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Stimuli-Responsive Hydrogel Hollow Capsules by Material Efficient and Robust Cross-Linking-Precipitation Synthesis Revisited Mikhail Motornov, Halyna Royter, Robert Lupitskyy, Yuri Roiter, and Sergiy Minko* Department of Chemistry and Biomolecular Science, Clarkson University, 8 Clarkson Avenue, Potsdam, New York 13699, United States ABSTRACT: Monodisperse stimuli-responsive hydrogel capsules were synthesized in the 100-nm-diameter to 10-μmdiameter range from poly(4-vinylpyridine) (P4VP) and poly(ethyleneimine) (PEI) through a simple, efficient two-step cross-linking-precipitation template method under conditions of a good solvent. In this method, the core shell particles were obtained by the deposition (heterocoagulation mechanism) of the cross-linked P4VP, PEI, or their mixtures on the surfaces of the template colloidal silica particles in nitromethane (for PEI) or a nitromethane acetone mixture (for P4VP and P4VP PEI mixtures) in the presence of cross-linker 1,4-diiodobutane. The cross-linked polymeric shell swollen in a good solvent stabilized the core shell colloids. This mechanism provided the conditions for the synthesis of core shell colloids in a submicrometer range of dimensions with an easily adjusted shell thickness (wall of the capsules) ranging from a few to hundreds of nanometers. The chemical composition of the shell was tuned by varying the ratio of co-cross-linked shell-forming polymers (P4VP and PEI). In the second step, the hollow capsules were obtained by etching the silica core in HF solutions. In this step, the colloidal stability of the hollow capsules was provided by ionized P4VP and PEI cross-linked shells. The hollow capsules demonstrate that the pH- and ionicstrength-triggered swelling and shrinking result in size-selective uptake and release properties. Cross-linked via quaternized functional groups, P4VP capsules undergo swelling and shrinking transitions at a physiologically relevant pH of around 6. The 200nm-diameter hollow capsule with 25-nm-thick walls demonstrated a factor of 2 greater capacity to accommodate cargo molecules than the core shell particles of the same dimensions because of an internal compartment and a combination of radial and a circumferential swelling modes in the capsules.
’ INTRODUCTION Methods for the fabrication of submicrometer polymer capsules include the layer-by-layer (LbL) deposition of oppositely charged polyelectrolytes onto the surfaces of template particles, the polymerization of monomers (typically using radical polymerization mechanisms) on the surfaces of template particles, and the self-assembly of amphiphilic block copolymers. These methods yield core shell particles. The next steps may involve crosslinking polymers to stabilize the polymer shell and dissolve the core. Many variations of these methods have been developed and reviewed.1 7 The template methods3 are of special interest because they involve a useful combination of two important properties: (1) regulation of the particle size and polydispersity using welldefined templates and (2) fabrication of particles with a complex structure via core shell templates. A versatile controlled precipitation method for the synthesis of hollow capsules was proposed by Mohwald et al.8,9 The method was based on polymer precipitation caused by changes in the solvent quality or the formation of insoluble polyelectrolyte complexes. The coagulated polymer was harvested by colloidal particles via the heterocoagulation mechanism. Then the synthesized core shell particles were transformed into hollow capsules by etching the template core. The major challenge of this method r 2011 American Chemical Society
is colloidal stability. The polymer precipitation on colloidal particles must be controlled to avoid aggregation. Under poor solvent conditions, several parallel processes may take place: aggregation of colloidal particles, coagulation of macromolecules, heterocoagulation (harvesting coagulated molecules by the colloidal particles), and aggregation of the core shell particles. As mentioned by Mohwald et al.,8 the conditions for the formation of the shell around the particles (a high concentration of polymer) and the avoidance of the formation of polymer aggregates (low concentration of polymer) are mutually exclusive. A partial solution of this problem was found by applying a dropwise technique: the polymer solution was added dropwise to the dispersion of colloidal particles so that the polymer concentration remained low. However, that did not solve the problem of core shell particle aggregation under poor solvent conditions for the polymer shell. The contradictive requirements to avoid aggregation when using coagulation precipitation result in a narrow window for optimal concentrations, which becomes even narrower with a decrease in particle diameter. The method is inappropriate for the efficient synthesis of submicrometer core shell particles Received: November 1, 2011 Published: November 04, 2011 15305
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Langmuir because of the core shell colloids' strong tendency to aggregate under poor solvent conditions. There are no published reports on coagulation precipitation methods for the synthesis of submicrometer hollow capsules with well-controlled diameter, polydispersity, thickness, and composition of the capsule walls. In this work, we developed the cross-linking-precipitation method for the fabrication of hollow capsules made of crosslinked stimuli-responsive hydrogels over a broad range of capsule dimensions. The key aspect in this work was to use a good solvent to cross-link the polymer. The cross-linked polymer was then harvested using template silica particles. After being harvested, the cross-linked polymer enveloped the template particles. The particles were stabilized by the swollen polymeric shell in a good solvent. The shell provides stability for the colloidal system even for nanoparticles, as explained below. In all steps of this method, colloidal particles were stabilized by the polymer in a good solvent (organic solvents) or by a highly ionized polyelectrolyte network in aqueous solution. The proposed method is an important addition to the synthetic toolbox of nanostructured materials because it eliminates some of the drawbacks of the other known methods. For example, LbL assembly requires multiple repetitive depositions and washing of oppositely charged ingredients that may be accompanied by the aggregation of submicrometer particles. Polymerization methods are limited by the number of monomers that can be polymerized by the selected polymerization mechanism and by the extraction of unreacted monomers. The selfassembly of block copolymer micelles and vesicles, followed by the degradation of the core, involves the use of complex block copolymers obtained by a multistep synthesis. This work provides a complementary methodology for the synthesis of hollow capsules. Advantages of the method include (1) simple (two-step) chemical synthesis using homopolymers or statistical copolymers with reactive functional groups (synthetic or natural), (2) easy control over the capsule dimensions, (3) high conversion of the ingredients, (4) synthesis of capsules from covalently cross-linked networks that can undergo quite substantial volumetric swelling shrinking transitions, and (5) easy regulation of the chemical composition of the capsules. Here, we describe examples of capsules made from poly(4-vinylpyridine) (P4VP), poly(ethyleneimine) (PEI), or their mixtures.
’ METHODS Materials. P4VP (Mw = 60 000 g/mol) and branched PEI (Mw = 25 000 g/mol) were purchased from Sigma-Aldrich. Nitromethane (NM) and methyl ethyl ketone (MEK) were purchased from Fisher Scientific. Tetraethyl orthosilicate (TEOS), ammonium hydroxide (28% solution in water), ethanol, acetone (AC), and 1,4-diiodobutane (DIB) were purchased from Sigma-Aldrich. Rose Bengal dye (RB) was purchased from Eastman Kodak Co. (Rochester, NY). Dextrans conjugated with dyes fluorescein isothiocyanate dextran (Mw = 20 000 g/mol) and rhodamine B isothiocyanate dextran (Mw = 70 000 g/mol) were purchased from SigmaAldrich. Silica particles (4 8 μm in diameter) were purchased from Cospheric (Santa Barbara, CA). All chemicals were used as received. Ultrapure water (resistivity >18.3 Ω/cm2) was prepared using a Millipore Milli-Q column system equipped with a Millipack filter with a 0.22 μm pore size at the outlet. Synthesis of Silica Particles (100 300-nm-Diameter Range). The silica particles were synthesized using the St€ober method.10 Ammonium hydroxide (19.0 mL) was added to 200.0 mL of ethanol under stirring. Then, 12.0 mL of TEOS was added in three portions of
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4 mL each. The particles were formed within 1 h, and the dispersion was left overnight with continued stirring. The particles were subsequently cleaned by three cycles of centrifugation and redispersion in pure ethanol using unltrasonication. The final concentration of the particles was 18 mg/mL, as determined by gravimetric analysis. Synthesis of P4VP Capsules. The core shell particles were synthesized using a controlled precipitation of P4VP onto silica particle templates. Several samples of silica particles, with diameters of 90 ( 5, 120 ( 5, and 200 ( 20 nm, were synthesized using the St€ober method by varying the reagent concentrations.10 Silica particles, 4 8 μm in diameter, were used as received. The silica particles rinsed in ethanol and isolated by centrifugation were redispersed in a 1:1 (v/v) mixture of NM and acetone (NM/AC). The core shell particles were synthesized by harvesting the precipitated cross-linked P4VP with the silica particles. The cross-linking of P4VP with DIB was conducted in an organic medium. P4VP was dissolved in a 1:1 (v/v) NM/AC mixture to yield a 0.5 wt % polymer solution. In the next step, a 1% suspension of silica particles in NM/AC was added dropwise to the polymer solution while stirring to avoid particle aggregation. The concentrations of P4VP and the silica particles in the NM/AC mixture were 0.25 0.5 and 0.50 wt %, respectively. It was important to note that these ratios were used for the 100 200 nm silica particles. For particles with a diameter of more than 1 μm, a smaller amount of polymer (0.12 wt %) was used. Afterward, DIB was added to the polymer and silica particle mixture. The concentration of DIB in the mixture was 0.1% vol. The controlled precipitation was conducted at 60 °C for 4 h under vigorous stirring. Upon completion, the particles were isolated and rinsed in NM/AC and ethanol by centrifugation. The gel capsules were produced by dissolving the silica core in a 0.5 wt % aqueous solution of HF for 1 h at room temperature. Particles with a diameter of more than 1 μm were etched at 55 °C with a 1% HF solution. The capsules were then washed by centrifugation and redispersed in Millipore water. Each step in the synthesis was monitored using DLS measurements. Synthesis of PEI Capsules. A 1 wt % silica particle suspension in NM (5.0 mL) was added to 10.0 mL of a freshly prepared PEI solution in NM (0.5 wt %) under stirring. This was followed by the addition of 4.8 mL of NM. The mixture was stirred for 15 min, and then 0.2 mL of DBE was added and the dispersion was left overnight (15 h) under stirring at room temperature. Finally, the particles were kept for 1 h in a water bath at 60 °C under stirring. The obtained core shell particles were cleaned by three cycles of centrifugation (RCF 6880g) and redispersion in pure NM. Transfer of the particles from an organic medium to an aqueous medium was conducted by centrifuging and redispersing in ethanol and Millipore water. The dissolution of the silica core was performed in a 1.0% aqueous solution of HF. The dispersion was stirred for 1 h at room temperature. The resultant polymer capsules were isolated from the HF solutions with two cycles of centrifugation and then were redispersed in Millipore water. The wall thickness was regulated by varying the silica particles/ PEI ratios. Synthesis of P4VP/PEI Mixed Capsules. The synthesis of P4VP/PEI mixed wall capsules was conducted using the same protocol as was described for the P4VP capsules, except that 5, 10, and 20% P4VP were replaced with PEI. Sample Characterization. TEM images were acquired using a JEOL JEM-1200EXII electron microscope (Japan). The samples were prepared on carbon-coated, 200-mesh copper grids obtained from Electron Microscopy Sciences (Hatfield, PA). DLS and zeta potential measurements were performed using a 90 Plus Particle Size Analyzer (Brookhaven Instruments Corp., Holtsville, NY) with a detection angle of 90°. The UV vis spectra were acquired using an Agilent 8453 singlebeam UV vis spectrophotometer (Agilent Technologies, Santa Clara, CA). The fluorescence spectra were acquired using an LS 55 fluorescence 15306
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Langmuir spectrometer (Perkin-Elmer, Waltham, MA). AFM studies were performed using a Dimension 3100 (Veeco, Plainview, NY) microscope. Tapping mode was used to map the capsules under ambient conditions. Tap300 silicon tips (BudgetSensors, Sofia, Bulgaria) with a nominal radius of less than 10 nm, a spring constant of 40 N/m, and a frequency of 300 kHz were used in the AFM experiments. RB and Dextran Uptake and Release. In experiments with the pH-triggered uptake and release of the RB dye, 0.7 mL of the aqueous solution of RB (1.0 mg/mL) was added to 1.5 mL of the capsule dispersion (0.4 mg/mL) in water at different pH levels, as explained below. The dispersion was stirred for 1 h and then centrifuged at RCF of 6880g for 30 min. The particles were redispersed in an ultrasonic bath of Millipore water. The centrifugation redispersion cycle was repeated five times. The pH of the dispersion was adjusted to 4. After the fifth cycle, the pH was raised to 6. After the particles were redispersed, the pH was raised to 7.4 and the dispersion was stirred for 1 h. This was then followed by a final centrifugation cycle. After each centrifugation, the supernatant was collected for spectral analysis. Fluorescein isothiocyanate dextran (Mw = 20 000 g/mol) was encapsulated at pH 3. The capsules in the aqueous dispersion were mixed with dextran solution to yield a 0.03 wt % dispersion of the capsules in a 1.80 mg/mL dextran solution. The mixture was incubated for 25 min, 55 min, and 12 h at room temperature. Upon incubation, the pH was adjusted to 7 and the mixture was centrifuged, followed by redispersion in water at pH 7. Fluorescent spectra were taken for both the supernatant and the dispersion. In the next step, the pH was decreased to 3 to swell the capsules and release the encapsulated dextran. We did not observe any differences in the amounts of dextran released after 10 min, 55 min, and 12 h. Therefore, we can conclude that the majority of the encapsulated dextran was released during the first 10 min upon swelling. The same protocol was used for rhodamine B isothiocyanate dextran (Mw = 70 000 g/mol).
’ RESULTS AND DISCUSSION Synthesis of Capsules. The method is explained in Figure 1a, b. A 0.5 wt % dispersion of colloidal (template) particles (silica, 100 nm in diameter) is coated with a polymeric network shell. The shell is formed during the cross-linking of the polymer (PEI, P4VP, or their mixtures) in its 0.25 wt % solution (in NM for PEI and NM/AC 1:1 blend for P4VP) via an alkylation reaction in the presence of a cross-linker (0.1 vol % DIB) by stirring for 4 h at 60 °C and then rinsing in NM (or NM/AC for PEI), and the ethanol and centrifuged particles are redispersed in an acidic aqueous solution. This method presents two challenges related to the colloidal stability of template particles and core shell particles in the polymer solution. If the colloidal stability is preserved, then the synthesis turns into a very robust and simple method for preparing hydrogel capsules after the templating core is dissolved in the HF solution. It is well known that, for the stabilizing polymer, the conditions for the colloidal stability of the particles can be approached in a good solvent. In the case of core shell structures where the shell is the polymeric network, the conditions of colloidal stability are similar to those for gel particles recently discussed elsewhere11 and explained below. In a good solvent, the crosslinking of the polymer leads to progressive increases in branching and molecular mass and then to phase separation because of the dramatic decrease in the entropy of mixing. The enthalpy of the polymer solvent interaction is only slightly changed in the cross-linking reaction so that the thermodynamic quality of the solvent remains (almost) unchanged. Alternatively, the enthalpy
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Figure 1. Synthesis of capsules in two steps: (a) cross-linking precipitation of P4VP in the presence of DIB on silica particle templates and (b) etching the silica core. Representative TEM images of (c) an original silica particle, (d) a core shell particle, and (e) a capsule. (f) DLS size distribution functions for these particles from left to right, respectively. (g) Hydrodynamic diameter (Dh) and ζ-potential changes as a function of pH for P4VP capsules. (h) A swollen capsule is open for the transport of the labeled dextran (20 kg/mol) in and out, but (i) the polymer is locked in a shrunken capsule.
of interaction (per polymer segment) between the polymer and solvent may even be improved by chemical changes in the polymer. Hence, when template particles harvest nucleated particles of the cross-linked polymers, the formed core shell colloids are stabilized by the steric stabilization mechanism provided by the network and dangling polymer tails of the network shell under good solvent conditions. In our experiments, we successfully tested the method for template particles with diameters ranging from 100 nm to 10 μm. Solvents have complex multiple effects on the formation of core shell colloids by the cross-linking-precipitation method. These effects include the solubility of the precursor polymer, the rate of the cross-linking reaction, and mechanisms to stabilize the nanogel particles (related to the solubility of the product). Obviously, solvent quality is important not only to the synthesis of core shell colloids but also to the effect of the solvent on the rate of cross-linking and coagulation of the polymer that is harvested by the colloids. At a very low rate of alkylation in the cross-linking reaction, the dissolved polymer is slowly harvested by particles and the conditions of polymer coagulation are not approached. In this case, the shell is formed by grafting the polymer to the particle surface rather than by harvesting the precipitated polymer. If the rate of cross-linking and thus the rate of coagulation is too high, then some fraction of the precipitated cross-linked polymer will not be harvested by the core shell colloids and instead will form hydrogel particles, as shown below, or coagulate and precipitate.8 However, at a high rate of cross-linking, the ratio between the rate of polymer coagulation and the rate of heterocoagulation can be adjusted 15307
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Figure 2. (a, c, e) AFM topographical images and (b, d, f) profiles for (a, b) P4VP core shell particles, (c, d) capsules, and (e, f) PEI capsules in the dry state. Note that the particle aggregates in dry samples are due to the effect of capillary forces during the drying step.
by varying the ratios of polymer and colloidal particle concentrations in the given solvent and by the solvent polarity. These parameters also can be used to regulate the thickness of the particle shell as explained below. The alkylation of P4VP and PEI results in quaternary compounds. Highly polar solvents favor the ionization of quaternary salts, thus providing a mechanism for electrostatic and steric stabilization of the core shell particles. Therefore, solvent plays one of the key roles in the crosslinking precipitation method. In our preliminary experiments with different polar solvents (NM, DMF, acetone, THF, chrloroform, and MEK), we identified that the best results were obtained when using solvents that provide high rates of alkylation crosslinking: NM, DMF, and their mixtures with MEK and AC. We discovered that the NM/AC mixture provides better stability for the colloidal dispersions of P4VP core shell particles, so the systematic study of capsule formation was conducted in NM for PEI and in a 1:1 NM/AC mixture for P4VP and P4VP/ PEI blends. The literature demonstrated that the rate of the alkylation reaction of amines with aliphatic alkyl halogens increases with increases in the Hildebrand solubility parameter, δ,12 and the dielectric constants, ε.13 Hildebrand solubility parameters and dielectric constants are 25.8 MPa1/2 and 35.9, respectively, for NM and 19.7 MPa1/2 and 20.7, respectively, for AC. Thus, these solvents and their mixtures provide a high alkylation reaction rate. At the same time, they cause the swelling of the cross-linked and quaternized polymeric shell because of the high polarity. Although δ and ε are quantitative characteristics of the solvents, the relation of these parameters to solvent quality is quite complex for such a polar environment. Therefore, these parameters provide some guidance in the selection of solvents, but
Figure 3. (a, b) TEM and (c) AFM topographical images of (a) PEI core shell particles and (b, c) dry capsules. Note that the particle aggregates in dry samples are due to the effect of capillary forces during the drying step.
the final selection was made on the basis of the experimental results. The capsules synthesized in these solvents were monodisperse: no changes in capsule distribution by size were observed when compared with the silica particle templates (Figure 1f). The internal diameter of the hollow capsule matched the diameter of the template particles in the dry state (Figure 1c e). The internal diameter was greater when the walls were swollen (Figure 1f). The thickness of the wall (typically ranging from 5 to 30 nm for submicrometer capsules to several hundred nanometers for micrometer capsules) was determined using DLS or AFM topographic profiles recorded for dry capsules (Figure 2). In contrast to the core shell precursors (Figures 2a,b), the capsules collapsed in the dry state so that the flattened capsules were as tall as the double-wall thickness (Figure 2c f). Similar behavior and properties were observed for PEI capsules. The SEM images of silica-PEI core shell particles and the SEM and AFM images of PEI capsules are shown in Figure 3. The wall thickness of the capsules was regulated by the ratio of the concentration of template particles and the concentration of the polymer (Table 1). For example, for the 300 nm PEI capsules, the thickest wall (30 nm) was observed for the 1:1 ratio of PEI/silica using 0.25 wt % of each component. When silica particles were present in excess (0.8:1), a thinner wall (10 nm) was observed. However, when PEI was present in high excess (2.5:1), the parallel formation of PEI gel particles took place, which was indicated by an increase in the polydispersity index (reduced second moment of distribution function) from 15308
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Table 1. Effect of the PEI/Silica Ratio on the Formation of Core Shell Particles PEI: silica,
diameter of particles,
thickness of the
polydispersity (reduced second
fraction of PEI that
sample
PEI, wt %
silica, wt %
wt/wt
Dh ( 10 nm
shell, ( 10 nm
moment of distribution function)
formed the shell, %
1
0.2
0.25
0.8:1
252
10
0.005
13
2
0.4
0.1
4:1
226
3
0.4
0.05
8:1
168
4
0.25
0.25
1:1
293
30
0.004
37
5
0.25
0.1
2.5:1
266
16
0.141
7
6
0.25
0.05
5:1
185
0.005 for 1:1 PEI/SiO2 to 0.25 for 4:1 PEI/SiO2. It is obvious that these conditions should be referred to a specific template particle size. The fraction of polymers consumed in the formation of the polymeric shell differs for different polymers and their mixtures. The conversion of the polymer to the network shell was estimated from the loaded amounts of silica and polymer, the diameter of the silica particles, and the shell thickness. It turned out that for 200 nm particles and 1:1 silica/polymer the conversion for P4VP was 95 ( 5% whereas for PEI (Table 1) and P4VP PEI mixtures it varied from 10 to 40%. The difference between P4VP and PEI is in their molecular mass and solubility in polar organic solvents. The solubility of PEI is higher than that of P4VP, so cross-linked PEI approached the phase-separation point at higher concentrations than did P4VP, which has a higher molecular mass than does PEI. An interesting behavior was found for the mixture of these two polymers. PEI contains primary amino-functional groups. Hence, PEI is more reactive with DIB than P4VP. We may speculate that DIB is consumed in the reaction with PEI, resulting in the delay of P4VP cross-linking in mixtures of these two polymers and thus a lower conversion. The composition of the mixed polymer capsules was regulated by the ratio of the P4VP and PEI polymers in solution. It was found that the ratio of the two polymers affected the wall thickness. When the fraction of the more-reactive PEI increased, the wall thickness increased as well. (Compare images a, b, and c in Figure 4). Thus, the wall thickness can be regulated by combinations of polymer and particle and P4VP/PEI ratios; for example, the walls of 10 μm capsules can be prepared in the 100 nm to 1 μm thickness range (Figure 5). Responsive Properties of Capsules. The walls of the capsules are made of weak polyelectrolytes. Hence, the capsules are responsive to changes in ionic strength (IS) and pH. They are positively charged over the entire studied range of pH (Figure 1g). The capsules swell upon protonation in an acidic pH range (the greatest swelling occurs at pH
