nanomaterials

Article
Polymeric Core-Shell Nanoparticles Prepared by
Spontaneous Emulsification Solvent Evaporation and
Functionalized by the Layer-by-Layer Method
Marta Szcz˛ech and Krzysztof Szczepanowicz *
Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8,
PL-30239 Krakow, Poland; nclapczy@cyf-kr.edu.pl
* Correspondence: ncszczep@cyf-kr.edu.pl; Tel.: +48-12-639-51-21; Fax: +48-12-425-19-23




Received: 18 February 2020; Accepted: 6 March 2020; Published: 10 March 2020


Abstract: The aim of our study was to develop a novel method for the preparation of polymeric
core-shell nanoparticles loaded with various actives for biomedical applications. Poly(caprolactone)
(PCL), poly(lactic acid) (PLA) and poly(lactide-co-glycolide) (PLGA) nanoparticles were prepared
using the spontaneous emulsification solvent evaporation (SESE) method. The model active
substance, Coumarin-6, was encapsulated into formed polymeric nanoparticles, then they were
modified/functionalized by multilayer shells’ formation. Three types of multilayered shells
were formed: two types of polyelectrolyte shell composed of biocompatible and biodegradable
polyelectrolytes poly-L-lysine hydrobromide (PLL), fluorescently-labeled poly-L-lysine (PLL-ROD),
poly-L-glutamic acid sodium salt (PGA) and pegylated-PGA (PGA-g-PEG), and hybrid shell composed
of PLL, PGA, and SPIONs (superparamagnetic iron oxide nanoparticles) were used. Multilayer shells
were constructed by the saturation technique of the layer-by-layer (LbL) method. Properties of our
polymeric core-shell nanoparticle were optimized for bioimaging, passive and magnetic targeting.

Keywords: drug delivery; polymeric nanoparticles; core-shell; magnetic targeting; passive targeting

1. Introduction
Numerous new chemicals have been developed to treat various complicated diseases effectively,
but at the same time, some of them produce serious side effects, proving that the benefit does not
always outweigh the risk [1,2]. Moreover, more than 40% of pharmacologically active compounds
exhibit poor solubility in water. However, they have been proven to be very effective in vitro but
cannot reach the side of action deeming them nearly worthless in vivo. While great progress has
been made in identifying drug targets along with designing and making better drug molecules,
there is still room to improve the drug-delivery systems and targeting [1,3]. Currently, we have a
broad set of pharmaceutical nanocarriers (liposomes, micelles, solid lipid nanoparticles, niosomes,
dendrimers, polymeric nanoparticles, etc.) at our disposal, along with the means to load them
with various drugs and adjust their surface properties to make them long-circulating, targeted,
stimuli-sensitive, and even multifunctional [4]. Polymeric nanoparticles have become an important
area of research in the field of drug delivery. The early nanoparticles were mainly formulated
from poly(alkylcyanoacrylate), currently the most widely used polymers for nanoparticles have
been: from natural proteins or polysaccharides e.g., chitosan, alginate; and synthetic polymers e.g.,
poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and their copolymers, poly(lactide-co-glycolide)
(PLGA) [1,5–8]. These polymers are known for both their biocompatibility and resorbability through
natural pathways [7]. The initial therapeutic effect of drug-loaded nanoparticles was relatively poor
due to rapid clearance of the particles by phagocytosis post-intravenous administration. In recent

Nanomaterials 2020, 10, 496; doi:10.3390/nano10030496 www.mdpi.com/journal/nanomaterials

Nanomaterials 2020, 10, 496 2 of 16

years, this problem has been solved by the proper surface modification of nanoparticles e.g., by grafting
their surface with many hydrophilic and flexible polymers. Presently, polyethylene glycol (PEG) is the
polymer most often used for nanomaterial functionalization. Alternative strategies replacing PEG with
poly-amino acids, e.g., poly-L-glutamic acid (PGA) has been also implemented lately [9–11]. One of the
powerful methods of surface modification is the sequential adsorption of charged nano-objects called
the layer-by-layer (LbL) method [11–14]. Advantages of the LbL method is the ease of manipulation
and the multifunctionality that comes from the possibility of modification of the multilayer shell
by organic molecules, polymers, inorganic nanoparticles, carbon nanotubes, antibodies, [15] by the
introduction of functional groups [16], lipids [17] or nanoparticles [18]. That multifunctionality can
be utilized for the preparation of targeted drug delivery systems. Targeting can be broadly classified
into three regimes; passive, active and physical one [19]. In the frame of physical pH-sensitive,
temperature-sensitive, redox potential-sensitive, ultrasound-sensitive, magnetic-sensitive systems are
included. Two of them: the passive targeting and physical one with magnetic-sensitive systems are
widely studied. Passive drug targeting based on an accumulation of drug through leaky vasculature
of a diseased area. It was found that under certain pathological states, such as tumors, infarcts, and
inflammation, the permeability of vascular endothelial increases and they become leaky. In such
regions with increased vascular permeability, nanoparticles can accumulate and exert their therapeutic
effect. This phenomenon is also known as an ‘enhanced permeability and retention’ (EPR) effect [19–21].
The concept is based upon conjugation of a drug molecule or drug nanocarriers with magnetic particles
and guiding and concentrating them towards the intended pathology site under the influence of an
external magnetic field [22–26]. In magnetic-sensitive systems, iron oxide nanoparticles referred to as
superparamagnetic iron oxide nanoparticles (SPIONs) with particle size 4–10 nm are used.
Nanoparticles (NPs) were initially developed as drug carriers, but they also received attention
as carriers of diagnostic agents. Moreover, both modalities, therapeutic and diagnostic can be on
one platform called theranostics. Theranostic nanoparticles may simultaneously monitor and treat
disease. Among the available imaging modalities including optical imaging (OI), magnetic resonance
imaging (MRI), computed tomography (CT), ultrasound (US), positron emission tomography (PET) or
single-photon emission computed tomography (SPECT), each has its own unique advantages [27–29].
Not only size but also size distribution, shape and surface of nanoparticles dictate their interactions
with biological systems. Therefore, all of them should be taken into account when designing
nanoparticles for drug delivery and imaging [30,31].
Based on the aforementioned data, the aim of the present study was to develop a novel method
of preparation of loaded polymeric core-shell nanoparticles for biomedical application. Polymeric
nanoparticles were prepared by the spontaneous emulsification solvent evaporation (SESE) method with
the following polymers: poly(caprolactone) (PCL), poly(lactic acid) (PLA) and poly(lactide-co-glycolide)
(PLGA). The model active substance was encapsulated in formed polymeric nanoparticles, then they
were modified/functionalized by the LbL method for bioimaging, passive and magnetic targeting
(Figure 1).
Nanomaterials 2020, 10, x FOR PEER REVIEW 3 of 16
Nanomaterials 2020, 10, 496 3 of 16

Figure 1. General
Figure scheme
1. General of proposed
scheme approach.
of proposed approach.

2. Materials
2. Materials andand Methods
Methods
2.1. Chemicals
2.1. Chemicals
Polymers: polycaprolactone (PCL, average Mw ~14,000), poly(lactic acid) (PLA, average
Polymers: polycaprolactone (PCL, average Mw ~14,000), poly(lactic acid) (PLA, average Mw
Mw ~60,000), and copolymer poly(lactide-co-glycolide) (PLGA, average Mw 4,000 to 15,000),
~60,000), and copolymer poly(lactide-co-glycolide) (PLGA, average Mw 4,000 to 15,000),
polyelectrolytes: poly-L-lysine hydrobromide (PLL, average Mw 15,000 to 30,000), poly-L-glutamic acid
polyelectrolytes: poly-L-lysine hydrobromide (PLL, average Mw 15,000 to 30,000), poly-L-glutamic
sodium salt (PGA, average Mw 15,000 to 50,000), Coumarin-6, docusate sodium salt (AOT), sodium
acid sodium salt (PGA, average Mw 15,000 to 50,000), Coumarin-6, docusate sodium salt (AOT),
chloride (NaCl), lissamine rhodamine B sulfonyl chloride (ROD) were received from Sigma-Aldrich
sodium chloride (NaCl), lissamine rhodamine B sulfonyl chloride (ROD) were received from Sigma-
(Poznan, Poland). Superparamagnetic iron oxide nanoparticles (SPIONs) were acquired from
Aldrich (Poznan, Poland). Superparamagnetic iron oxide nanoparticles (SPIONs) were acquired from
PlasmaChem (Berlin, Germany. Chloroform was purchased from Avantor Performance Materials
PlasmaChem (Berlin, Germany. Chloroform was purchased from Avantor Performance Materials
(Gliwice, Poland) while ultra-purified water was obtained using the Direct-Q 5UV purification system
(Gliwice, Poland) while ultra-purified water was obtained using the Direct-Q 5UV purification
from Millipore (Warsaw, Poland). All chemicals were used without further purification. Chemical
system from Millipore (Warsaw, Poland). All chemicals were used without further purification.
structures of the compounds are available in the Supporting Materials (Figure S1).
Chemical structures of the compounds are available in the Supporting Materials (Figure S1).
Pegylated polyanion PGA-g-PEG (g ~30% and PEG, Mw ~5000) was previously synthesized in
Pegylated polyanion PGA-g-PEG (g ~30% and PEG, Mw ~5000) was previously synthesized in
our lab [32] while fluorescently-labeled poly-L-lysine hydrobromide (PLL-ROD) was synthesized via
our lab [32] while fluorescently-labeled poly-L-lysine hydrobromide (PLL-ROD) was synthesized via
coupling of lissamine rhodamine B sulfonyl chloride according to the protocol described in [33].
coupling of lissamine rhodamine B sulfonyl chloride according to the protocol described in [33].
2.2. Polymeric Nanoparticles’ Preparation
2.2. Polymeric Nanoparticles’ Preparation
Polymeric nanoparticles were prepared by the SESE method [6,8] with some modifications. Briefly,
Polymeric nanoparticles were prepared by the SESE method [6,8] with some modifications.
certain amounts of polymer and surfactant AOT were dissolved in a solvent mixture comprising
Briefly, certain amounts of polymer and surfactant AOT were dissolved in a solvent mixture
chloroform and ethanol, and then the mixture was gently added into an aqueous solution containing
comprising chloroform and ethanol, and then the mixture was gently added into an aqueous solution
polycation (PLL) under stirring (500 rpm). After the formation of a stable nanoemulsion, the organic
containing polycation (PLL) under stirring (500 rpm). After the formation of a stable nanoemulsion,
solvent was evaporated either by increasing the temperature/under vacuum or by continuous stirring to
the organic solvent was evaporated either by increasing the temperature/under vacuum or by
finally form polymeric nanoparticles. Blank nanoparticles were prepared following a similar procedure.
continuous stirring to finally form polymeric nanoparticles. Blank nanoparticles were prepared
following
2.3. Druga similar procedure.
Encapsulation and Efficiency of Encapsulation (EE)
Drug-loaded
2.3. Drug polymeric
Encapsulation nanoparticles
and Efficiency were prepared
of Encapsulation (EE) according to the procedure described above.
Briefly, prior emulsification, selected drug at a various concentration ranging from 0.15 to 3 mg/mL was
Nanomaterials 2020, 10, 496 4 of 16

dissolved in the organic phase. To evaluate encapsulation efficiency, synthesized polymeric NPs were
ultracentrifuged and the amount of free unencapsulated drug in supernatant solution was evaluated.
Efficiency of encapsulation was calculated by the following formula:
   
drugtotal − drugsupernatant
EE =   × 100% (1)
drugtotal
   
where drugtotal—the total weight of drug and drugsupernatant—the weight of the free drug in the
supernatant (not encapsulated).

2.4. Modification and Functionalization—Formation of Polymeric Core-Multilayer Polyelectrolyte

Shell Nanoparticles
Prepared polymeric nanoparticles were further modified/functionalized by the multilayer shell
formation. The shell was formed by the layer-by-layer method using the saturation approach. A
fixed volume of polymeric NPs was added to the oppositely charged polyelectrolyte’s solution during
vigorous mixing and the consecutive layer formation was followed by the zeta potential measurements.
Then, the coating process was repeated with the use of the oppositely charged polyelectrolyte. The
multilayer shell was constructed from the following charged nano-objects: PLL, PLL-ROD or SPIONs
as the cationic one, and PGA as the anionic one. To create pegylated polymeric nanoparticles,
PLL-terminated polymeric core-shell NPs were coated with the layer of PGA-g-PEG using the same
procedure as described above. As a result, polymeric core-shell nanoparticles were prepared.

2.5. Nanoparticles’ Characterization

All synthesized polymeric nanoparticles were characterized by measurements of their size, size
distribution, zeta potential, concentration, and morphology observation. Fluorescent emission spectra
of the loaded polymeric NPs, as well as empty ones, were acquired to confirm drug encapsulation. For
evaluation of the stability of synthesized polymeric nanoparticles, their size was monitored in time
during storage in the preparation buffer.
The zeta potential, average size and size distribution of synthesized polymeric nanoparticles and
their coatings with a various number of layers were carried out on a Zetasizer Nano ZS instrument
(Malvern-Pananalytical, Malvern, UK). Additionally, the concentration and the size distribution of
polymeric NPs were determined using Nanosight NS500 instrument (Malvern-Panalytical, Malvern,
UK). The morphology of polymeric nanoparticles was analyzed by a cryo-scanning electron microscope
(cryo-SEM) Jeol JSM-7600F Field Emission Scanning Electron Microscope, FESEM (Jeol Ltd., Tokyo,
Japan) according to the protocol described previously [34]. Spectrofluorimetric measurements were
acquired using a HORIBA Jobin Yvon Fluorolog-3 spectrofluorometer equipment (HORIBA Jobin Yvon,
Longjumeau, France).

3. Results and Discussion

3.1. Polymeric Nanoparticles—Synthesis and Characterization

In the emulsification/solvent evaporation technique selected polymer is dissolved in an organic
solvent and this mixture is then dispersed into an aqueous solution to make oil in water nanoemulsion by
using a surfactant agent. After formation of the stable nanoemulsion, the organic solvent is evaporated
leading to formation of polymeric nanoparticles [8]. The high energy emulsification methods like
high-speed homogenization or sonication are commonly used for nanoemulsion preparation, therefore,
in our work, we decided to use an alternative approach, low-energy emulsification method [34],
nanoemulsification assisted by Ouzo effect. From the list of biodegradable and bioacceptable polymers,
the following were selected for our work: poly(caprolactone) (PCL), poly(lactic acid) (PLA) and
poly(lactide-co-glycolide) (PLGA). The oil phase was prepared by dissolving the selected polymer in
 poly(caprolactone) (PCL), poly(lactic acid) (PLA) and poly(lactide-co-glycolide) (PLGA). The oil
phase was prepared by dissolving the selected polymer in the easily evaporating solvent, chloroform,
with the addition of anionic, oil-soluble surfactant AOT. The concentration of the surfactant favorable
nanoemulsification was chosen from our previous work [35], and it was 33% w/v, while the
Nanomaterials 2020, 10, 496
concentration of the selected polymer in organic solvent was varied from 2.5 to 25 mg/mL.5 Such of 16

prepared oil phase was mixed with absolute ethanol (0.1 mL of the oil phase and 10 mL alcohol) and
then
the gently
easily added into
evaporating an aqueous
solvent, solution
chloroform, withcontaining
the additionpolycation
of anionic,(PLL) under
oil-soluble stirring (500
surfactant AOT.rpm).
The
concentration of the surfactant favorable nanoemulsification was chosen from our previous workof[35],
Since the formed nanoemulsion is stabilized by the AOT/PLL interfacial complex, the amount PLL
wasit also
and optimized
was 33% w/v, whileto minimize free, unadsorbed
the concentration of the selectedpolyelectrolyte, which solvent
polymer in organic is obligatory for further
was varied from
modification by the layer-by-layer method (Supporting Materials (SM) Figure
2.5 to 25 mg/mL. Such prepared oil phase was mixed with absolute ethanol (0.1 mL of the oil phase S2). The final step of
the 10
and preparation
mL alcohol) wasandevaporation
then gentlyof an organic
added into ansolvent
aqueous either by increasing
solution containingtemperature/in
polycation (PLL) vacuo
under or
by continuous stirring. Both approaches result in similar sizes of polymeric nanoparticles,
stirring (500 rpm). Since the formed nanoemulsion is stabilized by the AOT/PLL interfacial complex, the however,
evaporation
amount of PLLby wasthealsocontinuous
optimized stirring result
to minimize in unadsorbed
free, lower polydispersity indexwhich
polyelectrolyte, (PDI). Optimized
is obligatory
parameters
for including drug
further modification andlayer-by-layer
by the polymer concentrations are summarized
method (Supporting in the(SM)
Materials Supporting Materials
Figure S2). The
as Table S1. Characterization of polymeric nanoparticles formed under optimized
final step of the preparation was evaporation of an organic solvent either by increasing temperature/in conditions are
summarized in Table 1, while the example of size distribution measured by dynamic
vacuo or by continuous stirring. Both approaches result in similar sizes of polymeric nanoparticles, light scattering
(DLS) and
however, cryo-SEMbyimages
evaporation for moststirring
the continuous favorable
resultones are presented
in lower on Figure
polydispersity 2 andOptimized
index (PDI). Figure 3,
respectively.
parameters including drug and polymer concentrations are summarized in the Supporting Materials
as Table S1. Characterization of polymeric nanoparticles formed under optimized conditions are
Table 1. Polymeric nanoparticles’ characterization.
summarized in Table 1, while the example of size distribution measured by dynamic light scattering
(DLS) and cryo-SEM
Polymer images for
(optimized most favorable
Average ones are presented on Figures 2 and 3, respectively.
Polydispersity
Zeta Potential Concentration
concentration) size index (PDI)
Poly(caprolactone), Table 1. Polymeric nanoparticles’ characterization.
76 (±5) nm 0.134 (±0.027) 68 (±3) mV ~1 × 1011 nanoparticle/mL
PCL (10 mg/mL) Average Polydispersity
Polymer (Optimized Concentration) Zeta Potential Concentration
Size Index (PDI)
Poly(lactic acid),
80 (±7) nm 0.166 (±0.021) 71 (±4) mV ~1 × 10 nanoparticle/mL
11
~1 × 1011
PLA (2.5 mg/mL) PCL (10 mg/mL)
Poly(caprolactone), 76 (±5) nm 0.134 (±0.027) 68 (±3) mV
nanoparticle/mL
Poly(lactide-co- ~1 × 1011
Poly(lactic acid), PLA (2.5 mg/mL) 80 (±7) nm 0.166 (±0.021) 71 (±4) mV
glycolide), PLGA (5 77 (±2) nm 0.179 (±0.048) 78 (±2) mV ~1 × 1011 nanoparticle/mL
nanoparticle/mL
mg/mL) ~1 × 1011
Poly(lactide-co-glycolide), PLGA (5 mg/mL) 77 (±2) nm 0.179 (±0.048) 78 (±2) mV
nanoparticle/mL

25
PCL NPs
PLA NPs
20
PLGA NPs

15
Number (%)

10

0
1 10 100 1 000 10 000
Size (d.nm)

Figure 2. Size distribution of PCL, PLA and PLGA nanoparticles measured by dynamic light
Figure 2.(DLS).
scattering Size distribution of PCL, PLA and PLGA nanoparticles measured by dynamic light
scattering (DLS).
Nanomaterials 2020, 10, 496 6 of 16
Nanomaterials 2020, 10, x FOR PEER REVIEW 6 of 16

Figure
Figure 3. 3.Cryo-scanning
Cryo-scanningelectron
electron microscope
microscope (cryo-SEM)
(cryo-SEM)images
imagesofofobtained
obtainedPCL
PCL(A), PLA
(A), (B)(B)
PLA andand
PLGA (C) nanoparticles.
PLGA (C) nanoparticles.

AsAs
cancan
be be seen
seen in Figure
in Figure 2, obtained
2, obtained sizessizes are independent
are independent on theontype
the of
type of polymer
polymer which which
indicates
indicates that the final size of synthesized polymeric nanoparticles is governed by the
that the final size of synthesized polymeric nanoparticles is governed by the size of nanoemulsion size of
Nanomaterials 2020, 10, 496 7 of 16
Nanomaterials 2020, 10, x FOR PEER REVIEW 7 of 16

nanoemulsion
droplets used as adroplets
templateused as a templatesynthesis.
for nanoparticles’ for nanoparticles’
Optimization synthesis. Optimization
of the preparation of the
parameters
preparation
allow parameters
the formation allow thenanoparticles
of polymeric formation of polymeric nanoparticles
with average size ranged with average
from 70 tosize ranged
80 nm, with
from 70 to 80 nm, with a polydispersity index (PDI) below 0.2, indicating
a polydispersity index (PDI) below 0.2, indicating that monodisperse polymeric NPs could be that monodisperse
polymeric
produced NPs optimized
under could be produced under
condition. It isoptimized condition.
also revealed that theIt isaverage
also revealed
surfacethat the
zeta average of
potential
surface zeta potential of the formed polymeric nanoparticles was +72 mV (±4 mV) which provides
the formed polymeric nanoparticles was +72 mV (±4 mV) which provides the capability of further
the capability of further functionalization by the layer-by-layer method and is high enough to ensure
functionalization by the layer-by-layer method and is high enough to ensure electrostatic stability of
electrostatic stability of the systems. Therefore, systematic measurements of the average size and zeta
the systems. Therefore, systematic measurements of the average size and zeta potential of synthesized
potential of synthesized polymeric NPs confirmed the long-term stability of the tested systems. It can
polymeric NPs confirmed the long-term stability of the tested systems. It can be summarized that
be summarized that our nanoparticles were stable for at least four months (Figure 4). The
our nanoparticles were stable for at least four months (Figure 4). The concentration of synthesized
concentration of synthesized polymeric NPs determined by NTA (nanoparticle tracking analysis
polymeric NPs
technique) fordetermined by NTA
all nanoparticle types (nanoparticle tracking analysis technique) for all nanoparticle
was ~1 × 1011 nanoparticle/mL.
11
types was ~1 × 10 nanoparticle/mL.

A.
25
PCL NPs (aq.) after synthesis
PCL NPs (aq.) after 4 months
20
Number (%)

15

10

0
1 10 100 1 000 10 000
B.
25

PLA NPs (aq.) after synthesis

20 PLA NPs (aq.) after 4 months
Number (%)

15

10

0
1 10 100 1 000 10 000
C.
25
PLGA NPs (aq.) after synthesis
20 PLGA NPs (aq.) after 4 months
Number (%)

15

10

0
1 10 100 1 000 10 000
Size (d.nm)

Figure 4. Long-term stability of synthesized PCL (A), PLA (B), and PLGA (C) nanoparticles evaluated
by size-distribution measurements.
Nanomaterials 2020, 10, 496 8 of 16

3.2. Active’s Encapsulation

Among numerous pharmaceuticals, some are especially difficult to handle because of their
poor solubility in water-based biological fluids. Poor solubility results in poor bioavailability and
difficulty in maintaining drug therapeutic concentrations in the blood [4,36]. Therefore, a hydrophobic
fluorescent dye Coumarin-6 (C-6) was selected as a model active substance for our development.
Polymeric nanoparticles containing Coumarin-6 were prepared according to the procedure described
above. Prior to emulsification, Coumarin-6 was dissolved in the oil phase. To optimize the procedure,
the concentration of the model dye was varied from 0.15 to 3 mg/mL, and the optimal amount was
obtained when a signal from Coumarin-6 in supernatant solution after ultrafiltration was not observed.
Efficiency of encapsulation for the optimized condition was calculated and summarized in Table 2.
Characteristic emission spectra of Coumarin-6 loaded polymeric nanoparticles’ suspension (peak at
~500 nm, see SM Figure S3) can be observed and comparison of spectra of empty and loaded polymeric
NPs provided the evidence of successful encapsulation of the model substance.

Table 2. Encapsulation efficacy of Coumarin-6 loaded polymeric nanoparticles (NPs).

Optimized Coumarin-6 Final Coumrine-6

Encapsulation Polymer/drug
Concentration in the Oil Concentration in
Efficiency Ratio
Phase Nanoparticles’ Suspension
PCL 0.3 mg/mL 0.150 mg/L 98.9% 33
PLA 0.25 mg/mL 0.125 mg/L 98.7% 10
PLGA 0.25 mg/mL 0.125 mg/L 98.8% 20

3.3. Functionalization of Polymeric Nanoparticles—Multilayer Shell Preparation

The layer-by-layer adsorption of charged nano-objects is considered as a convenient method
to obtain core-multilayer polyelectrolyte shell colloidal particles and those types of particles have
been the subject of intensive research since their invention in 1998 by Shukorukov [12]. The main
advantage of the layer-by-layer method is simplicity of manipulation and the multifunctionality
that comes from the possibility of modification of the polyelectrolyte shell by various functional
species [15–18]. That multifunctionality can be utilized for the preparation of targeted drug-delivery
systems. Therefore, synthesized polymeric nanoparticles were modified by the LbL method. Three
types of multilayered shell were formed: two types of polyelectrolyte shell where biocompatible and
biodegradable polyelectrolytes poly-L-lysine hydrobromide (PLL), fluorescently-labeled poly-L-lysine
(PLL-ROD), poly-L-glutamic acid sodium salt (PGA), and pegylated-PGA with PGA-g-PEG were used;
and a hybrid shell where PLL, PGA, and SPIONs were used. Multilayer shells were constructed by
saturation technique of the LbL method. In the saturation technique, the rinsing step is omitted since it
is possible to add just enough charged nano-objects to completely coat all of the particles present in the
system so that there are little free unadsorbed nano-objects remaining in the aqueous phase [37]. This
is the most important advantage of the saturation technique because the rinsing of nanoparticulate
systems is problematic and associated with nano-objects’ loss.
In our research for each system, the saturation concentration was determined empirically by
monitoring changes of the zeta potential of polymeric nanoparticles during mixing with oppositely
charged nano-objects. The optimal amount was determined and corresponds to the point just before
reaching the plateau of the dependence of zeta potential on the added amount of charged nano-objects
and the value was usually close to the zeta potential of nano-objects in solution/suspension [38].
Since the results obtained for PCL, PLA, and PLGA were comparable for the following study, PCL
nanoparticles were selected as a representative one.
 Nanomaterials 2020, 10, x FOR PEER REVIEW 9 of 16

Multilayer shell of polyelectrolyte PLL, PGA, and PGA-g-PEG was formed on PCL nanoparticles
in order to prepare polymeric core-shell nanoparticles optimized for passive targeting. Since the zeta
potential of PCL NPs was positive (+68 ± 3 mV), the formation of a multilayered shell was started
Nanomaterials 2020, 10, 496 9 of 16
with the polyanion PGA using the saturation technique of LbL method. Next, layers were built with
PLL or PGA, and the external layer was built with pegylated-PGA. Details concerning determination
of the
3.3.1. saturation
Polymeric concentration
Core-Shell for PLL,for
Nanoparticles PGA and PGA-g-PEG
Passive Targeting are described in Supporting Materials
(Figures S4 and S5 respectively). Figure 5A illustrates a dependence of the zeta potential of polymeric
Multilayer
core-shell shell
NPs onofthepolyelectrolyte
adsorption ofPLL, PGA, and
subsequent PGA-g-PEG
layers. was zig-zag
The typical formed shape
on PCLcannanoparticles
be considered
in order
as the evidence of the formation of multilayer shell. The absolute values of zeta potentials the
to prepare polymeric core-shell nanoparticles optimized for passive targeting. Since zetaand
of PLL
potential
PGA-endedof PCLpolymeric
NPs was positive
core-shell(+68 ± 3 mV), the
nanoparticles formation
were higher of a multilayered
than 30 mV whatshell was started
provided sufficient
with the polyanion PGA using the saturation technique of LbL method.
electrostatic stabilization against aggregation or agglomeration during the layer-by-layerNext, layers were built process.
with
PLLTheor PGA, and the external layer was built with pegylated-PGA. Details concerning
external layer was formed with pegylated polyelectrolyte PGA-g-PEG (PGA with grafted PEG determination
of the saturation concentration
chains) and grafting of PEGfor
on PLL,
PGAPGA and PGA-g-PEG
decreases are described
the charge density in Supporting
of polyanion, Materials
therefore, covering
(Figures S4 and S5 respectively). Figure 5A illustrates a dependence of the zeta
polymeric core-shell NPs with pegylated-PGA should significantly decrease their zeta potential in potential of polymeric
core-shell
comparisonNPs on theregular
with adsorption of subsequent
polyanionic layers.layers. The typical
The measured zig-zag
zeta shape
potential of can be considered
pegylated polymeric
as the
core-shell NPs was close to zero (−3 ± 4 mV). Stability tests were performed to confirmof
evidence of the formation of multilayer shell. The absolute values of zeta potentials PLL
that PEG
andcorona
PGA-ended polymeric core-shell nanoparticles were higher than 30 mV what provided
at the polymeric nanoparticles’ surface provides sufficient steric stabilization. The average sufficient
electrostatic stabilization
size of obtained against
pegylated aggregation
polymeric or agglomeration
core-shell NPs was 155 nm during
(PDIthe layer-by-layer
< 0.2) (Figure 5B),process.
moreover,
Theanyexternal layer was formed with pegylated polyelectrolyte PGA-g-PEG
significant changes were not observed in the size distribution and zeta potential (PGA with grafted
at least 30PEGdays.
chains) and grafting of PEG on PGA decreases the charge density of polyanion,
That results indicate that stable monodisperse polymeric core-shell nanoparticles can be produced therefore, covering
polymeric core-shellcondition.
under optimized NPs with pegylated-PGA should significantly decrease their zeta potential in
comparison with regular
The properties of our polyanionic
polymericlayers. TheNPs
core-shell measured zeta potential
make them promisingofcandidates
pegylatedaspolymeric
a vehicle for
core-shell was close to zero (−3 ±
passive targeting which based on the spontaneous drug accumulation into‘leaky’
NPs 4 mV). Stability tests were performed confirm thatofPEG
areas blood
corona at the polymeric nanoparticles’ surface provides sufficient steric stabilization.
vessels. In those areas with increased vascular permeability, our polymeric core-shell nanoparticles The average size
of obtained pegylated
can extravasate andpolymeric
accumulatecore-shell
inside the NPs was 155 space.
interstitial nm (PDI < 0.2)
It was (Figure
also revealed5B),that
moreover, any of
the surface
significant changescore-shell
our polymeric were not observed in the size distribution
NPs was functionalized and zeta
by pegylation potential
which might at have
least 30
thedays. That of
capability
results indicate that stable monodisperse polymeric core-shell nanoparticles can
avoiding the binding with immune cells or proteins in blood circulation and circulating for a long be produced under
optimized
time to condition.
provide a sufficient level of target accumulation.

A.
80 core PCL/AOT/PLL

60
PLL PLL
Zeta potential [mV]

40

20

0
0 1 2 3 4 5 6 7
-20 PGA-g-PEG

-40 PGA PGA

-60
No. of layers
Figure 5. Cont.
Nanomaterials 2020, 10, 496 10 of 16
Nanomaterials 2020, 10, x FOR PEER REVIEW 10 of 16

B.
20
PEGylated PCL
core-shell NPs

15
Number (%)

10

0
0 1 10 100 1 000 10 000
Size (d.nm)
Figure 5. (A) Dependence of the zeta potential of polymeric core-shell nanoparticles on the adsorption
Figure
of 5. (A) Dependence
subsequent of thelayers,
polyelectrolytes’ zeta potential
(B) size of polymericof
distribution core-shell
pegylatednanoparticles on the adsorption
polymeric core-shell NPs.
of subsequent polyelectrolytes’ layers, (B) size distribution of pegylated polymeric core-shell NPs.
The properties of our polymeric core-shell NPs make them promising candidates as a vehicle
3.3.2. Polymeric
for passive Core-Shell
targeting whichNanoparticles for Magnetic drug
based on the spontaneous Targeting
accumulation in ‘leaky’ areas of blood
vessels. In those areas with increased vascular permeability,
A multilayer shell of polyelectrolytes PLL and PGA with embedded our polymeric core-shell
SPIONs nanoparticles
was formed on PCL
can extravasateinand
nanoparticles accumulate
order to prepareinside the interstitial space. It was
a magnetically-responsive also revealed
nanosystem. that
Before thetheexperiment,
surface of
our polymeric
SPIONs core-shell NPs
were characterized bywas functionalized
measurements theirby pegylation
size (6 ± 2 nm)which might
and zeta have the
potential capability
(~+50 of
mV). Since
avoiding the binding with immune cells or proteins in blood circulation and circulating for
the zeta potential of PCL NPs was positive (+68 ± 3 mV), formation of the hybrid multilayered shell a long time
to provide
was starteda with
sufficient level of
polyanion PGAtarget accumulation.
using the saturation technique of the LbL method. Next, layers were
built with SPIONs and PGA. Details concerning determination of the saturation concentration for
3.3.2. Polymeric Core-Shell Nanoparticles for Magnetic Targeting
PGA and SPIONs are described in the Supporting Materials (Figures S4A and S6 respectively). Figure
A multilayer
6A illustrates shell zig-zag
a typical of polyelectrolytes
dependencePLL and
of the PGA
zeta with embedded
potential of polymericSPIONs was NPs
core-shell formed on
on the
PCL nanoparticles
adsorption in orderlayers,
of subsequent to prepare
which a magnetically-responsive
can be considered as evidencenanosystem. Beforeincorporation
of successful the experiment, of
SPIONs
SPIONswereinto characterized
the multilayerbyshell.
measurements
The absolutetheir size (6of±zeta
values 2 nm) and zeta of
potentials potential
formed(~+50 mV). Since
polymeric core-
the zeta
shell potential
NPs of PCL than
were higher NPs was positive
30 mV which(+68 ± 3 mV),sufficient
provided formationelectrostatic
of the hybrid multilayered
stabilization shell
against
was started with
aggregation polyanion PGA
or agglomeration usingthe
during thelayer-by-layer
saturation technique
process.ofThe
the average
LbL method.
size ofNext, layers
the hybrid
were built with
polymeric SPIONs
core-shell and PGA. Details
nanoparticles obtainedconcerning
was 160 nm determination of the saturation
(PDI < 0.3) (Figure 6B). They concentration
were stable in
for PGA
stock and SPIONs
solution are described
for a period of severalin the Supporting Materials (Figures S4A and S6 respectively).
weeks.
Figure 6A illustrates a typical zig-zag dependence of the zeta potential of polymeric core-shell NPs on
the adsorption of subsequent layers, which can be considered as evidence of successful incorporation of
SPIONs into the multilayer shell. The absolute values of zeta potentials of formed polymeric core-shell
NPs were higher than 30 mV which provided sufficient electrostatic stabilization against aggregation or
agglomeration during the layer-by-layer process. The average size of the hybrid polymeric core-shell
nanoparticles obtained was 160 nm (PDI < 0.3) (Figure 6B). They were stable in stock solution for a
period of several weeks.
Nanomaterials
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2020, 496x FOR PEER REVIEW 1111
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1616

A.
80 core PCL/AOT/PLL
60
SPIONs SPIONs
Zeta potential [mV]

40

20

0
0 1 2 3 4 5 6 7
-20

-40 PGA
PGA PGA
-60
No. of layers

B.
20 PCL core-shell NPs
with SPIONs

15
Number (%)

10

0
0 1 10 100 1 000 10 000
Size (d.nm)

Figure 6. (A) Dependence of the zeta potential of polymeric core-shell nanoparticles on the adsorption
Figure 6. (A) Dependence of the zeta potential of polymeric core-shell nanoparticles on the adsorption
of subsequent polyelectrolytes’ layers with superparamagnetic iron oxide nanoparticles (SPIONs),
of subsequent polyelectrolytes’ layers with superparamagnetic iron oxide nanoparticles (SPIONs), (B)
(B) size distribution of magnetically-responsive polymeric core-shell NPs.
size distribution of magnetically-responsive polymeric core-shell NPs.

The proof of concept of the magnetic delivery capacity of hybrid nanoparticles is shown in
The proof of concept of the magnetic delivery capacity of hybrid nanoparticles is shown in
Figure 7 where our hybrid polymeric core-shell NPs were attracted by the magnetic field gradient
Figure 7 where our hybrid polymeric core-shell NPs were attracted by the magnetic field gradient
generated by a permanent magnet. The cuvette containing our magnetically-responsive polymeric
generated by a permanent magnet. The cuvette containing our magnetically-responsive polymeric
core-shell nanoparticles’ suspension was placed close to a permanent magnet. In the beginning, the
core-shell nanoparticles’ suspension was placed close to a permanent magnet. In the beginning, the
distribution of magnetically-responsive polymeric core-shell nanoparticles was homogeneous. After a
distribution of magnetically-responsive polymeric core-shell nanoparticles was homogeneous. After
few hours, as the result of attraction by the magnetic field, magnetically-responsive polymeric core-shell
a few hours, as the result of attraction by the magnetic field, magnetically-responsive polymeric core-
NPs were concentrated at the side of the cuvette located next to the magnet and the residual liquid
shell NPs were concentrated at the side of the cuvette located next to the magnet and the residual
became transparent. These results prove that changes in hybrid polymeric core-shell NPs’ distribution
liquid became transparent. These results prove that changes in hybrid polymeric core-shell NPs’
in the cuvette were caused by the magnetic field. Our magnetically-responsive hybrid polymeric
distribution in the cuvette were caused by the magnetic field. Our magnetically-responsive hybrid
core-shell nanoparticles may be very promising for future applications in magnetic drug delivery.
polymeric core-shell nanoparticles may be very promising for future applications in magnetic drug
delivery. Moreover, incorporation of SPIONs in polymeric core-shell nanoparticles opens a new
perspective on the application of that systems in theranostic (MRI or hyperthermia.
Nanomaterials 2020, 10, 496 12 of 16

Moreover, incorporation of SPIONs in polymeric core-shell nanoparticles opens a new perspective on

the application
Nanomaterials 2020,of
10,that systems
x FOR in theranostic (MRI or hyperthermia.
PEER REVIEW 12 of 16

Figure 7.
Figure 7. Effects
Effects of
of magnetic
magnetic field
field on
on synthesized
synthesized hybrid
hybrid polymeric
polymeric core-shell
core-shell nanoparticles
nanoparticles using
using aa
permanent magnet.
permanent magnet.

3.3.3. Polymeric Core-Shell

3.3.3. Polymeric Core-Shell Nanoparticles
Nanoparticles for
for Bioimaging
Bioimaging
One
One of of modalities
modalities that that have
have been
been used
used for
for non-invasive
non-invasive imaging imaging in in medicine
medicine is is optical
optical imaging.
imaging.
From
From the theoptical
opticalimaging
imagingtechniques
techniques available,
available, thosethosebased
basedon the
on thefluorescence
fluorescence has emerged
has emerged as theas most
the
powerful imaging techniques. Optical fluorescence depends on the
most powerful imaging techniques. Optical fluorescence depends on the inherent property of the inherent property of the fluorophore
that was usedthat
fluorophore for labeling.
was usedOrganic fluorescent
for labeling. Organicdyes fluorescent
are the mostdyes commonly
are theusedmost fluorophores.
commonly Dyes used
such as fluorescein isothiocyanate (FITC) and the carboxyfluorescein
fluorophores. Dyes such as fluorescein isothiocyanate (FITC) and the carboxyfluorescein diacetatesuccinimidyl ester (CFSE),
rhodamine (ROD) and ester
diacetatesuccinimidyl cyanine class of
(CFSE), dyes (NIR)
rhodamine have been
(ROD) used in various
and cyanine class of biological
dyes (NIR) applications,
have been
e.g.,
usedfluorescently-labeled
in various biologicalantibodiesapplications, ande.g.,
molecules that are used to antibodies
fluorescently-labeled stain cells or andorganelles
molecules [39,40]. In
that are
the case of our polymeric core-shell nanoparticles, rhodamine dye
used to stain cells or organelles [39,40]. In the case of our polymeric core-shell nanoparticles, was conjugated to polycation PLL
and that type
rhodamine dye of was
fluorescently
conjugated labeled polycation
to polycation PLL was andusedthatfortype
formation of the multilayer
of fluorescently shell.
labeled polycation
A multilayer
was used shell ofofpolyanion
for formation PGA shell.
the multilayer and fluorescently-labeled polycation PLL-ROD was formed
on PCL nanoparticles in order
A multilayer shell of polyanion PGA andto prepare fluorescently-labeled
fluorescently-labeled polymeric
polycation core-shell
PLL-ROD nanoparticles.
was formed
Since the zeta potential of PCL NPs was positive (+68 ± 3 mV), formation
on PCL nanoparticles in order to prepare fluorescently-labeled polymeric core-shell nanoparticles. of the multilayered shell was
started
Since the with polyanion
zeta potentialPGA using
of PCL NPsthewas
saturation
positive technique
(+68 ± 3 of mV),LbLformation
method. The next
of the layers were built
multilayered shell
with fluorescently-labeled polycation PLL-ROD or polyanion
was started with polyanion PGA using the saturation technique of LbL method. The next PGA. Details concerning determination
layers were
of the with
built saturation concentration forpolycation
fluorescently-labeled PGA and PLL-ROD PLL-ROD are or described
polyanionin PGA. the Supporting Materials
Details concerning
(Figures S4A and S7 respectively). Figure 8A illustrates a dependence
determination of the saturation concentration for PGA and PLL-ROD are described in the Supporting of the zeta potential of polymeric
core-shell
Materials NPs on the
(Figures S4Aadsorption of subsequent
and S7 respectively). layers8A
Figure and the typical
illustrates zig-zag shape
a dependence of can
the be
zeta considered
potential
as the evidence
of polymeric of formation
core-shell NPs onofthe multilayer
adsorption shell. Also here,layers
of subsequent the absolute
and the values
typical of zeta potentials
zig-zag shape can
of
be PLL-ROD
consideredand PGA-ended
as the evidence of polymeric
formationcore-shell
of multilayer NPs shell.
were Also higher than
here, the30absolute
mV, which values provided
of zeta
sufficient electrostatic stabilization against aggregation or agglomeration
potentials of PLL-ROD and PGA-ended polymeric core-shell NPs were higher than 30 mV, which during the layer-by-layer
process.
providedThe average
sufficient size of thestabilization
electrostatic fluorescently labeled
against polymericor
aggregation core-shell nanoparticles
agglomeration during the obtained
layer-
was 106 nm (PDI < 0.25) (Figure 8B). Moreover, any significant changes
by-layer process. The average size of the fluorescently labeled polymeric core-shell nanoparticles were not observed in the size
distribution
obtained wasand 106zeta
nm potential
(PDI < 0.25) at least
(Figure30 8B).
days.Moreover,
Fluorescent anyproperties
significantofchanges
polymeric were core-shell
not observed NPs
were confirmed by spectrofluorimetry, characteristic emission spectra (peak
in the size distribution and zeta potential at least 30 days. Fluorescent properties of polymeric core- at ~580 nm) of Rhodamine
can
shellbeNPsobserved and comparison
were confirmed of spectra of empty
by spectrofluorimetry, and loaded
characteristic polymeric
emission core-shell
spectra (peak NPs at ~580provided
nm) of
evidence
Rhodamine of the
can labeling
be observed of our andpolymeric
comparison nanoparticles
of spectra (Figure
of empty 9).and
Theloaded
resultspolymeric
indicate that stable
core-shell
monodisperse fluorescently-labeled polymeric core-shell NPs could
NPs provided evidence of the labeling of our polymeric nanoparticles (Figure 9). The results indicate be produced under optimized
condition.
that stable Incorporation
monodisperseoffluorescently-labeled
selected fluorophores in polymeric
polymeric core-shell
core-shell NPsnanoparticles
could be produced opens aunder new
perspective on the application
optimized condition. of systems
Incorporation in bioimaging
of selected or theranostics.
fluorophores in polymeric core-shell nanoparticles
opens a new perspective on the application of systems in bioimaging or theranostics.
Nanomaterials 2020,
Nanomaterials 10, 496
2020, 10, x FOR PEER REVIEW 13 of
1316
of 16

A.
80 core PCL/AOT/PLL
60
PLL-ROD
PLL-ROD
Zeta potential [mV]
40

20

0
0 1 2 3 4 5 6 7
-20

-40
PGA PGA PGA
-60 No. of layers
B.
20 PCL core-shell NPs
with PLL-ROD

15
Number (%)

10

0
0 1 10 100 1 000 10 000
Size (d.nm)

Figure 8. (A) Dependence of the zeta potential of polymeric core-shell PCL nanoparticles on the
Figure 8. (A) Dependence of the zeta potential of polymeric core-shell PCL nanoparticles on the
adsorption of subsequent polyelectrolytes’ layers with fluorescently-labeled polycation poly-L-lysine
adsorption of subsequent polyelectrolytes’ layers with fluorescently-labeled polycation poly-L-lysine
hydrobromide rhodamine (PLL-ROD), (B) size distribution of fluorescently-labeled polymeric core
hydrobromide rhodamine (PLL-ROD), (B) size distribution of fluorescently-labeled polymeric core
shell NPs.
shell NPs.
Nanomaterials
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Figure 9. Emission spectra of fluorescently-labeled polymeric core-shell PCL nanoparticles with

Figure number
different 9. Emission spectra layers
of PLL-ROD of fluorescently-labeled
and a picture of thepolymeric core-shell PCL nanoparticles with
prepared suspensions.
different number of PLL-ROD layers and a picture of the prepared suspensions.
4. Conclusions
4. Conclusion
A new type of polymeric core-shell (PCL, PLA, PLGA) nanoparticles was developed. The
nanoparticles
A new were
type formed via thecore-shell
of polymeric spontaneous(PCL,emulsification
PLA, PLGA)solvent evaporation
nanoparticles was (SESE) method.
developed. The
The model hydrophobic compound (Coumarin-6) was encapsulated in our polymeric
nanoparticles were formed via the spontaneous emulsification solvent evaporation (SESE) method.nanoparticles
and,
Themoreover, the formed compound
model hydrophobic polymeric NPs were further
(Coumarin-6) modified/functionalized/optimized
was for passive
encapsulated in our polymeric nanoparticles
and
and,magnetic targeting,
moreover, as wellpolymeric
the formed as for bioimaging,
NPs werebyfurther
the layer-by-layer (LbL) method. Polymeric
modified/functionalized/optimized for
core-shell nanoparticles with average size ranging from 100–200 nm and low
passive and magnetic targeting, as well as for bioimaging, by the layer-by-layer the polydispersity index
(LbL) method.
(PDI) were obtained.
Polymeric core-shellThe developed polymeric
nanoparticles with average core-shell NPs may
size ranging be considered
from 100–200 nm as aand
promising
low the
platform for future nanomedicine especially in targeted drug delivery, theranostics,
polydispersity index (PDI) were obtained. The developed polymeric core-shell NPs may be or bioimaging.
Further workas
considered will focus on bioanalysis
a promising including
platform for future biocompatibility, controlled
nanomedicine especially in release,
targetedand
drugactivity of
delivery,
the encapsulated
theranostics, or drug, cellular uptake,
bioimaging. Further localization, etc. on bioanalysis including biocompatibility,
work will focus
controlled release, and activity of the encapsulated drug, cellular uptake, localization, etc.
Supplementary Materials: The following are available online at http://www.mdpi.com/2079-4991/10/3/496/s1,
Figure S1: Chemical
Supplementary structures
Materials: Theoffollowing
polymersare and dyes. Figure
available onlineS2: Changes of the zeta potential
at www.mdpi.com/xxx/s1, Figureof S1:emulsion
Chemical
cores with the AOT/PLL ratio. Figure S3: Emission spectra of Coumarin-6 loaded polymeric nanoparticles and
structures of polymers and dyes. Figure S2: Changes of the zeta potential of emulsion cores with the AOT/PLL
their filtrates after ultracentrifugation. Figure S4: Changes of the zeta potential during formation of the (A)
ratio.
PGA Figure
layer S3: layer.
(B) PLL Emission spectra ofmarked
The condition Coumarin-6 loaded on
by an asterisk polymeric nanoparticles
both figures representsand theirstable
the first filtrates
sampleafter
ultracentrifugation. Figure
after overcharging. Figure S5: S4: Changes of the zeta potential during formation of the (A) PGA layer
of the zeta potential during formation of the PGA-g-PEG layer. The(B) PLL layer.
condition marked
The condition by an by
marked asterisk represents
an asterisk thefigures
on both first stable samplethe
represents after overcharging.
first stable sampleFigure S6: Changes of
after overcharging. the
Figure
zeta potential during formation of the SPIONs layer. The condition marked by an asterisk represents
S5: Changes of the zeta potential during formation of the PGA-g-PEG layer. The condition marked by an asterisk the first
stable sample after overcharging. Figure S7: Changes of the zeta potential during formation of the PLL-ROD layer.
represents the first stable sample after overcharging. Figure S6: Changes of the zeta potential during formation
The condition marked by an asterisk represents the first stable sample after overcharging. Table S1: Optimized
of the SPIONs
parameters layer. The condition
for nanoparticle’ synthesis.marked by an asterisk represents the first stable sample after overcharging.
Figure S7: Changes of the zeta potential during formation of the PLL-ROD layer. The condition marked by an
Author Contributions: Conceptualization, M.S. and K.S.; Data curation, M.S. and K.S.; Formal analysis, M.S. and
asterisk represents the first stable sample after overcharging. Table S1: Optimized parameters for nanoparticle’
K.S.; Funding acquisition, M.S.; Investigation, M.S. and K.S.; Methodology, K.S.; Supervision, K.S.; Validation,
synthesis.
K.S.; Visualization, M.S.; Writing—Original draft, M.S. and K.S.; Writing—Review and editing, M.S. and K.S. All
authors have read and agreed to the published version of the manuscript.
Author Contributions: Conceptualization, M.S. and K.S.; Data curation, M.S. and K.S.; Formal analysis, M.S.
and K.S.;This
Funding: research
Funding was funded
acquisition, in part
M.S.; by the National
Investigation, Science
M.S. and K.S.;Center of PolandK.S.;
Methodology, PRELUDIUM Project
Supervision, K.S.;
2016/23/N/ST5/02783 and by the statutory
Validation, K.S.; Visualization, research fund ofdraft,
M.S.; Writing—original ICSCM.S.
PAS.and
TheK.S.;
APCWriting—review
was funded by MDPI.
and editing, M.S.
and K.S.of
Conflicts All authorsThe
Interest: have read and
authors agreed
declare no to the published
conflict version of the manuscript.
of interest.
Funding: This research was funded in part by the National Science Center of Poland PRELUDIUM Project
2016/23/N/ST5/02783 and by the statutory research fund of ICSC PAS. The APC was funded by MDPI.

Conflicts of interest: The authors declare no conflict of interest.

Nanomaterials 2020, 10, 496 15 of 16

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