoming limitations associated with 2D HFF devices.
Remarkable efforts towards the design of a robust platform for high-
throughput production of PMs and in situ monitoring of the process of
nanoparticle formation were made by Chastek and Iida [147]. The
microfluidic channels had square cross-section and were machined
into an aluminium plate and then sealed with using a thin film of
transparent Kapton. Micelles were obtained by dilution of the block
copolymers with a selective solvent, which caused the copolymer to
self-assemble. A micro-well with a miniaturised stir bar was fabricated
to achieve good mixing of the reagents. A dynamic light scattering
(DLS) unit was integrated with the microfluidic platform, with laser
and detector fibre optic probes in direct contact with the fluid samples.
With this technique, authors investigated the temporal evolution
and the mechanisms of PM formation. Notably, they were able to
discriminate between the formation of discrete micelles or micelle
aggregates, depending on the length of the corona block. A future
development of this platform may integrate also on-line measurements
Table 4
Summary of the studies investigating microfluidic-based production of polymer NPs, hybrid NPs and complexes. PMMA: Poly(methyl methacrylate); LNPs: lipid
nanoparticles; SHM: staggered herringbone micromixer; ODN-LNPs: oligodeoxynucleotide lipid nanoparticles.
Nanomaterial type
Polymer nanoparticles
PMMA + ketoprofen
Hybrid nanoparticles and complexes
Lipid–polymeric hybrid nanoparticles
Lipid–polymeric hybrid nanoparticles
DNA polyplexes
pDNA polyplexes
siRNA-LNPs
siRNA-LNPs
siRNA-LNPs
ODN-LNPs
1517
of the molecular weight of the polymer products, which cannot be
quantified via DLS.
4.2.5. Polymer nanoparticles
A summary of the reviewed studies reporting microfluidic-based
production of polymeric nanoparticles is reported in Table 4.
An innovative approach to the production of polymer NPs in a
microfluidic system has been recently reported by Anton et al. [253],
based on an impact-jet micromixer [254]. The micromixer was
constituted of two main components, (i) the mixing plate, in which
microchannels converged towards a central hole, and (ii) the outlet
plate, which contained an outlet channel where the nanoprecipitation
occurred. With this technique, NP characteristics can be controlled by
varying (i) the flow rates of solvent and non-solvent, (ii) the polymer
concentration in the organic solvent, and (iii) the presence of
surfactants as stabilising agents. Polymeric (PMMA) NPs, with a mean
diameter of ~100 nm and a narrow size distribution, were produced
with this method, and loaded with a lipophilic drug (ketoprofen).
Notably, this setup may open new perspectives for large-scale
production of NPs, given (i) the possibility for online modification of
the NP size, (ii) control of the NP loading and monitoring of the
formulation, and (iii) higher polymer solution flow rates (i.e. up to
1 mL/min) compared to other microfluidic-based techniques.
4.2.6. Hybrid nanoparticles and complexes
A summary of the reviewed studies reporting microfluidic-based
production of hybrid NPs and complexes is reported in Table 4.
A range of studies have recently focused on the production of hybrid
nanoparticles by means of microfluidic-based strategies. The physico-
chemical and structural peculiarities of these formulations make them
suitable for a range of biomedical applications, including drug and
gene delivery, and medical imaging.
Lipid-polymeric hybrid NPs combine the unique characteristics of
polymeric NPs and liposomes, such as high encapsulation yield of
bioactive molecules, high stability in blood and slow release kinetics.
Moreover, temporally controlled release of different agents from
these NPs has been achieved, by encapsulating one agent in the
lipid layer and the other agent within the polymeric core [255].
This type of NPs generally comprises three functional components
[256]: (i) a hydrophobic polymer core for the encapsulation of
poorly water-soluble molecules; (ii) a lipid layer surrounding the
polymer core, providing a biocompatible shell and promoting retention
of encapsulated molecules; and (iii) a hydrophobic polymer sheath
layer to increase NP stability and lifetime in the haematic circulation.
Conventional batch methods for producing hybrid NPs are complex
and they usually require a two-step formulation process, consisting of
production of polymeric NPs first, followed by encapsulation of
polymeric NPs within liposomes. Intermediate steps, such as heating,
vortexing and long incubation periods introduce significant variability
in the characteristics of the formed NPs. In order to overcome this
Microfluidic strategy References
Impact-jet micromixer Anton et al. [253]
Tesla mixer Valencia et al. [255]
Microvortice mixer Kim et al. [257]
T-shaped mixer Ho et al. [264,265]
Various micromixer Debus et al. [261]
SHM mixer Chen et al. [258]
SHM mixer Belliveau et al. [259]
SHM mixer Leung et al. [267]
2-steps planar flow focusing mixer Koh et al. [268]
1518 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532

limitation, Valencia et al. [255] developed a PDMS-based microfluidic
mixer consisting of Tesla structures [260] (Fig. 7a), and operating at
Re N 20. NPs produced with this technique were monodisperse and
with a mean diameter of 40 nm. The rapid convective mixing obtained
within this microdevice allowed for uniform lipid coverage around the
polymeric core to be achieved, and the self-assembly of the polymeric
core occurred independently from the lipid component, which acted
as a stabiliser of the overall structure providing long-term stability.
Conventional production methods (i.e. diffusion-based) instead
result in the formation of a combination of liposomes, hybrid NPs
and polymeric NPs; as a consequence of the slow-mixing kinetics.
With using the same microfluidic device, authors demonstrated the
production of hybrid lipid-quantum dot (QD) nanoparticles, to be
potentially employed for biomedical imaging applications (Fig. 7a).
Notably, QDs are semi-conductive nanocrystals with excellent optical
properties; however, hydrophobicity and low colloidal stability make
them unsuitable candidates for clinical usage. To address this issue,
lipid coating of QDs has been achieved in this study, in order to improve
their stability and biocompatibility. Hybrid lipid-QD NPs produced with
this technique were homogeneous and monodisperse (mean diameter:
~60 nm) (Fig. 7a), thus opening the way for their potential application
in the field of nanomedicine. However, despite the production
capabilities of this method are significantly greater than those of
strategies relying on diffusive mass transfer only, the fluid flow
rates employed here (i.e. ~ 50 μL/min) may still limit its translation
into large-scale production systems.
Recently, a novel microfluidic-based strategy has been developed
by Kim et al. [257] for efficient producing hybrid lipid-polymer NPs
(Fig. 7b–c). The method does not rely on diffusive mixing between
species, but on the formation of microvortices (Fig. 7b) at relatively
high Reynolds number (Re ~ 75). Notably, diffusive mixing does not
allow for the formation of NPs requiring the assembly of precursors
in the aqueous phase (i.e. lipids) with precursors in the organic phase
(i.e. polymer), due to the associated slow kinetics. In this study, a
three-inlet architecture was employed and microvortices were formed
at the intersection between the inlets (Fig. 7b), resulting in rapid mixing
between the polymer (PLGA) and the lipid (lechitin + DSPE-PEG)
(Fig. 7c). Importantly, the flow pattern downstream from the intersection
displayed three-dimensional focusing features (Fig. 7c), preventing
polymer aggregation near the channel walls. NPs produced with this
method were generally monodisperse and small in size (i.e. diameter
range: 55–80 nm), particularly at the higher Reynolds numbers
(i.e. Re N 30) at which rapid and effective convecting mixing occurred,
as demonstrated by CFD simulations. Furthermore, NP size changes
were more sensitive to the fluidic conditions (i.e. increasing Re resulted
in a decrease of NP size) than the polymer-lipid composition, which was
indicative of the role played by the mixing velocity (Fig. 7c). Relevant
to applications, this technique allows for high NP productivity, which
is significantly greater than previous methods based on diffusive or
convective mixing [128,255].
4.2.7. Nanomaterial produced through complexation mechanism
Polymer complexes, referred to as polyplexes, have been also
produced by microfluidic technology. They are formed by electrostatic
interaction between negatively charged pDNA and positively charged
polymers (i.e. PEI) [261], and have been established as a non-viral
vector for intra-cellular delivery of a range of different nucleic acids.
Bulk production of polyplexes generally relies on pipetting, which
leads to poor control over NP characteristics [262] and is generally
restricted to relatively low concentration of polyplexes in solution,
limiting their application in the clinic [263]. Furthermore, depending
on the type of polymer employed and its molecular weight, the
characteristics of the formed NPs can vary significantly. In order to
achieve a more accurate control over NPs properties, formation of
polyplexes within microfluidic systems has been investigated in
recent studies. In this regard, a study by Ho et al. [264,265] reported
the formation of DNA polyplexes by using a PDMS-based microdevice
consisting of a T-junction. By combining quantum-dot-mediated

Fig. 7. Microfluidic-based production of hybrid NPs and complexes. (a) Schematic of a microdevice employed for producing hybrid lipid–polymeric and lipid–quantum dot NPs. Rapid
mixing is achieved by means of Tesla structures within the microfluidic domain. Representative TEM images of the obtained lipid–polymer (top) and lipid–quantum dots (bottom)
NPs are reported (reprinted with permission from [255]. Copyright 2010 American Chemical Society). (b–c) Schematic and cross-section views of a three-inlet microfluidic device
generating two symmetric microvortices and a 3D HFF pattern, employed for rapid and high-throughput production of lipid–polymer hybrid NPs. CFD simulations in (c) illustrate the
mass transfer process within the microfluidic environment. TEM images of NPs produced at Re = 75 (top) and Re = 150 (bottom) demonstrate control over NPs size. Scale bars =
100 nm (reprinted with permission from [257]. Copyright 2012 American Chemical Society). (d) Three-inlets microfluidic platform for producing siRNA–lipid NPs. Rapid mixing is
achieved within a microfluidic channel due to groove structures located on the channel bottom wall (e) (reprinted with permission from [258]. Copyright 2012 American Chemical
Society). (f) Schematic depiction of a siRNA–lipid NPs formulation strategy based on a staggered herringbone micromixer (SHM) which induces chaotic advection of the laminar inlet
streams. Effective mixing is achieved only few milliseconds after solutions are injected within the device (reprinted by permission from Macmillan Publishers Ltd on behalf of Cancer
Research UK: [259]. Copyright 2012).
 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532
fluorescence resonance energy transfer (QD-FRET) and microfluidic
technology, authors were able to characterise the kinetics of chitosan/
DNA polyplexes assembly, with high temporal resolution. For this
purpose, a FRET pair of 605QD (donor) and Cy5 (acceptor) were
labelled to pDNA and chitosan, respectively; and their molecular
interaction was monitored on-time via FRET signals. Notably, two
different stages of interaction were detected during the self-assembly
process: (i) a ‘fast cationic binding’ occurring in the millisecond range
and dominated by electrostatic interactions, and (ii) a ‘nano-assembly
flocculation’ stage (whose effect was detected at 1 s), which is a typical
feature of diffusion–reaction limited processes. This study represents
the first attempt to monitor the kinetics of DNA polyplexes in situ by
using a lab-on-a-chip platform; and the developed technique could be
potentially employed to investigate the formation mechanisms of a
wide range of lypoplex and polyplex systems.
Recent studies have been devoted to the development of more
elaborated microfluidic-based platforms for the production of
pDNA polyplexes. For example, in a work by Debus et al. [261] a
range of different chips with distinct architectures and mixing
lengths were fitted onto a baseboard. Polyplexes with PEI and
pDNA were produced within the microfluidic environment, under
well-controlled mixing conditions, and compared with those
produced by traditional pipetting procedure. Results from this study
further demonstrated the possibility of using microfluidic technology
for producing DNA polyplexes. Importantly, it was demonstrated that
the ratio of PEI to DNA had a significant influence on the properties of
the formed NPs, while other operating conditions were less important.
Relevant to application, the size of polyplexes prepared with this
technique was observed to be almost constant, in the range
140–160 nm, over a relatively wide range of complex concentrations.
In contrast, the size of polyplexes prepared by bulk method was
observed to be more variable. Notably, authors expanded the range
of potential applications by employing the developed lab-on-a-chip
platform to form siRNA and mRNA polyplexes, thus opening the
way for a range of further potential applications of microfluidics in
gene therapy.
Another type of complexation NPs produced by microfluidic
technology is represented by lipid nanoparticles–nucleic acids
complexes (or lypoplexes) (LNP). Recently, considerable attention
has been devoted to the formulation of LNPs encapsulating small
interfering RNA (siRNA), given the therapeutic potential of these
formulations [258,266,267]. Conventional methods for producing
LNPs ― such as extrusion ― generally suffer from the need for
labour-intensive multiple steps, which are sometime associated with
significant loss of expensive chemicals (i.e. siRNA). Other methods,
such as sonication, may instead damage the structure of nucleic acids,
thus compromising the therapeutic efficacy of the product. In order to
achieve precise control over the operating conditions, microfluidic-
based strategies have been recently implemented. For example, in a
recent study by Chen et al. [258], a PDMS-based microfluidic mixer
was developed for producing siRNA-LNPs (Fig. 7d–e). The microfluidic
device possessed three inlets for sequential injection of (i) siRNA
aqueous solution, (ii) alcohol solution of cationic lipids and (iii) buffer
solution to prevent inter-particle aggregation (Fig. 7d). Effective and
rapid chaotic mixing was achieved thanks to periodic trenches and
ridges which were asymmetrically arranged on the channel bottom
surface [104]. CryoTEM acquisitions revealed that LNPs produced by
microfluidics had a narrow size distribution, with a diameter in the
range 60–90 nm (at a flow rate of 0.3 mL/min); while LNPs produced
by bulk methods (i.e. pipet mixing) were polydisperse, with a diameter
N100 nm. Importantly, microfluidic-produced LNPs were capable of
encapsulating siRNA with an efficiency of ~80%. Increasing the flow
rate ratio resulted in the formation of smaller NPs, which is coherent
with the theory of vesicle formation and species mixing behaviour.
However, either NP diameter and encapsulation efficiency remained
approximately unchanged with increasing the flow rate above a critical
1519
value of 0.2 mL/min from all inlets. Based on this observation, authors
investigated simultaneous manual injection of the solutions (at an
estimated flow rate N0.3 mL/min) and were capable of producing
reproducible particle sizes. In this way, a reliable “equipment-free”
method of particle formation was established, representing a user-
friendly and cost-effective alternative to other laborious production
strategies.
Rapid mixing within a microfluidic device has been exploited by
Belliveau et al. [259] and Leung et al. [267] to form monodisperse
siRNA-LNPs (Fig. 7f). In these studies, a staggered herringbone
micromixer was developed which allowed for effective mixing to
be achieved, at relatively high fluid flow rates suitable for large-
scale production (i.e. ~ 2 mL/min). Authors employed a variety of
techniques and bio-physical assays to characterise the structure of
formed lypoplexes, including cryo-TEM, 31P NMR, membrane fusion
assays, density gradient measurements and molecular modelling
[267]. Results from this study revealed that siRNA-LNPs had an
electron-dense inner lipid core which contained siRNA duplexes
complexed to internalised cationic lipids, resulting in siRNA being
fully protected from external RNase. Furthermore, the interior of
these NPs exhibited a periodic structure of aqueous compartments,
some of which contained siRNA duplexes. Results from this study
have provided new insights into the mechanisms of formation of
siRNA-LNPs, to be exploited for the development of novel and more
sophisticated complexes.
Another type of lypoplex formulation which has been
produced by means of microfluidic technology is represented by
oligodeoxyribonucleotide (ODN)-LNP systems. The use of ODNs to
down-regulate oncogenes has been identified as a potentially useful
anticancer treatment; however, its application in the clinic has been
limited by its rapid clearance in the haematic circulation and the low
permeability through the cell membrane bilayer. Thus, complexation
of ODNs with cationic liposomes has been recently proposed as an
alternative strategy to overcome these limitations. In a study by Koh
et al. [268], the production of ODN-LNPs using a microfluidic device
has been reported for the first time. For this purpose, a five-inlet flow
focusing architecture was designed and fabricated by micromilling of
PMMA layers. A two-step HFF was achieved within the microdevice,
where the first step consisted of an ODN solution focused by two
streams of protamine solution. The obtained flow stream was then
focused by two streams of lipid solution in the second step. Using this
technique authors demonstrated continuous-flow production of ODN-
LNPs with a lower size and a narrower size distribution compared
to bulk methods (i.e. vortexing). Furthermore, zeta-potential of NPs
produced by microfluidics was slightly lower compared to the ones
obtained by bulk method. ODN encapsulation efficiency between
the two methods was comparable (N70%), with the microfluidic-
based strategy resulting in slightly better encapsulation performance
compared to bulk production. Regarding NP morphology, it was
revealed that complexes produced by microfluidics had more multi-
lamellar vesicles and onion-like structures and a reduced number
of large aggregates, compared to bulk technique. However, limitations
of this method include the need for off-chip procedures to be performed
(i.e. dialysis and filtration), and the wider NP size distribution compared
to methods based on faster mixing [258,259].
5. Microfluidic-based manipulation, immobilisation and in situ
analysis of nanoparticles and vesicular systems
In situ analysis of nanomaterial and vesicle characteristics is of
crucial importance to evaluate the efficacy and reliability of a given
production technique (i.e. quality control). However, manipulation
and immobilisation of nanoscale materials are non-trivial, and
may require the development of advanced and laborious-to-
operate technological platforms.
1520 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532

With the emerging of microfluidic-based technology and the related
potential to operate tiny fluid volumes, together with the possibility
to integrate different functions on a single lab-on-a-chip platform,
advances have been made towards the in situ manipulation and analysis
of nanomaterials. Notably, high temporal and spatial resolution analysis
of nanomaterial properties may provide innovative insights into the
mechanisms of nanoparticles and vesicle formation and, thus, allow
for the optimisation of current production techniques. The lack of
quality controls on the produced nanoparticles is indeed limiting the
translation of production techniques into the clinical or industrial
routine [269].
In the present paragraph, a brief overview on microfluidic-based
methodologies for nanoparticles and vesicular systems manipulation,
immobilisation and analysis are reported.
5.1. Nanoparticles and vesicular systems manipulation
The use of electric fields as a means to manipulate, sort or separate
micro- and nano-materials has been extensively studied, and is usually
referred to as dielectrophoresis (DEP) [270]. With this technique,
dielectric nanoparticles suspended in a fluid medium are exposed to a
non-uniform electric field. Thus, a net force is generated on the particle,
due the interaction of the particle's dipole with the spatial gradient of
the electric field [271]. Differences in the magnitude and direction
of this force can be exploited for a range of applications, including
NP separation, concentration and size-fractionation [272]. Furthermore,
Green and Morgan [273] demonstrated that DEP behaviour of sub-
micrometric particles is significantly influenced by the surface
properties of the particle, thus allowing for detection and quantification
of changes in particle surface composition. Notably, miniaturised
electrodes can be incorporated in microfluidic platforms for the
generation of electric fields within tiny fluid volumes [274,275];
opening the way for microfluidic-based DEP [271]. This technique
has been demonstrated to be capable of effectively manipulating
nanoscale model particles (i.e. latex beads) [275] ― down to a diameter
of 40nm [276] ― carbon nanotubes [277,278], DNA nanoparticles [279]
(Fig. 8a–b), silica nanoparticles [280] and vesicles [281]. With this
respect, sorting of liposomes using microfluidic-DEP has been
demonstrated by Cheng et al. [282]. In this work, a broadly distributed
liposome sample was sorted into different size ranges, at a relatively
high fluid velocity, by means of a DEP microsystem. In addition,
the negative DEP force employed in this study prevented undesired
attachment of nanoparticles to the channel walls. Accurate manipulation
of liposome position by DEP was achieved by Osaki et al. [281], in a
PMMA–glass microfluidic system. With this method, authors were
capable of accurately directing the liposomes towards a planar
bilayer membrane for investigating protein reconstitution mechanisms.
Furthermore, in a study by Vijayan et al. [283], the manipulation of
worm micelles using electric fields generated within a microsystem
has been reported. The easy-to-fabricate experimental chamber
was formed between a glass slide and a coverslip, with sealing at
the four edges. Electrodes were drawn onto the glass slide to
generate oscillating electric fields. Controlled displacement and
stiffening of the suspended micelles by means of the electric field
was demonstrated. Recently, lab-on-a-chip systems were developed
in order to achieve high-throughput nanoparticles manipulation by
DEPs, by means of an array of microelectrodes integrated with a
microfluidic environment [284]. These developments represent an
important step towards the miniaturisation of automated biochemical
analyses, to be potentially employed in diagnostic applications.
Despite DEPs representing an effective strategy for manipulating
nanomaterials, attention must be devoted to the effect of the applied
electric field on vesicle/nanoparticle integrity, particularly for applications
which require the encapsulation of bio-actives. Notably, local heating and
fluid convection may originate from electric field application, potentially
causing undesired alterations of nanoparticle properties.
In recent years, the potential of integrating optical and microfluidic
elements into a single platform has been investigated, particularly with
the aim of improving fluid and particle manipulation [285] (Fig. 8c).
The latter can be achieved by means of optical tweezers, or by indirect
optically-induced microfluidic effects. In a study by Kulin et al. [286] the
potential of optical tweezers to manipulate liposomes has been reported,
with the aim of facilitating liposomes fusion by UV exposure (Fig. 8d).
Importantly, liposomes were observed not to be disrupted by the strain
levels achieved during optical trapping and manipulation. However,
while it is particularly suitable for single-vesicle analysis applications,
this technique suffers from the low number of processed vesicles
compared to other continuous-flow strategies, thus limiting its
spectrum of potential applications. A continuous-flow optofluidic
strategy was instead developed by Yin et al. [287]. The microdevice
relied on planar fluidic and optical pathways for nanoparticle
manipulation and detection, and it could be potentially interfaced
with other well-established analytical techniques. Furthermore, due to
its planar geometry, this platform could be scaled up to a larger number
of intersecting waveguides.
The use of magneto-hydrodynamic (MHD) forces to manipulate
phospholiposomes within microfluidic channels has been reported
in a study by Son et al. [288]. Soft lithography was employed to
fabricate a network of PDMS microchannels, which was bonded to
a glass slide by O2 plasma treatment. Authors demonstrated active
control of liposomes trajectory by application of an external magnetic
field (i.e. possibility to direct the vesicles in selected outlet channels).

Fig. 8. On-chip nanoparticles manipulation. (a) Magnified view of a microelectrode array device employed to manipulate NPs by dielectrophoresis (DEP). Upon electric field application,
200 nm diameter NPs can be concentrated into the positive DEP high field regions, as illustrated in (b) (adapted with permission from [279]. Copyright 2008; John Wiley and Sons).
(c) Schematic illustration of the mechanism of transport of two different sizes of NPs by optical manipulation in a slot waveguide. The force Fprop corresponds to the radiation pressure
force responsible for optofluidic transport, while Ftrap is the trapping force holding NPs within the slot region (reprinted by permission from Macmillan Publishers Ltd on behalf of Cancer
Research UK: [285]. Copyright 2012). (d) Optically-driven fusion of two liposomes, one containing fluo-3 dye and the other containing calcium ions. Bright field (left) and fluorescence
(right) microscope images have been recorded. After fusion, the fluorescence intensity increases due to the reaction in which fluo-3 chelates the calcium ions (reprinted with permission
from [286]. Copyright 2003 American Chemical Society).
 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532
Furthermore, they identified two factors influencing liposomes flow,
namely, the charge density on liposome surface and the Lorentz force
generated by the transverse field. However, only nanoparticles with
sufficiently high charge could be effectively manipulated with this
technique, posing significant limitations to its widespread applicability.
On-chip mechanical methods to fractionate nanoparticles based on
their physical properties (i.e. size) have been implemented in a study
by Nomura et al. [289]. The microfluidic device consisted of a series of
micropillars which were capable of trapping vesicles based on their
diameter. Compared to the methodologies previously mentioned, this
technique did not require the application of external physical stimuli,
and it can be regarded as a gentle manipulation approach.
5.2. Nanoparticles and vesicular systems immobilisation and in situ
analysis
Recently, few studies have been reported about the implementation
of microfluidic strategies for trapping or immobilising nanomaterials,
in order to facilitate the analysis of their physical and chemical
characteristics in situ, by coupling with microscope-based and analytical
instrumentation. Among these, a hydrodynamic trapping technique has
been developed by Nuss et al. [290], by using a PDMS microfluidic device.
The microdevice consisted of a series of hydrodynamic traps made of
identical junctions, allowing for highly-efficient trapping performance
of vesicles with mean diameter in the range 2–40 μm. In situ real-time
analysis of vesicles behaviour and physical properties (i.e. permeability)
in response to changes of the fluidic environment characteristics could
be performed with this technique.
Recently, Khun et al. [291] presented a novel chemical method to
achieve long-term immobilisation of liposomes on a substrate. In this
technique, cholesterol–PEG–biotin was used as a linker, which
binded via avidin to biotinylated BSA (bBSA) previously absorbed
on a glass surface. The liposomes solution was flowed over the
functionalised substrate within a microfluidic channel. It has been
demonstrated successful immobilisation of liposomes having
different size, which remained stable for prolonged time and at
relatively high volumetric flow rates. This experimental method is
particularly suited for long-term investigations where fluids need to be
rapidly changed, for measuring fast kinetics and to study the effect of
fluid shear stress on liposome characteristics. Notably, the immobilised
vesicles could be released on-demand after the analysis. By coupling
with microscope-based technology, this method may allow for in situ
analysis of the temporal dynamics of membrane physical properties,
including the spatial distribution of lipid domains (i.e. cholesterol-rich
lipid rafts), membrane deformation and fluidity. This may be of help to
further explore the potential of lipid vesicles to replicate certain
characteristics of cell membrane dynamic behaviour under biomimetic
conditions [292,293].
Advances in the field of nanoparticle manipulation and analysis on-
a-chip have been recently reported by Issadore et al. [284]. The authors
developed a hybrid integrated circuit/microfluidic chip capable of
performing programmed manipulation, trapping and poration of lipid
vesicles, by means of electric fields. An array of micro-electrodes, driven
by a radio frequency (RF) voltage, allowed for a range of operations to
be performed efficiently. In particular, the chip could operate in two
distinct modes, (i) voltages at MHz frequencies were used to trap and
deform vesicles by DEP, and (ii) frequencies b 1 kHz were used to
electroporate or fuse the lipid bilayers. By making use of low-cost
integrated circuit technology, these microdevices could be employed
in the field of point-of-care diagnostics and biochemical detections of
analytes.
One of the greatest limitations to the commercialisation of lipid
vesicles is the lack of formulation stability, since small size variations or
changes in the surface chemistry can significantly compromise
their performance in vivo. As described previously, microfluidic-based
production strategies have been developed in recent years in order to
1521
achieve higher control over nanoparticles properties. However, the
large majority of liposomes formulations are still performed using
conventional macroscale methods, resulting in generally poor control
over the product quality. In order to facilitate product development, a
novel microfluidic-based device has been developed by Birnbaumer
et al. [269], which contained embedded dielectric microsensors capable
of quantifying liposomes formulation stability and composition from
measurement of their electric properties. With using this platform,
authors demonstrated that different liposomes membrane modifications,
including membrane-bound surface proteins, lipid-bilayer soluble drugs,
protein and dye encapsulation, could be detected and discriminated. The
developed lab-on-a-chip device represents a significant advancement
toward efficient screening of nanoparticles quality, thus opening the
way for its potential application in industrial production systems.
6. Effect of microfluidic environment on dynamics of
nanomaterial formation
As already explained in the previous sections, a controlled formation
of nanomaterial generally relies on an efficient solvent-displacement
process. This is realised by a fast solvent shifting, from organic to non-
solvent that triggers the precipitation process.
An estimation of the velocity with which this process proceeds can
be obtained considering the mixing time, τmix. It has been recognised
by different authors as a key parameter for the formation of NPs through
precipitation, since it directly affects the dimensional properties of the
produced nanomaterials [173,294]. A shorter mixing time can in fact
ensure a spatially homogeneous environment during the precipitation
process which in turn leads to uniform reaction condition and particle
formation kinetics.
Despite the processes for the production of organic nanomaterial
are well established, there is still a general lack of fundamental
understanding of the mechanism of nanomaterial formation
[118,143,176,295–297]. This particularly applies to the production of
nanomaterials within microscale reactors, where the microfluidic
environment and associated characteristics (i.e. flow rate ratio, channel
architecture and species residence time) have been demonstrated to
significantly affect nanomaterial output [176,255].
An effective way to achieve controllable, predictable and fast mixing
is the use of microreactors in which the architecture of the fluidic
domain allows the generation of a hydrodynamic focusing flow pattern.
The control over the mixing process by varying the width of the focused
stream, wf, provided a direct means to control NPs characteristics, as
demonstrated previously [126,169]. The theoretical mixing time for
flow focusing reactors (τmix) was estimated as
w2f
τmix ≈ ð10Þ
4D
where D represents the diffusion coefficient for the solvent. However,
this estimation represents only a largely simplified model; and many
other factors should be taken into account to understand the effect of
microfluidic environment on nanomaterial synthesis.
For instance, Capretto et al. [170] have demonstrated that the
dimensional properties of polymeric micelles are determined not only
by the flow rate ratio, as largely understood, but also by microrector
geometry and fluid physical properties (i.e. viscosity), all of which act
in concert to govern the mixing process and thus nanomaterial output.
Similar findings were reported by Jahn et al. [176] for the production of
lipid vesicles. Authors examined the role played by the total flow rate in
the fine tuning of vesicle dimension under certain focusing regimes.
Karnik et al. [128] have demonstrated a consistent decrease in NPs
size as the mixing time decreased far below the estimated aggregation
time for block copolymer NPs, which was putatively associated with
rearrangements of block copolymer during the growth process. These
observations were in contrast with previous results by Johnson et al.
1522 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532
[173] with CIJ mixers, where a minimal critical NP size was associated
with a breakpoint mixing time. Furthermore, they showed an effect of
species mixing time on the surface properties of NPs, as a result of the
smaller portion of hydrophilic block buried in nascent NPs. These
observations indicated that the mechanism of nanomaterial production
in a microfluidic environment might differ from that postulated for CIJ
and MVIM mixers, and that more investigations are required to open
the way for industrial applications of microfluidic-based production of
organic NPs.
In reason of what stated above, this section aims to summarize
the current literature on how the microfluidic environment influences
the dynamics of nanomaterial production leading to somewhat peculiar
results, as compared with conventional preparation methods. This
influence can be attributed to two main factors being the mixing
dynamics and the fluid shear stress.
6.1. Computational fluid dynamic (CFD) models for indirect investigation of
the nanomaterial formation dynamics
Microfluidic reactors are generally characterised by laminar
flow conditions which generate predictable and quantifiable species
concentration distributions. This characteristic can be leveraged by
indirectly investigating, through computational fluid dynamic
models, the dynamics of nanomaterial formation (i.e. self-assembly
or nanoprecipitation) and its dependence on the mixing dynamics.
In a similar way CFD models could also be used to investigate how
various system parameters (such as device geometry, flow rate
ratio, total flow rate, etc.) affect the mixing dynamics with the
ultimate goal of correlating device/system operational conditions
to nanomaterial output [170].
Many researchers have therefore tried to couple computational and
experimental results in an effort to unveil the mechanism through
which microconfinement affects nanomaterial characteristics. Capretto
et al. [170] used a CFD model that took into consideration the variation
of the diffusion coefficient of species according to the Stokes–Einstein
equation, which describes the relation between viscosity of the medium
and the diffusion coefficient of the species, and investigated its effect on
the microfluidic mixing process and polymeric micelles size. They
demonstrated that variation in viscosity and diffusivity during solvent
exchange affects mixing efficiency by modulating mass transport
between solvent and non-solvent streams. The extent of this effect
also depends on channel size and flow conditions. They therefore
concluded that the interfacial phenomena and fluid properties
significantly affect the nanoprecipitation process, alongside the effect
of reactor geometry and fluidic control. Similar findings which correlate
the effect of viscosity of solvent phase and nanoparticle dimension
were also recently reported for the production of hyaluronic acid
nanoparticles [298].
It should be also considered that the high viscosity difference
between the central focused stream and the two sheath fluids
represents a crucial feature to take into consideration when looking at
the mixing process. Multi-fluid flows have been characterised by the
tendency for the less viscous fluid (i.e. water) to place itself in regions
of higher shear stress (i.e. near the channel walls) thus enveloping the
high viscosity fluid [299]. In addition, species diffusion across the fluid
streams generates viscosity gradients which affect non-trivially both
fluid velocity field and mixing performance [299]. In this regard, it has
been demonstrated that reducing the flow rate ratio, the focused stream
becomes progressively more ensheathed by the non-solvent. This
causes a gradual increase of the surface-to-volume ratio of the interface
between the two phases, which likely accelerates the mixing process
resulting in smaller and less polydisperse nanomaterial output [172].
It should be noted that the analytical models (i.e. Eq. (10)) are
mostly based on mass flow balance and two-dimensional diffusion
mass transfer, hence they do not take into account the contribution of
additional influencing factors such as the velocity profile and SVR of
the mixing interface. In this regard, Jahn et al. demonstrated with the
aid of a CFD model that total flow rate could modulate liposome size
distribution [176]. Experimental results and CFD models [176,300]
have shown that two different regions can be identified in a focused
stream, each characterised by different mixing performance. The first
region is located at the channel junction, in the arrowhead-shaped
focusing region and is characterised by rapid convective-diffusive
mass transport. The second region is located downstream the channel
junction, in the focused stream region, and is characterised by mixing
dominated by molecular diffusion in the direction normal to the flow
streamlines. Through the variation of the total flow rate Jahn et al.
were able to influence the relative amount of liposomes assembling in
the two regions. This resulted in a fine modulation of mixing time
which in turn resulted in the variation of liposome size [176]. Capretto
et al. [172] expanded this view for polymeric micelles self–assembly,
and developed a mathematical tool to rule out the effect of velocity
field from CFD results, making it possible to investigate the role of
mixing process and residual solvent content during self-assembly in a
non-convoluted manner with fluid velocity field.
Taken together these findings have demonstrated that hydro-
dynamics and diffusive mixing in microchannels are highly coupled
phenomena. Viscosity, flow rate ratio, total flow rate, diffusivity and
fluid velocity profile contribute in concert to determine the mixing
process. An additional level of complexity is introduced when hybrid
nanoparticles are produced. In this case two species undergo the
process of precipitation/self-assembly influencing each other.
By using Pluronic F127 and β-carotene as model stabiliser and
hydrophobic drug, respectively, it was demonstrated the existence of
competitive reactions resulting in the formation of two types of
nanoparticles, i.e., either with or without loading β-carotene in the
core–shell structure [1]. The relative amount of the two types of
particles could be varied by varying the process parameters such as
concentration of the two species and fluidic conditions [1]. The authors
explained the results with the aid of a CFD model that simulated the
effect of NP formation on their diffusion and advection kinetics and by
introducing a new parameter representing the mismatching between
the aggregations of the two precipitant species which affected the
relative amount of the two particles produced. Interestingly, Valencia
et al. [255] have demonstrated that in the case of the production of
lipid–polymer hybrid NPs, the stabiliser self-assembly (i.e. lipids) does
not affect the precipitation of the polymer core; hence, the formation
of the hybrid NPs could be seen as a two separate stages process. Despite
the contrasting results obtained, the two groups similarly pointed out
the importance of controlling the amount of shell formation molecules
(i.e. Pluronic F127 or lipid) to avoid undesired formation of empty
polymeric micelles or liposomes.
In an effort to correlate mixing and precipitation dynamics, different
authors have used physical parameters such as cloud point [296] and
critical water concentration [1] as mixing criterion to quantify mixing
efficiency and the triggering of nanoprecipitation due to solvent-
displacement. This strategy has been found particularly useful in
understanding the mixing capability of a complex mixer in relation to
the variation of the process parameters [296].
6.2. Methods for direct investigation of nanomaterial formation dynamics
Beside the indirect analysis performed with CFD models, direct
techniques have also been used to investigate the nanomaterial
formation dynamics. Schutze et al. [301] have monitored the formation
of block copolymer NPs by using a fluorescent block copolymer and a
careful selection of chromospheres with an adsorption spectrum that
overlapped with the emission spectrum of the fluorescent polymer. In
this way the authors were able to spatially resolve particle formation
as well as incorporation of small guest molecules by microscopic
fluorescence spectroscopy analysis. Authors showed that mixing
process and particle formation occurred simultaneously, with the first
 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532
being the rate determining step. These results suggest that there is not
distinguishable lag time before the creation of a supersaturate solution
and the triggering of the aggregation. In addition, they demonstrated
that the payload find his way to the core of the particle gradually during
the particle formation process, rather than partitioning when the core–
shell structure is already formed.
Jahn et al. [302] have recently described a method that relies on
instantaneous immobilisation by means of ultra-rapid cooling with
propane jet-freezing (Fig. 9a). With this method the authors were able
to study the dynamic of formation of liposome by microscopic analysis
of the slices of frozen fluid domain taken at different distances from
channels junction (Fig. 9b–f). Different distances correspond to different
residence time and hence represent different stages along the assembly
process. The method was able to reveal the formation of transient
intermediate species and it was found a correlation between computed
concentration profile and distribution of liposomes across the cross
section. Of particular importance is that this method could be applied
without altering the chemical characteristics of the molecules of interest
as opposed to the fluorescent polymer method previously describe in
this section.
6.3. Effect of shear stress on nanomaterial characteristics
In microfluidic environment, due to the small channel dimension
and to the large surface-to-volume ratio, the wall effect becomes
relevant in terms of the relative amount of fluid volume that it is
influencing. In other words, due to the small dimension, the shear
field extends its effect across the whole cross section of the channel
and impacts a relatively large amount of fluid volume. Recently few
reports have shown how shear induced effects have an impact on
nanostructures formed in microfluidic environments. Using a classical
flow focusing device Hasani-Sadrabadi et al. [252] were able to exert a
control over the size and shape of polymeric NPs by tuning the flow
rate ratio (Fig. 10a–b). The authors have suggested that the formation
of elliptical NPs could be attributed to the shear field at the focusing
region that organizes the polymer chain in extended wormlike
structures. The rapid mixing then freezes this structures giving rise
to elongated particles. The particles became more elongated as the
flow rate ratio increased as a result of a longer mixing time that allows
the stretched wormlike structures to be formed. Notably, authors
Fig. 9. Schematic representation of the instantaneous immobilisation device which performs ultra-rapid cooling of the microfluidic domain with propane jet-freezing (a). Cryo-SEM
images of the slices of frozen fluidic domain: (b) low magnification image showing a clearly distinguishable focused stream at the centre of the fluidic domain; (b–f) high magnification
images showing the radial distribution of lipid vesicles (adapted with permission from [199]. Copyright 2013 American Chemical Society).
1523
revealed that NP aspect ratio is of crucial importance to obtain a
functional ion exchange nanocomposite. Thanks to the fine control
over NP characteristics, the fuel cell performance was observed to
improve significantly.
Wang et al. reported a procedure where a segmented gas–liquid
device was used to produce block copolymer micelles of various
morphologies [304] and vesicles (Fig. 10c–h) [303]. The authors
postulated that the unique shear stress field produced in the device,
characterised by shear hotspots at the plug corners, caused a shear-
induced coalescence of the just formed micelles, which led to the creation
of larger micellar structures; the latter, as results of the intramicellar
relaxation rearrangements, gave rise to various non-spherical micelles,
which became kinetically frozen when collected off-chip in a large excess
of water. These non-spherical micelles were mainly represented by
cylinders, however, due to the statistical nature of collision-coalescence
events at the hotspots, various shapes were created including linear
and looped circles, bilayer sheets and networks. Interestingly, the same
authors also showed that by changing the solvent environment it was
possible to direct the rearrangement step toward the formation of
vesicles rather than cylinders [303].
7. Assessing the biological performance of nanomaterials produced
by microfluidic technology: in-vitro and in-vivo tests
Evaluating the interaction between microfluidic-produced nano-
materials and biological systems (i.e. cells and tissues) is a necessary
pre-requisite to facilitate their potential translation into the clinical
environment. However, only few studies have been performed in this
sense ― both in-vitro and in-vivo ― and a brief summary of recent
findings is reported in this paragraph.
In a study by Capretto et al. [171] a DNA-binding drug (mithramycin)
has been encapsulated within Pluronic® block copolymer micelles (PM-
MTH) produced by HFF (Fig. 11a–b). Authors investigated the effect of
PM-MTH on erythroid differentiation of both human erythroleukemia
and human erythroid precursor cells in-vitro. A comparative inves-
tigation between micelles produced by microfluidic technology and
micelles produced by conventional bulk procedures was carried
out (Fig. 11b). Notably, PM-MTH showed a slightly lower toxicity
and a greater differentiative activity, when compared with the free
drug. Furthermore, PM-MTH produced by microfluidic technology
1524 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532

Fig. 10. Effect of shear stress on nanomaterial characteristics. (a–b) Aspect ratio of nanoparticles was controlled by varying the flow rate ratio (adapted with permission from [252].
Copyright 2012 American Chemical Society). (c–e) Schematic representation of the gas-flow reactor used for the production of block copolymer micelles of various morphologies.
(f) Shear stress field caused a shear-induced coalescence of the micelles, which led to the creation of larger micellar structures which later rearranged to give rise to a (g–h) mixture of
spherical and non-spherical micelles (adapted with permission from [303]. Copyright 2012 American Chemical Society).

were capable of upregulating preferentially the production of γ-
globin messenger ribonucleic acid, and increasing the accumulation
of foetal haemoglobin (HbF). The percentage of HbF-containing
cells and their HbF content were also increased without stimulating
the expression of α-globin, which is a known responsible for the
clinical symptoms associated with β-thalassemia. Results from this
study represent an important step toward a potential clinical translation
of the produced polymeric micelles, since an increase in HbF could
reduce or alleviate the symptoms of β-thalassemia and sickle cell
anaemia on the treated patients. In conclusion, observations from this

Fig. 11. Biological response induced by nanomaterials produced via microfluidic technology. (a–b) Effect of mithramycin on erythroid differentiation of K562 cells. Microscope images of
cells after benzidine staining (a), upon cell treatment with mithramycin encapsulated in polymeric micelles (i.e. produced by HFF), at varying mithramycin concentrations (mM). Scale
bar = 25 μm. The percentage of benzidine-positive cells is reported in (b) for control (absence of drug), MTH-PMs produced in batch, MTH-PMs produced by HFF, and free drug. Data
represent the average of three independent experiments (run in triplicate) ± standard deviation (microfluidics vs free mithramycin; P b 0.05) (republished with permission of DOVE
Medical Press, from [171], Copyright 2012; permission conveyed through Copyright Clearance Center, Inc.). (c–d) Osteogenic differentiation of hPDLSCs after exposure to PMs produced
by HFF. Differentiation potential of hPDLSCs was determined by evaluating, up to 21 days, the mineralisation of the cell cultures in the presence of control medium, osteogenic traditional
medium, PMs-batch and PMs-microfluidics. Alizarin Red staining for extracellular calcium deposition is reported. Furthermore, viability is determined by double staining assay with
Calcein-AM and propidium iodide (PI). Fluorescence photomicrographs show the presence of green fluorescence (Calcein-AM)-labelled live cells and the absence of red fluorescence,
(PI)-labelled dead cells (reprinted from [251], Copyright 2013, with permission from Elsevier). (e–g) Intracellular delivery of pDNA/PEI complexes produced by microfluidic HFF. pGFP
expression in NIH 3T3 cells at 4 days post transfection is reported for NPs production by batch method (e), HFF (f), and positive control (Lipofectamine) (g). (Scale bars = 500 μm).
(h–i) Confocal microscope images showing the intracellular distribution of PEI/pDNA complexes produced by microfluidic HFF, at 4 days post-transfection. pDNA is labelled with
YOYO-1 dye (green), and PEI is conjugated with rhodamine (red). Scale bars = 10 μm. (reprinted with permission from [305]. Copyright 2009 American Chemical Society).
 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532
study suggest that microfluidic approaches may be exploited for
producing PM-MTH, to be potentially employed as a novel therapeutic
protocol for β-thalassemia.
The same group later reported in-vitro co-delivery of dexamethasone
(Dex) and ascorbyl-palmitate (AP) to human periodontal ligament
mesenchymal stem cells (hPDLSCs), for induction of osteogenic
differentiation [251] (Fig. 11c–d). Drugs were co-encapsulated in
block copolymer micelles produced by a microfluidic HFF device.
Both microfluidic-based and bulk production techniques were
investigated for comparison. Notably, drugs encapsulated into PMs
resulted in a lower in-vitro cytotoxicity compared to the free drug. In
addition, PMs produced by HFF had a stronger osteogenic differentiative
activity, if compared with PMs prepared by bulk method (Fig. 11c–d).
This was attributed to the smaller size of PMs produced by microfluidic
technology, which resulted in a more efficient cellular internalisation of
the bio-actives. Results from this study suggested that PMs produced by
microfluidics may be employed as drug carriers for lipophilic drugs
acting as inducers of osteoblastic differentiation and bone tissue
regeneration, with potential application in tissue engineering and
repair.
Koh et al. [305] investigated the use of polyplexes produced by
microfluidic technology as non-viral vectors for intra-cellular gene
delivery (Fig. 11e–g). Polyethylenimine (PEI) and plasmid DNA
(pDNA) complexes (PEI/pDNA) were produced by means of an HFF
microdevice, which was constituted of microchannels milled into layers
of PMMA. Both transfection efficiency and cell viability were quantified
in-vitro, by using NIH 3T3 fibroblast cells and mouse embryonic stem
(mES) cells. The performance of polyplexes produced by microfluidic
technology was compared with that of bulk methods (i.e. vortexing).
Results showed that the microfluidic method allowed for improved
transfection efficiency compared to bulk methods, particularly at
the higher flow rate ratios (i.e. FRR = 6.7), which was assessed by
either pGFP expression and alkaline phosphatase (SEAP) activity, at 2
and 4 days post-transfection. This was attributed to the more uniform
NPs size distribution and the efficient complexation achieved
with HFF. Confocal laser scanning microscopy (CLSM) revealed the
spatial localisation of complexes within the cell (Fig. 11h–i). Notably,
complexes prepared by HFF accumulated at the perinuclear region of
the cells at 2 days post-transfection. However, at 4 days post-
transfection, the large majority of complexes disassociated and plasmids
entered the cell nucleus for transfection (Fig. 11i). Importantly, cells
treated with complexes produced by microfluidic technology had the
highest viability, likely due to more efficient complexation resulting in a
lower amount of free PEI. Results from this study thus demonstrate the
potential of microfluidic-based HFF methods for producing gene carriers
with high transfection performance and biological tolerability. However,
an accurate design of the optimal production technique must take
into consideration the effect of fluid shear stress on the integrity of
the encapsulated molecules. In this study, authors showed that the
hydrodynamic focused sample was characterised by increased number
of structural changes of the pDNA molecule (i.e. open circle structures)
compared to macroscale methods. Therefore, an optimal balance
between complexation efficiency, volume production and preservation
of actives' molecular integrity must be identified.
In a further study by Koh et al. [268], a multi-inlet microfluidic HFF
device has been employed to produce lipopolyplex (LP) containing
Bcl-2 antisense deoxyoligonucleotide (ODN). Modulation of oncogene
expression by means of ODNs has long been pursued as a therapeutic
route in cancer therapy; however, the use of free ODNs has been limited
by rapid clearance from the haematic circulation, opening the way for
the use of liposomes as drug vehicles. Results from this study showed
that complexes produced by HFF exerted greater downregulation effect
on Bcl-2 in K562 leukaemia cells in-vitro, compared to bulk methods.
This is likely due nanoparticles smaller size, narrower size distribution,
lower zeta potential, and more uniform structure than those produced
by conventional bulk methods. Furthermore, it has been demonstrated
1525
in this study that complexes produced by HFF resulted in higher
percentage of cells undergoing late stages of apoptosis (i.e. programmed
cell death in the cell's life cycle) compared to macroscale routes,
suggesting that different methods of lypoplexes production may
significantly influence cell death mechanisms.
Recently, Belliveau et al. [259] investigated the performance of
lipid nanoparticles (SLN)–siRNA systems produced via a staggered
herringbone micromixer. The gene silencing potency of SLN–siRNA
systems was studied in-vivo, by using a mice model. Importantly,
results from this study showed that LNP–siRNA systems produced by
microfluidic-based mixing had equivalent or improved performance,
when compared with the ones produced by traditional bulk techniques
(T-tube synthesis) using cationic lipids with optimised cationic lipid
proportions and siRNA/lipid ratios. Furthermore, it was demonstrated
that increasing the cationic lipid content (i.e. from 40 to 60 mol%)
at a fixed siRNA/total lipid ratio (i.e. 0.06 wt/wt), resulted in a
significant reduction of the siRNA dose level required to achieve
50% of gene silencing, ED50 (i.e. from 0.2 to ~ 0.03 mg siRNA/kg).
Further improvement in potency could be achieved by decreasing
the siRNA-to-lipid ratio (ED50 ~ 0.03 mg siRNA/kg, at siRNA/lipid
ratio of 0.01). Results from this study suggest that microfluidic mixing
allows for routine production of LNP–siRNA systems, characterised by
high siRNA encapsulation efficiency and equivalent or greater gene
silencing potency, compared to previous formulation techniques.
In a recent study by Wi et al. [196] delivery of gene using liposomes
has been investigated. Lipid vesicles encapsulating recombinant
expression plasmid pORF2 were produced by HFF, and their delivery
into PK-15 cells was studied in-vitro. The expression of Cap
protein was monitored by fluorescence microscopy as an indication
of successful transfection. Results demonstrated the capability of
liposomes produced by microfluidic technology to allow for effective
transfection of mammalian cells in-vitro; however, no quantitative
analysis of transfection efficiency has been reported in this study,
which could represent the subject of future investigations.
It is also worth of notice that many of the proposed advancements of
nanomaterial produced with microfluidic approaches, such as long
circulation and targeting properties, resulting from the small particle
size and size homogeneity, could be addressed only in in-vivo setting.
Considering the interesting results obtained so far with in-vitro test, it
is envisaged that more in-vivo tests will be performed in the near future.
8. Conclusion and outlooks
Advances in nanomedicine have dictated the need for developing
more robust and controllable procedures for the production of
nanoparticles [306,307]. Classical methods largely rely on bulk mixing
and tend to suffer from poor reproducibility from batch to batch and
difficulties to implement fast screening and optimise nanomaterial
properties. In this respect, continuous flow microfluidic reactors have
shown the capability to produce micro- and nanoparticles in a
controllable and reproducible manner offering a possible solution to
the aforementioned issues [106].
Compared to the intense research at the intersection between
inorganic nanoparticles and microfluidics, relatively little has been
done for the production of organic nanomaterials. Considering its high
potential in nanomedicine, the use of microfluidic-based reactors for
the production of various nanostructured drug delivery systems has
increasingly attracted attention from different fields.
The fluid dynamic environment within microfluidic domains has been
observed to have important implications on nanomaterials production.
Notably, unique features make microfluidics an appealing technique for
nanomaterial production, including: (i) an efficient and controllable
mixing which results in a homogeneous reaction environment; (ii) an
efficient temperature control and heat transfer; (iii) the possibility of
in situ monitoring the nanomaterial production process; (iv) the
possibility of tuning the characteristics of nanomaterials by controlling
1526 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532
the kinetics of the process; and (v) the opportunity to integrate post
synthesis processes and measurement systems on a single technology
platform. Those features have been leveraged by various researchers
to produce different categories of nanomaterial, including drug
nanoparticles, polymeric micelles, liposomes, niosomes, polymersomes
and hybrid nanoparticles. However, despite the breathing activity and
the very encouraging results so far, the field of microfluidic production
of nanomaterials is still at its early stage. Microfluidic reactors have thus
the potential to become a standard production tool for the formulation
of nanomedicine delivery systems with unprecedented degree of
controllability and homogeneity. In our opinion, various aspects should
be addressed in the future to advance this technology.
One of the most relevant shortcomings of microfluidic devices is
the relatively small production rate of nanomaterials. This is due to
the small dimension of the operating channels and the associated
technological restriction of mass flow rate attainable, due to the increase
in the pressure drop across the microchannels with increasing the flow
rate. Recently, various strategies to tackle this issue have been proposed
which have demonstrated nanoparticles production as high as 3 g/h.
They relies on new micromixer designs where either microvortices
[257], jet-impact [253] or multi-lamination strategies [308] have
been employed to attain high production rate without compromising
the controllability of mixing. It is envisaged that in the future more
researchers will try to address this challenge, because it represents
one of the most important issues which hinder the use of microfluidic
devices in industrial setting. Enhanced device throughput is often
achieved by parallelization of individual functional units [239].
However, it is often difficult to ensure identical physical conditions
within each unit, thus sometime resulting in lower control over NPs
properties [239]. It is thus important that increased production
throughput is accompanied by high quality products. In this respect,
soft lithography techniques may be employed to produce microchannels
architectures with finely controlled dimensional characteristics, allowing
for enhanced microchannel parallelization with accurate prediction of
the flow field within each segment of the microfluidic system. Recently,
Kang and coworkers [309] developed a multi-layered device comprising
of three PDMS layers and a glass slide, for high-throughput production of
polymeric NPs. Device architecture consisted of a tree-like structure
with N 100 output channels in which hydrodynamic focusing of a
solution of MPEG–PLGA in acetonitrile was achieved. Furthermore, the
microfluidic device was designed in such a way that NP self-assembly
occurred outside the PDMS channel, thus avoiding potential fouling on
the channel wall. NPs with average size in the range 50–200 nm were
obtained, characterised by significantly narrower size distribution
compared to batch production. Importantly, NP production rate was
improved compared to previous studies (polymer flow rate in the
range 0.5–2.0 mL/h) and represents a promising step forward towards
mass production of NPs.
Microfluidic reactors are also suitable for introducing in line
measurement systems which provide on time information on the
characteristics of the produced nanomaterials [143]. Such information
could be then employed to monitor the operation of the device and
hence quickly act to prevent or reduce the impact of any undesired
deviation on the nanomaterial batch. As already demonstrated [119],
this information could be fed into a control algorithm to generate
internal feedback which will automatically tune the reaction conditions
to maintain the nanomaterial critical characteristics. In addition, in line
measurement systems would provide a prompt information on the
characteristics of the nanomaterial produced reducing the time needed
during optimisation and screening processes. However, despite the
interesting applications of in line measurement systems, only limited
research has been devoted to this area and it is therefore possible that
in the near future new studies will address this topic. In particular,
the developed techniques sometime suffer from NP-specificity and
poor sensitivity which makes them unsuitable for robust and large-
scale NP characterisation and quality assay. In order to overcome
these limitations, promising advancements have been reported
by Tarabella et al. [310] in a recent study. Authors developed a
microfluidic-based organic electrochemical transistor (OET) capable of
sensing and real-time monitoring of liposome-based structures. The
microdevice demonstrated to be an efficient and sensitive detection
system, with a lowest detection limit of 10−7 mg/mL, which falls within
the range required for drug loading/drug delivery applications.
One of the major challenges in the field of NPs analysis and physico-
chemical characterisation is also represented by the difficulties
encountered in manipulating nanometre-sized bodies. In this review we
discussed the use of different physical forces (i.e. electro-magnetic, optical
and mechanical) for manipulating, trapping and sorting NPs. However,
challenges still remain to achieve high-throughput manipulation and
efficient interfacing with analytical and sensor systems. Given to its
contact-less nature, electrophoresis represents the most widely adopted
technique in this field, and recent studies have demonstrated its
capability to manipulate NPs in a continuous-flow format [311].
Contact-less manipulation methods are usually preferred to mechanical
manipulation, which can potentially result in compromised NP
integrity. However, the latter is usually simpler to implement and
does not require integration with external energy sources. Recent
advancements in the field of mechanical manipulation of NPs have
been reported in a study by Wang et al. [312]. Authors developed
ciliated micropillars for multi-scale “gentle” filtration of a liposomes
suspension. Cilia consisted of porous silicon nanowires which trapped
the suspended liposomes and could be subsequently dissolved in PBS
for recovery of the trapped sample. Notably, liposomes were integer
after device operation.
Another important feature of microfluidic reactors is the limited
requirement in terms of reagent consumption. This, together with the
possibility of in process variation of reaction conditions, including
reactant concentration and content, might provide the possibility to
create platform for rapid optimisation and high-throughput screening
of nanomaterial's biological activity. A step towards this paradigm has
been recently presented [313] utilising a continuous flow system.
However, considering the fast growing interest and technological
advancements in droplet microfluidics for screening applications
[226,314], it may be possible to see in the future the coupling of
microreactor technology and droplet-based analysis systems.
Relevant to the assessment of NP biological performance is the
development of devices capable of mimicking the physiological flow
dynamic environment. Recently, Kusunose et al. [315] reported a PDMS-
based micro-flow chamber for investigating the binding of liposomes to
a monolayer of endothelial cells, exposed to physiologically-relevant
fluid shear stress in the range 2.4–8.6 dyn/cm2. This platform could be
employed as an alternative to animal models for testing the performance
of NPs for drug delivery applications. Notably, the mechanisms of
interaction between NPs and cells, and the cellular processes that govern
intracellular drug delivery remain largely unclear. A recent investigation
by Sahay et al. [316] has opened new fascinating insights into the
mechanisms of cellular uptake of short interfering RNA (siRNA) loaded
in lipid NPs produced by microfluidic technology. Authors demonstrated
that the uptake efficiency can be improved by designing delivery vehicles
which can escape the recycling pathways (i.e. endocytosis).
Recently various authors have shown that beside nanoparticle size, NP
shape is also able to affect the process of cell internalisation [317–319]. In
this regard, it is important to remark that the fast mixing achievable in
microfluidics provide a way to “lock” the formed NP in non-equilibrium
morphology and crystal structures whit the aim to produce non-
conventional nanomaterial shapes. Investigations towards this direction
have been recently published with promising results [252] but still with
limited evidence on the underlying governing mechanisms. In particular,
Wang and coworkers [320] recently investigated the role played by
either chemical and fluidic conditions on the morphology of copolymer
self-assemblies produced using a two-phase gas–liquid segmented
microreactor. Authors highlighted peculiar characteristics of microfluidic
 L. Capretto et al. / Advanced Drug Delivery Reviews 65 (2013) 1496–1532
production compared to macroscale synthesis. This represents a very
fascinating area of research which could open a highway of possibility
not only limited to nanomedicine [252].
Furthermore, it must be highlighted that only few studies have
investigated the encapsulation of actives within NPs in a microfluidic
system or the related governing mechanisms. As a matter of fact, this
is crucial to design novel therapeutic strategies and it is envisaged that
it will represent the subject of future investigations, as manifested by
recent published studies. With this respect, Majedi et al. [321] employed
a T-shape 2D HFF device to produce hydrophobically-modified chitosan
NPs loaded with hydrophobic anticancer drugs (i.e. paclitaxel). Authors
investigated the effect of operating parameters on loading efficiency
and studied the kinetics of drug release from NPs.
Another important issue that should be addressed in the future is
represented by the necessity of performing additional post processing
steps, after the microfluidic nanomaterial production, to obtain samples
suitable for in-vivo and in-vitro tests. These include for instance, the
purification of the reaction mixture from toxic solvent through dialysis
[322], and the separation of the particles from the reaction mixture [323].
A fascinating area of future research is also represented by the design
and production of vesicles to be employed as models of biological
systems. Microfluidics could represent a powerful tool to generate
dimensionally controlled cellular models and incorporate sub-cellular
structures. In a recent study by Mijajlovic et al. [324], synthesis of 1-
palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles was
performed with using 2D HFF device. From the moment the POPC is
one of the fundamental components of cell membrane, the produced
vesicles could find application in the modelling of cells or organelles. A
study by Kamiya et al. [325] has recently reported the production of
cell-sized vesicles encapsulating smaller vesicles by multi-step pulsed
jetting. The produced “vesicles in a vesicle” could represent an artificial
cell model to be employed in the investigation of biological processes,
such as the intra-cellular traffic of vesicles.
In conclusion, despite the fact that the use of microfluidic reactor for
nanomaterial synthesis is not yet a mature technology, research carried
out in the last decades has established that microfluidics could facilitate
the production of nanomaterial for drug delivery purposes, offering a
novel method to improve and tune their properties. Considering the
rapid expansion of both the fields of nanomedicine and microfluidics,
the expansion of the knowledge at their intersection is likely to provide
new exciting avenues in the future for tailoring the properties of
nanomaterial through controlled microfluidic precipitation.
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