Journal of Nanoparticle Research

, Volume 9, Issue 5, pp 885–900 | Cite as

Formation of silk fibroin nanoparticles in water-miscible organic solvent and their characterization

  • Yu-Qing Zhang
  • Wei-De Shen
  • Ru-Li Xiang
  • Lan-Jian Zhuge
  • Wei-Jian Gao
  • Wen-Bao Wang
Article

Abstract

When Silk fibre derived from Bombyx mori, a native biopolymer, was dissolved in highly concentrated neutral salts such as CaCl2, the regenerated liquid silk, a gradually degraded peptide mixture of silk fibroin, could be obtained. The silk fibroin nanoparticles were prepared rapidly from the liquid silk by using water-miscible protonic and polar aprotonic organic solvents. The nanoparticles are insoluble but well dispersed and stable in aqueous solution and are globular particles with a range of 35–125 nm in diameter by means of TEM, SEM, AFM and laser sizer. Over one half of the ɛ-amino groups exist around the protein nanoparticles by using a trinitrobenzenesulfonic acid (TNBS) method. Raman spectra shows the tyrosine residues on the surface of the globules are more exposed than those on native silk fibers. The crystalline polymorph and conformation transition of the silk nanoparticles from random-coil and α-helix form (Silk I) into anti-parallel β-sheet form (Silk II) are investigated in detail by using infrared, fluorescence and Raman spectroscopy, DSC, 13C CP-MAS NMR and electron diffraction. X-ray diffraction of the silk nanoparticles shows that the nanoparticles crystallinity is about four fifths of the native fiber. Our results indicate that the degraded peptide chains of the regenerated silk is gathered homogeneously or heterogeneously to form a looser globular structure in aqueous solution. When introduced into excessive organic solvent, the looser globules of the liquid silk are rapidly dispersed and simultaneously dehydrated internally and externally, resulting in the further chain–chain contact, arrangement of those hydrophobic domains inside the globules and final formation of crystalline silk nanoparticles with β-sheet configuration. The morphology and size of the nanoparticles are relative to the kinds, properties and even molecular structures of organic solvents, and more significantly to the looser globular substructure of the degraded silk fibroin in aqueous solution. It is possible that the silk protein nanoparticles are potentially useful in biomaterials such as cosmetics, anti-UV skincare products, industrial materials and surface improving materials, especially in enzyme/drug delivery system as vehicle.

Keywords

silk fibroin nanoparticles organic solvents biomaterials drug delivery colloids 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Notes

Acknowledgements

We are grateful to Associate Professor Wang-Fang Shen for her help in the preparation of this manuscript. This work was partly supported by the National Basic Research Program of China (2005CB121000), the University Natural Science Funds of Jiangsu Province (04KJB180122) and Natural Science Funds of Jiangsu Province (BK2006053), P. R. China

References

  1. Ajisawa A.J. (1998) Dissolution of silk fibroin with calcium chloride/ethanol aqueous solution. J. Sericult. Sci. Japan 67(2):91–97Google Scholar
  2. Altman G.H., Diaz F., Jakuba C., Calabro T., Horan R.H., Chen J., Lu H., Richmond J., Kaplan D.L. (2003) Silk-based biomaterials. Biomaterials 24:401–416CrossRefGoogle Scholar
  3. Demura M., Asakura T., Nakamura E., Tamura H. (1989) Immobilization of peroxidase with a Bombyx mori silk fibroin membrane and its applicaton to biophotosensors. J. Biotechnol. 10:113–120CrossRefGoogle Scholar
  4. Habeeb A.F.S.A. (1996) Determination of free amino group in proteins by trinitrobenzenesulfonic acid. Anal. Biochem. 11:328–336Google Scholar
  5. Hermans P.H., Weidinger A. (1948) Quantitative X-Ray investigations on the crystallinity of cellulose fibers. A background analysis. J. Appl. Phys. 19(5):491–506CrossRefGoogle Scholar
  6. Inoue S., Matsunaga Y., Iwane H. (1986) Entrapment of phenyalanine ammonia-lyase in silk fibroin for protection from proteolytic attack. Biochem. Biophys. Res. Commun. 141:165–170CrossRefGoogle Scholar
  7. Ishida M., Asakura T., Yokoi M., Saito H. (1990) Solvent- and mechanical-treatment-induced conformational transition of silk fibroins studied by high-resolution solid-state 13C NMR. Macromolecules 23:88–94CrossRefGoogle Scholar
  8. Jin H.J., Kaplan D.L. (2003) Mechanism of silk processing in insects and spiders. Nature 424:1057–1061CrossRefGoogle Scholar
  9. Kuzuhara K., Asakura T., Tomoda R., Matsunaga T. (1987) Use of silk fibroin for enzyme membrane. J. Biotechnol. 5:199–207CrossRefGoogle Scholar
  10. Ladokhin A.S., Meyers R.A. (2000) Encyclopedia of Anal Chem. John Wiley & Sons Ltd., Chichester, pp 5762–5779Google Scholar
  11. Li M., Masayo O., Minoura N. (2003) Enzymatic degradation behavior of porous silk fibroin sheets. Biomaterials 24:357–365CrossRefGoogle Scholar
  12. Li M., Wu Z., Zhang C.J. (2001) Study on porous silk fibroin materials II Preparation and characteristics of spongy porous silk fibroin materials. Appl. Polym. Sci. 79:2192–2199CrossRefGoogle Scholar
  13. Lin W., Coombes A.G.A, Davies M.C., Davis S.S., Illum L.J. (1993) Preparation of sub-100 nm human serum albumin nanospheres using a pH-coacervation method. J. Drug Target. 1:237–243Google Scholar
  14. Mathur A.B., Tonelli A., Rathke T., Hudson S. (1997) Dissolution and characterization of Bombyx mori silk fibroin in calcium nitrate-methanol solution and the regeneration of films. Biopolymers 42(1):61–74CrossRefGoogle Scholar
  15. Matsumoto K., Uejima H.J. (1997) Regenerated protein fibers. I. Research and development of a novel solvent for silk fibroin. J. Polym. Sci. A: Polym. Chem. 35(10):1949–1954CrossRefGoogle Scholar
  16. Miller J.N. (1979) Recent advances in molecular luminescence analysis. Proc. Anal. Div. Chem. 16:203–209Google Scholar
  17. Minoura N., Tsukada M., (1990) Coagulation of silk fibroin. Japan Patent JP02–084503Google Scholar
  18. Mita K., Ichimura S., James T.C. (1994) Highly repetitive structure and its organization of the silk fibroin gene. J. Mol. Evol. 38:583–592CrossRefGoogle Scholar
  19. Monti P., Taddei P., Freddi G., Ohgo K., Asakura T. (2003) Vibrational, 13C-cross-polarization/magic angle spinning NMR spectroscopic and thermal characterization of poly (alanine–glycine) as model for silk Bombyx mori fibroin. Biopolymers 72:329–338CrossRefGoogle Scholar
  20. Mori H., Tsukada T. (2000) New silk protein: modification of silk protein by gene engineering for production of biomaterial. J. Biotechnol. 74(2):95–103Google Scholar
  21. Müller G.M., Leuenberger H., Kissel T. (1996) Albumin nanospheres as carriers for passive drug targeting: an optimized manufacturing technique. Pharm. Res. 13:32–37CrossRefGoogle Scholar
  22. Nakayama H. (1981) Immobilized protease and its preparation. Japan Patent JP56015687Google Scholar
  23. Nam J., Park Y.H.J. (2001) Morphology of regenerated silk fibroin: effects of freezing temperature, alcohol addition, and molecular weight. J. Appl. Polym. Sci. 81(12):3008–3021CrossRefGoogle Scholar
  24. Nazarov R., Jin H.J., Kaplan D.L. (2004) Porous 3-D scaffolds from regenerated silk fibroin. Biomacromolecules 5:718–726CrossRefGoogle Scholar
  25. Otoi K., Horikawa Y. (1980) Process for producing a fine powder of silk fibroin. US Patent 4, 233, 212Google Scholar
  26. Saito H., Tabeta R., Asakura T., Iwanaga Y., Shoji A., Ozaki T., Ando I. (1984) High-resolution 13C NMR study of silk fibroin in the solid state by the cross-polarization-magic angle spinning method Conformational characterization of silk I and silk II type forms of Bombyx mori fibroin by the conformation-dependent 13C chemical shifts. Macromolecules. 17:1405–1412CrossRefGoogle Scholar
  27. Sakabe H., Ito H., Miyamoto T., Noishiki Y., Ha W.S. (1989)In Vivo blood compatibility of regenerated silk fibroin. SEN-I GAKKAISHI 45:487–490Google Scholar
  28. Sano M., Mikami S., Sasaki N., Kusamoto N., Fukatsu F., Ubara A., Yasue T., Ohyama S. (1998) Process for producing fine silk fibroin powder. European Patent EP0875523A1Google Scholar
  29. Siamwiza M.N., Lord R.C., Chen M.C., Takamatsu T., Harada I., Matsuura H., Shimanouchi T. (1975) Interpretation of the doublet at 850 and 830 cm−1 in the Raman spectra of tyrosyl residues in proteins and certain model compounds. Biochemistry 14(22):4870–4876CrossRefGoogle Scholar
  30. Snyder S.L., Sobocinski P.Z. (1975) An improved 2,4,6-trinitrobenzenesulfonic acid method for the determination of amines. Anal. Biochem. 64(1):284–288CrossRefGoogle Scholar
  31. Tajima M., Tanaka T. (1994) Silk powder and its production, coating agent using silk powder and its production, and cleaning finishing sizing agent using silk powder and its use. Japan Patent JP06–306772Google Scholar
  32. Tamada Y. (2005) New process to form a silk fibroin porous 3D structure. Biomacromolecules 6:3100–3106CrossRefGoogle Scholar
  33. Toshio U., Kazuo K., Hiroyuki A. (2000) Properties of silk pigment and it’s application for cosmetics. Fragrance J. 28(4):15–21Google Scholar
  34. Trabbic K.A., Yager P. (1998) Comparative structural characterization of naturally- and synthetically-spun fibers of Bombyx mori fibroin. Macromolecules 31:462–471CrossRefGoogle Scholar
  35. Tsubouchi K. (1998) Process for preparing fine powder of silk fibroin. US Patent. US 5, 853, 764Google Scholar
  36. Tsukada M., Gotoh Y., Nagura M., Minoua N., Kasai N., Freddi G.J. (1994) Structural change of silk fibroin membranes induced by immersion in methanol aqueous solutions. J. Polym. Sci. Polym. Phys. Ed. 32:961–968CrossRefGoogle Scholar
  37. Vollrath F., Knight D.P. (2001) Liquid crystalline spinning of spider silk. Nature 410:541–548CrossRefGoogle Scholar
  38. Weber C., Coester C., Kreuter J., Langer K. (2000) Desolvation process and surface characteristics of protein nanoparticles. Int. J. Pharm. 194:91–102CrossRefGoogle Scholar
  39. Yamada H., Nakao H., Takasu Y., Tsubouchi K. (2001) Preparation of undegraded native molecular fibroin solution from silkworm cocoons. Mater. Sci. Eng. C 14:41–46CrossRefGoogle Scholar
  40. Yamaguchi K., Kikuchi Y., Takagi T., Kikuchi A., Oyama F., Shimura K., Mizuno S. (1989) Primary structure of the silk fibroin light chain determined by cDNA sequencing and peptide analysis. Mol. Biol. 210:127–139CrossRefGoogle Scholar
  41. Zhang Y.Q. (1998) Natural silk fibroin as a support for enzyme immobilization. Biotechnol. Adv. 16:961–971CrossRefGoogle Scholar
  42. Zhang Y.Q., Shen W.D., Gu R.A., Zhu J., Xue R.Y. (1998)a Amperometric biosensor for uric acid based on uricase-immobilized silk fibroin membrane. Anal. Chim. Acta 369:123–128CrossRefGoogle Scholar
  43. Zhang Y.Q., Shen W.D., Mao J.P. (2003) Culture medium using sericin as nitrogen source. China Patent CN1443840Google Scholar
  44. Zhang Y.Q., Zhu J., Gu R.A. (1998)b Improved biosensor for glucose based on glucose oxidase-immobilized silk fibroin membrane. Appl. Biochem. Biotechnol. 75:215–233Google Scholar
  45. Zhou C.Z., Confalonier F., Medina N., Zivanovic Y., Esnault C., Yang T., Jacquet M., Janin J., Duguet M., Perasso R., Li Z.G. (2000) Fine organization of Bombyx mori fibroin heavy chain gene. Nucleic Acids Res. 28:2413–2419CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • Yu-Qing Zhang
    • 1
  • Wei-De Shen
    • 1
  • Ru-Li Xiang
    • 1
  • Lan-Jian Zhuge
    • 2
  • Wei-Jian Gao
    • 2
  • Wen-Bao Wang
    • 2
  1. 1.Silk Biotechnol. Lab., School of Life ScienceSoochow University702-303 Room, No. 1 Hengyi Road, Dushuhu Higher Education Town, Suzhou, 215123P.R. China
  2. 2.Analytical CenterSoochow UniversitySuzhouP.R. China

Personalised recommendations