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Protamine-Templated Biomimetic Hybrid Capsules: Efficient and Stable Carrier for Enzyme Encapsulation† Yufei Zhang, Hong Wu,* Jian Li, Lin Li, Yanjun Jiang, Yan Jiang, and Zhongyi Jiang* Key Laboratory for Green Chemical Technology of Ministry of Education, School of Chemical Engineering and Technology, Tianjin UniVersity, Tianjin 300072, People’s Republic of China ReceiVed July 22, 2007. ReVised Manuscript ReceiVed September 30, 2007
Biosilica with a highly complex and intricate structure can be formed by a diatom under physiological conditions in the presence of silaffins. Herein, the biosilicification process in vivo is mimicked. A natural cationic polypeptide, protamine, was for the first time utilized in vitro to inspire and template silica formation at ambient temperature and neutral pH conditions. The silica-precipitating and templating effect of protamine was first tentatively elucidated. Then, this biomimetic silicification process was performed on the outer surface of liquid-core alginate (Alg) capsules in which β-glucuronidase (GUS) was preencapsulated. An alginate/protamine/silica (APSi) hybrid capsule with a distinct liquid core-solid shell structure was thus fabricated. The rigid, mesoporous silica shell dramatically inhibited the swelling of the capsule and effectively enhanced the mass transfer of substrates and products. Meanwhile, the biocompatible polysaccharide liquid core created a benign microenvironment and well preserved the three-dimensional structure of GUS. The stability of encapsulated GUS was significantly enhanced after silicification, and no loss in activity was found after 10 reaction cycles. Moreover, the relative activity of GUS encapsulated in APSi capsules reached 125%, not only exceeding that encapsulated in Alg capsules but also being higher than that of the free enzyme.
Introduction The structure-controllable materials synthesized under mild conditions are of growing interest to the materials research community and may find promising applications in biotechnologies involving enzyme encapsulation.1–4 If we turn our eyes to nature, we can find that such materials commonly exist in biological organisms formed via biomineralization.5,6 An intriguing paradigm is the intricate nanostructured biosilica, which constitutes the main component of the protective wall on a unicellular diatom. This biosilica is assembled inside specialized silica deposition vesicles in which some specific polypeptide-based biopolymers play a crucial role both in silica-precipitating and in structuredirecting procedures.7–9 The uniquely interesting feature of the biosilicification process is that the biopolymers act not only as catalysts to inspire precipitation at very low silicon †
Part of the “Templated Materials Special Issue”. * To whom correspondence should be addressed. Tel.: +86-22-27892143. Fax: +86-22-27892143 . E-mail:
[email protected] (Z.J.), wuhong2000@ gmail.com.
(1) Darder, M.; Aranda, P.; Ruiz-Hitzky, E. AdV. Mater. 2007, 19, 1309– 1319. (2) Ogasawara, W.; Shenton, W.; Davis, S. A.; Mann, S. Chem. Mater. 2000, 12, 2835–2837. (3) Lazos, D.; Franzka, S.; Ulbricht, M. Langmuir 2005, 21, 8774–8784. (4) Ye, P.; Xu, Z. K.; Che, A. F.; Wu, J.; Seta, P. Biomaterials 2005, 26, 6394–6403. (5) Reith, F.; Rogers, S. L.; Mcphail, D. C.; Webb, D. Science 2006, 313, 233–236. (6) Lian, B.; Hu, Q. N.; Chen, J.; Ji, J. F.; Teng, H. H. Geochim. Cosmochim. Acta 2006, 70, 5522–5535. (7) Sumper, M.; Brunner, E. AdV. Funct. Mater. 2006, 16, 17–26. (8) Kröger, N.; Deutzmann, R.; Sumper, M. J. Biol. Chem. 2001, 276, 26066–26070. (9) Poulsen, N.; Sumper, M.; Kröger, N. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 12075–12080.
concentration but also as templates entrapped during the silicification.10,11 This dual function makes a key contribution in the facile formation of inorganic–organic hybrid biosilica with delicate architectures. Biopolymer-mediated silicification in vivo provides a useful archetype for biomimetic silicification in vitro. Silaffins, natural biopolymers isolated from diatoms, were first identified to be able to catalyze the formation of silica nanospheres in vitro within seconds at mild conditions.12 Subsequently, various chemically synthesized analogues of silaffins, including 19 amino acid R5 peptide,10,13,14 homopolymers composed of key amino acids,11,15–18 long-chain polyamines,19–22 short-chain (10) Naik, R. R.; Tomczak, M. M.; Luckarift, H. R.; Spain, J. C.; Stone, M. O. Chem. Commun. 2004, 168, 4–1685. (11) Patwardhan, S. V.; Clarson, S. J. J. Inorg. Organomet. Polym. 2003, 13, 193–203. (12) Kröger, N.; Deutzmann, R.; Sumper, M. Science 1999, 286, 1129– 1132. (13) Brott, L. L.; Naik, R. R.; Pikas, D. J.; Kirkpatrick, S. M.; Tomlin, D. W.; Whitlock, P. W.; Clarson, S. J.; Stone, M. O. Nature 2001, 413, 291–293. (14) Luckarift, H. R.; Spain, J. C.; Naik, R. R.; Stone, M. O. Nat. Biotechnol. 2004, 22, 211–213. (15) Rodriguez, F.; Glawe, D. D.; Naik, R. R.; Hallinan, K. P.; Stone, M. O. Biomacromolecules 2004, 5, 261–265. (16) Tomczak, M. M.; Glawe, D. D.; Drummy, L. F.; Lawrence, C. G.; Stone, M. O.; Perry, C. C.; Pochan, D. J.; Deming, T. J.; Naik, R. R. J. Am. Chem. Soc. 2005, 127, 12577–12582. (17) Patwardhan, S. V.; Clarson, S. J. J. Inorg. Organomet. Polym. 2003, 13, 49–53. (18) Coradin, T.; Livage, J. Colloid Surf. B 2001, 21, 329–336. (19) Patwardhan, S. V.; Clarson, S. J. Polym. Bull. 2002, 48, 367–371. (20) Patwardhan, S. V.; Taori, V. P.; Hassan, M.; Agashe, N. R.; Franklin, J. E.; Beaucage, G.; Mark, J. E.; Clarson, S. J. Eur. Polym. J. 2006, 42, 167–178. (21) Yuan, J. J.; Jin, R. H. AdV. Mater. 2005, 17, 885–888. (22) Jin, R. H.; Yuan, J. J. Chem. Mater. 2006, 18, 3390–3396.
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amines23–25 and even monoamines,26,27 have been successively employed for silica precipitation. Furthermore, exogenous proteins such as gelatin and lysozyme are also demonstrated to possess a similar ability in inducing silica precipitation.28–32 Moreover, because the above silica-precipitating polymers can pre-self-assemble into different “shapes” or exhibit different sizes in solution, they have been used as templates to direct the final silica morphology.33 By variation of the molecular weight, concentration, and assembling condition of the polymers, spherical, hexagonal, and fibrous silicas with tunable pore diameters ranging from 1.5 to 8.0 nm have been fabricated.15,16,34,35 When the main similarities of the efficient silica-precipitating and templating polypeptides or polyamines are summarized, it can be found that they are all positively charged at neutral pH33 and have a long chain11,14,36 constructing into a certain conformation in the medium for silicification. Keeping these similarities in mind, protamine is conjectured to be an ideal candidate to inspire and template silica precipitation in vitro. Protamine is a 32 amino acid globular peptide containing 20-22 cationic arginine,37 satisfying the basic requirements to precipitate silica. In addition, its relatively low price, facile purification, and easy availability from fish, avian, and mammalian sperm nuclei38 ensure protamine a promising silica-precipitating reagent in practical use. For enzyme encapsulation where a rigorous restriction for the carrier structure and the encapsulation condition is required, the advantages of the biomimetic silicification approach over the traditional sol–gel method are obvious. A variety of enzymes such as butyrylcholinesterase,14 lysozyme,31 catalase, and horseradish peroxidase10 encapsulated during silica precipitation inspired by R5 peptide or poly-L-lysine (PLL) has been well proven to retain or even enhance their bioactivity. However, it seems to be a tricky problem that enzymes encapsulated in such silica precipitates have an inherent limitation because of the difficult collection of nanosized particles for reutilization. Also, a stable and (23) Roth, K. M.; Zhou, Y.; Yang, W. J.; Morse, D. E. J. Am. Chem. Soc. 2005, 127, 325–330. (24) Belton, D.; Paine, G.; Patwardhan, S. V.; Perry, C. C. J. Mater. Chem. 2004, 14, 2231–2241. (25) Belton, D.; Patwardhan, S. V.; Perry, C. C. Chem. Commun. 2005, 347, 5–3477. (26) Delak, K. M.; Sahai, N. Chem. Mater. 2005, 17, 3221–3227. (27) Belton, D. J.; Patwardhan, S. V.; Perry, C. C. J. Mater. Chem. 2005, 15, 4629–4638. (28) Coradin, T.; Marchal, A.; Abdoul-Aribi, N.; Livage, J. Colloid Surf. B 2005, 44, 191–196. (29) Allouche, J.; Boissière, M.; Hélary, C.; Livage, J.; Coradin, T. J. Mater. Chem. 2006, 16, 3120–3125. (30) Coradin, T.; Bah, S.; Livage, J. Colloid Surf. B 2004, 35, 53–58. (31) Luckarift, H. R.; Dickerson, M. B.; Sandhage, K. H.; Spain, J. C. Small 2006, 2, 640–643. (32) Coradin, T.; Coupe, A.; Livage, J. Colloid Surf. B 2003, 29, 189– 196. (33) Patwardhan, S. V.; Clarson, S. J.; Perry, C. C. Chem. Commun. 2005, 111, 3–1121. (34) Patwardhan, S. V.; Mukherjee, N.; Steinitz-Kannan, M.; Clarson, S. J. Chem. Commun. 2003, 112, 2–1123. (35) Hawkins, K. M.; Wang, S. S.-S.; Ford, D. M.; Shantz, D. F. J. Am. Chem. Soc. 2004, 126, 9112–9119. (36) Coradin, T.; Durupthy, O.; Livage, J. Langmuir 2002, 18, 2331–2336. (37) Raukas, E.; Mikelsaar, R. H. Bioessays 1999, 21, 440–448. (38) Nakano, M.; Kasai, K.; Yoshida, K.; Tanimoto, T.; Tamaki, Y.; Tobita, T. J. Biochem. 1989, 105, 133–137.
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biocompatible liquid pool environment may be preferred to a solid pore for the enzyme because it corresponds to the usual form of enzyme existence in nature, especially for our model enzyme, β-glucuronidase (GUS). In our previous study,39 GUS was encapsulated in sodium carboxymethylcellulose (CMC) core-alginate (Alg) membrane capsules and showed a high relative activity up to 88%. In the present study, a silica shell was elaborately fabricated onto the Alg capsule membrane through a biomimetic silicification process templated by protamine in order to inhibit the swelling of the capsule and enhance the operation stability. The structure characteristic, swelling degree, and mass-transfer property of the alginate/protamine/silica (APSi) hybrid capsules were systematically studied. Additionally, the catalytic activity and recycling stability of GUS encapsulated in the APSi capsules were investigated and compared with that of the enzyme in Alg capsules and in free form. Experimental Section Chemicals and Materials. A protamine sulfate salt from salmon (P4380) and β-glucuronidase (GUS) from Escherichia coli (G7396) were purchased from Sigma Chemical Co. Sodium silicate (23% SiO2) from Tianjin Jiangtian Chemical Co. Ltd. was used as a silica precursor. Sodium alginate (SA) and sodium carboxymethylcellulose (CMC) were obtained from Tianjin Reagent Chemicals Co. Ltd. Baicalin and baicalein standards for analysis were obtained from the National Institute for the Control of Pharmaceutical and Biological Products of China. Baicalin (purity g98%) used as a substrate was purchased from Sichuan Xieli Pharmaceutical Co. Ltd. All of the other chemicals were of analytical reagent grade. Synthesis of Protamine-Silica Nanocomposites. Protamine was suspended with a concentration of 5 mg/mL in a pH 7.0, 30 mmol/L Tris-HCl buffer solution. A freshly prepared 30 mmol/L sodium silicate solution was obtained by dissolving sodium silicate in water followed by acidification to pH 7.0 with HCl. A total of 10 mL of a protamine solution was then mixed with 40 mL of a sodium silicate solution. The mixture was allowed to react for 30 min. The resultant precipitates were collected by centrifugation, rinsed twice with deionized water to remove unreacted protamine and silicate, and lyophilized to dryness. Preparation of GUS-Containing APSi Capsules. GUS-containing Alg capsules were synthesized via an extrusion process that was carried out as detailed in our previous study.39 A Tris-HCl buffer (30 mmol/L, pH 7.0) was used as a universal solvent to dissolve all of the chemicals. CMC was dissolved in a 0.10 mol/L CaCl2 solution to give a final content of 2% (w/v). GUS was then added to the CMC-CaCl2 solution to get an enzyme content of 0.1 mg/mL. A total of 2 mL of the above CMC-CaCl2-GUS solution was extruded through a 0.45 mm injection needle into 40 mL of a stirred SA solution [1.0% (w/v)]. After 30 min, the mixture was diluted with 160 mL of deionized water before filtration. A total of 10 mL of a Tris-HCl buffer was used to remove the unreacted SA from the capsule surface. The capsules were then transferred into a 0.10 mol/L CaCl2 solution to be further crosslinked for another 10 min. About 200 such Alg capsules were recovered and rinsed with a Tris-HCl buffer twice. Subsequently, the capsules were placed in contact with 35 mL of the protamine solution (2 or 5 mg/mL) for a period of time (15-90 min). After filtration, capsules were added to 70 mL of the 30 mmol/L sodium (39) Jiang, Z. Y.; Zhang, Y. F.; Li, J.; Jiang, W.; Yang, D.; Wu, H. Ind. Eng. Chem. Res. 2007, 46, 1883–1890.
Protamine-Templated Biomimetic Hybrid Capsules silicate solution (dissolved in water and acidified to pH 7.0 with HCl). The silicification reaction was allowed to proceed for 2 h. The capsules were removed, rinsed with a Tris-HCl buffer, and stored at 4 °C. Dynamic Light Scattering (DLS). The average colloidal particle size of protamine dissolved in a pH 7.0, 30 mmol/L Tris-HCl buffer solution was measured using a Brookhaven Instruments BI200SM DLS system. An argon-ion vertically polarized 532-nm laser was used as the light source. Scanning Electron Microscopy (SEM) and Energy-Dispersive Spectroscopy (EDS). The morphology of nanocomposites and capsules was observed by SEM (Philips XL30 ESEM). The microchemical analysis and element mapping were conducted by EDS (Oxford) attached to the SEM. Transmission Electron Microscopy (TEM) and Selected-Area Electron Diffraction (SAED). TEM observation was performed on a JEM-100CXµ instrument, and the crystallinity of the nanocomposites was measured by SAED attached to the TEM. Fourier Transform Infrared (FTIR). FTIR spectra of nanocomposites and the capsule shell were obtained using a Nicolet560 spectrometer. A total of 32 scans were accumulated with a resolution of 4 cm-1 for each spectrum. Thermogravimetric Analysis (TGA). TGA of nanocomposites was performed in a Perkin-Elmer TGA/DTA thermogravimetric analyzer by heating to 800 °C at a rate of 10 °C/min under a nitrogen atmosphere. 29 Si MAS NMR. Solid-state 29Si MAS NMR spectra of the nanocomposites and the capsule shell were recorded on an Infinity Plus-300 MHz spectrometer. The sample was spun at 3 kHz. X-ray Photoelectron Spectroscopy (XPS). The elemental composition of the capsule surface was analyzed by XPS in a Perkin-Elmer PHI 1600 ESCA system with a monochromatic Mg KR source and a charge neutralizer. Pore-Size Distribution. The pore-size distribution of the capsule shell was determined by nitrogen adsorption–desorption isotherm measurements performed at 77 K on a Tristar 3000 gas adsorption analyzer. The samples were degassed at 313 K for 24 h prior to measurement. Pore-size-distribution curves were calculated based on the adsorption branch of nitrogen isotherms using the Barrett-Joyner-Halenda (BJH) method. Protamine Coating Amount. The amount of protamine coated onto the Ca-Alg membrane was determined by measuring the amount of protamine left in the coating solution, using a UV spectrophotometer (Hitachi U-2800) at λmax ) 225 nm. The calibration curve was plotted at a protamine concentration ranging from 15 to 500 µg/mL. Swelling Property. Fresh capsules with a weight of Wf were immersed into a pH 7.0, 30 mmol/L Tris-HCl buffer at 37 °C. The weight of the capsules was monitored until a constant value was reached (Ws). The swelling degree (Sw) was calculated as follows:
Sw (%) ) (Ws - Wf) ⁄ Wf × 100% Mass-Transfer Property. The mass transfer of the substrate, baicalin, from the solution to the capsules was investigated. Capsules were immersed in 20 mL of a well-stirred pH 7.0, 30 mmol/L Tris-HCl buffer solution containing 0.09 mmol/L baicalin. The measurement was performed at 37 °C. At designed time intervals, the baicalin concentration in the solution was determined by a high-performance liquid chromatograph (HPLC; HP1100, Agilent). The mass transfer was expressed by the following equation:40,41 (40) Coradin, T.; Livage, J. Mater. Sci. Eng. C 2005, 25, 201–205. (41) Coradin, T.; Mercey, E.; Lisnard, L.; Livage, J. Chem. Commun. 2001, 2496–2497.
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mass transfer (%) ) (C0 - Ct) ⁄ C0 × 100% where C0 and Ct are the baicalin concentrations in the buffer solution at time ) 0 and t, respectively. Circular Dichroism (CD). The secondary structure of GUS released from the capsule was determined using a Jasco J715 CD spectrophotometer in the far-UV region (195-260 nm) equipped with a 1.0 cm quartz cell and compared with that in free form. GUS was released into a pH 7.0, 5 mmol/L Tris-HCl buffer solution after 24 h of incubation in APSi capsules by cutting. Each spectrum was obtained by averaging six scans. Encapsulation Efficiency. Capsules were disrupted by cutting to release the GUS inside,42 and the encapsulation efficiency was determined by the following equation:
encapsulation efficiency (%) ) [GUS]capsule ⁄ [GUS]droplet × 100% where [GUS]capsule and [GUS]droplet are the concentrations of GUS in the final capsule and in the original CMC-CaCl2-GUS liquid droplet, respectively. The GUS concentration was determined by the micro-Bradford method using a UV spectrophotometer (Hitachi U-2800).39 Enzyme Activity and Recycling Stability of Encapsulated GUS. The activity of encapsulated GUS was evaluated by the bioconversion reaction of baicalin and calculated based on the amounts of baicalein produced. GUS-containing capsules were introduced into a beaker containing 20 mL of 0.09 mmol/L baicalin and 0.1% (w/v) Na2SO3, both dissolved in a Tris-HCl buffer (30 mmol/L, pH 7.0). Na2SO3 was used here as an antioxidant. The beaker was tightly sealed, and the reaction was performed at 37 °C under stirring. At different time intervals, the amount of baicalein in the reacting solution was measured by a HPLC (HP1100, Agilent).39 The relative activity of encapsulated GUS was represented by a ratio of the encapsulated enzyme activity to its free-form activity under identical reaction conditions. GUS-containing capsules were filtered after each reaction batch, thoroughly rinsed with a Tris-HCl buffer, and utilized in the next reaction cycle. The recycling stability of encapsulated GUS was evaluated by measuring the enzyme activity in each successive reaction cycle.
recycling efficiency (%) ) enzyme activity in the nth cycle × 100% enzyme activity in the 1st cycle Results and Discussion Biomimetic Silicification in the Presence of Protamine. To mimic the usual form of soluble silicon in nature and the unsaturated solution of silicic acid in the living environments of diatoms, a dilute solution of sodium silicate (30 mmol/L) was used as the silica precursor.33,43 No precipitate was observed in this dilute silicate solution even after being left standing for 24 h at neutral pH and room temperature. The addition of Alg to the above sodium silicate solution made no difference; still no precipitation occurred. When Alg capsules were put into the sodium silicate solution, the Ca-Alg membranes were thoroughly liquefied within 30 min. (42) Blandino, A.; Maciìas, M.; Cantero, D. Enzyme Microb. Technol. 2000, 27, 319–324. (43) Eglin, D.; Shafran, K. L.; Livage, J.; Coradin, T.; Perry, C. C. J. Mater. Chem. 2006, 16, 4220–4230.
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Zhang et al. Scheme 1. Schematic Representation of the Formation Process of the APSi Capsule: (a) Alg Capsule; (b) Alg/ Protamine Capsule; (c) APSi Capsule
Figure 1. (a) SEM photograph, (b) TEM photograph and SAED pattern, (c) FTIR spectrum, and (d) 29Si NMR of the protamine-silica nanocomposites.
At neutral pH, the electrostatic repulsive interaction between the negatively charged Alg and the also negatively charged silicate prohibited the occurrence of silica precipitation.44 In addition, the replacement of all of the Ca2+ ions by Na+ ions further liquefied the membrane of Alg capsules. Because Alg itself alone was proved to be unable to precipitate silica from a silicate solution, a cationic chemical with a certain structure is required to facilitate and template the formation of a desired silica shell on the Ca-Alg membrane. For this purpose, a new silica-templating reagent, protamine, was explored. Protamine is a peptide extracted from sperm nuclei and composed of 32 amino acids, 20-22 of which are arginines. Because of the high content of cationic arginines, protamine exhibits a high isoelectric point between 10 and 12, thus being positively charged at neutral pH. Its positive charge at pH 7.0 and long chain satisfy the two main characteristics for being a possible silica-precipitating reagent. As a matter of fact, precipitation occurred immediately upon the addition of protamine into the silicate solution. The morphology and particle size of the resulting silica precipitates were observed by SEM and TEM (Figure 1a,b). This analysis revealed silica aggregates composed of primary nanosilica particles of ca. 25 nm. The SAED analysis confirmed the amorphous nature of the silica precipitates (Figure 1b, inset). The FTIR spectrum of the precipitates (Figure 1c) showed not only typical peaks of silica (Si-O-Si and Si-OH) but also peaks of protamine (N-H and amide), indicating the entrapment of protamine within the silica matrix. The entrapped protamine was ca. 30 wt % of the total weight of precipitates according to the TGA (data not shown). The chemical structure of the silica at the atomic scale was studied by 29Si NMR, as shown in Figure 1d, helping in the analysis of the degree of silicate condensation catalyzed by protamine. The three peaks at -91.5, -100.6, and -110.3 ppm were attributed to Q2 [Si(OSi)2(OH)2], Q3 (44) Coradin, T.; Nassif, N.; Livage, J. Appl. Microbiol. Biotechnol. 2003, 61, 429–434.
[Si(OSi)3(OH)], and Q4 [Si(OSi)4] with relative percentages of 8%, 36%, and 56%, respectively. The high content of Q3 and Q4 adding up to 92% indicated a fairly high proportion of condensed silanols, revealing that protamine helped to form well-condensed silica networks. Formation of APSi Capsules. The formation process of APSi capsules is illustrated in Scheme 1. Spherical-shaped Alg capsules obtained by a common extrusion technique display a distinct core-membrane structure, and, more specifically, the liquid CMC core with GUS suspended inside is surrounded by a macroporous Ca-Alg membrane (Scheme 1a and ref 39). Alg is a linear polymer consisting of D-mannuronate (M block) and L-guluronate (G block). Although Ca2+ ions have been proven to be bound to most -COO– of G blocks,45 the -COOH groups of M and MG blocks still remain in a free state. Therefore, the capsules are negatively charged when immersed into the positively charged protamine solution at pH 7.0. The guanidyl groups of protamine are prone to be attracted by free carboxyl groups of Alg through electrostatic attractive interaction, thus realizing a facile deposition of protamine on the Alg capsule surface and creating a positively charged layer. Meanwhile, the smallsized protamine with a molecular weight of less than 10 000 Da46 may easily diffuse into the Ca-Alg membrane, causing cross-linking with Alg and replacement of Ca2+ ions (Scheme 1b).47 When these freshly formed Alg/protamine capsules are kept in contact with the sodium silicate solution, the free cationic guanidyl groups of protamine attract the silicate anions in the solution via electrostatic interactions, leading to an increased silicate concentration around the protamine molecules, inspiring and templating the silicification process. A silica outer shell is thus formed as the silicate condensation proceeds. In addition, it should be noted that, at the same time as the silicification, the Na+ ions in the solution go into the shell, further loosening or even liquefying (45) Morch, Y. A.; Donati, I.; Strand, B. L.; Skjak-Break, G. Biomacromolecules 2006, 7, 1471–1480. (46) Arellano, A.; Canales, M.; Jullian, C.; Brunet, J. E. Biochem. Biophys. Res. Commun. 1988, 150, 633–639. (47) Torre, M. L.; Faustini, M.; Norberti, R.; Stacchezzini, S.; Maggi, L.; Maffeo, G.; Conte, U.; Vigo, D. J. Controlled Release 2002, 85, 83– 89.
Protamine-Templated Biomimetic Hybrid Capsules
Figure 2. SEM photographs of capsules: (a) the surface of the Alg capsule (inset is the whole Alg capsule; scale bar 1 mm; (b) the surface of the APSi capsule (inset is the whole APSi capsule; scale bar 1 mm); (c) the surface of the Alg/protamine capsule; (d) cross section of the Alg capsule; (e) cross section of the APSi capsule (5 mg/mL protamine; coating time of 60 min).
the residual Ca-Alg membrane by replacing the Ca2+ ions,44,48 thus leaving the outer Alg/protamine and protamine/ silica shell intact (Scheme 1c). The precipitating and templating functions of protamine are tentatively discussed as follows. Precipitating Function of Protamine. The transparent and colorless Alg capsules became opaque when contacted with protamine and then became white when further immersed into a silicate solution. Freshly prepared Alg and APSi capsules showed no obvious difference in the size scale (ca. 3 mm), but after being freeze-dried, the Alg capsule shrank remarkably by 30% and its surface exhibited a typical Alg network structure (Figure 2a and inset). In comparison, the APSi capsule shrank only slightly and its surface was quite uniform (Figure 2b and inset). High contents of silicon and oxygen were detected on the APSi capsule surface (Figure 3a). Moreover, the FTIR spectrum of the APSi capsule shell was identical with that of protamine-silica nanocomposites except for the appearance of a typical peak of Alg (COO-) (Figure 3b). The 29Si NMR spectrum indicated relative percentages of 9%, 37%, and 54% for Q2, Q3, and Q4 (Figure 3c), respectively, confirming a similar chemical structure of silica on the capsule shell and in nanocomposites. All of this evidence supported the efficient precipitating function of protamine for silicification. At pH 7.0, the protamine molecules (pI ) 10-12) coated on the Alg capsule surface generate a positively charged layer (48) Darrabie, M. D.; Kendall, W. F.; Opara, E. C. Biomaterials 2005, 26, 6846–6852.
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that can adsorb and concentrate negatively charged silicates28,49 via electrostatic attractive interactions.15,16 With the increase of the silicate concentration on the capsule surface, the silicic acid polycondensation reaction of second order is remarkably accelerated.50 At the same time, hydrogen bonding between Si-O- of silicate and dNH2+ of protamine inspires the nucleophilic substitution of a Si-O- oxygen atom on another adjacent silicon atom, making some additional contribution to the acceleration of polycondensation.12,23,50,51 Templating Function of Protamine. When the morphology of the silica precipitates in solution (Figure 1a) and the silica shell on the APSi capsule (Figure 2b) are compared, it is easy to find the obvious difference between them. Silicification inspired by protamine distributed in a buffer solution led to aggregated protamine-silica precipitates consisting of amorphous nanoparticles, whereas silicification inspired by protamine precoated on capsules led to a fairly continuous and uniform hybrid shell. This difference strongly implied that silica morphology could be templated by protamine existing in different environments. In addition, the templating function of protamine seems quite different from that of the widely used PLL, where silica spheres obtained with the aid of 20 amino acid PLL exhibited a much larger size of ca. 500 nm16 under approximately the same silicate concentration. The preorganization of protamine molecules is supposed to be a determining factor for the final silica morphology and size. A protamine molecule adopts a globular conformation in a Tris-HCl solution.37,46 The folding of the polypeptide chain determined by β turns drives the amino end groups and carboxyl end groups to remain close together, exposing the hydrophilic terminal domains of the chain facing the solvent and burying the hydrophobic middle domain of the chain into the peptide interior.46 The average DLS diameter of protamine dissolved in a 30 mmol/L Tris-HCl buffer solution is about 3.5 nm (data not shown), which is in good agreement with the diameter of an individual hydrated protamine molecule (2-4 nm),37,46 indicating that protamine remains individual in a Tris-HCl buffer solution. No aggregation occurs in the presence of multivalent sulfate associated with the commercially available protamine and monovalent chloride introduced by a Tris-HCl buffer. Therefore, each protamine molecule can be regarded as a globular nucleating center to inspire silica formation. As the condensation proceeds, the silica particle grows up and aggregates with other adjacent particles through condensation of their surface silanol groups, ultimately resulting in protamine-silica composites with a diameter of ca. 25 nm. In contrast, when Alg capsules were introduced into a protamine solution, the Ca-Alg membrane serves as an interface for the spontaneous organization of globular protamine molecules, generating a protamine shell with high density and homogeneity (Figure 2c). During the subsequent silicification process, silica uniformly grows on the Alg/ protamine shell and fills up the interstitial spaces among (49) Coradin, T.; Roux, C.; Livage, J. J. Mater. Chem. 2002, 12, 1242– 1244. (50) Coradin, T.; Lopez, P. J. ChemBiolChem 2003, 4, 251–259. (51) Iler, R. K. J. Phys. Chem. 1952, 56, 673–677.
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Figure 3. (a) EDS, (b) FTIR spectrum, (c) 29Si NMR, and (d) nitrogen adsorption–desorption isotherms (the inset is the pore-size-distribution curve by the BJH method) of the APSi capsule shell (5 mg/mL protamine; coating time of 60 min).
Figure 4. (a) XPS spectra of the capsule shell: (1 and 2) inner and outer surfaces of the Alg/protamine capsule shell; (3 and 4) inner and outer surfaces of the APSi capsule shell. (b) High-resolution Ca spectra of the capsule: (i and ii) inner and outer surfaces of the Alg/protamine capsule shell; (iii and iv) outer and inner surfaces of the Alg capsule membrane (5 mg/mL protamine; coating time of 60 min).
protamine molecules, resulting in the continuous and uniform protamine/silica hybrid layer. This observation is consistent with those previously published oxide coating processes templated by different polymers and conducted on diverse surfaces. The formation of either an oxide thin film33,52,53 or a patterned oxide nanostructure13,54–56 is totally determined by the arrangement and coverage of templates. Only the templates with high surface density lead to the growth of a continuous and uniform film. (52) Pogula, S. D.; Patwardhan, S. V.; Perry, C. C.; Gillespie, J. W.; Yarlagadda, S.; Kiick, K. L. Langmuir 2007, 23, 6677–6683. (53) Kim, D. J.; Lee, K. B.; Chi, Y. S.; Kim, W. J.; Paik, H. J.; Choi, I. S. Langmuir 2004, 20, 7904–7906. (54) Coffman, E. A.; Melechko, A. V.; Allison, D. P.; Simpson, M. L.; Doktycz, M. J. Langmuir 2004, 20, 8431–8436. (55) Glawe, D. D.; Rodríguez, F.; Stone, M. O.; Naik, R. R. Langmuir 2005, 21, 717–720. (56) Tahir, M. N.; Théato, P.; Müller, W. E. G.; Schröder, H. C.; Janshoff, A.; Zhang, J.; Huth, J.; Tremel, W. Chem. Commun. 2004, 2848– 2849.
Hierarchical Structure of Hybrid Capsules. The composition and structure change of the shell during the hybrid capsule-forming process was analyzed by XPS and SEM. After protamine deposition, a majority of the N element was detected on the outer surface of the shell (Figure 4a, line 2), suggesting that most of the protamine molecules resided therein. The minor amount of the N element existed on the inner surface (Figure 4a, line 1) was caused by the diffusion of protamine inward. Only a trace of the Ca element was detected on the inner and outer surfaces (Figure 4b, lines i and ii), indicating that most of the Ca2+ ions in the Ca-Alg membrane (Figure 4b, lines iii and iv) had been replaced by protamine; thus, the Alg-protamine network was formed. The composition was changed again after Alg/protamine capsules were immersed in a sodium silicate solution. Concomitant with the appearance of the Si element on the outer surface, an increase in the O signal and a decrease in
Protamine-Templated Biomimetic Hybrid Capsules
Chem. Mater., Vol. 20, No. 3, 2008 1047
Figure 5. Effect of the coating time and protamine concentration on (a) the swelling degree of the APSi capsules and (b) the amount of protamine coated on the Alg capsules.
the N signal were found (Figure 4a, line 4). These results indicated that protamine was indeed encapsulated during silica precipitation, and the protamine/silica hybrid shell is clearly identified in Figure 2e. At the same time, the residual Ca-Alg membrane was further liquefied because of the replacement of Ca2+ ions by Na+ ions. As this replacement went on, some of the Alg redispersed into the core. However, in the region close to the outer surface where the protamine concentration was relatively higher, the Alg network crosslinked by protamine was maintained and exposed as a new inner surface. It was thus easy to understand that the shell thickness was reduced from 180 (Figure 2d) to 15 µm (Figure 2e) after the capsules were successively treated with protamine and sodium silicate solutions, and a two-layered shell composed of a Alg/protamine layer and a protamine/silica layer was finally constructed (Figure 2e). These results are in good agreement with the conjectured capsule formation process shown in Scheme 1. Swelling Property of the APSi Capsules. Swelling behavior is a characteristic property for evaluating the stability and lifetime of hydrogels. In this section, the effect of the protamine concentration and coating time on the swelling property of APSi capsules was studied. The swelling degree of Alg capsules was 114%. After the capsule was kept in contact with a 2 mg/mL protamine solution for 15 min, its swelling degree was considerably reduced to 93%. By an increase in the protamine concentration and/or a prolonging of the coating time, the capsule swelling could be further substantially suppressed. As shown in Figure 5a, the swelling of APSi capsules was completely inhibited when 5 mg/mL protamine and a coating time of 60 min were utilized. Increasing the protamine concentration and coating time could facilitate the association of protamine with Alg capsules, thus leading to an increase in the final total amount of coated protamine (Figure 5b). In addition, the density and homogeneity of protamine on the capsule surface along with the ability of capsules to precipitate silica were all increased (Table 1). DLS measurements had indicated that changes in the protamine concentration had no appreciable effect on the average colloidal particle size of protamine dissolved in a buffer solution (data not shown); therefore, variation in the protamine coating conditions exerted no pronounced influence on the pore size of the mesoporous silica shell. Average pore diameters of about 4 nm for the protamine/silica layer and 14-17 nm for the Alg/protamine layer were acquired
Table 1. Effect of Coating Conditions on the Atomic Percent of Silicon Precipitating on the Capsule Surface protamine concentration, coating time
protamine coated (mg)
Si atomic percent (%)
2 mg/mL, 60 min 5 mg/mL, 30 min 5 mg/mL, 60 min
28.2 45.3 68.1
9.2 17.8 22.8
Table 2. Encapsulation Efficiency and Relative Activity of Free and Encapsulated GUS encapsulated GUS encapsulation efficiency (%) relative activity (%)
free
Alg capsules
APSi capsules
100
77 88
69 125
(Figure 3d). In fact, the protamine/silica layer grew intact gradually with an increase of the protamine concentration and coating time. Therefore, under optimum conditions (5 mg/mL protamine; coating time of 60 min), the Alg capsules were entirely covered by the hybrid silica shell so that the swelling degree could be decreased as low as zero, and this is just the case in our study. Therefore, in the next section, only GUS-containing APSi capsules prepared under these optimum conditions were employed in the assessment of the catalytic activity and recycling stability. Catalytic Activity and Recycling Stability of Encapsulated GUS. GUS was initially encapsulated in the Alg capsules with a high encapsulation efficiency of 77%; about 8% of the GUS leaked out of the capsules during the subsequent 3 h silicification process (Table 2). CD spectra of GUS before encapsulation and after release from the APSi capsules overlapped almost entirely (Figure 6), indicating that the native GUS conformation was well preserved. This identity proved the favorable effect of the benign microenvironment provided by the CMC liquid core and the mild conditions associated with the silicification process. Furthermore, it was worth mentioning that the formation of the hybrid silica shell clearly speeded up the mass-transfer process (Figure 7). This acceleration might be attributed to the considerable decrease in the shell thickness (from 180 to 15 µm) that shortened the diffusion path for the substrate baicalin. At the same time, the transition of the pore diameter from macropore to mesopore would not affect the free movement of the substrate and product with a molecular weight of less than 500 Da. Moreover, the equilibrium partition coefficient of baicalin increased markedly from 0.30
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Figure 6. CD spectra of GUS dissolved in a pH 7.0, 5 mmol/L Tris-HCl buffer solution (black line) and released to a pH 7.0, 5 mmol/L Tris-HCl buffer solution after encapsulation in APSi capsules (red line).
Zhang et al.
Figure 8. Recycling stability of GUS encapsulated in (a) Alg capsules and (b) APSi capsules.
strength. The loss of GUS activity after 10 cycles was caused by the breaking of the capsules after multiple treatments under continuous stirring. Conclusions
Figure 7. Evolution of the mass transfer of baicalin with time in (a) Alg capsules and (b) APSi capsules.
to 0.61 on account of the favorable adsorption tendency of the silica shell. Because of the entirely preserved GUS conformation and the enhanced baicalin mass transfer, the relative activity of encapsulated GUS was substantially increased from 88% to 125% after silicification. It was also found that the relative activity of GUS encapsulated in APSi capsules even exceeded that of free GUS (Table 2) owing to the more biocompatible microenvironment and the increase of the local baicalin concentration within the liquid core of the capsules. No appreciable loss in activity was observed for GUS encapsulated in the APSi capsules after 10 repeated cycles, whereas the GUS encapsulated in the Alg capsules lost 42% of its initial activity after five cycles (Figure 8). The recycling stability was significantly enhanced by the biomimetic silicification on the capsule surface because of the notable decrease in the swelling degree and the enhanced mechanical
In this study, protamine was for the first time demonstrated to be capable of inspiring and templating the biomimetic silicification process efficiently. Protamine-templated silica deposited on the surface of GUS-containing Alg capsules resulted in the formation of APSi hybrid capsules with a GUS-CMC-Alg core and a two-layered shell involving an inner Alg/protamine layer and an outer protamine/silica layer. Under appropriate protamine-coating conditions, a protaminetemplated silica shell would grow intact and, accordingly, the swelling of the hybrid capsule was drastically inhibited. Consequently, the recycling time of GUS encapsulated in APSi capsules was enhanced from 4 to 10 times with no loss in enzyme activity compared with GUS encapsulated in Alg capsules. Moreover, the moderate mass-transfer and adsorption properties of the hybrid silica shell helped to increase the relative activity of GUS to 125%, which was not only significantly higher than that encapsulated in Alg capsules but also considerably higher than free GUS in a buffer solution. Acknowledgment. The authors are thankful for financial support from the Natural Science Foundation of Tianjin (Grant 06YFJMJC10600), the Cross-Century Talent Raising Program of Ministry of Education of China, the program for Changjiang Scholars and Innovative Research Team in University (Grant PCSIRT), the National Science Foundation of China (Grant 20576096), and the Program of Introducing Talents of Discipline to Universities (Grant B06006). CM701959E
