Synthesis of hollow polymeric nanoparticles for protein delivery via inverse miniemulsion periphery RAFT polymerization

Abstract

Hollow polymeric nanoparticles with a hydrophilic liquid core have been synthesized in a one-pot approach via a novel inverse miniemulsion periphery RAFT polymerization process. Successful encapsulation and release of a model protein is reported as a potential application.

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This journal is cThe Royal Society of Chemistry 2012 Chem. Commun., 2012, 48, 11103–11105 11103
Cite this:
Chem. Commun
., 2012, 48, 11103–11105
Synthesis of hollow polymeric nanoparticles for protein delivery via
inverse miniemulsion periphery RAFT polymerizationw
Robert H. Utama, Yi Guo, Per B. Zetterlund* and Martina H. Stenzel*
Received 23rd August 2012, Accepted 25th September 2012
DOI: 10.1039/c2cc36116g
Hollow polymeric nanoparticles with a hydrophilic liquid core
have been synthesized in a one-pot approach via a novel inverse
miniemulsion periphery RAFT polymerization process. Successful
encapsulation and release of a model protein is reported as a
potential application.
The administration of therapeutically active proteins has shown
great potential in the treatment of many diseases, including
cancer and diabetes. However, the delivery of proteins has been
extremely limited through the parenteral route due to the rapid
degradation of the protein. This has prompted the development
of suitable carriers, such as polymer conjugates,
1,2
lipids
3,4
and
nanoparticles,
5–7
that allow for a more robust delivery process.
Hollow polymeric nanoparticles offer several advantages in
comparison to other types of carriers, e.g. higher encapsulation
permassofpolymer,processversatility,
8,9
and (compared to
many hydrogels) their relatively small size, which allows the
penetration into cells.
10
The synthesis of protein-loaded poly-
meric nanoparticles is often challenged by denaturation of the
protein.
11,12
Miniemulsion polymerization is a technique where-
by polymerization is conducted within 50–500 nm droplets
dispersed in a continuous phase (usually water).
13,14
Synthesis
of hollow nanoparticles with a liquid hydrophobic
15–18
or a
hydrophilic
19–21
core utilising radical polymerization in minie-
mulsion has been reported. However, this process is deemed
inappropriate for protein encapsulation because the presence of
radicals can potentially cause protein denaturation.
22
Further-
more, limitations of the aforementioned process include: (i) the
formation of some solid or collapsed nanoparticles; (ii) the use of
non-biocompatible surfactants which prompt the necessity for
further purification steps; (iii) the dependency of the core size on
the polymerization due to the inward growth of the shell and (vi)
limited possibilities for further surface modification, thereby
reducing the versatility of the process and of the synthesized
nanoparticles. All of the above limitations can be overcome by
using the novel synthetic route reported herein.
The term miniemulsion periphery polymerization (MEPP) was
first coined by Wang and co-workers,
23–25
who reported the
synthesis of hollow nanoparticles by essentially crosslinking poly-
meric surfactant species on the outer periphery of oil droplets by
using metal coordination polymerization. We have built on this
concept, and have developed a technique for synthesis of hollow
nanoparticles with a hydrophilic interior by combining the inverse
miniemulsion concept with surface-initiated RAFT polymerization.
An inverse miniemulsion system was specifically chosen to enable
synthesis of hollow nanoparticles with a hydrophilic core suitable
for protein delivery. RAFT crosslinking polymerization is
employed to provide control and functionalization of the nano-
shell. The crosslinking polymerization process, which takes place at
the periphery of the droplets in the oil phase, will ensure the
formation of stable hollow nano-spheres, with the proteins being
encapsulated within the reaction free aqueous interior. As depicted
in Scheme 1, protein encapsulation and production of the nano-
particles are achieved in one-pot via this novel approach, which is
termed inverse miniemulsion periphery RAFT polymerization
(IMEPP).
Scheme 1 Schematic description of the synthesis of hollow nanoparticles
via inverse miniemulsion periphery RAFT polymerization (IMEPP) of
methyl methacrylate/ethylene glycol dimethacrylate using a poly(HPMA)-
b-poly(MMA) macroRAFT agent at 60 1C.
Centre for Advanced Macromolecular Design (CAMD), School of
Chemical Engineering, The University of New South Wales, Sydney,
NSW 2052, Australia. E-mail: p.zetterlund@unsw.edu.au,
m.stenzel@unsw.edu.au; Fax: +61-2-93856250;
Tel: +61-2-93854344
wElectronic supplementary information (ESI) available: Syntheses,
IMEPP processes, TEM images, GPC, DLS, fluorescence and UV-Vis
characterisations. See DOI: 10.1039/c2cc36116g
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11104 Chem. Commun., 2012, 48, 11103–11105 This journal is cThe Royal Society of Chemistry 2012
A typical IMEPP recipe comprised toluene (a good solvent for
the poly(MMA) block and non-solvent for poly(HPMA)) as the
continuous phase, water as the dispersed phase, an amphiphilic
block-copolymer fulfilling the dual roles of a colloidal stabiliser
and a macroRAFT agent, and sodium chloride as a lipophobe to
minimize Ostwald ripening. For the subsequent polymerization,
methyl methacrylate (MMA), ethylene glycol dimethacrylate
(EGDMA) and AIBN were used as a monomer, a cross-linker
and an initiator, respectively. Poly(N-(2-hydroxylpropyl meth-
acrylamide)-b-poly(methyl methacrylate)) (polyHPMA
19
-b-poly-
MMA
79
), synthesized via RAFT polymerization
26
(see ESIw),
was used as a stabiliser/macroRAFT agent. Note that the
hydrophilic–lipophilic balance (HLB) value of the stabiliser
(4.9) was, calculated using Griffin’s method, well within the
acceptable range for an inverse miniemulsion application.
27,28
The initial inverse miniemulsion was prepared by dissolving
the stabiliser, monomer, cross-linker and initiator in the organic
phase and the lipophobe in the aqueous phase. The combined
mixture was then ultrasonicated, followed by polymerization in
sealed glass ampoules under vacuum at 60 1C. The size and
stability of the droplets (prior to polymerization) were initially
monitored using varying amounts of dispersed phase, stabiliser,
lipophobe, and ultrasonication period. The combinations that
led to a suitable particle size and sufficient stability for the desired
application were used in all further experiments (Table 1).
Using the conditions listed in Table 1, an overall monomer
conversion of 51% was reached in 7 h. The polymerization was
accompanied by no changes in turbidity or viscosity, implying the
preservation of the original miniemulsion characteristics. However,
monomer conversions significantly beyond 51% led to a marked
increase in viscosity, consistent with inter-particle crosslinking.
The droplet/particle size distributions were monitored by
DLS, revealing similar monomodal distributions before and
after polymerization with number-average diameters (d
n
)of
170 and 220 nm, respectively, consistent with a miniemulsion
mechanism whereby the initial droplets are converted to polymer
particles (Fig. S2, ESIw). The high degree of preservation of droplet
identity further confirms the absence of significant inter-particle
crosslinking. Overall, the results also showed the efficacy of the
chosen block-copolymers in stabilising droplets and controlling
the subsequent RAFT polymerization.
TEM analysis of the polymerized miniemulsion (diluted
with toluene) revealed hollow nanoparticles with an average
diameter in the range of 190–210 nm, perfectly in line with the
DLS analysis (Fig. 1). The well-defined spherical morphology
provided strong evidence that the shell remained intact during
the post-treatment and drying. The relatively narrow particle size
distribution obtained by DLS analysis was reflected in the TEM
images by the uniformity of the particles in terms of their size and
shape (Fig. S4, ESIw). The hollow core was clearly visualized,
with the methacrylate-based shell exhibiting a lighter contrast
than the background. Based on the TEM images, the thickness of
the shell was found to be B20 nm. Theoretical calculations of the
shell thickness resulted in values in the range of 4–24 nm, in good
agreement with the TEM images (the minimum value obtained
by considering the amount and density of the reacted monomer
(including the PMMA ‘‘hairs’’), and the maximum value corres-
ponding to the total contour length of the chain extended
PMMA ‘‘hairs’’ (see ESIw).
In order to test the use of these hollow nanoparticles as potential
drug carriers, a model protein was incorporated into the inverse
miniemulsion recipe. Bovine serum albumin (BSA) was added to
the dispersed phase (2 wt%), while keeping all other parameters
constant. BSA can be denatured in various ways, including via
temperature and pH.
29–31
However, there has not been any
conclusive evidence that shows denaturation of BSA by ultra-
sonication. Therefore, the effects of ultrasonication on both the
activity and the structure of BSA were investigated.
The activity of BSA was tested by examining the catalytic
activity towards the hydrolysis of p-nitrophenyl ester.
31
The BSA
catalysed hydrolysis of p-nitrophenyl acetate was carried out for
17 h, using both ultrasonicated BSA and native BSA. Comparison
of the UV-Vis spectra of the two samples (Fig. S6, ESI;wsignal
intensity at 405 nm corresponding to the characteristic wavelength
of the product) revealed no differences, thus indicating the same
catalytic activity (Fig. S7, ESIw). The BSA structural integrity was
confirmed based on the tryptophan (Trp) fluorescence emission
profile.
32
The fluorescence emission profiles of both the native and
the sonicated BSA exhibited an emission maximum at 349 nm,
which corresponds to the intact Trp residues (Fig. S8, ESIw).
Subsequently, IMEPP in the presence of BSA was carried out.
BSA in the prescribed quantity was observed to have no
significant impact on the initial miniemulsion droplet size and
stability (Tables S1 and S2, ESIw). Consistent results with the
experiments without BSA were also obtained in the subsequent
polymerization, with an overall conversion of 22% achieved in
3 h. Measurement of d
n
before and after polymerization resulted
in 170 and 183 nm, respectively. The absence of any secondary
peaks in the particle size distributions (Fig. S3, ESIw) is consistent
with successful encapsulation.
TEM analysis showed that the nanoparticles exhibited
similar morphologies to the hollow nanoparticles without BSA.
Table 1 Optimized IMEPP recipe for synthesis of hollow polymeric
nanoparticles
Continuous phase Dispersed phase Quantity
Toluene 6.5 g
MacroRAFT
a
10 wt% relative to water
MMA [MMA] : [RAFT] = 200 : 1
EGDMA [EGDMA] : [RAFT] = 25 : 1
AIBN [AIBN] : [RAFT] = 0.5 : 1
DI water 10 wt% relative to toluene
NaCl 2 wt% relative to water
BSA
b
2 wt% relative to water
a
PolyHPMA
19
-b-polyMMA
79
,M
n,theo
=10600gmol
1
,PDI=1.26.
b
Bovine serum albumin.
Fig. 1 TEM images of hollow nanoparticles synthesized via IMEPP
of MMA and EGDMA.
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This journal is cThe Royal Society of Chemistry 2012 Chem. Commun., 2012, 48, 11103–11105 11105
The clearly visible additional dark features correspond to
encapsulated BSA (Fig. 2; 80% loaded spheres calculated
from Fig. S5, ESIw). Diameter measurement based on the
TEM images gave a result of 170 nm with a shell thickness of
B12 nm, which was again in agreement with the theoretical
minimum and maximum thicknesses of 3 and 16 nm, respectively.
The thinner shell obtained at lower conversion confirms that
the shell thickness is controlled by the monomer conversion.
Subsequent experiments also confirmed the possibility of higher
protein loadings. Protein fractions of up to 5 wt% were incorpo-
ratedwithnonoticeableaggregationandchangesinsizecontrol
and stability of the droplets (confirmed visually and by DLS).
To confirm the integrity of the nano-shells, purified nano-
particles were re-dispersed in 1,4-dioxane, a common solvent
for both the HPMA and the MMA block. Without cross-
linking, the hollow nanoparticles would disappear, resulting
in unimolecularly dissolved block copolymers. DLS analysis of
the re-dispersed nanoparticles showed the presence of stable
particles comparable with the particle size obtained from the
original (prior to re-dispersion) miniemulsion. In addition, the
absence of any precipitate confirmed the protection provided by
the shell on the encapsulated BSA. Consistent results obtained
from the DLS and TEM analyses showed the efficiency and
convenience of the IMEPP process in synthesising well-defined,
protein-loaded polymeric nanoparticles (Fig. 2).
To test the hollow particles as potential carriers for therapeutic
agents, the release of BSA was investigated during continuous
mixing of the protein-loaded particles in PBS-buffer solution.
HPLC analysis of the filtrate of the solution revealed that 41%
of the originally encapsulated BSA was released in 3 days. This
confirmed that the extent of crosslinking of the hollow particles
was sufficient to ensure structural stability, yet the crosslink density
was low enough to allow diffusion of BSA through the shell.
Subsequent fluorescence analysis of the encapsulated BSA revealed
a Trp emission peak at 355 nm. Comparison of this peak with that
of the native (355 nm) and denatured BSA (361 nm), together with
the very minor difference in catalytic activity to that of the native
BSA (87% of the original activity), confirmed the preservation of
the BSA structure, despite exposure to ultrasonication at elevated
temperature (Fig. S10 and S11, ESIw).
In summary, a convenient one-pot synthetic route to synthesize
hollow polymeric nanoparticles having a hydrophilic aqueous
core, suitable for protein delivery, has been developed. The
method is based on inverse miniemulsion periphery polymeriza-
tion (IMEPP) using a block-copolymer as a macroRAFT agent
and a stabilizer in the absence of a conventional surfactant.
Successful encapsulation and release of BSA was demonstrated,
revealing no detrimental denaturation of BSA during particle
synthesis. Further work to improve the stability of the nano-
particles against aggregation in aqueous media is underway.
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Fig. 2 TEM images of protein-loaded nanoparticles (dark spots are
encapsulated BSA) and number-based droplet/particle size distributions
before and after IMEPP (in toluene), and after re-dispersion in 1,4-dioxane.
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