Biodegradable Interpolyelectrolyte Complexes Based on Methoxy

Biomacromolecules 2008, 9, 2653–2661

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Biodegradable Interpolyelectrolyte Complexes Based on Methoxy Poly(ethylene glycol)-b-poly(r,L-glutamic acid) and Chitosan Kun Luo,† Jingbo Yin,*,† Zhijiang Song,† Lei Cui,*,‡ Bin Cao,† and Xuesi Chen*,§ Department of polymer materials, Shanghai University, 20 Chengzhong Street, Jiading, Shanghai, China, National Tissue Engineering Center of China, Shanghai, 20 QinZhou Street, China, and State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun, China Received July 11, 2008

We synthesized methoxy poly(ethylene glycol)-b-poly(R,L-glutamic acid) (mPEGGA) diblock copolymer by ringopening polymerization of N-carboxy anhydride of γ-benzyl-L-glutamate (NCA) using amino-terminated methoxy polyethylene glycol (mPEG) as macroinitiator. Polyelectrolyte complexation between mPEGGA as neutral-blockpolyanion and chitosan (CS) as polycation has been scrutinized in aqueous solution as well as in the solid state. Water-soluble polyelectrolyte complexes (PEC) can be formed only under nonstoichiometric condition while phase separation is observed when approaching 1:1 molar mixing ratio in spite of the existence of hydrophilic mPEG block. This is likely due to mismatch in chain length between polyanion block of the copolymer and the polycation or hydrogen bonding between the components. Hydrodynamic size of primary or soluble PEC is determined to be about 200 nm, which is larger than those reported in some literatures. The increase in polyion chain length of the copolymer leads to the increase in the hydrodynamic size of the water-soluble PEC. Formation of spherical micelles by the mPEGGA/CS complex at nonstoichiometirc condition has been confirmed by the scanning electron microscopy observation and transmission electron microscopy observations. The homopolymer CS experiences attractive interaction with both mPEGA and PGA blocks within the copolymer. Competition of hydrogen bonding and electrostatic force in the system or hydrophilic mPEG segments weakens the electrostatic interaction between the oppositely charged polyions. The existence of hydrogen bonding restrains the mobility of mPEG chains of the copolymer and completely prohibits crystallization of mPEG segments. In vitro culture of human fibroblasts indicates that mPEGGA/CS-based materials have potential in biomedical application, especially in tissue engineering.

Introduction Polyelectrolyte complexes (PEC) can be formed by the interaction of a polyelectrolyte with an oppositely charged polyelectrolyte in aqueous solution. If equimolar amounts of charged polymer chain units and oppositely charged ones, stoichiometric complexes are formed.1,2 Such complexes are usually insoluble in water. The mechanism and properties of polymer complexes were found to be influenced by the charge ration of anionic-to-cationic polymers, chain flexibility, temperature, the degree of neutralization, ionic strength, charge density on the macroions, and so on.3-9 However, using PEC as carrier systems, problems may occur due to secondary aggregation when approaching the 1:1 molar mixing ratio of anionic groups to cationic ones or addition of salt. Recently, water-soluble stoichiometric PEC have been described. This type of PEC can be formed when both the polyanion and the polycation or one of them are covalently bound to a nonionic hydrophilic block.10-15 In contrast to the complexes of homopolymers, which phase separate, the PEC of polyelectrolyteneutral block copolymers are water-soluble over the whole range * To whom correspondence should be addressed. E-mail: jbyin@ staff.shu.edu.cn (J. Y.); [email protected] (L. C.); [email protected] (X. C.). † Shanghai University. ‡ National Tissue Engineering Center of China. § Changchun Institute of Applied Chemistry, Chinese Academy of Sciences.

of compositions of the mixture, including the electroneutral stoichiometric complexes. One of the interests in reactions occurring between oppositely charged polymers in solution lies in their similarity to biological system.1 In the field of medicine and biology, PEC based on naturally or synthetically biodegradable polyelectrolytes have attracted increasing attention in the past few decades.16-24 PEC can be prepared in various forms such as film, hydrogel, microcapsule, or sponge, which can be used as drug carrier and scaffold in tissue regeneration. Prominent examples include various kinds of complexes of polycations with DNA, with applications as nonviral gene vectors, polymer films fabricated by layer-bylayer deposition of oppositely charged polyelectrolytes, and protein-polysaccharide complexes in foods.17,22,25,26 Poly(R,L-glutamic acid) (PGA) is unique in that it is composed of naturally occurring L-glutamic acid linked together through amide bonds rather than a nondegradable C-C backbone.27 The pendent-free γ-carboxyl group in each repeating unit of L-glutamic acid is negatively charged at a neutral pH, which renders the polymer water-soluble.27 The carboxyl groups also provide functionality for drug attachment. Its biodegradability and nontoxic features make PGA a promising candidate as a biomedical carrier.27 The performance of PGA can be further improved by modifying the architectural structure and composition of the polymer while maintaining its useful character, that is, water solubility, carboxyl functionality, biocompatibility, and biodegradability.28-30 For instance, poly-

10.1021/bm800767f CCC: $40.75  2008 American Chemical Society Published on Web 08/29/2008

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ethylene glycol (PEG) can be introduced to PGA in the form of block or graft copolymers.31-35 PEG and its derivatives have been widely used as biomedical materials because of their good solubility in aqueous and organic solvent, good biocompatibility, lack of toxicity, and immunogenicity.36 Inclusion of the functional side groups of -COOH in glutamic acid can help to improve their affinity to proteins and cells or to covalently or ionically combine with drugs, antibodies, or DNAs. Chitosan, composed of poly[β-1,4-2-amino-2-deoxy-D-glucopyranose] repeating units, has a biodegradable, nontoxic, and antibacterial properties and is the second most widespread biopolymer on earth after cellulose.37 Therefore, it sounds fantastic to provide a novel biomedical material that can combine the advantages of three biodegradable polymers -PEG, PGA, and CS together. In this paper, we synthesized methoxy poly(ethylene glycol)-b-poly(R,L-glutamic acid) (mPEGGA) diblock copolymer by ring-opening polymerization. mPEGGA is a typical neutral-block-polyelectrolyte composed of nonionic mPEG segments and polyanion PGA segments in aqueous solution while CS is a typical polycation under acidic condition, which indicates that PEC can be formed between mPEGGA and CS. Copolymerization and polyelectrolyte complexation were utilized to fabricating the novel biocomposite. A primary task also the main objective of the present study is to examine the PEC formation between mPEGGA and CS in dilute solution as well as in the solid state. Moreover, the cellular uptake of porous material derived from the PEC was visualized using confocal laser scanning microscopy (CLSM).

Experimental Section Materials. Methoxy polyethylene glycol (mPEG) with Mn ) 3000 supplied by Shanghai TaiJie Chemical Co., was purified by precipitation into diethyl ether from methylene dichloride (CH2Cl2) before use. Triphosgene and p-toluene sulfonylchloride was purified by recrystallization. CH2Cl2, pyridine, tetrahydrofuran (THF), N,N-dimethylformamide, ligroin, and ethyl acetate were dried over calcium hydride and distilled before use. Benzyl alcohol, L-glutamic acid, potassiumphthalimide, dichloroacetic acid, and hydrogen bromide (HBr) in acetic acid (25%) were used when received. CS was purchased from Jinan Haidebei Marine Bioenineering Co., China. To improve the degree of deacetylation, the following treatments were applied: A solution of CS in acetic acid (5%) was added dropwise into 10% NaOH solution. After this treatment, its Mv was found to be 2.1 × 105 Da and the degree of deacetylation was calculated to be 96% from the 1H NMR spectrum. N-Hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were purchased from Sigma. Synthesis of the Neutral-block-polyanion Copolymer. Aminoterminated mPEG (mPEG-NH2) was prepared by converting terminal hydroxyl groups of mPEG to more reactive primary amino groups in following steps. First, a solution of p-toluene sulfonylchloride (pTS; 4 g) in pyridine (35 mL) was added to a solution of mPEG (15 g) in CH2Cl2. The mixture was stirred at room temperature for 24 h. mPEGpTS was obtained by precipitation into diethyl ether. Second, potassiumphthalimide (2.4 g) was added to a solution of mPEG-pTS (14 g) in DMF and the mixture was stirred at 120 °C for 5 h under a nitrogen atmosphere. mPEG-phthalimide was obtained from filtration and precipitation to diethyl ether. Third, mPEG-phthalimide (8.2 g) was dissolved in ethanol (80 mL), 4 mL of hydrazine hydrate was added, and the mixture was stirred under reflux and a nitrogen atmosphere for 12 h. mPEG-NH2 was obtained by filtration and precipitation to diethyl ether: yield, 6.4 g. N-Carboxy anhydride of γ-benzyl-L-glutamate (NCA) was synthesized in THF in the presence of triphosgene by applying standard

Luo et al. procedures.32,37 The copolymer mPEG-b-poly(γ-benzyl-L-glutamate) (mPEGBG) was obtained by ring-opening polymerization of NCA in the presence of mPEG-NH2 as macroinitiator. In a typical procedure,35 to the solution of BLG-NCA in water-free chloroform under a nitrogen atmosphere, a designed amount of mPEG-NH2 in CH2Cl2 was added. The solution was stirred for 3 days at ambient temperature. mPEGBG was isolated by precipitation in ethanol and dried under vacuum. The γ-benzyl protection groups were removed in the presence of HBr.38,39 A total of 4 mL of HBr in acetic acid (25%) was added to a solution of mPEGBG (4.0 g) in dichloroacetic acid (40 mL) while stirring. After the reaction mixture was stirred at 35 °C for 1.5 h, the resultant mPEG-b-poly(R,L-glutamic acid) (mPEGGA) was isolated by precipitation from ether and centrifugation. The mPEGGA was dried under vacuum: yield, 2.1 g. Preparation of PEC in Dilute Solution. PEC in dilute solution was prepared according to the typical method with slight modification.6 The stock solutions of mPEGGA and CS are of concentration 0.5 mg/mL. To prepare the PEC, the solution of mPEGGA was added dropwise to the solution of CS. The final pH value of the mixture was adjusted by the addition of small amounts of 1 M NaOH or HCl, which was controlled with a pH meter (PHS-3TC, Shanghai Tianda Instrument Co., China). The mixture was stirred for 24 h at room temperature to ensure full complexation between mPEGGA and CS. Separation of Solid PEC from Solution. The solid complex aggregates were prepared by centrifugation method. The concentration of mPEGGA and CS solutions was 10 mg/mL. The mPEGGA solution (50 mL) was added dropwise into the CS solution (20 mL) under moderate stirring, and then the mixture was stirred for 24 h to ensure full complexation. The pH value of the resultant mixture was adjusted to 4.90. The formed PEC were separated and isolated as a solid at 4 °C using a laboratory centrifuge (3K30, Sigma) and dried under vacuum. Measurement. 1H NMR spectrum was measured by an AV-400 NMR spectrometer at room temperature, with CF3COOD as solvent and TMS as internal reference. Infrared spectra were obtained on a Nicolet Model 380 Fourier transform infrared spectrometer. The wavenumber range scanned was 4000-500 cm-1. A total of 64 scans at a resolution of 2 cm-1 were signal averaged. Samples were prepared by grinding with KBr and compressing the mixture to form disks. Turbidity was measured at 420 nm with a UV-vis spectrophotometer (Agilent 8453, U.S.A.) to monitor polycomplexes formation in solution at 25 °C as a function of mass ratio, pH value, and polyanion chain length. Viscometry of mPEGGA/CS systems obtained, as described above, was carried out using an Ubbelohde viscometer at 25 ( 0.05 °C. Quasi elastic light scattering measurements were performed at a scattering angle of 90° with a commercially available Zetasizer 3000 (Malvern Instruments Ltd., U.K.) equipped with a 10 mW He/Ne laser as light sources. The operating wavelength was 633 nm. Analysis of the autocorrelation function g(2) (t) was done automatically to yield the mean diffusion coefficient DT and, thence, from the Stokes/Einstein expression, the apparent mean hydrodynamic particle diameter, Dh. ζ-Potential measurements were performed at 25 °C using a Malvern 3000 ζ-Sizer instrument. Scanning electron microscopy (SEM) was performed on an electron microscope (JXA-840, JEOL, Japan). The sample for SEM observation was prepared by first dropping about 0.06 mL of the water-soluble PEC solution on a clean glass slide and then volatilizing the solution at room temperature and finally sputtering a thin gold layer. Transmission electron microscopy observation was performed on an electron microscope (JEM-200CX, JEOL, Japan) at an accelerating voltage of 200 kV. The sample were prepared by placing about 6 µL micelle solution on copper grid and dried under heating. The differential scanning calorimetry (DSC; Perkin-Elmer DSC-7) measurements were performed over the temperature ranges of -50∼150 °C at a heating rate of 10 °C/min under a nitrogen atmosphere. The results of the second scan are reported. The weight of samples was within 4-6 mg.

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Scheme 1. Synthesis of Diblock Copolymer mPEGGA

Cell Culture. Porous material derived from PEC was prepared for cell culture as follows.40 Briefly, NHS and EDC were added into aqueous solution of mPEGGA and the reaction was kept to proceed for 6 h. The molar ratio of carboxyl groups of mPEGGA to EDC to NHS was 1:1:1. The mixture was added into the CS solution (10 mg/ mL) under stirring. The reaction was allowed to proceed for 6 h at room temperature. After that, the reaction mixture was dialyzed first against phosphate buffer and subsequently against deionized water. The resultant porous material was obtained by frozen drying. Human fibroblasts, which were isolated from biopsy of resected foreskin were prepared following a typical method, as described by Ma et al.41 Cultured fibroblasts at passage 3 were used in this study. Porous material was sterilized by immersing in 75% ethanol for 0.5 h and washed three times in PBS, followed by washing with Dulbecco’s Modified Eagle Media (DMEM) containing 10% fetal bovine serum (culture medium). Fibroblasts at passage 3 was harvested and were prelabeled before seeding with fluorescent 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO) dye (Molecular Probes, U.S.A.) at 37 °C for 20 min following the manufacturer’s protocol. Labeled cell suspension in a volume of 50 µL was then seeded at a density of 50 × 106 cells/mL in each biomaterial. After incubation for 4 h, culture medium was added and a cell-biomaterial complex was cultured in a 5% CO2 incubator at 37 °C. After 2 weeks, fibroblasts anchoring in porous material were observed by CLSM (Leica SP50).

peaks of all intermediate products and the final product were the same as mPEG, which implied that the basic structure of mPEG did not change except for the conversion of terminal groups. The mPEGGA diblock copolymer was characterized by 1H NMR, as shown in Figure 2. Typical signals of both mPEG and PGA units were detected. The peak at 3.3 ppm is assigned to the protons of the mPEG unit, and the peak at 2.9

Figure 1. FT-IR spectra of mPEG (1), mPEG-pTS (2), mPEGphthalimide (3), mPEG-NH2 (4).

Results and Discussion Polymer Synthesis. mPEGGA’s synthetic route is outlined in Scheme 1. To quantitatively convert the end hydroxyl groups of mPEG to primary amino groups, a pathway was proposed following the sequence of reactions involving esterifization, phthalimide, and finally hydrazinolysis.42 The formation of mPEG-NH2 was confirmed by FT-IR measurement, as shown in Figure 1. For mPEG-pTS, the peak at 3300-3500 cm-1 for OH of mPEG disappeared and a characteristic peak could be observed at 815 cm-1 attributed to the δCH vibration of the benzyl. Then for mPEG-diphthalimide, a characteristic peak appeared at 1712 cm-1 (CdO) due to the presence of phthalimide. Subsequently, for the mPEG-NH2, a weak and broad peak appeared at 3400-3600 cm-1 assigned to terminal amino groups of mPEG, while the peak at 1712 cm-1 disappeared. The other

Figure 2. 1H NMR spectrum of mPEGGA.

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Figure 3. Dependence of turbidity on the content of mPEGGA in mPEGGA-CS mixture. X represents the mass content of mPEGGA in mPEGGA-CS mixture. The final pH of the mixture is 3.82.

ppm is attributed to the protons of terminal methoxy. The peaks at 1.7, 2.1, and 4.3 are characteristic of proton peaks of PGA homopolymer, which is in accordance with our previous result.43 So one can conclude that the resultant product is composed of mPEG and PGA components. Herein, two mPEGGA samples with different molecular weights were synthesized: mPEGGA (Mn ) 8160) and mPEGGA (Mn ) 18400). The PDI of two samples measured by GPC were 1.84 and 1.97, respectively. It should be pointed that only mPEGGA (Mn ) 8160) was used in following experiments except “polyanion chain length” section. Complex Formations in Dilute Aqueous Solution. Mass Content of mPEGGA in mPEGGA-CS Mixture on Polyelectrolyte Complexation. Turbidity measurement is a simple and direct indicator for complex formation. Typically, the formation of PEC is accompanied by drastic changes of the system turbidity of the solution.6,44 Figure 3 demonstrates a curve of turbidimetric titration of a solution of CS with a solution of mPEGGA. mPEGGA content in the mixture (X) was defined as following when a mPEGGA solution was added to a CS solution

X)

VmPEGGA × 100% VmPEGGA + VCS

(1)

where VmPEGGA and VCS are volume of mPEGGA and CS solutions, respectively. The concentrations of mPEGGA and CS are both 0.5 mg/mL, that is to say, X represents the mass content of mPEGGA in mPEGGA-CS mixture. On initial addition of the neutral-block-polyanion to the solution of the cationic polyelectrolyte, there is a slight tendency of increasing the turbidity with increasing X. The mixture of the solutions remains homogeneous without any macroscopic precipitation at X < 50%, which is the signature of self-assembly of the interpolymer complex formed. According to the literature, in this range, nonstoichiometric water-soluble micelles are formed, composed by a hydrophobic electroneutral PEC core surrounded a corona of mPEG segments and uncomplexed CS segments.13,45 However, with progressive addition of the anionic polyelectrolyte, a phase separation is observed when the mass ratio X exceeds a certain value, 50%, resulting in the abrupt increase in turbidity of the mixture. This observation can be explained on the basis of more efficient and more intimate ion-pairing between the polymers, accompanied by concomitant desolvation and insolubilization of the complex. The curve shows maximum at X ) 80%, which suggests stoichiometric PEC form. A dilute solution reduced viscosity study was also performed, as it is expected that the reduced viscosity of the mixture will

Figure 4. Dependence of reduced viscosity (0) and ζ-potential (b) on the content of mPEGGA in mPEGGA-CS mixture. X represents the mass content of mPEGGA in mPEGGA-CS mixture. The final pH of the mixture is 3.82.

be influenced by the polymer-polymer complexation. Generally, disruption of intermolecular bonds and formation of uncharged or weakly charged aggregates is expected to decrease the system viscosity.46 Figure 4 shows the reduced viscosities of the mixture of polymer solutions versus the mass content of mPEGGA. Over the whole range, the reduced viscosities of the mixture presented a negative deviation from unity. The negative deviation in viscosity is a result of the formation of soluble micelles and the decrease of number of polyelectrolyte molecules in continuous phase. It is seen that the minimum of viscosity and the maximum of turbidity appear at the same X point, 80%, which indicate the formation of stoichiometric PEC. The increase of of ηsp after X ) 80% is a proof for the absence of interactions between the neutral-block-polyanion in excess and PEC. This observation is typical for a system in which insoluble complexes precipitate out of solution and do not contribute to the viscosity of the mixture.15,46 Water-soluble nonstoichiometric PEC formed was further proved by ζ-potential measurements, as demonstrated in Figure 4.47 The charge of the colloidal particles formed changes from positive to electroneutral, as the polyelectrolyte mixture composition changes from a mixture in which CS is in excess, to the mixture of stoichiometric PEC and excess mPEGGA. The zero charge obviously corresponds to the charge neutralization composition or stoichiometric mixing ratio. Increasing the mPEGGA content from stoichiometric ratio, X ) 80%, has no significant effect on particle ζ-potential. Water-soluble micelles formed between the components were further examined by size measurement. The dependence of the particle size versus X is represented in Figure 5. The hydrodynamic size (Dh) is below 20 nm when the solution remains colorless and transparent. Clearly, in this case, polymer chains exist single molecules or unimers and there is practically no complex formation in the system. However, Dh reaches about 200 nm with the appearance of bluish solution at X ) 30%, which is much larger than unimers of mPEGGA and CS. The results confirm the formation of visible micellar complexes started at X ) 30%. The curve trend of the size is similar to that of the turbidity. However, it should be noted that the size of micelles varies little as a function of X increasing from 30 to 50%, which is a solid evidence for the structure of typical micelle. When X reaches 55%, drastic increase of the size indicates that the complex phase separate. Effect of pH Variation on Polyelectrolyte Complexation. The pH-dependent complex formation was examined using turbidity and size measurements in various pH values. Slight turbidity is observed at 2.5 < pH < 4.5 (Figure 6). However, there is a


Figure 5. Dependence of hydrodynamic diameter of PEC on the mPEGGA content in mPEGGA-CS mixture. X represents the mass content of mPEGGA in mPEGGA-CS mixture. The final pH of the mixture is 3.82.


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Figure 8. Influence of polyanion chain length on hydrodynamic size of water-soluble PEC. X represents the mass content of mPEGGA in mPEGGA-CS mixture. The final pH of the mixture is 3.85: (9) CS and mPEGGA (Mn ) 18400); (0) CS and mPEGGA (Mn ) 8160).

Figure 9. SEM image (a) and TEM image (b) of water-soluble PEC formed. mPEGGA content in the mixture is 35% and the final pH is 3.5.

Figure 6. Influence of pH value on turbidity of PEC solution. mPEGGA content in the mixture is 40%.

Figure 7. Influence of pH value on hydrodynamic size of PEC. mPEGGA content in the mixture is 40%.

sharp increase in the turbidity when pH is below 2.5 or above 4.5. Also, in this case, a similar shape of the curve describing the dependence of the hydrodynamic size can be seen in Figure 7. One can conclude that water-soluble micellar complexes are only formed in a limited pH range. At pH < 2.5, CS is completely protonated, while the degree of ionization of the mPEGGA is almost zero and PGA becomes a typical hydrophobic block. There is almost no complexation between the components. Also, hydrogen bonding between PGA segments with the mPEG segments may occur because of carboxyl groups along PGA can be regarded as a proton-donator when nonionized. One can suppose that both factors cause the insolubilization of mPEGGA. At 2.5 < pH < 4.5, CS and mPEGGA are both partially ionized, interpolyelectrolyte complexation takes place. Nonstoichiometric micelles are formed that consist of a hydrophobic PEC core surrounded by mPEG segments and uncom-

plexed CS segments. In this range, the micelle size varies little along with the change in the degree of ionization of the two polyelectrolytes. However, at pH > 4.5, mPEGGA is mostly ionized and complex formation is more efficient and more intimate between the polymers and the loose large size aggregates persist. Effect of Polyanion Chain Length on Water-Soluble PEC. Two mPEGGA samples with different molecular weights were synthesized in this work: mPEGGA (Mn ) 8160) and mPEGGA (Mn ) 18400). mPEG segments chain length is the same for the two samples so the increase in molecular weight of mPEGGA means the increase in the chain length of polyanion segments of the copolymer. When the polycation homopolymer was the host and a guest polyanion-neutral block copolymer was added to it, the increase of the polyanion chain length caused an increase in Dh of the primary PEC (Figure 8). This can be explained by the fact that mPEGGA with longer polyanion segments would have a lower amount of available ionized carboxyl groups per gram. Therefore, for mPEGGA with longer polyanion chain, polyelectrolyte complexation with CS would involve a higher number of mPEGGA molecules, which caused the increase in the size of PEC core and the resultant micelles. Morphology of the Nonstoichiometric Water-Soluble PEC. To further explore the structure of the water-soluble PEC, SEM, and TEM have been used to observe the morphology of the water-soluble PEC. Figure 9 displays water-soluble PEC micelles prepared by adding mPEGGA into CS dropwise at pH ) 3.4 and X ) 0.35. As illustrated in SEM image (Figure 9a), nonstoichiometric PEC formed spherical nanoparticles. The TEM image without any staining clearly shows a core-shell structure (Figure 9b). This can be explained by high contrast in density between the loose chain packing of shell and compact PEC core. Discussion for Polyelectrolyte Complexation BehaVior in Dilute Solution. In the present study, one can see that the complexation behavior of mPEGGA with CS is different from

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that of typical polyion complex or block ionomer complex. According to the literature, when a nonionic hydrophilic block is linked to one end of a polyelectrolyte, macroscopic phase separation of PEC is suppressed effectively and water-soluble stoichiometric PEC can be formed.10,12,14,48,49 In the core, the polyelectrolyte blocks and the oppositely charged species are tightly bound and form a dense coacervate microphase. The shell is made of the neutral chains and surrounds the core. Clearly, the formation of a corona of nonionic hydrophilic segments surrounding the core of water-incompatible segments is the reason for prevention of progressive aggregation of the core and for stabilization of the micelles. In the present study, CS is a host polyelectrolyte, whereas mPEGGA is a guest neutral-block-polyelectrolyte. It seems that water-soluble stoichiometric PEC can also be obtained as expected because chain length of nonionic hydrophilic segments of the copolymer is long enough. However, as described aforementioned, soluble PEC can only be obtained at nonstoichiometric conditions according to the classical guest-host theory of PEC. The nonstoichiometric micellar PEC is composed by a hydrophobic PEC core surrounded a corona of mPEG segments and uncomplexed CS segments. Macroscopic phase separation takes place instead of the formation of water-soluble stoichiometric PEC, which is similar to the complexation behavior of two homopolyelectrolytes. To explain the unexpected phenomenon, we present two possible reasons: (1) Mismatch in chain length of polyionic segments of the copolymer and the polycation. General information can be concluded from many literature sources reporting water-soluble stoichiometric PEC: The chain length of homopolymer is closed to that of polyion segments of copolymer, which means that a stoichiometric micelle involve almost equimolar amounts of polyanions and polycations. Some work has been devoted to the research in chain length recognition.11,45,50 For example, Harada and co-workers had found that molecular recognition based on length occur between oppositely charged pairs of flexible and randomly coiled block copolymers.50 Prokop and co-workers reported nanoparticulate PEC system with desirable characteristics for use in biological systems and found that PEC formulations from precursors with similar low molecular weights yielded dispersions with suitable physicochemical characteristics due to efficient ion pairing.45 In Kabanov’s work, two polymers with different molecular weights were used to participate in polyelectrolyte complexation.11 The contour length of one of the homopolymers was approximately 24 times higher than that of the polyion segments of the copolymer. By contrast, the contour length of the other hompolymer was almost 2 times smaller than that of the polyion segments of the copolymer. An obvious increase in turbidity was observed with increasing the molecular weight of the homopolymer, which means the PEC was more hydrophobic.11 However, the molecular weight of the homopolymer (CS) used in the present study was about 2.1 × 105 kDa, which was approximately 70 times higher than that of polyanion segments of the mPEGGA copolymer. Phase separation instead of formation of stoichiometric water-soluble PEC occurred between mPEGGA and CS may suggest that the similar chain length of the polyanion and polycation would favor the formation and stability of polyion complex micelles. Therefore, we hypothesize the mismatch in chain length of the polycation and ionic segments of the neutral-polyanion copolymer should be responsible for failure in the formation of water-soluble stoichiometric PEC in this work. This hypothesis can be enriched in the “hydrodynamic size measurement” described previously. The hydrodynamic size of nonstoichiometric PEC based on mPEG-

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GA and CS is determined to be above 200 nm, which is higher than that of typical micellar PEC (below 100 nm) formed by oppositely charged polymers according to many literatures.12,51,52 One can readily understand that the size of the aggregated structure is too large to fit to well-defined micelles when approaching stoichiometric molar ratio. Moreover, the influence of molecular weight or chain length of the homopolymer on the copolymer/homopolymer polyelectrolyte complexation will be scrutinized in our further study. (2) Hydrogen bonding between mPEG segments of the copolymer and CS. Strong hydrogen bonding can occur between mPEG and CS as reported by some authors,53-55 which usually causes the miscibility of the system. That means hydrogen bonding besides electrostatic forces can also exist between mPEGGA and CS, which inhibits the formation and stability of the water-soluble stoichiometric micelles. This hypothesis can be further enriched in following DSC experiment to some extent. Scheme 2 gives a schematic presentation for the unusual complexation behavior between mPEGGA and CS in dilute solution. As is clearly shown in Scheme 2, only nonstoichiometric water-soluble micelles can be formed between mPEGGA and CS. Phase separation is observed under stoichiometric conditions, which should attribute to the unmatched polyion chain lengths or hydrogen bonding between the components. Characterization of PEC in the Solid State. FT-IR Analysis. We performed FT-IR and DSC experiments to find the role of uncomplexable groups-mPEG in polyelectrolyte complexation. FT-IR spectra of mPEGGA, CS, and the complex can be seen in Figure 10. The spectrum of pure CS shows the presence of the characteristic absorption bands at 3438, 2935, 1579, 1155, 1043, and 1012 cm-1. The broadband at 3438 cm-1 can be due to the OH-stretching, which overlaps with NHstretching in the same region. The peaks at 2935 cm-1 correspond to CH-stretching. The bands at 1579 cm-1 are NHbending. The absorption bands at 1155 cm-1 (antisymmetric stretching of the CsOsC bridge), 1043 cm-1, and 1012 cm-1 (skeletal vibrations involving the CsO stretching) are characteristic of CS’s saccharide structure. The spectral features of pure CS are in agreement with our previous results.56 In the spectrum of pure mPEGGA, the absorption band at 1710 cm-1 has been assigned to CdO stretching vibration from carboxylic groups. In most published work, the band always shifted to a lower wavenumber because of electrostatic interaction and the peaks of CdO group remained independent in complexes.57,58 However, our recent work shows that the peak of CdO group disappear in the complex PGA/CS which reflect strong interaction between PGA and CS.59 In the present study, the FT-IR spectra suggest that the peak of CdO group at 1710 cm-1 still exists in the complex mPEGGA/CS but weaken compared to the pure mPEGGA. DSC Result. DSC is often used for the characterization of polymeric blends driven by hydrogen bonding.56,60 In cases for which the components form a complex through hydrogen bonds, the mobility of the polymer can be greatly restrained, and Tg of the system is not only above the weight averaged Tg, but also higher than Tg of either component.61 It appeared that the principle also applies to PEC.62 However, in some cases, Tg of PEC can not be obtained due to high density of ionic crosslinking.63 Moreover, some authors reported other DSC results.64 Typically, DSC trace in a PEC system is utilized to examine the shift of Tg reflecting the function of complexation.65,66 As reported previously, the transition in DSC trace for CS is difficult to be detected because of low sensitivity of DSC measurement.64,67 Also, Tm of CS is not easy to be found because of rigid-rod



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Scheme 2. Schematic Presentation of Complexation Behaviors of mPEGGA and CS in Aqueous Solution

polymer backbone having strong inter- and intramolecular hydrogen bonding.68 In the present study, the objective is to find out the variation of mPEG crystallinity after polyelectrolyte complexation. To eliminate the effect of moisture, we present the result in the second heating run, as shown in Figure 11. mPEG segments in mPEGGA copolymer exhibits a typical endothermic effect at 33 °C, which is lower than Tm of mPEG (55 °C) homopolymer. That is to say, mPEGGA diblock copolymer was phase separated. This result is agreement with

Figure 10. FT-IR spectra of mPEGGA, CS, and solid PEC.

Figure 11. DSC thermogram of mPEGGA, CS, and solid PEC in the second heating run.

those reported in some literature.69,70 It is likely that Tg of mPEG is too low to be detected. However, the mPEGGA/CS complex isolated from solution does not exhibit the melting effect typical for mPEG, which means the crystallinity of mPEG segments diminishes. Discussion for Polyelectrolyte Complexation in the Solid State. As shown above, electrostatic force as a driving force in a typical PEC system is not only the factor in mPEGGA-CS system. It could be concluded from FT-IR and DSC results that the homopolymer CS experiences attractive interaction with both mPEG and PGA blocks within the copolymer. Clearly, the former is hydrogen bonding while the latter is Coulombic force. Competition between the two noncovalent forces greatly affects physicochemical characterization of the PEC in the solid state, which can readily explain the FT-IR and DSC results. Electrostatic interaction between PGA block and CS is weakened due to the existence of hydrogen bonding within the system. Alternative possible reason is that mPEG block is hydrophilic which may hamper the formation of hydrophobic PEC. The formation of interassociated hydrogen bonds between the components usually induces miscibility in a blend.71 At the same time, the crystallization of the crystalline component is greatly suppressed. In the PEC based on mPEGGA and CS hydrogen bonding as an important role completely prohibit the crystallization of mPEG segments of the copolymer. In Vitro Cellular Uptake of Porous Material Derived from the PEC. Polyelectrolyte-based micelles have shown attractive potential in wound healing,19 entrapping enzyme,72 drug delivery,73,74 and other biomedical fields. However, this type of micelles are formed and stabilized, usually only when polymer concentration is relatively low or a proper amount of salt are added, which limits its broad application. On the contrary, more intensive work on application has been devoted to solid PEC with some modification.23,57,75,76 The cellular uptake abilities of PEG, PGA, or CS-based materials have been tested in some literatures.77,78 Teramura and co-workers have reported fantastic result on behavior of some PEG-based materials immobilized on a cell membrane.77 Lin and co-workers found that PEC based on poly(γ-glutamic acid) and CS could

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of human fibroblasts implies that mPEGGA/CS-based materials have potential in biomedical application especially in tissue engineering. Acknowledgment. The work has been supported by the National Natural Science Foundation of China (Project Number 50473061) and Shanghai Science and Technology Committee (Project Number 05JC14067). Dr. Weijun Yu and Ms. Yanyan Lou from Instrumental Analysis Research Centre of Shanghai University are acknowledged for their help in TEM experiment and quasi elastic light scattering measurement.

References and Notes

Figure 12. CLSM image of cells after culture with porous material derived from PEC. The green represents cell and the blue represents porous material derived from PEC.

effectively reduce the transepithelial electrical resistance of Caco-2 cell monolayers derived from human colorectal adenocarcinoma. In this section, in consideration of the fact that the PEC prepared by simple mixing would undergo hydrolysis in biological fluid, its stability was improved by the cross-linking method, as described previously. Figure 12 represents the CLSM image of the human fibroblasts cultured for 2 weeks in porous material derived from the PEC. The merged image reveals that a great number of fibroblasts were adhered to the material even though distributed unevenly, which implies that mPEGGA/CS formulation is at least a suitable biomedical candidate material especially scaffold for tissue engineering. Clearly, fabricating an outstanding porous material is a key to achieve homogeneous distribution of cells and how the components in the system mPEGGA/CS contribute to the cellular uptake is still a question, which is required to be clarified in the near future.

Conclusion Biodegradable mPEGGA diblock copolymer was synthesized by ring-opening polymerization of NCA using amino-terminated mPEG as macroinitiator. Only nonstoichiometric water-soluble micelles can be formed between mPEGGA and CS. Phase separation is observed under stoichiometric condition. Unmatched polyion chains or hydrogen bonding between the components should be attributed to the unexpected phenomenon. The increase in the polyanion chain length of the copolymer results in the increase in the size of the water-soluble PEC. The core-shell structure of the spherical micelle was proved by TEM and SEM observations. DSC and IR results indicate that the presence of uncomplexable segments-mPEG of the copolymer plays an important role in the system copolymer/homopolymer even though they do not participate in polyelectrolyte complexation. Competition of hydrogen bonding and electrostatic force in the PEC weakens the electrostatic interaction between the components. Hydrogen bonds between mPEG and CS in polyelectrolyte complexation restrain the mobility of mPEG chains and destroy its crystalline domain. In vitro culture

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