pH-Induced Self-Assembly and Capsules of Sodium Alginate

Jun 17, 2005 - Facile Preparation and Enhanced Capacitance of the Polyaniline/Sodium Alginate Nanofiber Network for Supercapacitors. Yingzhi Li , Xin ...
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Biomacromolecules 2005, 6, 2189-2196

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pH-Induced Self-Assembly and Capsules of Sodium Alginate Yi Cao,† Xiaochen Shen,† Ying Chen,† Jian Guo,† Qi Chen,† and Xiqun Jiang*,†,‡ Laboratory of Mesoscopic Chemistry and Department of Polymer Science and Engineering, College of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China, and Jiangsu Provincial Laboratory for Nanotechnology, Nanjing University, Nanjing, 210093, China Received March 2, 2005; Revised Manuscript Received April 11, 2005

In this investigation, we used a kind of polyelectrolyte, sodium alginate, as a model biomacromolecule to investigate the aggregation behaviors in aqueous solution after partial protonation of carboxylate groups in the alginate molecules. It is demonstrated that the alginate assemblies with core-shell structure can be generated by the partial protonation of carboxylate groups in sodium alginate chains using the protons released gradually from the reaction of K2S2O8 with water at 70 °C in aqueous solution. The partial cross-linked alginate assemblies are pH sensitive and can change to hollow structure in the medium with relatively high pH value. This approach avoids use of block or grafted copolymers as the precursors or any other template to prepare assemblies and capsules, and provides a functional surface for subsequent chemical reaction at the surface (e.g., for binding biomolecules and for surface grafting). Such unique assemblies are also expected to be useful in biomedical fields. Introduction The synthesis of nanosized polymeric assemblies such as micelles, nanospheres and nanocapsules in aqueous solution has attracted great interest due to their applications in biologic and medical areas such as diagnostic testing, bioseparations, controlled releasing of drugs, and gene therapy.1-3 One typical approach to construct them in aqueous solution is mixing oppositely charged polymers,4 polymer/surfactant,5 or polymer/small counterion.6 The electrostatic force interaction between a pair of oppositely charged polymers, polymer/ surfactant, or polymer/small counterion drives the formation of polymeric assemblies. Recently, several groups have reported a novel method to prepare pH-sensitive micelles from double-hydrophilic watersoluble AB diblock or graft copolymers.7,8 These copolymers, which contain at least one polyelectrolyte block, can dissolve molecularly in water in a certain pH range and aggregate spontaneously to form micelles upon an appropriate change in the pH value. The difference in protonation or deprotonation of the polyelectrolyte block in a certain pH range drives the micellization of the copolymer in aqueous solution and determines the pH-responsive behavior of the micelles.9,10 Among these approaches, in most cases, the micelles are comprised of block or graft copolymers, with the pHsensitive solvent-phobic part forming the core and the solvent-philic part forming the corona. On the contrary, in nature, a variety of protein and polypeptide biomacromolecules consisting of hydrophobic, hydrophilic, and/or charged amino acid residues on the backbone chain can self-assemble to aggregates in aqueous medium with hydrophobic amino acids as the core and hydrophilic or charged amino acids as * To whom correspondence should be addressed. Fax: 86 25 83317761. E-mail: [email protected]. † College of Chemistry and Chemical Engineering, Nanjing University. ‡ Jiangsu Provincial Laboratory for Nanotechnology, Nanjing University.

the shell. To mimic this behavior of the biologic macromolecules, a number of theoretic studies and computer simulations on the aggregation of protein-like copolymers consisting of uncharged monomer units and charged ones in aqueous medium have been reported.11 Furthermore, a theoretic model of two-letter copolymers comprised of two types of monomer units, H (hydrophobic) and P (hydrophilic or polar), has been proposed.12 These investigations show that the copolymers with a heterogeneous, nonaltering sequence of H and P units along the copolymer chain can form stable, finite aggregates with H-core/P-shell structure. A few experimental studies also determined that a random copolymer could aggregate to form unimolecular micelles in aqueous solution.13 Although advances have been made in understanding aggregation behavior of individual protein-like copolymers in aqueous medium, few experimental studies have investigated the multichain aggregation of two-letter copolymers with random distribution of two types unit along the backbone chains.14 In this paper, we used the polyelectrolyte, sodium alginate, as a model biomacromolecule to investigate the aggregation behaviors in aqueous solution. We chose alginate based on the following reasons: (1) alginates are biocompatible unbranched binary copolymers of (1f4)-linked residues of β-D-mannuronic (M) and R-L-guluronic (G) acids (the pKa values for them are 3.38 and 3.65, respectively),15 which have been widely used as a kind of the desired biomaterials in many fields such as cell immobilization, tissue engineering and drug delivery;16 (2) the hydrophilic and hydrophobic units along a molecule chain can be altered by the protonation and deprotonation of carboxyl groups in the backbone chain; (3) the carboxyl groups existing in alginate can be used for further chemical modification such as cross-linking reaction. The dissociated carboxyl groups in alginate chains are protonated smoothly by the protons which are released

10.1021/bm0501510 CCC: $30.25 © 2005 American Chemical Society Published on Web 06/17/2005

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Scheme 1. Chemic Structure of Sodium Alginate Used, with Chair Conformationa

a

M, β-D-mannuronate; G, R-L-guluronate. FG ) 0.65 and FGG ) 0.53.

gradually upon the thermal decomposition and reaction of potassium persulfate (K2S2O8) in water. By this means, alginate molecules can form regular core-shell type aggregates in aqueous solution without the aid of any other homopolymer, copolymer, or surfactant. Such core-shell structure assemblies can be fixed by cross-linking the carboxyl groups in sodium alginate chains at room temperature. Moreover, the cross-linked assemblies obtained have a pH-dependent structure and can change between a coreshell structure to a hollow structure depending on the pH change of medium. Experimental Section Materials. Sodium alginate, purchased from Beijing Stock of China Medicine Company, was refined twice by dissolving it in distilled water, filtered, precipitated with ethanol, and finally dried in a vacuum at 60 °C. Its chemical structure is shown in Scheme 1. The viscous-average molecular weight of sodium alginate used was 170 kDa, determined by viscometric methods.17 The alginate was analyzed by 1H NMR spectroscopy at 70 °C using a Bruker DRX-500 (500 MHz) spectrometer.18 The molar fraction of guluronic acid residues (FG) and sequence parameters given as the diad (FGG, FMM, FGM, and FMG) was determined by NMR to be FG ) 0.65, FM ) 0.35, FGG ) 0.53, and FMM ) 0.23. Potassium persulfate (K2S2O8) was recrystallized from deionized water. Pyrene was recrystallized two times from benzene. Preparation of Sodium Alginate Assemblies. A total of 0.25 g of sodium alginate was dissolved in 100 mL of deionized water under magnetic stirring, and the temperature was raised to 70 °C. The desired amount of K2S2O8, based on the content of carboxylate groups in alginate, was added to the system. With the decomposition of K2S2O8 and the reaction of K2S2O8 with water, protons were released gradually to the solution, and the dissociated carboxyl groups of sodium alginate were partially protonated. We sampled the solution at different time points after K2S2O8 was added and cooled the samples to 0 °C with ice water. Cross-Linking of Assemblies and Preparation of Capsules. When sodium alginate assemblies were formed in the aqueous solution, the desired amount of 2,2′-(ethylenedioxy)bis(ethylamine), as a cross-linker, was added into the aqueous solution in the presence of 1-(3-dimethylaminopropyl)-3ethylcarbodiimide methiodide at room temperature, and the cross-linking reaction was allowed to take place for 10 h. The cross-linked product was dialyzed against distilled water for 72 h to remove the byproducts. Then, the aqueous solutions with different pH values were prepared. In each

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aqueous solution, the product was dialyzed for 48 h. The size and morphology of the samples were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). pH Value Measurement. The pH measurements were performed with a Delta 320 pH-meter (Mettler Toledo Instruments Co., Ltd.). Each sample was measured three times, and the results shown are the mean pH value. Fluorescence Measurement. Pyrene was chosen as the fluorescent probe to study the onset of assembly of sodium alginate solution. Pyrenes (6.0 × 10-7 molar) were dissolved in 10 mL of 0.25% (w/w) alginate solutions in the presence of NaCl (0.05 M). The pH value of the solutions was adjusted by HCl and NaOH. Steady-state fluorescent spectra were measured using a Perkin Elmeter LS50B fluorescence spectrophotometer in the right-angle optical geometry with a bandwidth of 0.5 mm. The excitation wavelength was 390 nm. Dynamic Light Scattering (DLS) Measurement. The mean sizes of the assemblies were measured by DLS using a Brookheaven BI9000AT system (Brookheaven Instruments Corporation, USA). All DLS measurements were done with a wavelength of 633.0 nm at 25 °C with an angle of detection of 90°. Each sample was measured three times, and the results shown are the mean diameter for two replicate samples. The zeta potential of the assemblies was obtained with Zetaplus (Brookheaven Instruments Corporation, USA). The results were the average of three runs. Transmission Electron Microscopy (TEM). The morphologies of the samples were observed by TEM (JEOL JEM-100S, Japan). Samples were placed onto copper grid covered with carbon and dried at room temperature. They were examined without being stained. Scanning Electron Microscopy (SEM). The morphology of the samples was examined by SEM (SX-40 scanning microscope, AKA SHI, Japan). The sample was placed onto the hydrophilic surface of the glass substrate which was treated with a solution of H2O2 and H2SO4, washed with distilled water, and dried at room temperature. Results and Discussion Self-Assembly of Alginate Induced by pH. It has been shown that fluorescence technique is a powerful tool for the investigation of many micellar properties such as micelle formation, micelle structure, and the kinetics of micelle formation.22 Using pyrene, a polarity-sensitive molecule, as a fluorescent probe, it is possible to obtain information on the aggregation of amphiphiles in aqueous solution from a shift of the (0, 0) band from 334 to 337 nm in the excitation spectra of pyrene.19 In our case, we chose pyrene as the fluorescent probe to study the self-assembly of alginate at different pH values and to see if the sodium alginate chains can form the hydrophobic domains after protonation of carboxyl groups. Fluorescence excitation spectra of the pyrene probe dissolved in 0.25% (w/w) alginate aqueous solutions in the presence of 0.05 M NaCl were measured, and the ratio (I337/I334) of the intensity at 337 nm to that at 334 nm of the solution was estimated at different pH values.

Capsules of Sodium Alginate

Figure 1. Ratios of I337/I334 in pyrene excitation spectra as a function of pH in 0.25% (w/w) sodium alginate aqueous solution in the presence of 0.05 M NaCl.

Figure 1 displays the intensity ratio of I337/I334 of alginate aqueous solutions as a function of pH value. It can be seen that, beyond the pH value of 6.6, the I337/I334 ratio is practically constant around 0.62, indicating that the location of pyrene probe is in the hydrophilic environment. As the pH value decreases from 6.6 to 4.1, the I337/I334 ratio increases substantially and reaches a maximum at pH of 4.1, suggesting that pyrene is progressively solubilized into the hydrophobic environment. Below the pH value of 4.1, the I337/I334 ratio keeps the constant of 1.47, indicating that the pyrene probe locates primarily in the hydrophobic environment. The behavior observed indicates that the alginate molecules can form hydrophilic-hydrophobic aggregates in aqueous solution depending on the pH of the medium. Here, we postulate that this pH dependence of “aggregation” as a signature for self-assembly caused by the partial protonation of dissociated carboxyl groups in the alginate main chain. Preparation of Regular Assemblies of Sodium Alginate. Based on the result of fluorescence measurement, the partial protonation of dissociated carboxyl groups in the alginate main chain was selected to prepare alginate assemblies. The partial protonation of dissociated carboxyl groups in sodium alginate was achieved by the decomposition of the desired amount of K2S2O8 in sodium alginate aqueous solution. Since the thermal decomposition of K2S2O8 will dissociate symmetrically into two sulfate free-radicals which can react with water to produce hydrogen sulfate ions and donate the protons to aqueous solution,20 alginate can obtain the protons upon the decomposition of K2S2O8 in aqueous solution. Figure 2 shows pH variation caused by the decomposition of K2S2O8 in water as a function of heating time at different temperatures and concentrations. It can be seen that the pH value of the aqueous solution decreases gradually from a pH of 7 to a pH of less than 3 in the time period of 400 min. As the K2S2O8 concentration in the solution increases from 1.018 to 1.187 g L-1, the final pH value in the given time period decreases from 2.66 to 2.54 accordingly. At the same time, the decomposition rate of K2S2O8 increases when the temperature of the solution increases from 60 to 70 °C. This result indicates that the protons are gradually released by the decomposition of K2S2O8 into sulfate free-radicals and the reaction of sulfate free-radicals with water. The total amount of protons released to the solution can be controlled by the heating time and the K2S2O8 concentration. Also, the protonation rate can be adjusted through the decomposition

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Figure 2. pH variation caused by the decomposition and reaction of K2S2O8 in water as a function of heating time at different temperatures and concentrations.

Figure 3. Hydrodynamic diameters (Dh) of sodium alginate assemblies and the solution pH value as a function of heating time. (a) R ) 0.6, (b) R ) 0.7, (c) the hydrodynamic diameter distribution of assemblies formed at K2S2O8 decomposition time (t) of 140 and 400 min with R of 0.7.

temperature. Thus, we use K2S2O8 as a mediate proton-donoragent to prepare assemblies of alginate by partial protonation of dissociated carboxyl groups in alginate main chains. The assemblies of alginate were prepared by dissolving sodium alginate in deionized water at 70 °C followed by adding the desired amount of K2S2O8. Figure 3 shows the average hydrodynamic diameters of alginate assemblies measured by DLS, together with the variation of pH in

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medium, as a function of heating time. In Figure 3a, the initial K2S2O8 concentration is 1.018 g/L and the molar ratio, R, of the maximum possible amount of protons released upon the decomposition and reaction K2S2O8 in water to the total carboxylate groups of sodium alginate is 0.6, which means that the maximum possible protonation degree of carboxylate groups in given sodium alginate solution is 60%. Before the decomposition of K2S2O8, the pH value of alginate aqueous solution is 8.5, and no alginate aggregate was detected by DLS. With the decomposition of K2S2O8 at 70 °C, the pH value of the solution decreases from an initial value of 8.5 to 3.4. Meanwhile, the assemblies occur and the hydrodynamic diameter of the assemblies decreases substantially from 1070 nm at reaction time of 15 min (pH ) 5.5) to 320 nm at 400 min (pH ) 3.4). It is apparent that the shrinkage in assembly size is attributed to the protonation of the carboxyl groups in alginate chains. In other words, the number of dissociated carboxylic groups in alginate chains decreases with the decomposition of K2S2O8, which makes alginate lose its hydrophilicity to some extent. Initially, sodium alginate in aqueous solution would be in an extended, random coil conformation because of plenty of negative charges in chains. When some dissociated carboxylic groups in alginate chains are gradually protonated, the occurrence of hydrophobicity segments in alginate chains and hydrogen bonds between protonated carboxylic acid groups drives the formation of alginate assemblies. With the progressive decrease of pH, the hydrophobic segments in the alginate chains increase and the hydrophilic segments decrease, resulting in the substantial decrease in the size of the alginate assemblies. It has been reported that protonating the alginates with higher content of guluronic acid moieties gives rise to alginic gels with a higher aggregate density than the alginates with lower content of guluronic acid moieties because guluronic acids are protonated and form hydrogen bonds between acid groups more easily.21 Figure 3b shows the size of alginate assemblies as a function of the decomposition time of K2S2O8 at 70 °C with the initial K2S2O8 concentration of 1.187 g/L and R of 0.7. Similar to the behavior observed in Figure 3a, as the pH value decreases from 8.5 to 3.4, the assembly size decreases from 1000 nm (at 15 min) to 320 nm (at 140 min). However, when the pH value continues to decrease to 3.2 (at 400 min), the assembly size rises from 320 to 540 nm. This can be explained as follows. With the decrease in pH, the charges at the surface of the assemblies decrease. If the electrostatic repulsions are strong enough to keep the assemblies stable, hydrophobic associations are mostly intra-assembly. If the electrostatic repulsions among different assemblies are not strong enough, some of the assemblies may move close and aggregate with each other, resulting in the apparent increase of assembly size. This is confirmed by Figure 3c, which shows the hydrodynamic diameter distribution of assemblies formed at K2S2O8 decomposition time of 140 and 400 min. Compared with a unimodal size distribution of assemblies at 140 min (pH ) 3.4), the hydrodynamic diameter distribution of assemblies at 400 min (pH ) 3.2) is a bimodal size distribution. One of the peaks occurs at 320 nm with a little broadening in the peak width and another one appears at

Cao et al. Table 1. Size of Assemblies Prepared at Different Temperature temperature (°C)

〈Dh〉 (nm)

polydispersity index (PDI)

60 70 80 90 droppinga

349 ( 17 319 ( 6 335 ( 14 380 ( 26 412 ( 29

0.27 0.19 0.23 0.30 0.31

a The assemblies prepared by direct addition of 0.1 M HCl to sodium alginate solution at room temperature. (the molar ratio R of HCl and the carboxylate groups of sodium alginate is 0.6 and the concentration of sodium alginate is 0.25% (w/w)).

about 700 nm. The second peak may be attributed to the aggregation of assemblies which causes the assembly size to increase for more than two times. Thus, it is safe to conclude that the increase in assembly size when the pH value decreases from 3.4 to 3.2 may result from the coalescence of assemblies due to the decrease of charges in the shell of assemblies. Table 1 lists the minimum hydrodynamic diameter of alginate assemblies prepared by the decomposition and reaction of K2S2O8 in water at various temperatures with R of 0.6. It can be seen that at a temperature of 70 °C the minimum assembly size is 319 nm, and the hydrodynamic diameter polydispersity index of assemblies (PDI) obtained from µ2/Γ2 is 0.19. When the temperature of the system decreases to 60 °C or increases to 80 °C, the minimum size and PDI of assemblies show a slight increase. However, when the temperature reaches 90 °C, the size of assemblies shows a significant increase, only a little smaller than that formed by direct addition of HCl to sodium alginate aqueous solution at room temperature. These phenomena suggest that the assembly formation is also related to the protonation rate of carboxylate groups in the alginate chains, which determines the formation rate of hydrophobic segments and assembly behavior of alginate molecules. For example, in the case of the addition of HCl to sodium alginate aqueous solution at room temperature, the protonation rate of carboxylate groups is fastest, and the assembly size and PDI also are largest. In addition, the morphology of assemblies prepared by direct addition of HCl to sodium alginate solution at room temperature is comparatively irregular. Thus, it seems that, at an appropriate protonation rate of carboxylate groups or decomposition temperature, the assemblies prepared are smaller in size and more regular in shape. In addition, like many other preparation methods of nanosized polymeric assemblies, the concentration of polymer plays an important role to avoid precipitations. In our experiment, we found that the optimum concentration of sodium alginate was 0.25%. If the concentration of sodium alginate was increased from 0.25% (w/w) to 0.5% and the initial K2S2O8 concentration was 1.018 g/L, the diameter of resulting assemblies increased greatly from 320 to 754 nm after the reaction time reached 400 min at 70 °C. Moreover, these assemblies precipitated after 24 h of storage at room temperature. With further increasing the concentration of sodium alginate to 1%, only a gel of alginate was obtained after the reaction. However, when we lowered the concentration of sodium alginate to 0.1%, there were no significant

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Figure 4. Morphologies of the assemblies prepared by the decomposition of K2S2O8 in water, with R of 0.6, at 70 °C for different heating time (t). (a) TEM image, t ) 15 min, pH ) 5.5; (b) TEM image, magnification of a typical assemblies as seen in (a); (c) TEM image, t ) 80 min, pH ) 4.0; (d) TEM image, t ) 400 min, pH ) 3.4; (e) SEM image of assemblies as shown in (d).

changes in the size of resulting assemblies compared with the case of alginate concentration of 0.25%. It is also interesting that a rather large assembly diameter of around 1000 nm was obtained at a reaction time of 15 min as shown in Figure 3, panels a and b. Even if the sodium alginate was fully ionized in water, the radius of gyration of sodium alginate with weight-average molecular weight 190 kDa was found to be only 50 nm.22 This means that a single alginate chain could not possibly give an assembly with the size of 1000 nm. Therefore, it is reasonable to conclude that the observed assembly in our case is a multichain aggregate and the assembly size shrinks with the increase of protonation degree in sodium alginate chains. To investigate the morphology of the formed alginate assemblies and the assembly structure, TEM observation was carried out. Figure 4 shows the morphology of the assemblies prepared by the decomposition and reaction of K2S2O8 in water at 70 °C, with R of 0.6. When the decomposition time of K2S2O8 is 15 min (pH ) 5.5), the alginate assemblies with the core-shell micelle-like structure are observed, as shown in Figure 4a. The diameter of the assemblies is 722 ( 80 nm, and the core size and shell thickness are found to be 149 ( 37 and 322 ( 37 nm, respectively. Compared to the data measured in aqueous solution by DLS, the assembly size characterized by TEM is a little smaller due to the dry state in TEM observation. At a higher magnification image shown in Figure 4b, the core and shell of the alginate assemblies is clearly seen. It is notable that such giant core-

shell micelles-like assemblies are seldom observed in common polymeric micelle system. Based on the ζ potential of such assemblies (-54.88 ( 4.46 mV), which is proportional to the effective charge at the surface of assemblies, and the observation by TEM, the swelling shells, shown in Figure 4, panels a and b, should be comprised of the negatively charged sodium alginate segments, whereas the cores should be comprised mainly of the alginic acid segments. Since the core is charge-neutral, it should be somewhat hydrophobic compared with the anionic shell. Furthermore, as there is strong hydrogen bond interactions in alginic acid,21,23 the hydrogen bonds should be also present in the cores of these assemblies. Thus, with the increase of the protonation degree of alginate, the core size should increase, whereas shell thickness should decrease. This is confirmed by Figure 4c. When K2S2O8 has decomposed for 80 min (pH ) 4.0), the size of assemblies is found to be 342 ( 41 nm, much smaller than that at pH value of 5.5, whereas the core diameter of the assemblies increases to 184 ( 24 nm and the shell thickness of the assemblies decreases from 322 ( 37 nm (pH ) 5.5) to 79 ( 8.5 nm thick. The swelling of the assembly shell decreases markedly. Obviously, this can be attributed to a decrease of hydrophilic sodium alginate moieties, and an increase of alginic acid moieties in the backbone chains with the decrease of pH value. Meanwhile, the ζ potential of assemblies decreases from -54.88 ( 4.46 to -31 ( 3.62 mV. Therefore, it is safe to conclude that the core and the shell are comprised of protonated and ionized

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Table 2. Size of Cross-Linked Micelles with Different Cross-Linking Degreea cross-linking degreeb

〈Dh〉 (nm)

polydispersity index (PDI)

10% 20% 30% 40% 50% 60%

513 ( 25 413 ( 21 359 ( 18 326 ( 2 448 ( 6 560 ( 22

0.25 0.25 0.19 0.11 0.22 0.27

a The cross-linked samples are dialyzed in the water at pH 3.4 for 2 days to remove all of the impurities. b This refers to the molar ratio of amine groups in the cross-linker to carboxyl groups of sodium alginate. It reflects only the maximum possible degree of cross-linking.

alginate segments, respectively. Figure 4d shows the morphology of the assemblies as K2S2O8 has decomposed for 400 min (pH ) 3.4). At this stage, about 60% of the carboxyl groups in the sodium alginate chain are protonated since K2S2O8 in the solution is totally decomposed, which can be proved by the horizontal slope at the curve of pH variation shown in Figure 3a. It can be seen that the assemblies with the diameter of 180 ( 21 nm in the dry state become more compact in shape and the swollen shell is almost disappeared. Figure 4e displays the SEM image of assemblies as seen in Figure 4d at the hydrophilic surface of glass substrate. The size of assemblies in SEM image is found to be 285 ( 48 nm, which is larger than that in the TEM image, suggesting the shrinkage of assemblies at the hydrophilic and rough surface of glass substrate is smaller than that at the hydrophobic surface of carbon in grid during the drying process of samples. After close examination, it is also found that the center region of assemblies is somewhat concave compared to the edge region of assemblies, indicating the core of alginate assemblies is comparatively soft. However, the assemblies prepared at this stage are not stable enough. They trend to further assemble and form larger supermolecular structure after storage for several days in aqueous solution. Cross-Linked Alginate Assemblies. To improve the stability of the alginate assemblies in aqueous solution, the alginate assemblies at minimum assembly size (pH ) 3.4) were cross-linked by a cross-linker, 2,2′-(ethylenedioxy)bis (ethylamine), in the presence of coupling reagent, 1-(3dimethylaminopropyl)-3-ethylcarbodiimide methiodide (EDC). Table 2 displays the size of the assemblies measured by DLS after cross-linking. Since the cross-linker, 2,2′-(ethylenedioxy)bis (ethylamine), shows the basic property, the addition of the cross-linker into alginate assemblies solution caused some increase in assembly size but had not affected the crosslinking reaction in the given system. From Table 2, it can be seen that after the cross-linking reaction the assembly size decreases initially with the increase in the degree of crosslinking and reaches a minimum value, 326 nm, at the calculated molar ratio of cross-linker to repeat units of alginate (denoted as CMR) of 40%. Progressively increasing the amount of cross-linker results in an increase in the assembly size. It is well-known that EDC is extensively used as a coupling reagent in the condensation reaction between an acid group and amine group and can react with both carboxylic acids and carboxylate groups.8,24 However, the reaction rate with carboxylate groups is faster due to the

Figure 5. (a) TEM image of the cross-linked assemblies at pH ) 3.4; (b) TEM image of the cross-linked assemblies at pH ) 7.0.

Figure 6. Mean hydrodynamic diameter of cross-linked assemblies as a function of pH.

electrostatic effect between the reacting moieties.25 Thus, in our case, the cross-linking reaction may mainly take place at the assembly shell consisting of the negatively charged sodium alginate segments. This is confirmed by the increase in ζ potential after the cross-linking reaction. As the CMR is below 40%, the cross-linking reaction happens in the intraassemblies, whereas when CMR is above 40%, the crosslinking reaction may take place not only in the intraassemblies but also inter-assemblies, resulting in the increase in the average size of assemblies. Figure 5a shows the TEM image of cross-linked alginate assemblies at pH 3.4, with the molar ratio of cross-linker to repeat units of alginate being 40%. It can be seen that the structure of the cross-linked assemblies becomes considerable compact and the swollen shell is no longer present. In addition, the shape of cross-linked assemblies is not as round as the non-cross-linked assemblies, suggesting that the crosslinking reaction may not take place homogeneously in the assemblies. An interesting feature of the cross-linked assemblies is their response to environmental pH variation. Figure 6 displays the size change of cross-linked alginate assemblies at the cross-linking degree of 40%. The size was measured by DLS after dialysis against the aqueous solution with different pH values. It can be seen that a sudden increase in

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Figure 7. Schematic representation of pH-induced formation of sodium alginate assemblies, and the transition from the compact structure to hollow structure.

the assembly diameter between pH ) 4.5 and 8.0 occurs, and the size of cross-linked assemblies expands almost 2-fold. In addition, the integration of cross-linked assemblies is still maintained upon pH ) 10.1. This monotonic increase in the size with the increase of pH value in the medium most likely arises from the swelling of assemblies and the electrostatic repulsive inside assemblies. To further investigate this issue, TEM observation was carried out. Figure 5b shows the TEM image of the cross-linked assemblies at pH ) 7.0. It is found that the compact structure of crosslinked assemblies is transformed to hollow capsule structure in the medium of pH ) 7.0. A significant contrast between the center and the shell of the capsules indicates that a cavity in the assembly center is formed, which may result from the increase in assembly size after dialysis in relatively high pH medium. On the other hand, for non-cross-linked samples, the size of assemblies also increases with the increase of pH value in medium, as shown in Figure 3a and b. However, at high pH (pH > 7), a DLS measurement indicated that there was almost no assembly in the non-cross-linked system and the disintegration of the assemblies occurred, which are agreement with the result in Figure 1. This result indicates that the cross-linking reaction of alginate assemblies is effective. In addition, it is found that the cross-linked sample does not return to the size of the original non-cross-linked sample at the same pH values by comparison of Figures 3 and 6. Except in the case of pH 3.4 where the diameters of cross-linked and non-cross-linked assemblies are almost equal (326 ( 2.3 nm for cross-linked sample and 322 ( 8.1 nm for non-cross-linked sample), in the other cases such as pH ) 3.8, 4.5, and 5.4, the diameter of cross-linked samples is much smaller than that of non-cross-linked samples. This result suggests that after cross-linking reaction the assembly swelling is much suppressed. The pH-induced variation in the structure of cross-linked assemblies can be explained as follows. As shown in Figure 4, panels c and d, the alginate assemblies prepared around pH ) 4 are comprised of the cores with large volume fraction and the shell with small volume fraction. Once such assemblies were shell-cross-linked and were dialyzed against aqueous solution at pH ) 7.0, owing to the ionization of carboxylic acid groups the hydrogen bonds between carboxylic acids in the cores will be destroyed and the cores will disintegrate. Since the shells of assemblies are crosslinked, the structure of assemblies can be maintained but the expansion of assemblies is inevitable. Thus, the cavity in the center of the assemblies occurs, and the hollow capsule structure is formed. A proposed scheme of the pH-induced self-assembly of sodium alginate and transition of cross-linked assembly to hollow capsule is shown in Figure 7. First, gradually partial

protonation of carboxylate groups in sodium alginate chains causes the self-assembly of sodium alginate chains and forms the assemblies with the core-shell structure. Subsequently, the cross-linking reaction fixes the shape of the assemblies, which makes the assemblies yield a hollow capsule structure when they are in the medium with relatively high pH value. Conclusion In this investigation, we used a kind of polyelectrolyte, sodium alginate, as a model biomaterial to investigate the aggregation behaviors in aqueous solution after partial protonation of carboxylate groups in the alginate molecules. It is demonstrated that the alginate assemblies with coreshell structure can be generated by the smooth and partial protonation of carboxylate groups in sodium alginate chains using the protons released gradually from the decomposition and reaction of K2S2O8 in water at 70 °C. The partial crosslinked alginate assemblies are pH sensitive and can change to hollow structure at the medium with relatively high pH value. This approach avoids use of block or grafted copolymers as the precursors or any other template to prepare assemblies and capsules, and provides a functional surface for subsequent chemical reaction at the surface (e.g., for binding biomolecules and for surface grafting). Such unique assemblies are also expected to be useful in biomedical fields. Acknowledgment. This work has been supported by Natural Science Foundation of China (No. 20374026). References and Notes (1) (a) Kwon, G. S.; Okano, T. AdV. Drug DeliVery ReV. 1996, 21, 107. (b) Allen, C.; Maysinger, D.; Eisenberg, A. Colloids Surf. B Biointerfaces 1999, 16, 3. (c) Rosler, A.; Vandermeulen, G. W. M.; Klok, H. A. AdV. Drug DeliVery ReV. 2001, 53, 95. (2) Webber, S. E. J. Phys. Chem. B 1998, 102, 2618. (3) (a) Thurmond, K. B.; Remsen, E. E.; Kowalewski, T.; Wooley, K. L. Nucleic Acids Res. 1999, 27, 2966. (b) Miller, A. D. Curr. Med. Chem. 2003, 10, 1195. (4) (a) Harada, A.; Kataoka, K. Macromolecules 1995, 28, 5294. (b) Harada, A.; Kataoka, K. Science 1999, 283, 65. (c) Kabanov, A. V.; Bronich, T. K.; Kabanov, V. A.; Yu, K.; Eisenberg, A. Macromolecules 1996, 29, 6797. (5) (a) Bronich, T. K.; Kabanov, A. V.; Kabanov, V. A.; Yu, K.; Eisenberg, A. Macromolecules 1997, 30, 3519. (b) Kabanov, A. V.; Bronich, T. K.; Kabanov, V. A.; Yu, K.; Eisenberg, A. J. Am. Chem. Soc. 1998, 120, 9941. (c) Bronich, T. K.; Popov, A. M.; Eisenberg, A.; Kabanov, V. A.; Kabanov, A. V. Langmuir 2000, 16, 481. (d) Thunemann, A. F.; Beyermann, J.; Kukula, H. Macromolecules 2000, 33, 5906. (6) Mckenna, B. J.; Birkedal, H.; Bartl, M. H.; Deming, T. J.; Stucky, G. D. Angew. Chem., Int. Ed. 2004, 43, 5652. (7) Forster, S.; Abetz, V.; Muller, A. H. E. AdV. Polym. Sci. 2004, 166, 173. (8) Dou, H.; Jiang, M.; Peng, H.; Chen, D.; Hong, Yan. Angew. Chem., Int. Ed. 2003, 42, 1516. (9) (a) Lee, A. S.; Gast, A. P.; Bu¨tu¨n, V.; Armes, S. P. Macromolecules 1999, 32, 4302. (b) Lobb, E. J.; Ma, I.; Billingham, N. C.; Armes, S. P.; Lewis, A. L. J. Am. Chem. Soc. 2001, 123, 7913.

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