Article pubs.acs.org/Macromolecules
Inverse Miniemulsion Periphery RAFT Polymerization: A Convenient Route to Hollow Polymeric Nanoparticles with an Aqueous Core Robert H. Utama, Martina H. Stenzel,* and Per B. Zetterlund* Centre for Advanced Macromolecular Design (CAMD), The University of New South Wales, Sydney NSW 2052, Australia S Supporting Information *
ABSTRACT: The recently developed [Chem. Commun. 2012, 48, 11103−11105] inverse miniemulsion periphery RAFT polymerization (IMEPP) approach to prepare hollow polymeric nanoparticles (∼200 nm) with an aqueous core has been explored in detail. The method is based on an amphiphilic macroRAFT agent acting as stabilizer of water droplets in an organic continuous phase while also mediating cross-linking chain growth in a controlled/living manner on the outer periphery of the droplets. The macroRAFT agent comprised a hydrophilic block of poly(N-(2-hydroxypropyl)methacrylamide) and a hydrophobic block of either polystyrene or poly(methyl methacrylate), and the cross-linked shell was formed on polymerization of styrene/ divinylbenzene or methyl methacrylate/ethylene glycol dimethacrylate, respectively. The effects of various reaction parameters on the resulting hollow nanoparticles have been systematically investigated, and it has been demonstrated that the shell thickness can be tuned based on initial stoichiometry and monomer conversion. This method is particularly relevant for encapsulation of proteinssuccessful incorporation of proteins (bovine serum albumin) into the miniemulsion did not negatively affect the droplet size and stability.
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INTRODUCTION
greatly influences the size and size distribution of the resulting nanoparticles. A selection of miniemulsion polymerization techniques have been used to synthesize hollow polymeric nanoparticles. The use of radical polymerization within monomer droplets comprising a high weight fraction of an inert hydrophobe such as hexadecane is well documented. This approach has led to the creation of micrometer-sized17−21 and nano-sized capsules22,23 via concentration-induced phase separation (selfassembly of phase-separated polymer, SaPSeP) resulting in a core and a polymeric shell (i.e., the polymer migrates to the oil−water interface). Controlled/living radical polymerization (CLRP) in a dispersed system has also been applied, allowing the controlled insertion of functional groups, which ultimately enables postmodification. Previous works in synthesizing hollow polymeric nanoparticles via NMP, ATRP, and RAFT polymerization have been reported and reviewed.24 The versatility of RAFT polymerization in creating nanoparticles with various internal structures and functionalities is well-known and has been exploited extensively.25−27 Previous works on interfacial RAFT miniemulsion polymerization28−31 (including inverse miniemulsion polymerization32) have demonstrated successful creation of hollow polymeric nanoparticles (180−300 nm). Klumperman and co-workers30
Owing to their capability to encapsulate, protect, and release various molecules, hollow polymeric nanoparticles have been employed extensively in many areas, e.g., drug delivery,1,2 cosmetics,3,4 food applications,5 and wastewater treatment.6 When compared with other nanocarrier systems such as hydrogels, dendrimers, and liposomes, hollow polymeric nanoparticles offer a range of advantages due to their greater design versatility, size control, and loading capacity. The array of approaches to synthesize hollow polymeric nanoparticles, such as via a sacrificial template,7−9 self-assembly,10 and miniemulsion polymerization,11−13 has been expanding rapidly in recent years. Increased emphasis has been placed on developing more robust synthetic pathways enabling one to tune the particle size and the shell thickness as well as incorporate different functionalities. The miniemulsion polymerization pathway to produce hollow polymeric nanoparticles has gained significant interest, predominantly due to its compatibility with a range of polymerization techniques. Miniemulsion polymerization is a process whereby polymerization is conducted within 50−500 nm droplets dispersed in a continuous phase; i.e., particles are formed by direct transformation of monomer droplets into polymer particles. Stabilization of the droplets from coalescence (by use of surfactants) and Ostwald ripening (by use of an ultrahydrophobe) are keys in obtaining well-distributed nanoparticles.14−16 The initial droplets act as a soft template that © XXXX American Chemical Society
Received: January 30, 2013 Revised: February 25, 2013
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Aldrich) were deinhibited by passing through a column of activated basic alumina. Deinhibited monomers were stored at below 4 °C and used within 7 days. 2,2′-Azobis(isobutyronitrile) (AIBN) was recrystallized twice from methanol. Deionized (DI) water was produced by a Milli-Q reverse osmosis system and had a resistivity of 19.6 mΩ cm−1. The RAFT agent, 4-cyanopentanoic acid dithiobenzoate (CPADB), was synthesized according to the literature.42 Analyses. Size exclusion chromatography (SEC) was performed using a Shimadzu modular system, comprising an SIL-10AD autoinjector, an LC-10AT pump, a DGU-12A degasser, a CTO-10A column oven, and an RID-10A differential refractive index detector. A column arrangement consisting of a Polymer Laboratories 5.0 μm bead size guard column (50 × 7.8 mm), followed by four linear PL column (300 × 7.8 mm, 500, 103, 104, and 105 Å, 5 μm pore size) was used for the analysis. N,N-Dimethylacetamide (DMAc, 0.03% w/v LiBr, 0.05% w/v 2,6-dibutyl-4-methylphenol(BHT)) was used as the mobile phase at a constant temperature of 50 °C and a constant flow rate of 1 mL min−1. The SEC system was calibrated using linear polystyrene standards, ranging from 500 to 106 g mol−1 (Polymer Laboratories). Chromatograms were processed using Cirrus 2.0 software (Polymer Laboratories). Gas Chromatography (GC). GC was conducted to measure monomer conversions on a Shimadzu GC-17A with AT-WAX column (Altech 30 m, 0.25 mm i.d., film thickness 0.25 μm) with H2 as carrier gas (66 mL min−1) and a temperature program of 5 min at 120 °C, 40 °C min−1 to 200 °C, and 5 min at 200 °C. THF, with DMF as the internal standard (1000:1, THF:DMF), was used as the solvent. Nuclear Magnetic Resonance (NMR). NMR was utilized to analyze the structure of the synthesized compounds as well as to determine the monomer conversion. 1H NMR spectroscopy was carried out using a Bruker Avance III 300 MHz, equipped with an autosampler system. Chemical shifts are reported in parts per million (ppm), relative to the residual solvent peak. The theoretical molecular weight (Mn,th) was calculated according to the following equation:
demonstrated that the surface activity of the initiator radicals/ oligomers as well as the rate of chain growth were crucial factors in confining the chain growth to the interfacial region to obtain hollow structures (with a liquid core of e.g. hexadecane as above). Alternatively, Luo and co-workers32 achieved preferential polymerization at the interface by utilizing surface-active RAFT agents. However, one of the drawbacks of these RAFT miniemulsion polymerization techniques is that formation of unwanted solid or collapsed nanoparticles is difficult to avoid.29,33 Previous methods are less suitable for the encapsulation of various sensitive biological molecules or compounds bearing sensitive functionalities because the polymerization occurs within the encapsulated reservoir itself. Moreover, although necessary to provide miniemulsion stability, the presence of surfactants would generally require extra purification steps. Several groups have utilized different chemistries where the synthesis of the polymeric nanoshell is performed on the outer periphery of the droplet, i.e., away from the sensitive payload. Both interfacial condensation34,35 and “click” polymerizations36−38 have been employed with appropriate pairs of functionalized molecules with opposite polarity to localize the reaction at the droplet interface.34−38 Wang and co-workers39,40 synthesized a polymeric nanoshell on the outer side of the droplet via metal coordination polymerization at the periphery of miniemulsion droplets stabilized by organometallic surfactants. Recently, we published a communication describing a novel, one-pot approach to create hollow nanoparticles with an aqueous corea process referred to as inverse miniemulsion periphery RAFT polymerization (IMEPP).41 An amphiphilic block copolymer (synthesized via RAFT polymerization) fulfills the dual role of colloidal stabilizer and macroRAFT agent, thereby eliminating the need for commonly used surfactants. The subsequent formation of the polymeric nanoshell was achieved through RAFT cross-linking polymerization, whereby chains grow from the periphery of the droplet, thus providing a reaction-free environment within the aqueous core. In the present paper, the synthesis of hollow polymeric nanoparticles with either poly(styrene) or poly(methyl methacrylate) cross-linked shells via IMEPP is described. A number of process parameters have been investigated, e.g., the nature and amount of the block copolymer (stabilizer/ macroRAFT) as well as the amount of encapsulated material in the form of the model protein bovine serum albumin. Moreover, it is demonstrated that the shell thickness can be conveniently tuned by adjustment of the stoichiometry and the monomer conversion.
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M n,th =
[monomer] × conversion × M monomer + MRAFT [RAFT]
(1)
MRAFT denotes the molecular weight of the RAFT agent or the macroRAFT agent. Dynamic Light Scattering (DLS). DLS analyses were run on a Zetasizer Nano ZS (Malvern), with a 4 mV He−Ne laser operating at λ = 632 nm and noninvasive backscatter detection at 173°. Measurements were conducted in a quartz cuvette at 25 °C, with 30 s equilibration period prior to each set of measurements. For a given sample, a total of three measurements were conducted. In each measurement, the number of runs, attenuator, and path length used were automatically adjusted by the instrument, depending on the quality of the sample. The presented results are averages of the three measurements. Transmission Electron Microscopy (TEM). TEM was conducted using a JEOL1400 TEM operating at an accelerating voltage of 120 kV. Images were recorded via the Gatan CCD imaging software. All TEM samples were prepared by dropping a 1 mg mL−1 emulsion on a Formvar-supported copper grid. Excess solvent was drained using filter paper after 1 min. UV−Visible Spectroscopy (UV−vis). UV−vis spectra were recorded using a CARY 300 spectrophotometer (Bruker) equipped with CART temperature controller. All spectra were recorded in a quartz cuvette, using the baseline correction mode. MacroRAFT Stabilizer Synthesis. RAFT Polymerization of HPMA Using CPADB RAFT Agent. HPMA (1 g, 6.984 × 10−3 mol), CPADB (0.04 g, 1.4 × 10−4 mol) as RAFT agent, and AIBN (0.0115 g, 6.98 × 10−5 mol) as initiator were dissolved in DMAc (5.68 mL) to give a [monomer]:[RAFT]:[initiator] ratio of 50:1:0.5. The solution was thoroughly purged with nitrogen gas for 30 min before being placed in an oil bath at 70 °C for 7 h. After polymerization, the reaction was stopped by placing the solution in an ice bath for 30 min. The polymer was isolated by precipitation in diethyl ether to yield poly(HPMA) as a highly viscous red liquid. The monomer conversion
EXPERIMENTAL PART
Materials. All materials were reagent grade and used as received, unless otherwise specified: N-(2-Hydroxypropyl)methacrylamide (HPMA, Polysciences), sodium chloride (NaCl, Univar), bromine (>99.99%, Sigma-Aldrich) 1,3,5-trioxane (Sigma-Aldrich), toluene (99.5%, Univar), N,N-dimethylacetamide (DMAc, 99.9%, SigmaAldrich), diethyl ether (Et2O, 99%, Univar), methanol (Ajax Chemicals), tetrahydrofuran (THF, anhydrous, 98%, Sigma-Aldrich), N,N-dimethylformamide (DMF, >99.8%, Sigma-Aldrich), 1,4-dioxane (99.5%, Sigma-Aldrich), and n-hexane (95%, Univar) were used without further purification. Deuterated NMR solvents (CDCl3, DMSO, and D2O) were purchased from Cambridge Isotope Laboratories. Styrene (Sty, >99%, Sigma-Aldrich), divinylbenzene (DVB, 80%, Sigma-Aldrich), methyl methacrylate (MMA, 99%, SigmaAldrich), and ethylene glycol dimethacrylate (EGDMA, 98%, SigmaB
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was determined to be 38% via 1H NMR (Mn,th = 3000 g mol−1, Mn,SEC = 6300 g mol−1 (with respect to PS standards), PDI = 1.15). 1H NMR (300 MHz, D2O): δ (ppm) = 3.9 (1nH, −CH2(OH)CHCH3), 3.2 (2nH, −NHCH2(OH)CH−), 1.9−1.7 (2nH, CH2 of the main chain), 1.2−1.1 (3nH, −(OH)CHCH3), 1−0.9 (3nH CH3 of the main chain), where n is the degree of polymerization. Chain Extension of Poly(HPMA) with Styrene. Poly(HPMA) (0.35 g, 1.13 × 10−4 mol) was employed as macroRAFT agent and mixed with styrene (11.77 g, 0.113 mol) and AIBN (5.56 mg, 3.39 × 10−5 mol) in DMAc (6.49 mL) to give a [monomer]:[macroRAFT]: [initiator] ratio of 1000:1:0.3. The solution was thoroughly degassed in an ice bath for 30 min before being placed in an oil bath at 60 °C for 6 h. The polymerization was stopped by placing the solution in an ice bath for 30 min. The final solution was then precipitated in methanol to yield a brittle, pink solid. The monomer conversion was 12% by 1H NMR (Mn,th = 15 500 g mol−1, Mn,SEC = 19 300 g mol−1 (with respect to poly(Sty) standards), PDI = 1.24). 1H NMR (300 MHz, CDCl3): δ (ppm) = 7.2−6.2 (aromatic ring of styrene repeating unit), 2.95 (1nH, H of the main chain), 1.6−1.1 (2nH, CH2 of the main chain). Chain Extension of Poly(HPMA) with MMA. Poly(HPMA) macroRAFT (0.195 g, 7.22 × 10−5 mol), MMA (1.45 g, 1.4 × 10−2 mol), and AIBN (0.0024 g, 1.44 × 10−5 mol) were dissolved in DMAc (14 mL) to give a [monomer]:[RAFT]:[initiator] ratio of 200:1:0.2. The solution was then purged with nitrogen for 30 min, before placing it in an oil bath at 70 °C for 6 h. The polymerization was stopped by immersing the solution in an ice bath for 30 min. The polymer was isolated by precipitation in diethyl ether to yield poly(HPMA-bMMA) (40%) as a brittle, pink solid (Mn,th = 11 000 g mol−1, Mn,SEC = 15 200 g mol−1 (with respect to poly(Sty) standards), PDI = 1.22). 1H NMR (300 MHz, CDCl3): δ (ppm) = 3.6 (3nH, −COOCH3), 2.1 (2nH, CH2 of the main chain), 1−0.8 (3nH, CH3 of the main chain). Miniemulsion Stability Testing. A representative miniemulsion preparation process is as follows: Toluene was mixed with the macroRAFT block copolymer (stabilizer) in a 20 mL glass bottle to create the continuous (organic) phase of the miniemulsion. In a separate container, distilled water and NaCl (lipophobe) were mixed to create the dispersed phase and subsequently added to the organic phase. If bovine serum albumin (BSA) were to be incorporated, it was dissolved in the aqueous phase. The mixture was then transferred into a standardized ultrasonication vessel to ensure equivalent exposure to sonication power in all cases. Ultrasonication (Branson 450 sonifier, 70% amplitude, 5 mm tip diameter) was conducted for 5 min with the sample container immersed in an ice bath. The miniemulsion recipes are listed in Table 2. Inverse Miniemulsion Periphery RAFT Polymerization. For the cross-linking reaction via inverse miniemulsion periphery RAFT polymerization, a third solution consisting of monomer, cross-linker, and initiator was prepared and subsequently added to the continuous phase prior to the addition of the dispersed phase. The resulting mixture, prepared in accordance with the procedure described above, was transferred into a glass ampule to undergo repeated cycles of nitrogen purging/evacuation. The ampule was subsequently flamesealed under vacuum. Polymerization was then carried out at 60 °C with constant shaking in an oil bath. After polymerization, 1 mL of the emulsion was collected for 1H NMR and gravimetric analyses. DLS and TEM analyses were carried out on the diluted emulsion solution (0.1 mL of emulsion solution in 2 mL of toluene). Nanoparticles were collected from the raw emulsion by de-emulsifying the system using nhexane followed by centrifugation (7000 rpm for 10 min) and drying under reduced pressure at 40 °C. Quantification of Pendant Conversion. The conversion of pendant unsaturations (originating from the cross-linker EGDMA) was estimated by bromination. Initially, a calibration curve correlating bromine concentration and intensity was established (see Supporting Information). Synthesized hollow nanoparticles were initially dispersed in DMSO (5 mL) to give an opaque solution. A control bromination of the macroRAFT stabilizer was conducted in DMSO (5 mL) to investigate any possible bromination of the stabilizer itself. To obtain a smooth baseline (dispersed nanoparticles refract light, leading to an ascending baseline) and to ensure the applicability of Beer’s law, the
spectra of the dispersed nanoparticles in DMSO were used as the baseline for their corresponding subsequent analysis. Into these solutions bromine was added. UV−vis analysis was then conducted to obtain the initial concentration of bromine within the sample. These solutions were stirred for 2 h while immersed in an ice bath. Finally, UV−vis analysis was carried out after the reaction to determine the remaining quantity of bromine. The pendant conversion was calculated from the following equation:
⎛ n − nBrBC ⎞ pendant conversion = ⎜1 − Br ⎟ × 100 nEGDMA ⎠ ⎝
(2)
where nBr is the number of moles of Br2 reacted, nBrBC is the number of moles of Br2 reacted with the block copolymer (macroRAFT) (determined from the control experiment), and nEGDMA is the number of moles of EGDMA units. Theoretical Calculation of Shell Thickness. The maximum and minimum shell thicknesses were calculated as follows: Theoretical Maximum Shell Thickness. The contour length of the primary chain of the cross-linked polymer that forms the shell (i.e., not including the hydrophilic segment) is calculated based on eq 3:
Lcont = Nb sin(0.5Θ)
(3)
where N is the number of C−C bonds in the main polymer chain, b is the C−C bond length (0.154 nm),43 and Θ is the C−C bond angle within the polymer backbone (109°). The overall thickness would then be the sum of the contour length of the hydrophobic segment of the initial block copolymer and the primary chain of the cross-linked block (the latter formed during the actual miniemulsion polymerization). Theoretical Minimum Shell Thickness. The diameter of the “naked” initial (before polymerization) droplet (dd) is calculated by subtracting the maximum contour length of the hydrophobic segment (Lcont × 2, accounting for both sides of the droplet) from the initial droplet diameter (dn) as obtained by DLS (corresponding to the diameter of the droplet plus the contribution of the hydrophobic segments). The “naked” droplet volume is given by
Vdroplet =
3 4 ⎛ dd ⎞ π⎜ ⎟ 3 ⎝2⎠
(4)
Subsequently, the number of droplets in a given amount of dispersed phase is calculated according to total number of droplets =
Vdipersed phase Vdroplet
(5)
The volumes occupied by the monomer consumed in the miniemulsion polymerization (VIMEPP) and by the hydrophobic segment of the macroRAFT stabilizer (Vhydrophobic) (both were calculated based on the density of the corresponding monomer) are then added to Vdispersed phase to give
Vtotal = Vdipersed phase + VIMEPP + Vhydrophobic
(6)
The average volume of a single particle (Vp) can be calculated using eq 7:
Vp =
Vtotal total number of droplets
(7)
The shell thickness is finally calculated via eq 8: shell thickness =
d p − dd 2
(8)
where dp is the diameter corresponding to Vp. C
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Scheme 1. Synthetic Route to Poly(HPMA-b-Sty) and Poly(HPMA-b-MMA) MacroRAFT Stabilizers Followed by Schematic Description of Synthesis of Hollow Polymeric Nanoparticles via Inverse Miniemulsion Periphery RAFT Polymerization
Table 1. Synthesis Parameters and Molecular Weight Data of the Synthesized Poly(HPMA) MacroRAFT Agent and the Amphiphilic Block Copolymers (Stabilizers and MacroRAFT Agents) Poly(HPMA-b-Sty) and Poly(HPMA-b-MMA)
a
monomer
[monomer]/[RAFT]/[AIBN]
time (h)
conv (%)
Mn,thb (g mol−1)
Mn,SEC (g mol−1)
PDI
HPMA styrenea MMAa
50:1:0.5 1000:1:0.3 200:1:0.2
7 6 6
38 12 40
3 000 15 500 11 000
6 300 19 300 15 200
1.15 1.24 1.22
P(HPMA)19 was used as the macroRAFT agent. bCalculated using the conversion obtained from 1H NMR.
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RESULTS AND DISCUSSION
HLB = 20
MacroRAFT Stabilizer Synthesis. One of the main aims of the present synthetic pathway to hollow polymer nanoparticles is to eliminate the need for nonionic surfactants (commonly employed in inverse miniemulsions) by use of an amphiphilic block copolymer (Scheme 1). The block copolymer must possess the characteristic of a typical nonionic surfactant commonly used in an inverse miniemulsion by having segments with opposite polarities in the right ratio. This parameter is normally expressed numerically as the hydrophilic−lipophilic balance (HLB). Typically calculated using the Griffin’s method (eq 9), the HLB is obtained from the molecular weight ratio of the hydrophilic segment (Mh) and the total molecular weight (M):44,45
Mh M
(9)
In the case of inverse miniemulsion (water-in-oil), the HLB value is normally in the range of 3−8. The synthesis parameters for the homopolymerization and the chain extension polymerization are summarized in Table 1. N-(2-Hydroxypropylmethacrylamide) (HPMA) was chosen for the hydrophilic segment of the block copolymer (macroRAFT agent) as it is water-soluble, nonimmunogenic, and nontoxic. The RAFT polymerization of HPMA with CPADB as the RAFT agent yielded poly(HMPA) with 19 repeating units. Chain extension of this macroRAFT agent was conducted with two different hydrophobic monomers: styrene (poly(HPMA19b-Sty120)) and methyl methacrylate (poly(HPMA19-b-MMA79)) D
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(number of repeating units according to Mn,th based on conversion from 1H NMR). The synthesized block copolymers were also further analyzed by SEC (Figure 1)all molecular weight data are summarized
Table 2. Recipes Employed for Investigation of Initial (before Polymerization) Miniemulsion Characteristics parameter varied
continuous phase
stabilizer quantity
dispersed phase
lipophobe
dispersed phase
2.6 g
macroRAFT
varied H2 O
0.26 g
NaCl
2.6 mg
toluene macroRAFT
2.6 g 0.026 g H2 O
varied
NaCl
2.6 mg
toluene macroRAFT
2.6 g 0.026 g H2 O
encapsulated agent
Figure 1. Molecular weight distributions (normalized to peak height) of (a) poly(HPMA), (b) poly(HPMA-b-MMA), and (c) poly(HPMAb-Sty). Refer to Table 1 for the corresponding Mn and PDI values.
in Table 1. The chain extensions resulted in a clear shift to higher molecular weight without significant tailing for both Sty and MMA. The HLB values were 3.8 or 4 for poly(HPMA19-bSty120) and 5 or 4.8 for poly(HPMA19-b-MMA79), calculated based on Mn,th and Mn,SEC, respectively. These HLB values fall well within the recommended range of 3−8 (discussed above). Miniemulsion Preparation. Toluene was chosen as the continuous phase in all recipes because it is a good solvent for poly(Sty) and poly(MMA) and a nonsolvent for poly(HPMA). In addition, the monomers for the miniemulsion polymerizations, i.e., Sty and MMA, are also readily soluble in toluene. The initial water-in-toluene miniemulsions utilizing macroRAFT stabilizer and NaCl as lipophobe all had a milk-like appearance. The influence of a number of experimental parameters (amount of stabilizer, dispersed phase, lipophobe, and encapsulated agent; Table 2) on colloidal stability, droplet size, and size distribution of the initial miniemulsion (before polymerization) was studied via DLS. Increasing the stabilizer content from 10 to 40 wt % (relative to dispersed phase) resulted in the number-average diameter (dn) decreasing, as expected, from 198 to 147 nm (Table 3). The stabilizer quantity of 10 wt % was chosen for all subsequent investigations. The amount of dispersed phase was varied between 10 and 30 wt % (relative to the continuous phase), resulting in an increase in dn from 191 to 255 nm (Table 3). This is expected because the higher the amount of dispersed phase, the lower is the amount of stabilizer available per interfacial area at a fixed droplet size. The lipophobe NaCl was added to prevent Ostwald ripening. In the systems with 0.5, 1.0, and 1.5 wt % NaCl (relative to dispersed phase), the volume distribution was bimodal, in addition to a very high dv value indicating the presence of large droplets. While the number distribution remained monomodal also at low NaCl content, dn decreased as the amount of NaCl was increased. Monomodal droplet size distributions (by
amount
toluene
0.26 g
toluene
2.6 g
macroRAFT
0.026 g H2 O
0.26 g
NaCl
2.6 mg
BSA
varied
notes
10, 20, 30, 40 wt % of dispersed phase 10 wt % of continuous phase 2 wt % of dispersed phase 10 wt % of dispersed phase 10, 20, 30 wt % of continuous phase 2 wt % of dispersed phase 10 wt % of dispersed phase 10 wt % of continuous phase
10 wt % of dispersed phase 10 wt % of continuous phase 2 wt % of dispersed phase 2, 5, 10 wt % of dispersed phase
Table 3. DLS Results Showing the Number- and VolumeAverage Diameters (dn and dv) for Various Miniemulsion Recipes (before Polymerization)a Effect of Stabilizer Content parameter changed 10 20 30 40
wt wt wt wt
% % % %
dn (nm)
stabilizer 198 ± 3 stabilizer 171 ± 2 stabilizer 168 ± 3 stabilizer 147 ± 2 Effect of Dispersed Phase Quantity
parameter changed
dn (nm)
10 wt % dispersed phase 191 ± 2 20 wt % dispersed phase 223 ± 3 30 wt % dispersed phase 255 ± 5 Effect of Lipophobe (NaCl) Contenta parameter changed
dn (nm)
0.5 wt % lipophobe 395 ± 9 1 wt % lipophobe 385 ± 2 1.5 wt % lipophobe 353 ± 3 2 wt % lipophobe 318 ± 4 Effect of Encapsulated BSA Content
dv (nm) 243 212 206 207
± ± ± ±
10 3 4 3
dv (nm) 256 ± 6 307 ± 7 368 ± 4 dv (nm) 2411 3124 3154 450
± ± ± ±
78 159 112 11
parameter changed
dn (nm)
dv (nm)
2 wt % BSA 5 wt % BSA 10 wt % BSA
266 ± 5 183 ± 11 221 ± 11
426 ± 9 322 ± 9 447 ± 16
a
The ultrasonication power used for this series only was somewhat lower than in all other cases throughout this paperhence the droplets are larger. aRefer to Table 2 for the recipe of each experiment.
E
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Table 4. Inverse Miniemulsion Periphery Polymerization Recipes Used for the Synthesis of Hollow Polymeric Nanoparticles with Styrene- and Methyl Methacrylate-Based Shells continuous phase
dispersed phase
toluene (g) macroRAFT (g)b styrene (g) DVB (g) MMA (g) EGDMA (g) H2O (g)a NaCl (g)c polymerization time (h) conversion (%) pendant conversion (%) temperature (°C) sonication period (min)
R1
R2
R3
R4
R5
R6
R7
13 0.13h 0.85d 0.133d
13 0.13g
13 0.13g
13 0.13g
13 0.13g
13 0.13g
13 0.13g
1.3 0.026 24 14
0.237e 0.059e 1.3 0.026 7 51
60 5
60 5
0.237e 0.059e 1.3 0.026 1 12 29 60 5
0.237e 0.059e 1.3 0.026 3 25 57 60 5
0.237e 0.059e 1.3 0.026 6 48 66 60 5
0.532f 0.105f 1.3 0.026 3 29 64 60 5
0.532f 0.105f 1.3 0.026 24 72 84 60 5
a 10 wt % toluene. b10 wt % H2O. c2 wt % H2O. d[M]0:[CL]0:[RAFT]0:[I]0 molar ratio of 1000:125:1:0.5. e[M]0:[CL]0:[RAFT]0:[I]0 molar ratio of 200:25:1:0.5. f[M]0:[CL]0:[RAFT]0:[I]0 molar ratio of 450:45:1:0.5. gPoly(HPMA-b-MMA) macroRAFT used. hPoly(HPMA-b-Sty) macroRAFT used.
Figure 2. Size distributions (DLS) based on number and volume of the Sty/DVB (a, b) and MMA/EGDMA (c, d) of the initial droplets (thick solid lines), raw emulsion after polymerization (thin solid lines), and redispersed particles (dashed lines (a and b in THF; c and d in dioxane)).
number and volume) were obtained when a sufficient amount of NaCl (2 wt %) was used (Table 3). To test whether the incorporation of a foreign moiety would affect the stability and size distribution of the droplets, the water-soluble protein BSA was introduced. DLS analysis could initially confirm the presence of the protein within the aqueous dropletsif BSA had leached out of the aqueous phase, it would have been subject to immediate precipitation and
aggregation. Varying the amount of BSA between 2 and 10 wt % (relative to dispersed phase) did not result in a clear trend in size/size distribution (Table 3). The results obtained from these investigations confirmed that the amphiphilic block copolymer is indeed suitable as stabilizer in these inverse miniemulsions. Although all of the above systems exhibited sufficient kinetic stability, the composition comprising toluene as the continuous phase, 10 F
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Figure 3. TEM images of hollow polymeric nanoparticles for the Sty/DVB system (a, b) and the MMA/EGDMA system (c, d).
size compared to the raw emulsion (442 vs 308 nm) may have been caused by larger droplets/particles (due to coalescence and Ostwald ripening) being unable to maintain their integrity in THF if the level of cross-linking is insufficient. TEM analysis of the diluted raw emulsion solution (Figure 3a,b) revealed a mixture of both hollow and solid nanoparticles of different sizes. The diameter of the smaller hollow polymeric nanoparticles was ∼240 nm (Figure 3a), while the larger hollow nanoparticles were ∼600 nm (Figure 3b). The latter were presumably formed as a result of coalescence and/or Ostwald ripening. However, regardless of size, the particle shells exhibited similar thicknesses of ∼33 nm (measured from the TEM image). The observed spherical solid particles of ∼80 nm are believed to be cross-linked micelles. It is likely that not all of the initial block copolymers are located at the oil−water interface (depending on individual block lengths, some may be located in the continuous phase), and moreover, release of block copolymer into the continuous phase may be promoted by a reduction in droplet interfacial area due to coalescence/ Ostwald ripening. If block copolymers are located in the continuous phase in sufficiently high concentration (above cmc), a micellar structure with the hydrophilic chain protected within the core of the micelle surrounded by the hydrophobic segment would be preferred. Subsequent RAFT polymerization would generate cross-linked micelles. Miniemulsion polymerizations of the system MMA/EGDMA were subsequently explored. The polymerization proceeded at a much higher rate, reaching 51% conversion in 7 h (determined gravimetrically). DLS analysis of the raw emulsion, before and after polymerization, revealed dn = 170 and 220 nm and dv = 210 and 290 nm, respectively (Figure 2). The increase in droplet/particle size was markedly smaller than for the Sty/ DVB system, and the distributions were narrower, indicating relatively successful preservation of the well-distributed system prior to polymerization.
wt % of water as dispersed phase, 10 wt % RAFT block copolymer as stabilizer, and 2 wt % NaCl was selected for the subsequent polymerizations. This inverse miniemulsion system was found to give droplets diameters
