ISSN 0965545X, Polymer Science, Ser. A, 2009, Vol. 51, No. 6, pp. 719–729. © Pleiades Publishing, Ltd., 2009.
Singlecomponent LayerbyLayer Weak Polyelectrolyte Films and Capsules: Loading and Release of Functional Molecules1 V. A. Kozlovskayaa, E. P. Kharlampievaa, I. ErelUnalb, and S. A. Sukhishvilib a
b
Department of Materials Science and Engineering, Georgia Institute of Technology, Atlanta, GA 30332 Department of Chemistry, Chemical Biology and Biomedical Engineering, Stevens Institute of Technology, Hoboken, NJ 07030 email:
[email protected]
Abstract—Poly(carboxylic acid) hydrogel films and hollow capsules undergo reversible size changes in response to variations in pH and/or ionic strength. The films and capsules were obtained from hydrogen bonded polyNvinylpyrrolidone/poly(carboxylic acid) layerbylayer films by chemical crosslinking of the polyacid, followed by pHinduced removal of polyNvinylpyrrolidone. Surfaceattached hydrogel films present attractive matrices for reversible pHstimulated loading and/or controlled release of large amounts of synthetic or natural macromolecules including proteins. By varying acidity of poly(carboxylic acids), the hydrogel swelling and the corresponding values of pH for encapsulation/release of functional molecules could be tuned in a wide range from pH 5 to 10. In addition, the capsules are capable of entrapping macro molecules by “locking” the capsule wall with an electrostatically associating polycation, followed by the release of the encapsulated macromolecules at high salt concentrations. DOI: 10.1134/S0965545X09060170 1
INTRODUCTION
One of the important trends in modern biotechnol ogy, biomedical engineering and personal care tech nologies is a need in designing of “smart” materials capable of delivering in a controlled way a variety of functional molecules such as proteins, drugs, fra grances, or cosmetic ingredients. These intelligent materials should either reside at a solid surface (such as of an implant), or be dispersed in a medium as micro or nanocarriers loaded with a functional cargo. Polymers are an obvious choice as surface coat ings or/and encapsulation materials, and many syn thetic or natural polymers have been used as matrices for trapping and release of functional compounds from biodegradable coatings at stent surfaces [1, 2] degrad able solid porous particles [3], or natural polymer hydrogels for cell encapsulation [4, 5]. Often, a response of polymer matrices to environmental stim uli, such as pH or temperature is required. Examples include potential control of the release rate of func tional compounds in response to local environment, such as acidity in tissues associated with cancer or bac terial infection, or a possibility to control release of chemicals by cooling or by direct or remote heating of the polymer matrix. In this paper, we describe our approach to engineer pHresponsive weak polyelectrolyte ultrathin hydro gels, which are either attached to solid surfaces, or comprise the polymer wall of a hollow delivery con 1 The text was submitted by the authors in English.
tainer. Our strategy is based on using hydrogen bonded layerbylayer (LbL) multilayers as precursor films for engineering environmentally responsive matrices. The choice of the LbL technique provides us with two major advantages: (1) the possibility to con formally coat solid substrates of any shape, and (2) the fine and convenient control of the polymer coating thickness afforded by the stepbystep nmscale depo sition procedure. Specifically, we will focus on weak polyelectrolyte surface hydrogels and hollow capsules as matrices for controlled permeation, loading and release of functional chemical and biological mole cules. The potential of hydrogels as protein and drug carrying materials has been earlier explored for bulk hydrogels [6–8], while the area of ultrathin functional hydrogels as smart surface matrices or containers remained largely uncharted. While at surfaces, hydrogels form a soft three dimensional matrix that can provide an ideal environ ment for hosting a variety of functional molecules such as drugs or proteins. Relatively thick, hundreds of nanometers, surfacebound hydrogels were earlier obtained by radiationinduced attachment of polymer thin films to functionalized surfaces [9–12] but this approach can not be universally applied to complex surfaces. Ultrathin surface hydrogels comprised of two polymer components synthesized via thermal and photocrosslinking of hydrogenbonded multilayers were reported by Rubner and coworkers [13]. Serizawa et al. has engineered LbL surface hydrogels using sequential chemical crosslinking during poly
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mer assembly [14, 15], and studied pH and ionic strengthregulated adsorption of charged dyes within such hydrogel coatings [16]. We summarize our results on singlecomponent surfaceattached hydrogels derived from hydrogenbonded multilayers, and show that such hydrogels provide an excellent pHdriven control of loading and release of functional molecules. We also review our work on constructing hollow poly mer containers with hydrogel ultrathin polymeric walls, which are capable of controlling permeability, and therefore loading and release of macromolecules to/from such containers in response to environmental pH. During the last decade, hollow LbL polymeric capsules composed of oppositely charged polyelectro lytes have been intensively studied [17–19] as poten tially useful carriers for therapeutic agents [20]. An important feature of such containers is the avail ability of internal volume within such capsules, which can be filled with water or organic solvent [21] and serve as a depot for delivery of chemicals, or for sens ing [22, 23] and catalysis [24, 25] applications. To impart LbL capsules with novel functions, natural polyelectrolytes and biomacromolecules [26, 27] have been exploited for capsule wall construction. Several types of pH responsive capsule systems were also recently produced using electrostatic LbL assembly. pHControlled pore formation and healing for poly(styrene sulfonate)/poly(allylamine) hydrochlo ride (PSS/PAH) capsules was described by Antipov et al. [28]. PSS/PAH and poly(methacrylic acid)/PAH (PMAA/PAH) capsules were shown to demonstrate a reversible size change under the extreme pH conditions of 11.2 and 2.8, respectively [29]. Lvov and coworkers also performed selfassem bly of tannic acid with strong and weak polycations and produced capsules with the lowest permeability at pH 5–7 [30]. The potential of hollow multilayer cap sules to encapsulate large amounts of chemicals and to controllably release the capsule content has also been explored [31, 32]. The pHdependent permeability of the capsule wall was utilized for controlled inclusion and release of materials [33, 34]. Here, we demonstrate the potential of surface attached hydrogels and hollow hydrogelwall capsules produced from hydrogenbonded multilayers for a pH/salttriggered release of macromolecules. Our approach is based on our previous findings that hydro genbonded multilayers such as polyNvinylpyrroli done/PMAA (PVPON/PMAA) or poly(ethylene oxide)/PMAA (PEO/PMAA) disintegrate at pH val ues below physiological [35]. Selective chemical crosslinking of the polycarboxylic acid component of the capsule wall with a diamine crosslinker, followed by release of a neutral polymer from the film render the capsule wall stable at physiological pH. Further more, we show that pH swelling profiles of these sur face hydrogels and capsules can be finely tuned by
choosing various poly(cabroxylic acid)s for hydrogen bonded selfassembly, as well as by varying the density of crosslinks. Such tuning is important as various applications may require systems with particular swell ing/release characteristics including those with swell ing profiles opposite under acidic or basic conditions. For example, in oral drug delivery, swelling and subse quent release of a drug is expected to occur upon pH change from 1–4 to ~7.5 [36], while bacterial infec tions are associated with local pH lowering from pH 7.5 to pH 6.5–5 [37]. EXPERIMENTAL Materials. PMAA (Mw = 150 kDa), poly(acrylic acid) (PAA; Mw = 360 kDa), poly(2ethylacrylic acid) (PEAA; Mw = 304 kDa), PAH (Mw = 70 kDa), PVPON, (Mw = 55 kDa), FITC labeled dextrans with molecular weight of 500, 150, 70, and 4 kDa, hydro chloric acid, sodium hydroxide, sodium chloride, dibasic and monobasic sodium phosphate, 1ethyl3 (3dimethylaminopropyl)carbodiimide hydrochlo ride (EDC), Nhydroxysulfosuccinimide sodium salt (NSS), ethylenediamine (EDA), lysozyme (Lys), pan creas ribonuclease A (RNase), and heparin were pur chased from SigmaAldrich. All chemicals were used without any further purification. Quaternized poly(4 vinylpyridine) with 90% degree of quaternization (QPVP90) was synthesized according to the literature using poly(4vinylpyridine) with Mw 200 kDa (Sigma Aldrich) [38]. D2O with 99.9% isotope content was purchased from Cambridge Isotope Laboratories and was used as received. The SiO2 template particles were purchased from Polysciences Inc. as 10% dispersions in water and were of 4.0 ± 0.2 microns in diameter. Dialysis of capsules was performed in 100 µl Micro DispoDialysers (SpectrumLabs, Canada). Millipore (MilliQ system) filtered water with a resistivity 18.2 MΩ cm–1 was used in all experiments. Fluorescent labeling of PMAA. The labeling of PMAA was performed as described elsewhere [39]. Specifically, PMAA was dissolved in 0.1 M phosphate buffer solution and the solution pH was adjusted to 5 with 0.1 M sodium hydroxide solution. The polymer solution was mixed with EDC and NSS, 5 mg/ml each, and stirred for 30 min. The activation step was followed by adding fluoresceinylEDA solution at pH 6 with constant stirring for 10 hours in darkness. FluoresceinylEDA was synthesized as described ear lier [40]. The labeled PMAA was dialyzed against phosphate buffer (pH 7, 0.1 M) for at least 7 days and then pure deionized water for 12 h. The molecular weight cutoff of dialysis tubing was 25 kDa. The dialy sis was interrupted after no traces of fluorescence could be determined in the dialysis water. The dialyzed polymer solution was lyophilized and 0.2 mg/ml solu POLYMER SCIENCE
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tions of labeled PMAA at pH 2 were prepared and used for capsule preparation. Attenuated total reflection fourier transform infra red spectroscopy (ATRFTIR). In situ ATRFTIR deposition and crosslinking experiments were done with a Bruker Equinox55 Fourier transform infrared spectrometer equipped with a narrowband mercury cadmium telluride detector. The experiments were performed using a homebuilt flowthrough cell. The PVPON/PMAA multilayers were deposited onto a flat surface of oxidized Si. The oxidation of the surface, priming with the first layer, multilayer deposition, and calculation of the amount adsorbed has been previ ously described in the literature [41]. Confocal laser scanning microscopy (CLSM). Confocal images of capsules were obtained with an LSM 5 PASCAL laser scanning microscope (Zeiss, Germany) equipped with CApochromat 63×/1.2 water immersion objective. Capsules were visualized through deposition of the FITClabeled PMAA within the last two bilayers of PVPON/poly(carboxylic acid) capsules. The excitation wavelength was 488 nm. Crosslinking of hydrogenbonded multilayers on Flat Substrates. In a typical ATRFTIR experiment, 10 layers of PVPON/PMAA (below the number of lay ers will be denoted as a bottom index, e.g., (PVPON/PMAA)10 were constructed at pH 2. Condi tions of the crosslinking reaction are detailed below. Preparation of hydrogel capsules. Deposition of hydrogenbonded multilayers of PVPON/PMAA on particulate substrates has been described previously [42]. Briefly, 0.2 mg/ml polymer solutions were used within a typical deposition time of 15 min. Hydrogen bonded multilayers were deposited directly onto silica microparticles at pH 2 starting from PVPON. Each deposition cycle was followed by washing three times with a water solution with pH adjusted to 2. Suspen sions were settled down by centrifugation at 1200 rpm for 1 min to remove the supernatant. Deposition, washing, and redispersion steps were performed in a shaker (Fisher Scientific) at 1600 rpm. To make the capsules visible in CLSM, the labeled PMAA was deposited in the last two bilayers. When a desired num ber of layers were deposited, crosslinking was per formed. The detailed procedure included activation of the carboxylic groups with 5 mg/ml solution of EDC and NSS at pH 5 followed by reaction with 5 mg/ml solution of EDA at pH 5.8 for different period of times. After several washings of the particle suspension in phosphate buffer at pH 4.6 the cores were dissolved by shaking the particle dispersion for 4 h in 8% aque ous HF solution yielding hollow capsules. After that the dispersion of capsules with crosslinked PMAA walls was dialyzed in a buffer at pH 8.0 to ensure a removal of PVPON. Capsules were then transferred to POLYMER SCIENCE
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pH 4.6 by dialysis against phosphate buffer solution for 4 h. Preparation of capsules for CLSM. In studies of the pH and ionicdependence of the size of crosslinked polyacid capsules using CLSM, capsules were fixed at the surface of LabTek chambered coverglass. The procedure included the addition of a drop of a disper sion of hollow capsules to the chamber. The chambers of the LabTek coverglass were then sequentially filled with buffer solutions at a certain pH and ionic strength. Scanning electron microscopy (SEM). SEM analy sis was performed using a LEO 982 DSM instrument with a fieldemission gun at an operation voltage of 1 or 5 kV. A drop of a capsule suspension was applied to a precleaned silicon wafer and measurements were conducted after specimens were allowed to dry for two hours. Film thickness measurements. Thickness measure ments of dry films as well as film swelling were done using a custommade phasemodulated ellipsometer. Before film deposition, silicon wafers were first cleaned as described elsewhere [42]. To enhance sur face adhesion of the subsequently grown multilayer to a silicon wafer, two polymer bilayers were first depos ited as a precursor film. First, the surface of a silicon wafer was exposed to 0.2 mg/ml PAH solution in 0.01 M phosphate buffer at pH 9 which additionally contained 0.1 M NaCl. At the second polymer adsorp tion step, PMAA was allowed to selfassemble from 0.2 mg/ml solution in 0.01 M phosphate buffer at pH 5. This cycle was repeated until 2 bilayers of PAH/PMAA were deposited at the surface. Hydro genbonded PVPON/PMAA films were then depos ited from 0.2 mg/ml polymer solutions at pH 2. After depositing 20 or 40 bilayers, films were crosslinked as described above. Wafers containing crosslinked mul tilayers were then exposed to pH 8 for 30 min to remove remains of PVPON and activation agents, transferred to pH 4.6 and dried. For measurements of dry films, samples were dried under a stream of nitro gen. Studies of film swelling were performed using a cylindrical flowthrough liquid cell. Detailed descrip tion of the ellipsometry setup and swelling measure ments are described elsewhere [43]. RESULTS AND DISCUSSION Surfaceattached PMAA LbL Hydrogels Preparation and swelling of PMAA surface attached hydrogels. The formation of surfaceattached PMAA layered hydrogels was first studied using in situ ATRFTIR (data not shown). The hydrogenbonded PVPON/PMAA multilayers were deposited at pH 2 and resulted in a total dry thickness of 38 nm for (PVPON/PMAA)10 film. Using in situ ATRFTIR,
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we have earlier confirmed that an activation of carbox ylic groups within the hydrogenbonded film with watersoluble EDC and an addition of EDA resulted in the formation of amide bonds, and that PVPON was completely released from the film after the exposure of the crosslinked film to pH 8.0 [44]. This observation was in good agreement with the earlier finding of com plete dissociation of PVPON/PMAA multilayers at pH values higher than 6.9 [45]. The bilayer thickness of PVPON/PMAA film deposited at pH 2 was ~ 4 nm. After crosslinking PVPON/PMAA multilayers, films were exposed to pH 8 for 30 min to remove residual amounts of PVPON and the activation agents, transferred to pH 4.6 and dried, the dry film thickness decreased by ~45%, as was demonstrated for (PVPON/PMAA)20 and (PVPON/PMAA)40 films. Release of PVPON from the PVPON/PMAA films was also monitored by ellipsometry.
Swelling of (PMAA)10 surface hydrogels followed by phase modulated ellipsometry is shown in Fig. 1 [46]. Exposure of dry films to 0.01 M phosphate buffer at pH 4.6 resulted in an increase of film thickness from 38 to 80 nm. Based on the reported pKa values of PMAA (between 6 and 7) [39], the polyacid does not carry electric charge at pH 4.6, and the observed thick ness increase is due to hydration of uncharged PMAA segments. Using data for PMAA hydrogel swelling at pH 4.6, the molecular weight between crosslinks, Mc, was estimated for PMAA with molecular weight of 150 kDa [46] using the Flory equation for nonionic gels. However, because the swelling was constrained later ally, it was necessary to revise the Flory equation for the case of onedimensional swelling as described in our previous study [44]. Application of the Flory equa tion for the hydrogel swelling data shown in Fig. 1 yielded Mc = 960 Da, implying that there are about 11 PMAA monomer units between crosslinks. As will be shown below, this estimate includes both types of crosslinks, i.e., covalent and ionic ones. Swelling in this pH range occurs due to increased ionization of the carboxylic groups of PMAA. Specifically, strong hydrogel swelling also occurred at pH < 4.5 where car boxylic groups of PMAA are largely protonated. This type of swelling indicated that PMAA hydrogels also contained amino groups and, thus these hydrogels are amphoteric in nature. Inclusion of the basic groups within hydrogels occurred because a fraction of EDA used as a crosslinker during hydrogel synthesis reacted with PMAA chains through one end only. The presence of oneend attached amino groups in the crosslinked PMAA network was confirmed in our previous paper [47]. At neutral pH values, protonated amino groups of EDA are included in ionic pairs with carboxylate groups, and ionic crosslinks +
NH 3 /COO– coexist with covalent ones within the hydrogel. At pH < 4.5, hydrogels swell because the protonation of carboxylic groups involved in ionic +
NH 3 /COO– crosslinks occurs, and the hydrogel acquires positive charge. The hydrogel swelling was highly reversible and reproducible with increasing or decreasing pH. In the following sections, we explore how the amphoteric nature of the PMAA hydrogels can be used for controlled loading and release of charged compounds. Inclusion of macromolecules: Lys and heparin. Loading of proteins and heparin within the PMAA hydrogel was studied in situ using two complementary techniques – ATRFTIR and ellipsometry. Lys, a pro tein with an isoelectric point of 11.5 [48], was selected as a model protein which is positively charged at neu tral pH values. The kinetics of Lys adsorption within (PMAA)5 and (PMAA)10 hydrogels was fast, and the adsorbed amount saturated after ~15 min when POLYMER SCIENCE
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The amount of Lys bound with the (PMAA)5 hydrogel at pH 7.5 was 54 mg/m2 or 10 times larger than the maximum monolayer capacity calculated for endon oriented monolayer of this protein based on Lys molecular size of 3 × 3 × 4.5 nm [41] and the pro tein density of 1.37 g/cm3. The high capability of the (PMAA)5 hydrogels to absorb Lys indicates the inclu sion of the protein within the whole hydrogel thick ness. This result indicates that the hydrogel mesh size is large enough to allow transport of Lys globules through the hydrogel matrix. Above we have calculated the number of monomer units between crosslinks to be 11, when both ionic and covalent crosslinks were taken into account. Using our estimate that only a half of these crosslinks are covalent, we conclude that the dis tance of 22 monomeric units between covalent crosslinks allows diffusion of Lys within the hydrogel. Further confirmation of penetration of Lys within the bulk of the film was obtained in ellipsometry, which Vol. 51
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Fig. 3. pH dependence of amounts of Lys (open triangles) and heparin (filled triangles) absorbed within (PMAA)5 hydrogel as inferred from in situ ATRFTIR and con firmed with ellipsometry; pH values were supported by 0.01 M phosphate buffer solutions.
0.1 mg/ml protein solutions were used. Fig. 2 shows that ~98% of Lys loaded at pH 7.5 is released when the gel is exposed to pH 4, when carboxylic groups became completely protonated. Such good correlation of the amount of Lys loaded within the film with the charge density of the PMAA hydrogels points to essentially electrostatic, nature of protein interactions with the hydrogel matrix. The similar trend of reversible bind ing of positively charged protein within (PMAA)5 hydrogels was obtained with RNase (data are not shown).
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Fig. 2. Reversible pHcontrolled absorption and release of Lys into/from surfaceattached (PMAA)5 hydrogel quan tified by in situ ATRFTIR. The dry thickness of (PMAA)5 hydrogel was 23 nm (including a 10nm precursor layer) as quantified by ellipsometry. The equilibrated values of cali brated amount adsorbed are shown after the hydrogel was brought in contact with 0.1 mg/ml Lys solutions at various pH values from 3 to 7.5; pH values were supported by 0.01 M phosphate buffer solutions.
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showed that dry thicknesses of Lysloaded (PMAA)n films scaled linearly with the number of PMAA layers within bare hydrogel matrix. Negatively charged macromolecules such as hep arin could also be included within the amphoteric hydrogels. Amount of heparin loaded at pH 3 was pro portional to PMAA hydrogel thickness, and about twice as much heparin was included in (PMAA)10 compared to (PMAA)5 films as monitored by ellip sometry (data not shown). Fig. 3 contrasts the inclu sion of heparin and Lys within the surface hydrogel at various pH values. As in the case of Lys, heparin adsorption was irreversible towards dilution with buffer solutions at a constant pH. However, heparin was effectively released from the PMAA hydrogel upon an increase in pH, i.e., when the hydrogel car boxylic groups deprotonated and acquired negative charge. In a similar way, at physiological pH values of 7.5, the most abundant plasma protein albumin could not be included into the (PMAA)5 matrix (experi ments were done with 0.1 mg/ml bovine serum albu min), because of both the size and the charge consid erations. Hydrogel Capsules Preparation of PMAA hydrogel capsules. In this work we took advantage of the use of silica particles whose decomposition in acidic solutions of hydroflu oric acid is highly compatible with high stability of hydrogenbonded PVPON/PMAA films in acidic media. It is known from the literature that dissolution of other widely used cores such as melamine formal dehyde or polystyrene particles could either cause multilayer shell defects due to osmotic stress during
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Fig. 4. SEM images of PVPON/PMAA capsules dried from deionized water (a). The second image (b) shows the PMAAcrosslinked capsule dried from deionized water, after PVPON component was removed through dialysis at pH 8.
core dissolution [49] or could modify the polymeric shell properties due to deposition of residual material within the wall [50]. Following the procedure devel oped for hydrogels attached to flat substrates, PVPON/PMAA coretemplated films were activated and crosslinked at pH 5 and 5.8, respectively, and then produced hollow capsules were extensively dia lyzed at pH 8 to ensure complete removal of PVPON and residual amounts of EDC/NSS moieties from the crosslinked multilayers. Figure 4 compares SEM images of (PVPON/PMAA)7 capsules before cross linking (Fig. 4a) and PMAA crosslinked capsules (Fig. 4b) after PVPON was released and the EDC/NSS moieties were hydrolyzed via dialysis at pH 8, when these capsules were dried from deionized water (pH ~ 5.5−6). Both noncrosslinked and cross linked capsules collapse exhibiting folded structures upon drying. However, compared to noncrosslinked hydrogenbonded capsules which have pronounced sharp folds, PMAA crosslinked capsules demonstrate
folds which are much smoother, evidently due to the soft, hydrogellike structure of the capsule shell. Variations of size of PMAA hydrogel capsules with pH. Fig. 5 illustrates (PMAA)7 capsule swelling in a wider pH region from 2 to 12 [47]. Three distinct regions in swelling/deswelling behavior of capsules are clearly seen. Swelling in region II (from pH 5 to 8) is explained by increased ionization of PMAA carboxy lic groups. Capsule swelling in this region is accompa nied by drastic changes in the mechanical properties of the capsules [51]. As in the case of surface hydrogels, there is also an unusual swelling behavior in regions I (pH < 4.5) and III (pH > 9). These results can be ratio nalized through the presence of basic groups supplied from the oneendattached EDA crosslinker mole cules. To prove the presence of positive charges provided by primary amino groups within (PMAA) hydrogel capsule wall, ζpotential of both crosslinked and noncrosslinked capsules was measured. The ζ potential of noncrosslinked hydrogenbonded PVPON/PMAA and PMAA capsules derived from the former ones by their crosslinking with EDA were investigated at different pH. Fig. 6 shows that for non crosslinked capsules ζpotential was negative over the pH range from 2.8 to 5.8. Previously, we have reported that for hydrogenbonded layers of PVPON/PMAA and PEO/PMAA [42] deposited on colloidal particles, ζpotential remained negative in a wide pH range. In drastic contrast, with crosslinked hydrogel PMAA capsules described here, a decrease in pH results in switching of capsule charge from negative to positive. Note that the amide groups supplied by covalent crosslinking of EDA with carboxylic groups have low basicity (pKb ~ −0.5) and do not carry positive charge in the studied pH range. Instead, a positive charge originating from primary amino groups of the one endreacted crosslinker molecules is responsible for the observed pHinduced charge reversal. Interest ingly, at pH > 9, where additional capsule expansion occurred (Fig. 5), ζpotential is also acquired more negative values (Fig. 6). These facts are consistent with deprotonation of primary amino groups and resultant dissociation of ionic crosslinks in this pH range. The existence of the ionic crosslinks was also con firmed by FTIR [47] as well as by the fact that the addition of sodium chloride results in expansion of capsules at pH 5 where carboxylic groups of PMAA network do not carry charge [47]. Figure 7, top panel on the left demonstrates that that capsules crosslinked for 5, 18, and 22 h display similar swelling regimes, while the degree of swelling drastically decreases with increasing crosslinking time, indicating the increased number of crosslinks within the capsule wall. This behavior is typical to slab hydrogels, and reflects the higher number of EDA POLYMER SCIENCE
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crosslinks introduced within the capsule wall with longer crosslinking times. One would expect that the mesh size of the hydrogel will correspondingly decrease with the crosslinking time. The effect of crosslinking density on capsule permeability to model dextran molecules is described below. Varying the poly(carboxylic acid). We further aimed to tune pH swelling of transitions by varying the nature of the poly(carboxylic acid)s used in fabrication of the initial hydrogenbonded capsules. Bottom panel in Fig. 7 (left) demonstrates that when the polyacid was changed to PAA or PEAA, the overall shape of swelling profiles for polyacid capsules of all types remained similar. However, the region of the minimum swelling of the crosslinked capsules shifted towards lower (for the PAA capsules) or higher (for the PEAA capsules) pH values compared to that for the PMAA capsules. When exposed to higher pH from its minimum swell ing state, the PAA capsules started swelling at pH ~ 5 which is close to its reported pKa of 4.5 [52, 53]. In case of the PEAA crosslinked capsules, the swelling occurred at pH ~ 9.2 which is more than 2 pH units higher than the pKa of PEAA (its reported pKa is 7) [54]. This can be explained by the fact that PEAA is significantly more hydrophobic, and higher charge POLYMER SCIENCE
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densities are needed to expand the polymer chain from its compact conformation. A similar modulation of the pHinduced conformational change was reported for poly(methacrylic acidcoethacrylic acid) copoly mers with increasing the ethacrylic acid content [54]. The difference in the swelling amplitude for PAA, PMAA, PEAA crosslinked capsules seen in Fig. 7 is due to different crosslinking degrees of the systems, resulting from different pH values used during capsule crosslinking. Right panels in Fig. 7 illustrate the effect of increased hydrophobicity of the polyacid on capsule surface morphology. One can see that while the surface of (PAA)7 crosslinked capsules is smooth (Fig. 7a), it becomes rougher for (PMAA)7 capsules (Fig. 7b). The surface of airdried (PEAA)7 crosslinked capsules is very lumpy and grainy (Fig. 7c), similar to that of the PEAA films attached to the planar substrate. This can be attributed to changes in polymer conformations when hydrophobicity of poly(carboxylic acid)s is increased from PAA to PMAA and PEAA. Hydropho bicity of PMAA has been reported to result in compact hydrophobic clusters [55, 56] within PMAA chains in aqueous solutions at low pH values. Such clusters were
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(pH 8 and 2), but turned impermeable to this macro molecule at pH 5 where capsule wall expansion and capsule size were the smallest. The pHcontrolled selective permeability could be used for loading of macromolecules within the hollow capsules. Specifi cally, when the (PMAA)7 capsules were exposed to 1 mg/ml solution of FITCdextran with 70 kDa at pH 8 or 2, loading of the dextran molecules within the hollow capsule occurred after 30 and 15 min, respec tively, and loaded materials remained “trapped” within the capsule after capsules were transferred to 0.01 M buffer at pH 5. As seen from the CLSM images in Fig. 9a, the capsules are impermeable for the FITC dextran molecules. The capsule interior remained dark at least for 24 h, suggesting that permeability of the capsule to 70 kDa FITCdextran became negligi ble under these conditions.
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Fig. 6. ζpotentials of noncrosslinked hydrogenbonded (PVPON/PMAA)6 (open circles) and 16h crosslinked (PMAA)7 capsules (filled circles) as a function of pH. The pH values of aqueous capsule suspensions were adjusted using HCl or NaOH solutions.
not observed for PAA. More hydrophobic PEAA has limited solubility in water at low pH, and was depos ited from methanol solutions. When exposed to aque ous solutions, PEAA presumably phaseseparated to yield hydrophobic domains, whose aggregation gave rise to the grainy morphology observed in Fig. 7c. pHtriggered encapsulation and release: molecular sieving effect. At pH ~ 8 in 0.01 M buffer solutions, the 5 h crosslinked (PMAA)7 capsules were readily per meable to FITCdextrans of all studied molecular weights. However, an increase in crosslinking density afforded selective permeability of macromolecules through the hydrogel wall. Specifically, the 16 h cross linked capsules remained permeable to FITCdex trans with Mw = 70 kDa (Fig. 8a) at pH 8, but remained impermeable for FITCdextran with Mw of 500 kDa at both pH 8 (Fig. 8b) and pH 2 (not shown). Such selective permeability is consistent with a decrease in the capsule wall mesh size for the 16h crosslinked capsules as compared to 5h crosslinked capsules (110 Å at pH 8 and low ionic strength [44]) so that the capsule wall could efficiently retain 500 kDa FITCdextran whose hydrodynamic radius (Rh) is 160 Å [57]. At the same time, the mesh size remained large enough to allow 70 kDa FITCdextran molecule with Rh ~ 80 Å [58] to permeate through the capsule wall. Importantly, 16h crosslinked capsules remained in the “open state” for the 70 kDa FITCdextran in its highly swollen state at both high and low pH values
Encapsulation and release using polycation binding to the PMAA capsule wall. An alternative strategy to encapsulation and release of macromolecules with the PMAA hollow capsules is “locking” the capsule wall through forming a complex with a polycation. Figure. 9 illustrates the feasibility of this approach. The (PMAA)7 capsules were first soaked in 1 mg/ml 500 kDa FITCdextran solution at pH 8 for 120 min to fill up the capsules with the material. Then the FITC dextran solution was replaced with 0.1 mg/ml solution of QPVP90 at pH 8. QPVP90 was allowed to interact with dextranloaded (PMAA)7 capsules for 15 min, after that the solution was replaced by phosphate buffer at pH 8. This was accompanied by a drastic decrease of the capsule size from 7.3 to 3.0 µm, con sistent with the similar shrinking of bulk charged hydrogels caused by a formed complex with oppositely charged linear macromolecules [59]. As seen from the fluorescence profile in the CLSM image in Fig. 9a, the capsules remained filled with the FITCdextran mol ecules. The capsule interior remained fluorescent over two weeks, suggesting that permeability of the capsule to 500 kDa FITCdextran wall became negligible after QPVP90 was bound to PMAA in the capsule wall. This experiment demonstrates the feasibility of entrapping large macromolecules within the capsule via formation of an interpolymer complex between the anionic capsule wall and a linear polycation. It is known that the stability of PMAA multilayer com plexes with QPVP having a high charge density (quat ernization degree ≥84%) is dramatically decreased at high pH values in the presence of high concentrations of small ions [60]. We utilized 0.6 M NaCl solution in 0.01 M phosphate buffer at pH 8 to “unlock” capsules by destroying the electrostatic complex between QPVP90 and the anionic hydrogel capsule wall. The destruction of the PMAA/QPVP90 complex occurred due to competition of salt ions and polyelec trolyte charged units for ionic pairing [61]. The fluo rescence profile in the CLSM image of Fig. 9b shows POLYMER SCIENCE
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Fig. 7. Left, top: pHDependence of the diameter of the (PMAA)7 capsules crosslinked for 5 (circles), 18 (diamonds), and 22 h (triangles). Left, bottom: pHdependent swelling of crosslinked (PAA)7, (PMAA)7, and (PEAA)7 capsules crosslinked for 22 h (squares, circles, and triangles, respectively). Right: AFM topography images of type I capsules: (PAA)7 (a), (PMAA)7 (b), (PEAA)7 (c). Capsules were deposited on silicon wafers from aqueous solutions at pH 4, 5, and 7, respectively, and airdried. Scan area is 5.6 µm.
5 µm
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Fig. 8. CLSM images of (PMAA)7 capsules crosslinked for 16 h and incubated in 1 mg/ml FITCdextran solutions of different molecular weights at pH 8 (0.01 M phosphate buffer): 70 kDa for 3 min (a) or 500 kDa for 30 min (b).
that FITCdextran was completely released from the capsule interior after salt treatment. CONCLUSIONS In summary, we have demonstrated that onecom ponent crosslinked films and capsules of a weak POLYMER SCIENCE
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poly(carboxylic acid) can be produced from hydro genbonded multilayers after selective crosslinking of carboxylic groups of the polyacid with ethylenedi amine. These hydrogel films and capsules show revers ible swelling in response to changes in solution pH. By varying acidity of poly(carboxylic acid)s, the hydrogel swelling and the corresponding values of pH for
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Intensity, a.u.
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4000 3000 2000 1000
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Fig. 9. CLSM images of (PMAA)7 capsules consecutively exposed to 1 mg/ml FITCdextran 500 kDa solution for 120 min, and to 0.1 mg/mg QPVP90 for 15 min and washed with 0.01 M buffer at pH 8 (a), as well as after additional exposure of FITCdex tranloaded capsules shown in panel (a) to 0.6 M NaCl for 1 h (b).
encapsulation/release of functional molecules could be tuned in a wide range from pH 5 to 10. Hydrogel surface films present versatile matrices capable of loading large amounts of macromolecules including proteins, and that loaded molecules can be later released in response to pH variations. The capsules are useful for entrapping and storing macromolecules by pHcontrolled loading of the macromolecules at basic or acidic pH with subsequent capsule “closing” at pH 5. The release of the encapsulated macromole cules can be achieved by varying the pH values. Also, the capsules can be used for entrapment and storage of macromolecules by “locking” the capsule wall with electrostatically associating polycations at high pH. The release of the encapsulated macromolecules in this case can be achieved under high salt concentra tions when PMAA/polycation binding is suppressed. The capability of surface hydrogels and capsules to include and then release macromolecules in response to environmental stimuli holds a significant promise for applications in biomedicine and biotechnology. REFERENCES 1. H. H. Cho, D.W. Han, K. Matsumura, et al., Bioma terials 29, 884 (2008). 2. S. Prabhu and S. Hossainy, J. Biomed. Mater. Res. 80A, 732 (2007). 3. J.M. Rabanel and P. Hildgen, J. Microencapsulation 21, 413 (2004). 4. D.Q. Wu, Y.X. Sun, X.D. Xu, et al., Biomacromole cules 9, 1155 (2008). 5. J. Lukáš, V. Pale c kov a, J. Mokrý Karbanová, and B. Dvo r ánková, Macromol. Symp. 172, 157 (2001). 6. Y. Osada, J. P. Gong, and Y. Tanaka, J. Macromol. Sci., Rev. Macromol. Chem. 44, 87 (2004). 7. N. A. Peppas and W. Leobandung, J. Biomater. Sci., Polym. Ed. 15, 125 (2004).
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