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Ahlin Grabnar Pegi

Title: The manufacturing techniques of drug-loaded polymeric


nanoparticles from preformed polymers

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Journal of Microencapsulation, 2011; ??(?): 113


2011 Informa UK Ltd.
ISSN 0265-2048 print/ISSN 1464-5246 online
DOI: 10.3109/02652048.2011.569763

The manufacturing techniques of drug-loaded polymeric


nanoparticles from preformed polymers
Ahlin Grabnar Pegi and Kristl Julijana
Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia

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Abstract
Over the past few decades, nanoparticle (NP) formulation has been the subject of extensive research. The
choice of a suitable NP formulation technique is dependent on the physicochemical properties of the drug,
such as solubility and chemical stability. Different NP manufacturing methods enable modification of the
physicochemical characteristics such as size, structure, morphology and surface texture, but also affect the
drug loading, drug entrapment efficiency and release kinetics. This review covers an update on the state of
art of the manufacturing of polymeric NPs from preformed polymers. Both, conventional methods for NP
preparation, such as spontaneous formulation and emulsification-based methods, and new approaches in
NP technology are presented. A comparative analysis is given for polymer, drug and solvent nature, toxicity,
purification, drug stability and scalability of the method. The information obtained allows establishing
criteria for selecting a method for preparation of NPs according to its advantages and limitations.
Keywords: nanoparticles, polymer, formulation, preparation methods, drug delivery

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Introduction

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NPs composed of polymeric materials have been extensively investigated for their use in delivery and controlled
release of low molecular weight drugs, peptides and nucleotides via oral, topical and parenteral routes (Table 1).
Different polymers, both synthetic and natural, have been
developed to improve site-specific delivery, controlled
release and drug-loading capabilities. The most common
types of polymer used for the production of NP carrier
systems are Food and Drug Administration approved
hydrophobic materials such as poly-lactic acid (PLA),
poly-(lactic-co-glycolic acid) (PLGA), polycaprolactone
(PCL) and hydrophilic polymers such as chitosan, albumin,
gelatine and alginate. Advanced polymer chemistry and
engineering are leading to the production of increasingly
intricate polymer structures, including multivalent polymers, branched polymers, graft polymers, dendrimers,
dendronized polymers, block copolymers, star-like
polymers and hybrid glyco- and peptide polymeric derivatives. Their advantages include better defined chemical
composition, tailored surface multivalency and defined
three-dimensional architecture. Synthetic hydrophobic
polymers have the advantage of sustaining the release of

Over the past few decades, nanoparticle (NP) formulation


has been the subject of extensive research. Given the variety of fields of application of such colloidal objects from
nanomedicine, drug delivery and cosmetics, to printing ink
and petroleum sciences, a thorough knowledge of the
formulation processes is essential in order to achieve
the given purposes and needs (Anton et al., 2008). The
acceleration in the discovery of new therapeutic moieties
(chemical, biological, genetic) has led to an increasing
demand for delivery systems capable of loading,
protecting, transporting and selectively targeting those
agents to desired sites (Egle et al., 2008).
Nanoengineering may be achieved at various levels
during formulation of a drug within a carrier. Examples
include modulation of the drug environment at the
molecular level, alteration of the physicochemical characteristics of the drug (e.g. solubility and structural integrity),
control of drug-diffusive mobility and modification of the
bulk and surface chemistry of the drug nanocarrier
(Jayagopal and Shastri, 2007).

Address for correspondence: Pegi Ahlin Grabnar, Faculty of Pharmacy, University of Ljubljana, Askerceva 7, 1000 Ljubljana, Slovenia. Tel: 386 1 4769 621.
Fax: 386 1 4258 031. E-mail: ahlinp@ffa.uni-lj.si
(Received 8 Nov 2010; accepted 28 Feb 2011)
http://www.informahealthcare.com/mnc
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Table 1. Properties of nanoparticles as an ideal drug delivery system.


General for drug delivery
Non-toxic, biodegradable and biocompatible
Amenable to small molecules, peptides, proteins or nucleic acid
(genes, antisense drugs)
Minimal NP excipient-induced drug alteration (protein denaturation,
chemical degradation/alteration)
Modulation of drug-release profile
Scalable and cost-effective manipulating process
Addressing the drug delivery problems
Solving the issues related to solubility
Overcoming the poor bioavailability of the drugs
Overcoming the poor stability
Benefits for therapy
Able to control the bioavailability, biodistribution, tissue uptake and
pharmacokinetics of entrapped drug
Enhance drug efficacy for prolonged intervals
Smaller drug doses and decreased dosing variability
Diminished toxicity
Reduction of the side-effects of an existing therapeutics
Improved patient compliance
Extension of the patent lifetime for already marketed drugs

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the incorporated drug over a period of days to several


weeks, in contrast to natural hydrophilic polymers which
have a relatively short duration of drug release. But
synthetic polymers are limited by the use of organic
solvents and in some cases relatively harsher formulation
conditions. In addition to short duration of drug release,
natural hydrophilic polymers are also subject to poor
batch-to-batch reproducibility (des Rieux et al., 2006).
Several methods have been employed to load drug molecules into polymeric NP and selection of a particular one
depends on the nature of the polymer and the properties of
the drug. They can be classified into two main categories:
bottomup and topdown techniques. The bottomup
techniques, such as emulsion or microemulsion polymerization, interfacial polymerization and precipitation
polymerization, employ a monomer as a starting point.
These are so-called polymerization-based methods. They
can present drawbacks such as inadequate biodegradability of the product and the presence of toxic residues. To
avoid this problem and to achieve particle systems of high
biocompatibility, preparation techniques involving the use
of purified natural macromolecules or preformed synthetic
polymers have been developed. These are the so-called
topdown techniques.
The various NP manufacturing methods enable modification of the physical characteristics such as size, structure,
morphology, surface texture, and also of drug loading, drug
entrapment efficiency and release kinetics. All these
changes depend on a range of parameters involved directly
in the manufacturing processes. Therefore, an understanding of the physicochemical phenomena involved during NP
formation and their quantitative effects on NP properties is
very important in the controlled engineering of particles
(Galindo-Rodriguez et al., 2004).
Several reviews on conventional NP technology have
been already published (Vauthier-Holtzscherer et al.,

1991; Allemann et al., 1993; Quintanar-Guerrero et al.,


1998; Bala et al., 2004). Vauthier-Holtzscherer et al. summarize the different preparation methods of polymer NPs,
with an emphasis on scaling up NP production and on
processing NPs after formulation, such as purification, sterilization, lyophilization and concentration (Vauthier and
Bouchemal, 2009). The aim of this review is to highlight
the diversity of methods that can be applied to produce
polymer NPs from preformed polymers. As well as providing updates of conventional methods for NP preparation,
such as spontaneous formation and emulsification-based
methods, we focus on new approaches in NP technology.
This includes supercritical technology, electrospraying,
premix membrane emulsification and the aerosol flow
reactor method. Finally, a comparative analysis is given
for polymer, drug and solvent nature, toxicity, need for
purification, drug stability and scalability of the method.
The information obtained allows establishing criteria for
selecting a method for preparation of NPs according to its
advantages and limitations.

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Conventional methods for NP preparation

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Conventional methods for NP preparation basically follow


two approaches, one based on the spontaneous formation
of NPs and the other on emulsification (Figure 1).

Spontaneous formation of NPs


NPs can be formed spontaneously by taking advantage of
the solubility or gelling properties of a dissolved polymer.
Based on its solubility properties, the general principle is
to prepare a solution of the polymer and then induce a
phase separation by adding a non-solvent of the polymer
(nanoprecipitation) or by salting-out or coacervation.
Based on the gelation properties of the polymer, NPs
from a hydrophilic polymer, such as alginate or chitosan,
can be prepared by the spontaneous method called ionic
gelation.

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Nanoprecipitation or solvent displacement


Nanoprecipitation was first described by Fessi et al. (1986).
It involves the use of an organic solvent that is completely
miscible with the aqueous phase, such as acetone, ethanol
or methanol (Fessi et al., 1989). The organic phase containing the hydrophobic polymer and drugis added dropwise to
an aqueous solution of stabilizer under stirring (Figure 2).
The hydrophobic polymer and drug are immediately
precipitated in the form of NPs. After NP formation, the
solvent is removed under reduced pressure. The molecular
mechanism of instantaneous particle formation involves
complex interfacial hydrodynamic phenomena and has
been explained by interfacial turbulences between two
liquid phases which are governed by the Marangoni
effect (Quintanar-Guerrero et al., 1998).
The process of nanoprecipitation is simple and transposable to the industrial scale. It requires only gentle

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The manufacturing techniques of drug-loaded polymeric nanoparticles

Formulation of NP from preformed polymers


SPONTANEOUS FORMATION

phase separation

nanoprecipitation

EMULSIFICATION TECHNIQUES

O/W or W/O/W emulsification

ionic gelation

coacervation

solvent evaporation

W/O emulsification

solvent displacement

solvent diffusion

salting-out

Figure 1. Conventional methods for preparation of NPs from preformed polymers.

AQUEOUS PHASE
(water + stabilizer)

ORGANIC PHASE
(solvent + polymer + drug)

Precipitation

Nanoparticles

polymer/solvent

Water

NP

Figure 2. Nanoprecipitation or solvent displacement method.

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stirring and involves no high stress shear. Its disadvantage


is that hydrophilic drugs remain in solution in the aqueous
phase, so that the method is limited to hydrophobic drugs
that are highly soluble in polar solvents, but only slightly
soluble in water (Alonso, 1996). It is also difficult to choose
a polymer/drug/solvent/non-solvent system that allows
sufficient entrapment efficiency and drug loading.
Generally, only NP dispersions with low concentrations of
polymer can be produced. Elimination of residual solvent
may be difficult, but this is a general drawback encountered in NP preparation from preformed hydrophobic
polymers.
Recently, a modified nanoprecipitation method was
optimized so as to allow hydrophilic as well as hydrophobic
compounds to be entrapped within NPs using two miscible
non-aqueous solvents in the absence of water (Bilati et al.,
2005a). Bilati et al. showed that solvents such as DMSO
(instead of acetone or ethanol) and non-solvents such as
methanol or ethanol (instead of water) can be used to prepare PLA or PLGA NP with a size ranging from 130 to
560 nm (Bilati et al., 2005b).
Coacervation or controlled desolvation
The second approach for spontaneous NP formation is
appropriate for macromolecules, such as proteins

(Leo et al., 1997). Gelatine or albumin NPs can be produced


by the slow addition of a protein precipitating agent, like
neutral salt (sodium sulphate, ammonium sulphate) or
alcohol (ethanol), to the protein solution (Figure 3). The
addition of a desolvating agent shrinks the macromolecule
coil, which becomes smaller and smaller, until the phase
separation from the solvent occurs and protein aggregates
in nanometric size are formed. The process is followed by
chemical cross-linking with glutaraldehyde to stabilize the
NPs. The main advantages of this process are the absence
of toxic organic solvents and its simplicity compared with
emulsification techniques. The method is not appropriate
for highly water-soluble drugs because they interact more
strongly with water than with the macromolecule and are
readily washed out of the particles. The disadvantage of the
method is also the use of stabilizing agents that may react
with the drug and may increase the toxicity of NP formulations (Marty et al., 1978; Irache and Espuelas, 2006;
Langer, 2006).
Ganciclovir-loaded albumin NPs with a diameter
between 200 and 400 nm (Merodio et al., 2001) and legumin NPs between 250 and 300 nm (Irache et al., 1995) were
prepared by coacervation and cross-linking with glutaraldehyde. Human serum albumin NPs were prepared in a
size ranging between 150 and 280 nm.
Ionic gelation
NPs can be prepared from hydrophilic ionogenic polymers,
such as alginate or chitosan (Kristl et al., 1993), by a spontaneous method termed ionic gelation. Such NPs can be
used for peptide and nucleic acid delivery.
Alginate NP can be formed by injecting alginate solution
into calcium chloride solution. Low concentration of the
solutions is used, in order that particles are formed in the
pre-gel state (Rajaonarivony et al., 1993). Ionic gelation is
often followed by polyelectrolyte complexation with oppositely charged polyelectrolytes such as polylysine or chitosan. This complex forms a membrane of polyelectrolytes
on the surface of the gel particles, which increases the
mechanical strength of the particles.
Chitosan NPs can be formed after dropwise addition of
chitosan dissolved in acidic aqueous medium to specific
polyanionic (sodium triphosphate (TPP) or dextran sulphate) solution. All concentrations of polymer solutions
have to be very low. The controlled gelation and formation

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Desolvating
agent

AQUEOUS PHASE
(water + albumin)

Turbidimetry
control

ORGANIC PHASE
(oil)

Crosslinking

Aqueous solution
of protein

Nanoparticles

Emulsification

Figure 3. Coacervation or controlled desolvation.

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of NPs are the consequences of the formation of inter- and


intramolecular cross-links between chitosan amino groups
and the polyanion (Calvo et al., 1997; Janes et al., 2001b ;
Fernandez et al., 2006).
Ionic gelation is very mild process useful for
encapsulation of macromolecules like proteins, peptides
and oligonucleotides. It does not require any special equipment and can be performed at room temperature. The
main difficulty of the method is to identify cross-linking
conditions that will yield the NPs.

Emulsification-based methods for NP preparation


A considerable number of NP formulation methods are
based on nano-emulsion templates (Anton et al., 2008).
Emulsification methods for NP preparation are derived
from microparticle preparation techniques. They require
the formation of an emulsion as the first step of the procedure and this homogenization step is the determining
factor in obtaining submicron particles. It can be achieved
by the use of high pressure homogenizers, microfluidizers
and sonicators.
Emulsification-based methods differ depending on the
properties of the polymer and on the miscibility of the
organic solvent with the aqueous phase. When hydrophobic polymers are used some methods involve volatile
and water-immiscible solvents which can be extracted by
simple evaporation, leading to polymer precipitation
(emulsification-solvent evaporation method, double emulsion technique). In other methods, partially or fully water
miscible organic solvents are used and polymer precipitation occurs as a result of controlled diffusion processes
(salting-out and emulsification-diffusion methods).
W/O emulsification
W/O emulsification technique, described in 1972 by
Scheffel et al., was the first procedure to produce
microspheres and nanospheres from natural hydrophilic
macromolecules, such as albumin, for medical application
(Scheffel et al., 1972). It is suitable for the incorporation of
hydrophilic drugs. An aqueous solution of macromolecule
is emulsified at room temperature in a vegetable oil and
homogenized, by either high pressure homogenization
(Kramer, 1974) or ultrasonication (Figure 4; Morimoto
et al., 1981). W/O emulsion is formed which is then

W/O emulsion

a) Heated oil (120C) or


b) Cross-linking agent

Albumin nanospheres
Figure 4. W/O emulsification technique.

added dropwise to a large volume of preheated oil


(4120 C) under stirring. This leads to immediate vapourization of the water contained in the droplets, with irreversible denaturation of the albumin which coagulates in the
form of solid NPs. The dispersion is then allowed to cool
and submitted to several washings with large amounts of
organic solvent (e.g. ether, ethanol, acetone) for complete
removal of the oil. In this procedure, the crucial factors
in nanosphere production include the emulsification
energy and the stabilization temperature (Alonso, 1996).
The hardening step by heat denaturation is likely to be
harmful to heat-sensitive drugs. To avoid the heat denaturation process, an alternative is to add a cross-linking agent
(2,3-butanedione or formaldehyde) for the chemical
hardening of the macromolecule nanodroplets. The main
problem of this method is the purification of the resulting
NPs with organic solvent such as diethyl ether.
It is very difficult to produce small nanospheres with
narrow-size distributions by the W/O emulsification technique, due to the intrinsic instability of the emulsion prior
to hardening by heat or cross-linking (Irache and Espuelas,
2006). This method therefore leads preferentially to formation of microparticles. However, Nakagawa et al. (1987)
have described a method to prepare bovine serum albumin
nanospheres with a diameter of 170 nm that relies on steric
stabilization of the albumin droplets during cross-linking.
Emulsification-solvent evaporation
The emulsification-solvent evaporation technique is the
oldest method used to form NPs from preformed polymers
and was first reported by Gurny et al. (1981). Hydrophobic
polymer and drug are dissolved in an organic solvent which
is volatile and water immiscible (e.g. ethyl acetate, chloroform, methylene chloride; Figure 5). This solution is then

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The manufacturing techniques of drug-loaded polymeric nanoparticles


AQUEOUS PHASE
(water + stabilizer)

ORGANIC PHASE
(solvent + polymer + drug)

Salt (only in
salting out
method)

protein or a peptide drug is the rapid diffusion of the molecule into the outer aqueous phase during the emulsification. This can result in poor entrapment efficiency, i.e. drug
loading. Therefore, it is critical to ensure immediate formation of a polymer membrane during the first water-in-oil
emulsification (Zambaux et al., 1998).

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Emulsification

O/W emulsion
Water
addition
3
SOLVENT
EVAPORATION

SOLVENT
DIFFUSION

SALTING
OUT

Nanoparticles
Figure 5. Emulsification-solvent evaporation (1), emulsification-solvent
diffusion (2) and salting-out method (3).

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emulsified in an aqueous stabilizer solution (PVA, poloxamer). Emulsification is carried out by sonication or under
high-energy homogenization to reduce the size of the
emulsion droplets and an O/W emulsion is formed. The
organic solvent is then removed by evaporation at room
temperature under stirring or under reduced pressure,
which leads to polymer precipitation and NP formation.
Two factors contribute to rapid solvent loss high volatility
of the solvent and the increased tendency of the polymer to
precipitate as it becomes more and more exposed to water.
As the precipitation process takes control over diffusion,
the solvent is expelled from the particles. The physicochemical properties of a drug are decisive in influencing
its loading and distribution between particles and aqueous
medium (Ahlin et al., 2000a, 2000b, 2003). The method
provides NPs with high drug entrapment efficacy, although
it is limited to lipophilic drugs that are soluble in the same
solvent as the polymer. Hydrophilic drugs are distributed
into the external water phase and not retained in the polymer phase. Limitations are imposed by the scale up of the
high energy requirements in homogenization and the use
of toxic chlorinated solvents.
The double emulsion solvent evaporation technique is a
modification of the emulsification-solvent evaporation
method and can be employed to entrap small hydrophilic
drugs and proteins. An aqueous drug solution is emulsified
in an organic polymer solution, usually by sonication. This
pre-emulsion is then added to an aqueous phase containing a stabilizer. This results in W/O/W emulsion formation
which is followed by evaporation of the organic solvent.
The polymer precipitate and NPs are formed. The main
problem with encapsulating a hydrophilic molecule like a

Emulsification-solvent diffusion
The emulsification-solvent diffusion method (Leroux et al.,
1995) involves the use of an organic solvent that is partially
water-soluble, for example benzyl alcohol, propylene carbonate, ethyl acetate and others. Polymer and drug are
dissolved in this organic solvent, which is then emulsified
in an aqueous phase containing stabilizer to form an O/W
emulsion (Figure 5). A large amount of water is then added
to the system in order to overcome the miscibility ratio of
organic solvent. This causes the solvent to diffuse into the
external phase. The polymer precipitates as a result of
the diffusion of organic solvent into the water, leading to
the formation of NPs. The solvent can be eliminated by
cross-flow filtration. The method is suitable for hydrophobic drugs, such as estrogen (Kwon et al., 2001), enalaprilat
(Ahlin et al., 2002) and ibuprofen (Galindo-Rodriguez et al.,
2005). The advantages of the method are the use of low
toxicity solvents (benzyl alcohol) and high batch-to-batch
reproducibility. High entrapment efficiency (generally
470%) has been reported. The disadvantage of the
method is the large volume of water that has to be eliminated from the suspension.
The double emulsion solvent diffusion method combines
the advantages of two well-established techniques, double
emulsion solvent evaporation and emulsification solvent
diffusion, and it is suitable for incorporation of hydrophilic
drugs (Kocbek et al., 2007; Cohen-Sela et al., 2009). The
double emulsion system minimizes escape of the hydrophilic drug to the aqueous medium, and ethyl acetate is
more acceptable pharmaceutically and considered to be
less toxic than dichlormethane. The preparation technique
results in improved formulation characteristics over those
of the classical method, including smaller size, lower size
distribution, higher drug entrapment efficiency and more
biocompatible ingredients (Tween and Pluronic rather
than PVA). The authors explained these findings by the
different mechanisms of NP formation. The dichloromethane used in the double emulsion method is relatively
poorly soluble in water and the solvent does not diffuse
extensively through the aqueous phase. NPs are formed
during evaporation once the critical concentration for polymer precipitation is reached, with a single particle formed
from each emulsion droplet. On the other hand, ethyl acetate, used in the double emulsion solvent diffusion method,
is partially water-miscible, and the solvent diffuses freely
via the aqueous phase, creating regions of local supersaturation near the interface (Cohen-Sela et al., 2009).
A double emulsification-solvent diffusion technique has
been demonstrated to maintain protein function (Cegnar
et al., 2004). Maintenance of protein activity during the
fabrication process involves a delicate balance between
energy input (mechanical stirring, homogenization and

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sonication) and particle size. A synergistic effect between


mechanical stirring and ultrasound was shown to produce
NPs of 300 nm in diameter, while maintaining 85% of the
starting activity of cystatin protein (Kristl et al., 2003;
Cegnar et al., 2004).

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Salting-out
The emulsification-solvent diffusion method can be
improved by salting-out effects. In the salting-out technique a water-miscible solvent, like acetone, ethanol or
N-methyl-2-pyrrolidone, is used instead of chlorinated solvents. It was first proposed by Bindschaedler et al. (Ibrahim
et al., 1992; Kristl et al., 1996; Bindschaedler et al., 1988).
The mixing of organic and aqueous phases is prevented by
saturating the aqueous phase with electrolytes such as
magnesium acetate, magnesium chloride or calcium chloride. An organic solution of polymer and drug is emulsified
into an aqueous phase, which contains salt and colloidal
stabilizer, to form an O/W emulsion (Figure 5). The emulsion is then diluted with sufficient water to enhance the
diffusion of acetone into the aqueous phase, thus inducing
polymer precipitation. Both the solvent and the salting-out
agent are then eliminated by cross-flow filtration. The
method does not require an increase of temperature and
avoids the use of organic chlorinated solvents and large
amounts of stabilizer during formulation. High drug loadings can be achieved, depending on the solubility of the
drug in acetone and on the nature of the salting-out agent.
The disadvantages are exclusive application to lipophilic
drugs and the extensive NP washing steps. The use of acetone and large amounts of salts may raise some concern
about recycling of the salts and about compatibility with
active compounds.
The application of emulsification techniques is greatly
limited by disadvantages such as working with toxic solvents (dichloromethane, chloroform) and the requirement
of high energy apparatus (ultrasound probe or homogenizer). In contrast, methods for spontaneous formation of
NPs do not require a source of external energy or organic
solvents, except in nanoprecipitation where less toxic
organic solvents such as acetone (Class 3 according to
the ICH specifications) are used (Table 2).

electrohydrodynamic atomization and aerosol flow reactor


is that the production of dried NP powders is possible without any extra drying step.

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SCF technology

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New approaches for NP preparation

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More recently, very attractive new techniques have been


established as useful alternatives to conventional methods
for NP preparation. These techniques have the same basic
approach as conventional preparation methods: the
formation of droplets and precipitation of polymer and
drug after the solvent is evaporated. The most important
advantage of these methods is that they are more
technically sophisticated and are therefore useful for
industrial production. They include supercritical fluid
(SCF) technology, electrohydrodynamic atomization,
premix membrane emulsification and technologies using
membrane reactor, aerosol flow reactor or vibrating nozzle
device. Another advantage of the SCF technology,

A SCF can be defined as a solvent at a temperature above


its critical temperature, at which the fluid remains a single
phase, regardless of pressure. Supercritical carbon dioxide
(SC CO2) is the most widely used SCF because of its mild
critical conditions (low critical temperature Tc 31.1 C
and moderate critical pressure pc 73.8 bars), low toxicity,
non-flammability and low price. The main advantages of
such techniques include mild processing conditions, the
possible sterilizing properties of SC CO2, the ability to produce NPs in the form of dry powders and feasibility of
scale-up. The SCF technology comprises several processes
for micro/nanoparticle production which are selected
according to the solubility of the drug in the SCF. The
latter can be solvents or antisolvents for the drug and the
polymer. The most common processing techniques involving SCFs are rapid expansion of the supercritical solution
(RESS) and the gas/supercritical anti-solvent technique
(GAS/SAS; Figure 6).
In RESS, the polymer and drug are dissolved in an SCF
and the solution then rapidly expanded through a small
nozzle into a region with lower pressure. The solvent
power of the SCF dramatically decreases and the solute
eventually precipitates. Because solubility in SCF can be
up to a million times higher than that under ideal gas conditions, the rapid expansion from supercritical pressure to
ambient pressure results in extremely high supersaturation
and, consequently, in homogeneous nucleation of the
solute, which leads to narrow size distributions in the
products (Byrappa et al., 2008).
There are several variations of the RESS process, and the
most interesting is the rapid expansion of a supercritical
solution into a liquid solvent (RESOLV). It consists of spraying the supercritical solution into a liquid an aqueous
solution containing a stabilizer. The stabilizers utilized
are mainly surfactants like polysorbates, poloxamers and
lecithins which minimize particle aggregation. Operating in
this manner makes it possible to stunt the particles growth
in the precipitator, thus improving RESS process performance (Sun et al., 2005; Meziani et al., 2006). PLA NPs
loaded with retinyl palmitate were successfully prepared
by RESOLV. Using 0.1% Pluronic F127 as an aqueous
stabilizer solution, NPs with an average size range of
40110 nm were obtained (Sane and Limtrakul, 2009).
The GAS/SAS process has been developed in order to
achieve nanosizing of the hydrophobic materials that
cannot be processed by the RESS technique owing to
their poor solubility in SCF. The method employs a liquid
solvent (methanol) that is completely miscible with the SC
CO2, to dissolve the polymer. Polymer and drug are dissolved in a liquid solvent and the extract of the liquid solvent by SCF leads to instantaneous precipitation of the
polymer (because the polymer is insoluble in the SCF),

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Hydrophobic
Hydrophilic
Hydrophilic

Hydrophilic
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic

Spontaneous formation
Nanoprecipitation
Coacervation
Ionic gelation

Emulsification-based methods
W/O emulsification technique

Emulsification-solvent evaporation technique


Double emulsification-solvent evaporation technique
Emulsification-solvent diffusion method
Double emulsification-solvent diffusion method
Salting-out technique

Hydrophobic
Hydrophilic
Hydrophobic
Hydrophilic
Hydrophobic

Hydrophilic

Lipophilic
Hydrophilic
Hydrophilic

Drug nature

Class 3/Class 2
Class 3/Class 2
Class 3
Class 3
Class 3

Class 3/Class 2

Class 3
No organic solvent
No organic solvent

Solvent nature

Organic solvent for purification (ethylether)


Hardening agents
Residual solvents
Residual solvents
Residual solvents
Residual solvents
Residual solvents and salts

Residual solvents
Hardening agents (glutaraldehyde)
Low toxicity

Toxicity

Low
Low
Moderate
Moderate
High

High

Low
Moderate
Low

Purificationa

Moderate
Moderate*
High
Moderate*
High

Moderate

High
Moderate
High

Drug stablityb

Yes
Yes
Yes
Yes
Yes

NR

Yes
NR
NR

Scalability

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Notes: NR, no reference available.


Low purification means that there are few steps involved; ba high probability that the drug will remain stable during preparation of NP.
*Proteins can be denatured by high shear rate.

Polymer nature

Method

Table 2. Comparison of criteria suggested for the selection of NP preparation method.

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Drug and polymer


dissolved in SCF

High
pressure
vessle

Rapid expansion of
SCF after leaving
the nozzle and
polymer
precipitation

NP in
collection
chamber

High
pressure
collection
chamber

Expanded
solution

Supercritical
anti-solvent
GAS/SASS

RESS

Figure 6. SCF technologies: RESS and gas/supercritical anti-solvent technique (GAS/SAS) (adapted from Ginty et al. (2005) with permission from
Elsevier).

500

resulting in the formation of NPs (Byrappa et al., 2008).


A modified supercritical antisolvent with enhanced mass
transfer (SAS-EM) process was successfully employed to
fabricate PLA NPs with paclitaxel. The application of ultrasonication to the SAS process greatly enhances the mixing
between the solvent and antisolvent phases, resulting in
particles smaller than 1 mm (Lee et al., 2008).

Syringe pump
(solvent + polymer + drug)

High voltage
power supplies

Particle
collector

Air inlet

4
Heater

Electrospraying or electrohydrodynamic atomization


Discharge electrode

505

510

515

520

525

530

Electrospraying is a method of liquid atomization by means


of electrical forces (Jaworek, 2007). A typical electrohydrodynamic atomizer (electroatomizer; Figure 7) consists of
a capillary nozzle, usually made from a fine hypodermic
needle and a ring extractor electrode. The capillary nozzle
is connected to a high-voltage supply, while the ring electrode is grounded. A strong electric filed thus builds up at
the capillary outlet. The liquid, which flows out from the
capillary nozzle, forms a meniscus, which becomes elongated in this electric field and disintegrates into droplets
due to electrical forces. The solvent from the electrosprayed droplets evaporates, and the remaining solid material forms NPs (Jaworek and Sobczyk, 2008).
Many modes of spraying are distinguished in the literature, depending on the form of the meniscus, the pattern of
motion of the jet and a way it disintegrates into droplets.
For NP preparation, the most important mode of spraying
is the cone-jet mode. In this mode, the liquid meniscus
assumes the form of a regular, axisymmetric cone with a
thin jet (5100 mm in diameter) at its apex stretching along
the capillary axis.
Electrospraying is a single-step, low-energy, low-cost
material processing technology which can operate under
atmospheric conditions. Due to its properties, it is considered as an effective route to nanotechnology (Ding et al.,
2005). The advantage of electrospraying over mechanical
atomizers is that droplets can be extremely small, from
hundreds of micrometers down to several tens of nanometres, and the charge and size of the droplets can be
controlled to some extent by electrical means, i.e. by

Figure 7. Electrospraying or electrohydrodynamic atomization (adapted


from Ciach (2006) with permission from Elsevier).

adjusting the flow rate and voltage applied to the nozzle


(Jaworek, 2008). Droplet size can be decreased by decreasing the size of the capillary and by decreasing the liquid
flow rate and increasing liquid conductivity or surface tension. The size distribution of the droplets can be nearly
monodisperse.
Xie et al. successfully produced biodegradable polymeric NPs by the electrohydrodynamic atomization
method. Under fixed nozzle and ring voltage difference, a
flow rate of 0.1 mL/h produced particles as small as 200 nm.
Higher flow rates could still produce nanometre sized particles if a higher salt concentration was used to increase
solution conductivity (Xie et al., 2006).

535

540

545

Membrane reactor
The membrane reactor (contactor) controls the addition of
one reactant (the organic phase) to another reactant (the
aqueous phase; Figure 8). This process can be compared to
the membrane emulsification process, where the oil (or the
water phase) permeates through the membrane pores to
form droplets in water (or oil phase) for the preparation of
O/W or W/O emulsions (Charcosset and Fessi, 2005b). The
organic phase is pressed through the membrane pores,

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Tangential flow
of the aqueous
phase

Nanoparticles

Organic phase
(solvent + polymer + drug)
Figure 8. Membrane reactor method (adapted from Charcosset and Fessi
(2006) with permission from Elsevier).

Coarse
emulsion

Porous glass
membrane

Nanoemulsion

Figure 9. Premix membrane emulsification.

Aerosol flow reactor

560

565

570

575

allowing the formation of small droplets. The reaction


occurs between the droplets of the organic phase and the
aqueous phase flowing tangentially to the membrane
surface.
Charcosset et al. have shown that PCL NPs as small as
260 nm can be obtained with acetone as organic phase, a
1000 Da nanofiltration membrane, a transmembrane
pressure of 3 bar and a cross-flow rate of 1.7 m s1
(Charcosset and Fessi, 2005b). The membrane contactor
method could be also used for the preparation of
nanocapsules (Charcosset and Fessi, 2005a) and solid
lipid nanoparticles (SLN; Charcosset et al., 2005; Ahmed
El-Harati et al., 2006).
The main advantage of the membrane contactor is the
high flux obtained with microfiltration membranes, making
possible scale-up for industrial applications. Other advantages are the versatility of the preparation of either nanocapsules or nanospheres, by methods involving
polymerization of dispersed monomers or dispersion of
preformed polymers (nanoprecipitation), and control of
the average NP size by appropriate choice of the membrane
(Charcosset and Fessi, 2006).

Premix membrane emulsification

580

585

590

595

Premix membrane emulsification is a two-step procedure


which combines emulsion emulsification and premix
membrane emulsification (Figure 9). The coarse emulsion
was obtained by low-speed rotor-stator homogenization
and then poured into the premix reservoir. Uniform-sized
nanodroplets were achieved by extruding the coarse emulsion through a Shirasu porous glass membrane with high
pressure. The nanodroplets were stirred overnight to
evaporate the organic solvent and the obtained resulting
NPs were washed and collected.
Wei et al. prepared PLA NPs and found that several factors play a key role in obtaining uniform-sized NPs, including the type of organic solvent, the volume ratio of oil phase
to external water phase, the pore size of the microporous
membrane and the transmembrane pressure.
This novel method has the advantages of high productivity, simplicity and easy scale-up by increasing the surface
area of the membrane or by connecting membranes in
parallel in the apparatus (Wei et al., 2008).

Aerosol flow reactors are similar to spray dryers. They operate by atomizing a solution of polymer and drug in a
common solvent or solvent mixture (water, ethanol, etc.)
via a collision-type air jet atomizer into an inert carrier gas
which is then passed through a heated tubular chamber
(Figure 10; Peppas et al., 2007). The solvent is evaporated
in a controlled manner using a heated tubular laminar flow
reactor, and the particles produced are collected as a dry
powder using a low-pressure impactor (Eerikainen et al.,
2004). Compared to conventional spray drying, the aerosol
flow reactor method provides better control of the thermal
history and residence time of each droplet and product
particle, due to the laminar flow in the heated zone of
the reactor where droplet drying and particle formation
take place (Eerikainen et al., 2003). In spray drying, hot
gas is used as a source of heat to evaporate the solvent
and the spray-drying chamber is only used as a place for
heat transfer to occur and is not heated by itself.
The temperature of the gas changes across the chamber
as heat transfer occurs between the cold feed and the
hot gas. On the contrary, in the aerosol flow reactor
method, the droplets, which are generated by a jet
nebulizer, ultrasonic nebulizer or electrospray, are already
suspended in the carrier gas before they are fed into
the tubular flow reactor that is located in an oven and
maintained at a constant temperature. The carrier gas
flows evenly in the tubular reactor at a constant rate,
controlled temperature field and non-circulating flow
(Peppas et al., 2007).
The method is a simple and efficient one-step
process that can produce particles directly within a
desirable particle size range with consistent and controlled
properties (Raula et al., 2007). It produces dry particles
directly without the need for further purification, and
no additives are required (Eerikainen et al., 2004;
Raula et al., 2004).
Eerikainen et al. reported that corticosteroid-loaded
Eudragit NPs with mean diameter of 90 nm can be produced by an aerosol flow reactor method using ethanol.
The composition of the NPs and drying temperature did
not affect the particle size distribution, particle morphology
or structure. The problems of drug leaking and uneven
drug distribution, which are commonly encountered in
emulsion processes, are avoided using this novel method
(Eerikainen and Kauppinen, 2003).

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NANOPARTICLES
ORGANIC PHASE

COLLECTION
DILLUTION

VIBRATION
SYSTEM
PULSATION CHAMBER

Heating zone

NOZZLE
PARTICLE
FORMATION
ELECTRODE

Carrier gas
mixed with
droplets
DROPLET
GENERATION

STROBOSCOPE

solvent + drug
+ polymer
AQUEOUS PHASE

Figure 10. Aerosol flow reactor method (adapted from Eerikainen


et al. (2003, 2004) with permission from Elsevier).

Vibrating nozzle device


645

650

655

660

665

670

The vibrating nozzle device provides the means of


controlling encapsulation of drugs, animal cells, plant
cells, micro-organisms and enzymes, and is used mainly
for alginate bead preparation. It enables a high rate of production of a wide variety of micrometer-sized products
from both aqueous and organic solvents, with very
narrow bead size distributions. Zvonar et al. (2009) used
such a vibrating nozzle device for preparing NPs with high
drug loading and entrapment efficiency. The technology is
based on the principle that a laminar jet of the organic
phase containing drug and polymer, is forced through the
nozzle, allowing the formation of small droplets due to the
superimposed vibration (Figure 11). When passing through
an electrical field between the nozzle and the electrode, the
surface of the droplets becomes charged. Due to electrostatic repulsion, the one-dimensional droplet chain is
transformed in a funnel-like multiline stream, which prevents droplets from hitting one another. When droplets
enter the aqueous solution of stabilizer, the polymer precipitates and NPs with entrapped drug are formed. The
highly reproducible process can be carried out under
mild, non-toxic conditions and can easily be scaled up
using a multinozzle device. The process can also be carried
out under aseptic conditions, which opens up the possibility of preparing NPs for parenteral applications (Heinzen
et al., 2000).

Discussion and concluding remarks

675

This review provides an update on the state of art of


manufacturing polymeric NPs from preformed polymers.
NPs are promising drug delivery systems for small molecular weight drugs, as well as macromolecules such as

Figure 11. A schematic drawing of the vibrating nozzle device (adapted


from Zvonar et al. (2009)).

proteins, peptides or genes. The selection of a specific


method for preparation of NPs using preformed polymers
is usually determined by the drugs physicochemical characteristics, particularly, its solubility and stability under
operational conditions. One of the most challenging tasks
in the development of protein pharmaceuticals is dealing
with the physical and chemical stability of proteins when
they are incorporated into drug delivery systems. The
chemical and physical stability of proteins can be affected
by environmental factors such as pH, ionic strength, temperature, high pressure, non-aqueous solvents, metal ions,
detergents, adsorption, agitation and shearing (Frokjaer
and Otzen, 2005; Wang, 2005). Most of these factors are
present in common manufacturing processes, including
sterilization and lyophilization, which may damage the
proteins, therefore appropriate parameters should be
applied (Almeida and Souto, 2007). The method chosen
should also consider other aspects such as drug release
profile, entrapment efficiency, method feasibility, the
generation of contaminants and the need for subsequent
purification steps, solvent nature, the water volume
required and time consumption. Likewise, the feasibility
of scaling-up and cost should be considered
(Mora-Huertas et al., 2010).
Table 2 gives a comparative analysis of some of the criteria mentioned previously taking into account the information on NP research available in databases. As can be
observed, there is no ideal method because each one has its
advantages and limitations. In general terms, both,
lipophilic and hydrophilic drugs can be incorporated in
NPs. For the incorporation of lipophilic molecules,
methods such as nanoprecipitation, emulsification-solvent
evaporation, emulsification-solvent diffusion or salting-out

680

685

690

695

700

705

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710

715

720

725

730

735

740

745

750

755

760

765

can be chosen. On the contrary, if the hydrophilic drug has


to be incorporated, methods such as double emulsification-solvent evaporation, double emulsification-solvent
diffusion, coacervation, ionic gelation or W/O emulsification can be considered. All procedures, where organic
solvents are required, use solvents with low toxic potential,
except single and double emulsification-solvent evaporation technique where Class 3 solvents (methylene chloride)
are more frequently applied.
In relation to drug stability, the deleterious effects of
emulsification methods on protein stability have been
addressed by several authors (Crotts and Gwan Park,
1997; Morlock et al., 1997; Crotts and Park, 1998; Gasper
et al., 1998; Johansen et al., 1998; van de Weert et al., 2000).
The formation of NPs is a complex process, and the protein
is subjected to potentially destabilizing conditions, from
intense mechanical stress of emulsification to exposure to
hydrophobic interfaces in the emulsion and the solvent
removal process (Johnson, 2000; Cegnar et al., 2004).
Emulsion droplets are formed by the input of energy,
which may be mechanical (homogenizer) or ultrasonic
(ultrasonic probe). Application of the energy source is
accompanied by one or more processes including high
shear, cavitation and high temperature. Losing protein
activity depends on a range of parameters involved directly
in the manufacturing processes, therefore appropriate
preparation parameters should be applied to avoid damaging of the protein. The nanoprecipitation method does
not rely on shear stress to produce NPs, but takes advantage of differences in the interfacial tension, a phenomenon
also designated as the Marangoni effect. However, the use
of this technique may still expose the protein to organic
solvent during NP preparation (Cai et al., 2008). In the
coacervation technique, protein molecules undergo stress
as a result of pH changes and also of the cross-linking step
with glutaraldehyde. Glutaraldehyde is indiscriminate and
cross-links the NP matrix as well as the encapsulated protein or peptide drug. This lack of selectivity accounts for
possible loss in bioactivity of protein drugs encapsulated in
NPs that utilize chemical cross-linking (Johnson, 2000).
Once NP dispersions are obtained, purification is
needed to remove impurities and excess of reagents
involved during manufacture. Depending on the method
of preparation, impurities include organic solvents, oil,
salts, excess of surfactants or stabilizing agents and large
polymer aggregates. Evaporation at room temperature or
under reduced pressure is the most common approach to
remove large quantities of volatile organic solvents and
a part of water from NP dispersions prepared by
emulsification-solvent evaporation, nanoprecipitation
and salting-out method (Allemann et al., 1992; Govender
et al., 1999; Jaiswal et al., 2004). If a solvent with high
boiling point is used in emulsification diffusion method,
then cross-flow microfiltration (tangential filtration) is
method of choice for purification (Dalwadi et al., 2005).
This method can be also scaled up for industrial
applications (Quintanar-Guerrero et al., 1998).
In relation to method feasibility, it is only possible to
make an approximation taking into account laboratory

11

experiment and pilot scales. Although the preparation of


NPs by different methods is almost completely mastered at
a lab scale level, the transformation from the lab production to an industrial scale lack of information in the
literature. Scale up of methods for the production of NPs
were described for four types of methods: the emulsification-solvent diffusion, salting-out, emulsification-solvent
evaporation and the nanoprecipitation method. Pilotscale production of ibuprofen-loaded NP, prepared by
the emulsification-solvent diffusion, salting-out and
nanoprecipitation methods, was relatively well achieved.
In general, NP characteristics drug loading, residual
PVAL and morphology were reproduced at lab- and
pilot-scales. However, the scale-up process induced a
slight reduction in the size and drug loading of NP.
Compared to the emulsion-based methods, less time is
required for preparing a pilot-batch by nanoprecipitation
(Galindo-Rodriguez et al., 2005). Control of emulsion droplet size is very important factor in the preparation of NPs by
emulsification-solvent evaporation method. The high-pressure emulsification-solvent evaporation (HPESE) process
produces small, monodisperse NPs combined with a high
encapsulation efficiency, easy control and reproducibility.
The process can be automated and scaled up for producing
large amount of NPs (Jaiswal et al., 2004).
In spite of the methods advantages and limitations
mentioned above, it is possible to identify trends in
research into NP preparation method selection.
Therefore, taking into account a general review of the available information in electronic databases on NP preparation, the nanoprecipitation is the most used method for
incorporation of hydrophobic molecules. On the contrary,
if the objective of research is hydrosoluble molecule
entrapment,
the
method
preferred
is
double
emulsification.
While nanotechnology is expected to produce new colloidal carriers, it is also expected to revolutionize the way
the current drug delivery systems are produced. Current
approaches have been used successfully in producing a
variety of NPs containing pharmaceutically active ingredients, but the processing methods require significant
improvement. For example, some current methods need
improvement in drug loading and production scale-up.
Despite the numerous methods available to produce NPs
on a small scale, there are still problems in establishing
large scale production methods. This is considered to be
one of the reasons preventing their successful introduction
to the clinic and the market.

770

775

780

785

790

795

800

805

810

815

Declaration of interest
The authors report no conflicts of interest.

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