Facile Preparation of Novel Peptosomes through Complex Self

pubs.acs.org/Langmuir © 2009 American Chemical Society

Facile Preparation of Novel Peptosomes through Complex Self-Assembly of Hyperbranched Polyester and Polypeptide Bo Guo, Zengqian Shi, Yuan Yao, Yongfeng Zhou,* and Deyue Yan* School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, PR China Received April 17, 2009. Revised Manuscript Received May 7, 2009 This work reported for the first time a facile template-free method to prepare polypeptide-based vesicles (peptosomes) through one-step complex self-assembly of carboxyl-terminated hyperbranched polyester and cationic poly-L-lysine (PLL). The preparation of such peptosomes, named complex peptosomes (CPs) here, is very simple just by mixing two kinds of polymer aqueous solutions together. The CP size can be readily controlled from nanosize to microsize through the adjustment of polymer concentration. The resulting nanosized CPs show unexpected size stability independent of a broad solution pH range, long-term storage stability, and almost no cytotoxicity and have demonstrated great potential to be used as the carriers in drug delivery.

Introduction Polymer vesicles self-assembled from amphiphilic block copolymers have been widely studied for their potential applications in drug delivery, gene therapy, biosensors, nanoreactors, and model systems of biomembranes.1-16 Among them, polypeptide-based vesicles, specifically denoted as “peptosomes”,17,18 have attracted progressive interest for their great potentials in drug delivery as a result of the intrinsic biocompatibility and biodegradability of polypeptides. Many research groups have obtained peptosomes from the self-assembly of a variety of polypeptide-based block copolymers such as poly(styrene)-block-poly(isocyanide), poly (styrene)-block-poly(3-(isocyano-L-alanyl-amino-ethyl)- thiophene), *E-mail: [email protected], [email protected]. (1) Zhang, L.; Eisenberg, A. Science 1995, 268, 1728. (2) Zhang, L.; Eisenberg, A. Science 1996, 272, 1777. (3) Soo, P. L.; Eisenberg, A. J. Polym. Sci., Part B: Polym. Phys. 2004, 92, 423. (4) Discher, B. M.; Won, Y.-Y.; Ege, D.; Lee, J. C.-M.; Hammer, D. A. Science 1999, 284, 1143. (5) Discher, D. E.; Eisenberg, A. Science 2002, 297, 967. (6) Discher, B. M.; Hammer, D. A.; Bates, F. S. Curr. Opin. Colloid Interface Sci. 2000, 5, 125. (7) Ahmed, F.; Discher, D. E. J. Controlled Release 2004, 96, 37. (8) Antonietti, M.; Fo¨rster, S. Adv. Mater. 2003, 15, 1323. (9) Rodrguez-Hernandez, J.; Checot, F.; Gnanou, Y.; Lecommandoux, S. Prog. Polym. Sci. 2005, 30, 691. (10) Hamley, I. W. Soft Matter 2005, 1, 36. (11) Zhou, Y. F.; Yan, D. Y. Angew. Chem., Int. Ed. 2004, 43, 4896. (12) Zhou, Y. F.; Yan, D. Y. Angew. Chem., Int. Ed. 2005, 44, 3223. (13) Zhou, Y. F.; Yan, D. Y. J. Am. Chem. Soc. 2005, 127, 10468. (14) Graff, A.; Sauer, P.; van Gelder, P.; Meier, M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5604. (15) Nardin, C.; Widmer, M.; Winterhalter, M.; Meier, M. Eur. Phys. J. E 2001, 4, 403. (16) Chiu, D. T.; Wilson, C. F.; Karlson, A.; Danielson, A.; Lundqvist, A.; Stroemberg, F.; Ryttsen, M.; Davidson, F.; Nordholm, S.; Orwar, O.; Zare, R. N. Chem. Phys. 1999, 247, 133. (17) Kukula, H.; Schlaad, H.; Antonietti, M.; Fo¨rster, S. J. Am. Chem. Soc. 2002, 124, 1658. (18) Kimura, S.; Kim, D.-H.; Sugiyama, J.; Imanishi, Y. Langmuir 1999, 15, 4461. (19) Cornelissen, J. J. L. M.; Fischer, M.; Sommerdijk, N. A. J. M.; Notle, R. J. M. Science 1998, 280, 1427. (20) Vriezema, D. M.; Hoogboom, J.; Velonia, K.; Takazawa, K.; Christianen, P. C. M.; Maan, J. C.; Rowan, A. E.; Nolte, R. J. M. Angew. Chem., Int. Ed. 2003, 42, 772. (21) Checot, F.; Gnanou, Y.; Lecommandoux, S.; Klok, H.-A. Angew. Chem., Int. Ed. 2002, 41, 1340. (22) Checot, F.; Brulet, A.; Oberdisse, A. J.; Gnanou, Y.; Mondain-Monval, O.; Lecommandoux, S. Langmuir 2005, 21, 4308.

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poly(butadiene)-block-poly(L-glutamate), poly(L-glutamic acid)block-poly(L-lysine), and poly(L-lysine)-block-poly(L-leucine).17,19-25 However, the preparation of polypeptide block copolymers requires complicate synthesis tactics and cumbersome work. Therefore, a relatively simple method of constructing peptosomes is greatly needed. Very recently, a noncovalent method denoted as template selfassembly was utilized to prepare polypeptide-based vesicles or hollow spheres (capsules). The reported templates can be either hollow or solid. For the former, liposomes mixed with a certain number of charged lipids were used as templates to adsorb polypeptides with opposite charge onto their surface through electrostatic interactions. For example, Volodkin,26,27 Yaroslavov,28 and Paleos29 have coated negatively charged liposomes with commercially available PLL or poly-L-arginine. Fujimoto,30 Frisch,31 and Fournier32 have deposited alternating PLL and poly(glutamic acid) (PGA), PLL and poly-L-aspartic acid, or PLL and poly(acrylic acid) layers on the anionic liposome templates through a layer-by-layer technique. For the latter, solid templates were employed to adsorb polypeptides on their surface and then removed to prepare polypeptide-based microcapsules. For example, Caruso33-36 and Yu37 have used mesoporous silica spheres as (23) Rodrguez-Hernandez, J.; Checot, F.; Lecommandoux, S. J. Am. Chem. Soc. 2005, 127, 2026. (24) Holowka, E. P.; Pochan, D. J.; Deming, T. J. J. Am. Chem. Soc. 2005, 127, 12423. (25) Bellomo, E. G.; Wyrsta, M. D.; Pakstis, L.; Pochan, D. J.; Deming, T. J. Nat. Mater. 2004, 3, 244. (26) Volodkin, D.; Mohwald, H.; Voegel, J.-C.; Ball, V. J. Controlled Release 2007, 117, 111. (27) Volodkin, D.; Ball, V.; Schaaf, P.; Voegel, J.-C.; Mohwald, H. Biochim. Biophys. Acta 2007, 1768, 280. (28) Yaroslavov, A. A.; Rakhnyanskaya, A. A.; Yaroslavova, E. G.; Efimova, A. A.; Menger, F. M. Adv. Colloid Interface Sci. 2008, 142, 43. (29) Tsogas, I.; Tsiourvas, D.; Nounesis, G.; Paleos, C. M. Langmuir 2005, 21, 5997. (30) Fujimoto, K.; Toyoda, T.; Fukui, Y. Macromolecules 2007, 40, 5122. (31) Ciobanu, M.; Heurtault, B.; Schultz, P.; Ruhlmann, C.; Muller, C. D.; Frisch, B. Int. J. Pharm. 2007, 344, 154. (32) Germain, M.; Grube, S.; Carriere, V.; Richard-Foy, H.; Winterhalter, M.; Fournier, D. Adv. Mater. 2006, 18, 2868. (33) Wang, Y. J.; Caruso, F. Adv. Mater. 2006, 18, 795. (34) Wang, Y. J.; Bansal, V.; Zelikin, A. N.; Caruso, F. Nano Lett. 2008, 8, 1741. (35) Yu, A. M.; Wang, Y. J.; Barlow, E.; Caruso, F. Adv. Mater. 2005, 17, 1737. (36) Yu, A. M.; Gentle, I. R.; Lu, G. Q.; Caruso, F. Chem. Commun. 2006, 2150. (37) Yu, A. M.; Gentle, I. R.; Lu, G. Q. J. Colloid Interface Sci. 2009, 333, 341.

Published on Web 05/19/2009

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Letter Scheme 1. (a) Complex Self-Assembly Process of Hx-COOH and PLL at Different Polymer Concentrations, (b) Formation of Giant CPs by the Fusion of Small CPs, and (c) Molecular Structures of PLL and H20-COOH

the templates to adsorb monolayer or alternating multilayer polypeptides, and then the silica templates were removed by hydrofluoric acid. Haynie and co-workers38 have coated colloid CaCO3 particles with alternating PLL and PGA layers, and CaCO3 templates were then removed easily by adding ethylenediaminetetraacetic acid (EDTA). They also used the melamine formaldehyde (latex) particles as the templates, which was removed under a strongly acidic condition (pH 1.6), to alternatively adsorb positive and negative cysteine-containing polypeptides that can be reversibly cross-linked or cleaved by oxidizing or reducing reactions.39 In general, liposomes are readily available and biocompatible; however, they are delicate structures with limited stability, which will certainly inhibit the stability of the finally obtained polypeptide-based vesicles.40 Polypeptide-based hollow capsules through hard templates are more stable; however, an additional template-removal step is needed for the preparations, which is often performed under harsh conditions and frequently leads to the inactivation of loaded proteins. Herein, we reported for the first time a noncovalent, templatefree method of preparing polypeptide-based vesicles through the one-step complex self-assembly of anionic hyperbranched polymers and the cationic PLLs. Such vesicles can be looked as an extension of “peptosomes” and are denoted as complex peptosomes (CPs) here. The biggest advantage of such CP system lies in the facile preparation process just by mixing two kinds of polymer aqueous solutions together. In addition, the CP size can be readily (38) Haynie, D. T.; Zhi, Z. L. Chem. Commun. 2006, 147. (39) Haynie, D. T.; Palath, N.; Liu, Y.; Li, B. Y.; Pargaonkar, N. Langmuir 2005, 21, 1136. (40) Yaroslavov, A. A.; Kiseliova, E. A.; Udalykh, O. Y.; Kabanov, V. A. Langmuir 1998, 14, 5160.

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controlled from nanosize to microsize through the adjustment of polymer concentration (shown by Scheme 1). The resulting nanosized CPs show unexpected size stability independent of a broad solution pH range and can be kept in water without aggregates or sediments for at least 6 weeks, especially after chemical cross-linking. Furthermore, the CPs display almost no cytotoxicity according to the cell viability tests using human HeLa cells.

Experimental Section The details of the experiments are provided in the experimental section of the Supporting Information.

Results and Discussion Previously, we reported a type of pH-responsive, negativecharged hyperbranched polymer named Hx-COOH through the one-step esterification of the commercially available hydroxyterminated hyperbranched polyester of Boltorn Hx (x = 20, 30, 40) with succinic anhydride. The obtained carboxyl-terminated Hx-COOH (Scheme 1) could self-assemble into size-controllable polymer vesicles from 200 nm to 10 μm in water when the solution pH was adjusted from 5.5 to 2.0. However, when the solution pH was increased above 6, Hx-COOH formed unimolecular micelles of around 10 nm in diameter. Herein, H40-COOH was first dissolved in water at pH 7 to get a transparent unimolecular micelle solution. Then, the PLL aqueous solution at pH 7 was added to the as-prepared H40-COOH solution to induce the complex self-assembly. For the self-assembly process, the weight 41

(41) Shi, Z. Q.; Zhou, Y. F.; Yan, D. Y. Macromol. Rapid Commun. 2008, 29, 412.

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Figure 1. Typical morphologies of the complex assemblies at concentrations of (a-c) 1 mg/mL and (d-f) 10 mg/mL. (g-j) TEM images of cross-linked vesicle intermediates in fusion. The scale bars in the insets of a, c, e, f represent 50 nm, 50 nm, 2 μm, and 1 μm, respectively.

ratio of PLL to H40-COOH was kept at 1:1 while the total polymer concentration (PLL and H40-COOH) was varied. When the polymer concentration was 1 mg/mL, a blue opalescence appeared instantaneously. However, when the concentration was increased to 10 mg/mL, the solution became cloudy. Such turbidity changes indicated the formation of aggregates with different sizes in solution. The morphologies of the aggregates were investigated by transmission electron microscopy (TEM), cryo-TEM, scanning electron microscopy (SEM), and optical microscopy. Figure 1a-c show the micrographs of the complex assemblies at a polymer concentration of 1 mg/mL. In the cryo-TEM image (Figure 1a), the contrast difference between the aggregate skin and the inner pool (inset) proves that the aggregates are vesicular in nature and the average vesicle size is around 100 nm. Figure 1b,c shows the SEM and TEM images, respectively, of the cross-linked vesicles through the reaction between PLL and glutaraldehyde, and it also supports the formation of regular spherical vesicles around 100 nm. More interestingly, when the polymer solution concentration was increased from 1 to 10 mg/mL, the size of the aggregates increased substantially and could be observed directly under the optical microscope. As shown in Figure 1d, the polymers have self-assembled into giant vesicles around 5 μm. Figure 1e shows the fluorescent image of the vesicles in the presence of rhodamine B, and strong red-fluorescent vesicles indicate that the vesicles can effectively encapsulate hydrophilic drugs such as rhodamine B. The inset in Figure 1e shows the laser scanning confocal microscope (LSCM) image of green-fluorescent vesicles with the PLLs labeled by fluorescein isothiocyanate (FITC), which proves the vesicle structure as evidenced by a significant decrease in fluorescence intensity from the peripheral ring toward the center of the 6624 DOI: 10.1021/la901366g

sphere. Figure 1f shows the SEM and TEM images (inset) of the cross-linked giant vesicles, which also provide solid evidence to support the formation of micrometer-sized vesicles. Thus, the complex self-assembly of H40-COOH and PLL will give rise to polymer vesicles, and the vesicle sizes can be controlled from nanometer to micrometer through the simple adjustment of polymer concentration. Because of the polypeptide-based characteristics and the complex self-assembly process, we called such vesicles complex peptosomes (CPs). Generally, the size of polymer vesicles was determined by the hydrophilic fractions11,42 or the solvent compositions.5,20,43 However, such size-control mechanisms seemed not to be applicable in the CP system reported here because the mole ratio of H40-COOH to PLL (similar to hydrophilic fractions) was kept constant and no solvent variance was employed. To determine the concentration-dependent size-variable mechanism of the CPs, we tried to entrap the vesicle intermediates by adding the cross-linker as soon as the two polymer solutions were mixed together in order to track the complex self-assembly process of the giant CPs (GCPs). Figure 1g-j shows some TEM images of the cross-liked vesicle intermediates selected from a large number of micrographs, which display successive fusion sequences of CPs including membrane contact (Figure 1g), the formation of a fusion pore (Figure 1h), the symmetric expansion of the fusion pore (Figure 1i), and complete fusion (Figure 1j). Figure S1 coincidentally demonstrates three aggregated vesicles in an early stage of the formation of fusion pores. Therefore, it can be concluded that the formation of GCPs is attributed to the fusion of small CPs (42) Azzam, T.; Eisenberg, A. Angew. Chem., Int. Ed. 2006, 45, 7443. (43) Terreau, O.; Bartels, C.; Eisengerg, A. Langmuir 2004, 20, 637.

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Figure 2. Zeta potential measurements. (a) Plots of zeta potential vs pH of the aqueous solution of H40-COOH (9), SCP (2) and PLL (b). (b) Plot of zeta potential versus the weight ratio of PLL to H40-COOH of the SCPs, where the concentration of H40-COOH was kept at 1 mg/mL.

(SCPs). In addition, it should be noted that the fusion process proceeded quickly in our system with a higher concentration and only the GCPs were observed after holding the solution for 1 h. The outside surface properties of the obtained CPs were investigated by the measurement of the ζ potential with H40-COOH and PLL solutions as controls. Figure 2a shows the plots of the ζ potential versus pH for these three different systems. The ζ potential of H40-COOH is negative because the end succinic acid groups are deprotonated. PLL shows positive potentials at pH 7 as indicated by the decrease in ζ potentials. Accordingly, the ζ potential of the CPs is positive and much closer to that of PLL over the whole pH region, which indicates that the outside surface of the CPs consists of PLL chains. Taking into account the ζ-potential results and the concentration-dependent vesicle size, we propose the complex self-assembly mechanism as shown in Scheme 1. In this work, the weight ratio of PLL to H40-COOH (Rw) is kept at 1:1, so the amount of PLL is about twice that needed to neutralize H40-COOH because the stochiometric charge equivalency is around 0.40 (Rw, the theoretical value is 0.46) according to the electrophoretic titration curve (Figure 2b). When mixing the two polymer solutions together, the electrostatic complex between the succinic acid groups of Langmuir 2009, 25(12), 6622–6626

Figure 3. DLS measurments. (a) DH of SCPs and cross-linked SCPs before and after 6 weeks of storage at 4 °C. (b) DH of SCPs versus pH.

H40-COOH and the ε-amine groups of PLL occurred to form ion pairs, which dramatically decreased the hydrophilicity of H40-COOH. Thus, the totally neutralized H40-COOH molecules were spontaneously segregated into the hydrophobic clusters, which induced the conjugated PLLs to form the hydrophilic shells with excess ε-amine groups to prevent macroscopic phase separation and stabilize complexes in aqueous dispersions. Then, the microphase-separated complexes further aggregated into vesicles through a process analogous to that of linear block ionomer complexes44-47 and amphiphilic hyperbranched multiarm copolymers.8,11,48 In dilute solution, only the small, stable polymer vesicles were obtained. When the polymer concentration was increased, more small vesicles were formed, and they were further agglomerated and then fused into larger ones. The obtained SCPs have demonstrated excellent storage stability according to the dynamic light scattering (DLS) measurements. As shown by the DLS profile in Figure 3a, SCPs have a mean hydrodynamic diameter (DH) of 135.4 nm and a narrow size (44) Kabanov, A. V.; Bronich, T. K.; Kavanov, V. A.; Yu, K.; Eisenberg, A. J. Am. Chem. Soc. 1998, 120, 9941. (45) Bronich, T. K.; Ouyang, M.; Kavanov, V. A.; Eisenberg, A.; Szoka, F. C.; Kabanov, A. V. J. Am. Chem. Soc. 2002, 124, 11872. (46) Gohy, J.-F.; Mores, S.; Varshney, S. K.; Jrme, R. Macromolecules 2003, 36, 2579. (47) Gu, C. F.; Chen, D. Y.; Jiang, M. Macromolecules 2004, 37, 1666. (48) Zhou, Y. F.; Yan, D. E. Chem. Commun. 2009, 1172.

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distribution (PDI = 0.076) that is a little larger than that measured by TEM or SEM (Figure 1b,c). After 6 weeks of storage at 4 °C, almost no change in the DLS profile but a small decrease in DH to 118.2 nm was observed. After cross-linking, the vesicles (DH = 117.7 nm, PDI = 0.062) slightly decreased in size, which possibly arose from the shrinkage of PLL shells caused by crosslinking, and demonstrated better storage stability. In contrast, GCPs did not have such good storage stability as SCPs, and they were separated from the solution and deposited on the bottom of vessels under the effect of gravity within 2 days. However, the cross-linked GCPs possessed good stability against all kinds of extreme environments such as strong acid or alkaline, good solvents (acetone or DMSO), drying, and rehydration (Figure S2). We attributed the great storage stability of SCPs to the better hydration of the vesicle shells in dilute solution, which prevents the polymer vesicles from aggregating as a result of a stronger repulsive hydration force. The SCPs also show special pH stability. Generally, the peptosomes have been demonstrated to respond to pH or to ionic strength with a change in DH due to the polyelectrolyte nature of polypeptide blocks.23,24 For the peptosomes prepared from zwitterionic diblock copolypeptides of PGA-b-PLL, named “schizophrenic” vesicles by Lecommandoux et al., there exists a reversible inside-out phase inversion in the vesicles at pH 10, and at intermediate pH values, the vesicles disassemble and eventually precipitate through an electrostatic complex.23 However, in our CP system, although both the cationic PLLs and anionic H40-COOHs were involved, we found only a small size fluctuation (Figure 3b) at pH 4-8, which indicates that the SCPs are stable over a broad pH range. We attributed the pH stability to the strong complex interactions between PLLs and H40-COOHs, which tightly locked the vesicle structure. Only when the solution pH was decreased to below 4 or increased to above 8 was the deconstruction of the complex interactions observed (Figure 3b) as a result of the strong protonation of H40-COOHs or the deprotonation of PLLs, respectively. Such a broad pH window is desirable for drug carriers to realize sustained release independent of environmental pH. For in vivo applications, drug delivery carriers must possess good biocompatibility. Accordingly, we evaluated the influence of SCPs on cellular metabolic activity using the WST-1 assay. Human cervical carcinoma HeLa cells were treated with H40-COOHs, SCPs, and cross-linked SCPs, respectively, at a concentration of 0.5 mg/mL for 24 h. The data in Figure 4 show the cell viabilities compared to the control sample that is arbitrarily taken to be 100%. After culturing for 24 h, quite a number of cells cultured with H40-COOH (63 ( 2% viability) died, but only a few cells cultured with SCPs (92 ( 6% viability) or crosslinked SCP (97 ( 10% viability) died. The remarkable increase in

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Figure 4. Viability of HeLa cells cultured with H40-COOH, SCPs, and cross-linked SCPs measured by a WST-1 assay after seeding for 24 h. HeLa cells cultured without any polymers added at the same time interval were used as controls.

cell viability of SCPs compared with that of H40-COOHs suggests that the introduction of PLL into the supramolecular assemblies greatly reduces the cytotoxicity of the SCPs.

Conclusions We have successfully prepared a new type of peptosome through the complex self-assembly of anionic hyperbranched polyesters and cationic poly-L-lysines, which have demonstrated several advantages including facile preparation, controllable size, biocompatibility, and high pH or storage stability. In addition, the SCPs have a size similar to that of viruses or lipoproteins (10-200 nm). Such characteristics make the complex peptosomes very attractive in drug delivery application, and the results will be reported in due course. Acknowledgment. We thank the National Basic Research Program (2007CB808000), the National Natural Science Foundation of China (50633010, 20774057, 20874060, and 50873058), the Program for New Century Excellent Talents in University (NCET-07-0558), the Basic Research Foundation (07DJ14004) of Shanghai Science and Technique Committee, and the Shanghai Leading Academic Discipline Project (B202) for financial support. Supporting Information Available: Experimental section, vesicle intermediates in fusion, and stability evaluation of cross-linked GCPs. This material is available free of charge via the Internet at http://pubs.acs.org.

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