Peptide-based hydrogel nanoparticles as effective drug delivery agents

Bioorganic & Medicinal Chemistry 21 (2013) 3517–3522

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Peptide-based hydrogel nanoparticles as effective drug delivery agents Rafael Ischakov, Lihi Adler-Abramovich, Ludmila Buzhansky, Talia Shekhter, Ehud Gazit ⇑ Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel

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Article history: Available online 21 March 2013 Keywords: Drug delivery Fmoc-FF Hydrogel nanoparticles Emulsion Self assembly Vitamin E-TPGS

a b s t r a c t Peptide-based hydrogel nanoparticles represent a promising alternative to current drug delivery approaches. We have previously demonstrated that the Fmoc-FF aromatic dipeptide building block can self-assemble in aqueous solutions to form nano-scaled ordered hydrogels of remarkable mechanical rigidity. Here, we present a scalable process for the assembly of this peptide into hydrogel nanoparticles (HNPs) aimed to be utilized as potential drug delivery carriers. Fmoc-FF based HNPs were formulated via modified inverse-emulsion method using vitamin E-TPGS as an emulsion stabilizer and high speed homogenization. The formed HNPs exhibited two distinguishable populations with an average size of 21.5 ± 1.3 and 225.9 ± 0.8 nm. Gold nanoparticles were encapsulated within the hydrogel nanoparticles as contrast agents to monitor the formation of the assemblies and their ultrastructural properties. Next, we demonstrated a robust experimental procedure developed and optimized for the formulation, purification, storage and handling procedures of HNPs. Encapsulation of doxorubicin (Dox) and 5-flourouracil (5-Fu) within the HNPs matrix showed release kinetics of the drugs depending on their chemical structure, molecular weight and hydrophobicity. The results clearly indicate that Fmoc-FF based hydrogel nanoparticles have the potential to be used as encapsulation and delivery system of various drugs and bioactive molecules. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction The engineering of customizable, biocompatible and biodegradable nanoparticles (NPs) for delivery of various water soluble/ insoluble pharmaceutically-active molecules is a key challenge in the field of nano-medicine.1–3 The following properties are desirable for the biomedical applications of nanoparticles: (i) facile and reproducible synthesis of NPs using simple technique with few steps; (ii) NPs size and their surface properties should be readily controlled to achieve both active and passive targeting, while avoiding recognition and rapid clearance by reticuloendothelial system; (iii) methods of preparation should allow encapsulation of various types of drugs and bioactive molecules with high yield and avoid chemical reactions that may affect the therapeutic efficiency of the encapsulated entities; and (iv) display properties favorable for controlled and sustained release at the target site Abbreviations: ddH2O, double distilled H2O; Dox, doxorubicin; DLS, dynamic light scattering; DMSO, dimethyl sulfoxide; Fmoc-FF, N-fluorenylmethoxycarbonyldi-phenylalanine; 5-Fu, 5-flourouracil; HBS, Hank’s buffered saline; HNPs, hydrogel nanoparticels; HPLC, high-performance liquid chromatography; PBS, phosphate buffered saline; TEM, transmission electron microscopy; Vitamin E-TPGS, vitamin E D-a-tocopheryl, polyethylene glycol 1000 succinate. ⇑ Corresponding author. Tel.: +972 3 640 9030; fax: +972 3 640 5448. E-mail addresses: [email protected], [email protected] (E. Gazit). 0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmc.2013.03.012

and improving pharmacokinetic properties of the encapsulated drug.4,5 Different types of materials are used for the preparation of nanoparticles including polymers such as polylactic acid or poly (lactic-co-glycolic acid) (PLGA),6,7 phospholipids (liposomes),8 organic–inorganic composite particles9,10 and even virus based nanoparticles (VNPs)11 characterized by different pharmacokinetic properties of NPs. Over the past few years polymer-based hydrogel nanoparticles (HNPs) had gained considerable interest as effective drug delivery vehicles.5 Similar to conventional bulk hydrogels, HNPs are environmentally friendly, easily synthesized, soft, elastic and biocompatible materials which mainly consist of aqueous content (>99%) making them the material of choice for nanomedical applications.5 Various types of synthetic polymers [e.g., polyvinyl alcohol and polyethylene oxide] and natural polymers [e.g., chitosan and gelatin] may be used as building blocks for HNPs formation, offering variable controllability and reproducibility.1,2,5,12–14 For example, by selecting polymers with different surface properties or diverse bulk erosion rates, it is possible to control the release rate of the encapsulated drug. In addition, HNPs can be designed to release their load in response to environmental changes, such as pH and temperature.15,16 However, there are also some drawbacks as NPs prepared using those polymers are often fabricated via complex techniques that involve the use of extreme

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temperatures and pHs, as well as different types of chemicals and cross-linkers that exhibit low biocompatibility.17 Natural or synthetic self-assembled peptides represent an additional type of gel forming molecules. These low-molecular-mass, biocompatible, building blocks have the ability to self-assemble into well ordered hydrogel that can be utilized towards medical applications.18,19 The considerable interest in peptide based hydrogels and HNPs has gained momentum in recent years due to their numerous advantages over polymeric gelators: (i) peptide based hydrogel and HNPs are formed through molecular self-assembly, without the use of potentially hazardous chemicals such as cross-linkers that may affect its biocompatibility; (ii) usually nontoxic degradability in vivo, due to the fact that the building blocks of the HNPs are peptides composed of simple naturally-occurring amino acids; and (iii) short peptide building blocks are easy to manufacture in large quantities, and can also be chemically and biologically decorated, giving an ability to design ultrastructures with improved targeting and prolonged in vivo stability. Therefore, those useful characteristics have motivated us in designing peptide-based colloidal HNPs carriers for drug delivery applications. We have previously demonstrated that under defined conditions, the aromatic dipeptide, N-fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF), self-assembles in aqueous solutions to form peptide nano-fibrils that spontaneously form a rigid gel.20,21 The unique mechanical properties of the Fmoc-FF hydrogel are useful for the development of stable HNPs without the need for chemical

cross-linking due to spontaneous facile molecular self-assembly process. In the present study we utilize this aromatic peptide building block towards formation of a new type of hydrogel nanoparticles (HNPs) aimed to be used as nano-carriers for controlled drug delivery. Fmoc-FF based nanoparticles were formed using inverse emulsion technique. In order to stabilize those HNPs in aqueous solutions D-a-tocopheryl, polyethylene glycol 1000 succinate (vitamin ETPGS), a biocompatible and biodegradable surfactant, was applied. In order to define the most favorable conditions for HNPs formulation, a series of parameters were evaluated and examined with respect to physical stability in physiological buffer; size and zeta potential; post-formulation modifications for prolonged shelf-life; encapsulation efficiency and controlled drug release kinetics. The overall results suggest that the physical and chemical properties of Fmoc-FF based HNPs allow them to serve as a novel platform for drug delivery and other bionanotechnological applications. 2. Results and discussion 2.1. Preparation and characterization of HNPs The formation of HNPs using the Fmoc-FF peptide as a building block is based on the ability of this aromatic dipeptide to self assemble and form hydrogel with unique mechanical properties.20,21 As illustrated in Figure 1a modified inverse (water-in-oil) emulsion

Figure 1. HNPs formulation. (a) Schematic illustration showing the process of HNPs preparation by modified inverse emulsion method; (b) transmission electron microscopy (TEM) image of the HNPs formed; (c) size distribution of HNPs as measured by dynamic light scattering showing two peaks at 21.5 ± 1.3 (lower peak) and 225.9 ± 0.8 nm (higher peak).

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technique was used for HNPs fabrication. This is a widely used welldefined, simple and efficient strategy for NPs preparation.22,23 In order to fabricate the hydrogel nanoparticles, the peptide was first dissolved into its monomeric state in organic solvent (DMSO), followed by dilution in water, and its drop-wise addition into organic phase (mineral oil) containing vitamin E-TPGS as a surfactant. Vitamin ETPGS was chosen as a HNPs stabilizer due to several characteristics such as remarkable biocompatibility, biodegradability and low immunogenicity.24,25 Moreover, vitamin E-TPGS may provide hydroxylic functional groups for the attachment of targeting units such as antibodies and aptamers. The heterogeneous water/oil mixture was then subjected to homogenization. The increase in the energy input of the homogenization process allows the dipeptide mixture to be disperses and self-assemble into particle aggregates.26 In order to promote the gelation process and allow formation of the surfactant monolayer around the aqueous core of the HNPs the suspension was gently stirred for 2 h at 4 °C. Finally, HNPs were purified using centrifugation and a series of washings with hexane. The collected HNPs were resuspended in water or PBS prior to their use. The physical dimension of the nanoparticles is one of the most important parameters in NPs design, influencing their bio-distribution, clearance kinetics, and in vivo efficacy. Therefore, efficient control on the HNP’s size is of a substantial interest for basic research as well as for clinical applications. We were able to control the size of HNPs by optimizing the parameters of the emulsion process, such as the stirring speed and the volume ratio of the two liquids. Since the viscous resistance during the process of homogenization absorbs most of the applied energy, the comminution energy necessary to produce smaller nanoparticles involves additional shear forces. Therefore, in order to obtained smaller diameter NPs higher amounts of energy should be applied.26,27 The hydrodynamic size, polydispersity and the zeta potential of the obtained nanoparticles were analyzed using dynamic light scattering (DLS). The DLS analysis revealed two distinguishable populations of HNPs with an average size of 21.5 ± 1.3 and 225.9 ± 0.8 nm (Fig. 1c). This phenomenon is known as bimodal size distribution, and is well described in the review by Jafari et al.28 This bimodal particle size distribution is probably a result of equilibrium between two opposite processes, droplet fragmentation and droplet re-coalescence that occurs due to overall conditions used in this process, such as surfactant type and the speed of the homogenization. Furthermore, we analyzed the HNPs using transmission electron microscopy (TEM) analysis, demonstrating well-defined, discrete spherical structure with various sizes up to 100 nm in diameter (Fig. 1b). This variation between DLS and

a

TEM data attributed to the swelling of the nanoparticles in the aqueous environment during the DLS analysis, in comparison to the dry environment in the TEM analysis. The zeta potential values of the HNPs were in average of 25 ± 3 mV. HNPs negative charge could be attributed to the presence of carboxyl groups in the peptide building block. Another factor that may contribute to the negative zeta potential may be the coating by the vitamin E TPGS.25 2.2. Encapsulation of gold nanoparticles by HNPs One of the main characteristics of HNPs is its >99% water content; therefore, to confirm that the obtained particles indeed contain water, we added 20 nm gold nanoparticles dispersed in aqueous solution as probes. Due to their high contrast in TEM analysis, gold nanoparticles were utilized to visualize the hydrophilic core of HNPs and practically in confirming the ability of HNPs to encapsulate inorganic nanoparticles in the hydrogel matrix. As examined using TEM analysis, the hydrophilic colloidal gold nanoparticles are encapsulated inside the HNPs core and were not found outside the particles in the organic solvent surroundings (Fig 2a and b). This HNPs formulation process was applied using magnetic stirrer which has a much lower energy input than that of the high speed homogenizer. Therefore, the HNPs in Figure 2a appeared to be larger in diameter compared to those in Figure 1b. Since the size and size distribution of the particles are heavily influenced by the energy impute of the homogenization process the obtained size of HNPs were larger than that obtained while using high speed homogenizer. 2.3. HNPs post-formulation purification and storage stability Potential pharmaceutical applications of HNPs require the application of a lyophilization method to provide prolonged shelf-life, easier, simplified storage and transportation conditions. It is well-known that the stresses produced by the process of lyophilization may cause aggregation and fusion of the HNPs suspension upon freezing and sublimation.29 Generally, prior to lyophilization, cryoprotectants are added in to the HNPs solution to protect their fragile structure from destruction. Here we choose to use, t-butyl alcohol (t-BuOH) as cryoprotector.30,31 Prior to lyophilization, the two populations of HNPs have a mean particle diameter of 21.5 ± 1.3 and 225.9 ± 0.8 nm. After freeze-drying, HNPs with the addition of t-BuOH resulted in complete recovery in appropriative aqueous medium and exhibited the similar ultrastructural morphology and size distribution (Fig. 3a and b). The

b

Figure 2. Encapsulation of gold nanoparticles. (a) TEM image of gold nanoparticle clusters encapsulated by the HNPs; (b) schematic illustration of gold nanoparticles encapsulated by the HNPs.

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a

b

Figure 3. Post formulation stability of HNPs. (a) DLS analysis of HNPs before and after freeze-drying; (b) TEM images of HNPs after freeze-drying with and without cryoprotection. In all cases vitamin E-TPGS was present in the preparation.

Figure 4. Encapsulation and sustained drug release of HNPs. (a) Confocal microscopy of encapsulation of doxorubicin by HNPs; (b) release profiles of doxorubicin (red line) and of 5-Fu (black line) from HNPs at 37 °C in HBS measured by HPLC.

need to use cryoprotectants may be avoided as vitamin E-TPGS can also serve as a stabilization agent and not only as surfactant upon freeze-drying.25 Therefore, we intended to confirm the ability of vitamin E-TPGS to serve as a HNPs cryoprotector. We lyophilized the HNPs without the t-BuOH and compared them to the cryoprotected particles. Figure 3a and b demonstrates that the vitamin E-TPGS coated HNPs lacking the cryoprotection, have similar diameters and morphology as the cryoprotected HNPs with only slight variations between the samples. These results indicated that after formulation, the particles could be readily turned into

dry form, and resuspended prior to use. In addition, HNPs could be freeze-dried without cryoprotectants due to the presence of the vitamin E-TPGS layer on the HNPs surface. 2.4. Drug encapsulation and in vitro sustained release Next we used the HNPs for encapsulation and sustained release of two widely used chemotherapeutics with distinct chemical structures, molecular weights and hydrophobicity, namely doxorubicin (Dox) and 5-fluorouracil (5-Fu). The intrinsic fluorescence of

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Dox molecule was used to validate its encapsulation in HNPs. The red fluorescence of doxorubicin reveals its presence inside HNPs (Fig. 4a). Then, the drug loading efficiency and release kinetics of Dox-loaded and 5-Fu-loaded HNPs were determined by dialysis in HBS at 37 °C under gentle stirring, to mimic physiological conditions. The concentration of the released drug was measured in solution over time, and quantified using analytical HPLC. The resulted release profiles of Dox and 5-Fu are presented in Figure 4b. It should be noticed that the release profile of 5-Fu loaded HNPs differs from that of the Dox loaded nanoparticles. The release of 50% of 5-Fu from the HNPs was within 5 h, and after 12 h the kinetics of release reached a plateau. Conversely, Dox release from the HNPs matrixes was relatively slower, such that 50% of the drug was released only after 20 h, with 80% release within 55 h. This difference in release kinetics between 5-Fu and Dox might be attributed to their dissimilar chemical structural characteristics. We propose that aromatic interactions and hydrogen bond formation between the peptide building block and the drug may play a crucial role in its efficient encapsulation. In its structure, the Dox molecule contains extended aromatic moiety compared to 5-Fu. Under such hypothesis, the binding and intercalation of Dox between the aromatic amino acids, and the Fmoc moiety of the peptide (FmocPhe-Phe-OH) is more efficient compared to 5-Fu and affects the release kinetics.32 In addition, due to high water content of HNPs it is well-known that the release rate of low molecular weight drugs usually cannot be controlled and their diffusion through nanoparticle’s matrix is much faster with a noticeable burst release.33,34 The molecular weight of 5-Fu is much lower (130 g/mole) than that of Dox (580 g/mole) therefore it is expected that the release kinetics of 5-FU would be much faster than that of Dox. 3. Conclusion We designed and developed a new class of self-assembled aromatic peptide-based HNPs consisting of Fmoc-FF core, and vitamin E-TPGS monolayer as an outer shell. HNPs were formulated by few simple and reproducible steps using modified inverse emulsion technique utilizing the ability of Fmoc-FF building block to form hydrogels via assembly in water without the need of cross-linking or any other covalent bonding. The obtained HNPs were characterized and evaluated for their size, zeta-potential and post-formulation stability. It is assumed that the encapsulation ability of nanoparticles depends on the physicochemical characteristics of the encapsulated molecules which affect their release kinetics. The obtained results clearly indicate that these HNPs may be suitable as a potential drug delivery system. However, further studies are needed to evaluate the potential use of this novel type of peptide based HNPs in nano-medical applications. 4. Experimental 4.1. Fmoc-FF based HNPs preparation Fmoc-FF (Bachem, Switzerland), based Hydrogel Nanoparticles (HNPs) were prepared using self-assembly and modified inverse emulsion technique in which vitamin E-TPGS (Sigma Aldrich, Israel) was used as an emulsion stabilizer. In brief, HNPs were formulated by the dilution of Fmoc-FF/DMSA stock solution (100 mg/mL) in ultra-pure water (Biological Industries, Israel) to a final peptide concentration of 10 mg/mL. The resulting solution was added drop-wise into 50 mL slightly warmed (35 °C) mineral oil (Holland Moran Ltd., Israel) containing vitamin E-TPGS at concentration of 0.4% wt/v and homogenized using high speed homogenizer. Next, nanoparticles were allowed to self-assemble for 2 h with continues stirring at 4 °C. Upon the completion of

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the self assembly process, the resulting suspension was mixed with hexane at a concentration of 20% (v/v), and centrifuged to obtain phase separation. Supernatant was removed and HNPs were washed with 1 mL hexane twice more to remove the remaining residues of mineral oil and vacuum dried for 2 h in desiccators to remove traces of hexane. The obtained HNPs were used immediately, or stored at 4 °C for later use. 4.2. Freeze-dried HNPs preparation Upon preparation, the HNPs suspension was frozen and lyophilized using a bench-top freeze dryer to obtain the HNPs in a dried powder form. While t-BuOH was used as a cryoprotectant agent, aqueous HNPs suspension was mixed with a 50% (v/v) of aqueous cryoprotectant for 1 h; this mixture was then rapidly frozen using liquid nitrogen and lyophilized. 4.3. Nanoparticle characterization Nanoparticles size diameter, polydispersity, and surface charge were measured using ZetaPALS dynamic light scattering (DLS) (Malvern Instruments Ltb. Worcestershire, UK) with appropriate viscosity and refractive index settings. The temperature was maintained at 25 °C during the measurement. 4.4. Confocal microscopy To visualize the encapsulation of doxorubicin (Dox) (Sigma Aldrich Israel Ltd., Israel) by the HNPs the sample of Dox loaded HNPs were placed on a specimen slide, and viewed under Zeiss LSM 510 confocal microscope. The fluorescence emission spectrum of Dox (Excitation/Emission, 540 nm/600 nm) allowed it to be visualized in the red wavelength. 4.5. Transmission electron microscopy (TEM) A total of 500 lL of HNPs was prepared and suspended in PBS and placed on a 400-mesh copper grid. After 2 min, the excess of fluid were removed. Negative staining was obtained by covering the grid with 10 lL of 2% uranyl acetate in water. After 2 min, excess uranyl acetate solution was removed. Samples were viewed using a JEOL 1200EX TEM operating at 80 kV. 4.6. Encapsulation of gold nanoparticles For the preparation of HNPs encapsulating gold nanoparticles (Sigma Aldrich, Israel) we applied the same method as described above. However in that case HNPs were formulated in presence of hydrophilic inorganic gold nanoparticles using magnetic stirrer for homogenization. Colloidal gold particles with a diameter of 20 nm were diluted to the final concentration of 100 lL/mL; then the Fmoc-FF peptide was dissolved in DMSO and added in to the solution and emulsified. The obtained HNPs were examined using TEM. 4.7. In vitro drug release of doxorubicin and 5-fluorouracil from NPs For the preparation of drug containing HNPs, Dox or 5-fluorouracil (5-Fu) (Sigma Aldrich, Israel) were dissolved in Fmoc-FF/ DMSO solution at a final concentration of 5% w/w drug to peptide and emulsified. Drug containing HNPs were suspended in 1 mL Hank’s buffered saline (HBS, pH 7.4). The mixture was placed in a dialysis membrane with a molecular weight cut-off of 12–

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14 kDa; the dialysis membrane was placed in 22 mL of HBS at 37 °C dialyzed while gently stirring. At predetermine time intervals, 22 mL of solution outside the dialysis bag were replaced with 22 mL of fresh HBS and concentrations were evaluated at wavelength of 480 nm for Dox and 265 nm for 5-Fu using high performance liquid chromatography (HPLC). The change in the concentration of Dox and 5-Fu was deduced from a calibration curve of Dox and 5-Fu in HBS. The release kinetics and the halflives of the drugs were calculated by fitting the data with an exponential decay model using Origin software with R2 = 0.977 for Dox and R2 = 0.994 for 5-Fu. 4.8. High performance liquid chromatography (HPLC) Analytical HPLC was used for detection of the amount of drug released from HNPs. UltiMateÒ 3000 system (Dionex) equipped with 3000 pump, VWD-3000 UV–vis detector and ChromeleonÒ 6.80 software was used. The column used is LiChroCARTÒ 250  4.6 mm PurospherÒ STAR (5 lm) C-18 RP (reverse phase). Chromatographic conditions: flow: 1.0 mL/min, linear water (buffer A)/acetonitrile (ACN) (buffer B) gradient (buffer A—100% water, 0.1% TFA; buffer B—100% ACN, 0.1% TFA). With retention time of 7.8 min for 5-Fu and 14.8 min for Dox. Acknowledgments This work was supported in part by Grants from the Israeli National Nanotechnology Initiative and Helmsley Charitable Trust for a focal technology area on Nanomedicines for Personalized Theranostics. We thank Yaacov Delarea for help with TEM experiments, and members of the Gazit laboratory with special thanks to Yaron Bram and Aviad Levin for helpful discussion. References and notes 1. Cho, K.; Wang, X.; Nie, S.; Chen, Z. G.; Shin, D. M. Clin. Cancer Res. 2008, 14, 1310. 2. Hans, M. L.; Lowman, A. M. Curr. Opin. Solid State Mater. Sci. 2002, 6, 319. 3. Chakraborty, M.; Jain, S.; Rani, V. Appl. Biochem. Biotechnol. 2011, 1.

4. MacKay, A.; Chen, M.; McDaniel, J.; Liu, W.; Simnick, A.; Chilkoti, A. Nat. Mater. 2009, 8, 993. 5. Gonçalves, C.; Pereina, P.; Gama, M. Materials 2010, 3, 1420. 6. Acharya, S.; Sahoo, S. K. Adv. Drug Delivery Rev. 2011, 63, 170. 7. Nobs, L.; Gurny, R.; Allemann, E. Eur. J. Pharm. Biopharm. 2004, 58, 483. 8. Puri, A.; Loomis, K.; Smith, B.; Lee, J.; Yavlovich, A.; Heldman, E.; Blumenthal, R. Crit. Rev. Ther. Drug Carrier Syst. 2009, 26, 523. 9. Esumi, K.; Sarashina, S.; Yoshimura, T. Langmuir 2004, 20, 5189. 10. Shapira, A.; Livney, Y. D.; Broxterman, H.; Assaraf, Y. Drug Resist. Updat. 2011, 14, 150. 11. Manchester, M.; Singh, P. Adv. Drug Delivery Rev. 2006, 14, 1505. 12. Nayak, S.; Lyon, L. A. Angew. Chem., Int. Ed. 2005, 44, 7686. 13. Hamidi, M.; Azadi, A.; Rafiei, P. Adv. Drug Delivery Rev. 2008, 60, 1638. 14. Chan, J. M.; Zhang, L.; Yuet, K. P.; Liao, G.; Rhee, J. W.; Langer, R.; Farokhzad, O. C. Biomaterials 2009, 30, 1627. 15. Wang, X. Q.; Zhang, Q. Eur. J. Pharm. Biopharm. 2012, 82, 219. 16. Chen, H.; Gu, Y.; Hub, Y.; Qian, Z. PDA J. Pharm. Sci. Technol. 2007, 61, 4303. 17. Soppimath, K. S.; Aminabhavi, T. M.; Kulkarni, A. R.; Rudzinski, W. E. J. Control Release 2001, 70, 1. 18. Bawa, R.; Fung, S. Y.; Shiozaki, A.; Yang, H.; Zheng, G.; Keshavjee, S.; Liu, M. Y. Nanomed. Nanotech. Biol. 2012, 8, 647. 19. Adhikari, B.; Banerjee, A. J. Indian Inst. Sci. 2011, 91, 471. 20. Orbach, R.; Adler-Abramovich, L.; Zigerson, S.; Mironi-Harpaz, I.; Seliktar, D.; Gazit, E. Biomacromolecules 2009, 10, 2646. 21. Mahler, A.; Reches, M.; Rechter, M.; Cohen, S.; Gazit, E. Adv. Mater. 2006, 18, 1365. 22. Myers, D. Surfactant Science and Technology; VCH: New York, 1988. 23. Sasaki, Y.; Akiyoshi, K. Chem. Rec. 2010, 10, 366. 24. Mu, L.; Feng, S. S. J. Control Release 2002, 80, 129. 25. Feng, S. S.; Zeng, W. T.; Lim, Y. T.; Zhao, L. Y.; Win, K. Y.; Oakley, R.; Teoh, S. H.; Lee, R. C. H.; Pan, S. R. Nanomedicine 2007, 2, 333. 26. Abismaïl, B.; Canselier, J. P.; Wilhelm, A. M.; Delmas, H.; Gourdon, C. Ultrason. Sonochem. 1999, 6, 75. 27. Hecht, L. L.; Wagne, C.; Landfester, K.; Schuchmann, P. H. Langmuir 2011, 27, 2279. 28. Jafari, S. M.; Assadpoor, E.; He, Y.; Bhandari, B. Food Hydrocolloids 2008, 22, 1191. 29. Abdelwahed, W.; Degobert, G.; Stainmesse, S.; Fessi, H. Adv. Drug Delivery Rev. 2006, 58, 1688. 30. Peters-Libeu, C.; Newhouse, Y.; Krishnan, P.; Cheung, K.; Brooks, E.; Weisgraber, K.; Finkbeiner, S. Acta Cryst. 2005, 61, 1065. 31. Cingolani, G.; Moore, S. D.; Prevelige, P. E.; Johnson, J. E. J. Struct. Biol. 2002, 139, 46. 32. Mart, R. J.; Osborne, R. D.; Stevens, M. M.; Ulijn, R. V. Soft Matter 2006, 2, 822. 33. Yu, C. Y.; Zhang, X. C.; Zhoua, F. Z.; Zhang, X. Z.; Cheng, S. X.; Zhuo, R. X. Int. J. Pharm. 2008, 357, 15. 34. Yu, C. Y.; Jia, L. H.; Yin, B. C.; Zhang, X. Z.; Cheng, S. X.; Zhuo, R. X. J. Phys. Chem. 2008, 112, 16774.