Pharmaceutical Research

, Volume 21, Issue 2, pp 344–353 | Cite as

Uptake and Cytotoxicity of Chitosan Molecules and Nanoparticles: Effects of Molecular Weight and Degree of Deacetylation

  • Min Huang
  • Eugene Khor
  • Lee-Yong Lim


Purpose. To evaluate the effects of molecular weight (Mw) and degree of deacetylation (DD) on the cellular uptake and in vitro cytotoxicity of chitosan molecules and nanoparticles.

Methods. Chemical depolymerization and reacetylation produced chitosans of Mw 213,000 to 10,000 and DD 88-46%, respectively. Chitosan was labeled with FITC and transformed into nanoparticles by ionotropic gelation. Uptake of chitosan by confluent A549 cells was quantified by fluorometry, and in vitro cytotoxicity was evaluated by the MTT and neutral red uptake assays.

Results. Nanoparticle uptake was a saturable event for all chitosan samples, with the binding affinity and uptake capacity decreasing with decreasing polymer Mw and DD. Uptake fell by 26% when Mw was decreased from 213,000 to 10,000, and by 41% when DD was lowered from 88% to 46%; the uptake data correlated with the ζ potential of the nanoparticles. Uptake of chitosan molecules did not exhibit saturation kinetics and was less dependent on Mw and DD. Postuptake quenching with trypan blue indicated that the cell-associated chitosan nanoparticles were internalized, but not the cell-associated chitosan molecules. Chitosan molecules and nanoparticles exhibited comparable cytotoxicity, yielding similar IC50 and IC20 values when evaluated against the A549 cells. Cytotoxicity of both chitosan entities was attenuated by decreasing polymer DD but was less affected by a lowering in Mw.

Conclusions. Transforming chitosan into nanoparticles modified the mechanism of cellular uptake but did not change the cytotoxicity of the polymer toward A549 cells. Chitosan DD had a greater influence than Mw on the uptake and cytotoxicity of chitosan nanoparticles because of its effect on the ζ potential of the nanoparticles.

molecular weight degree of deacetylation chitosan uptake cytotoxicity 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    K. A. Janes, M. P. Fresneau, A. Marazuela, A. Fabra, and M. J. Alonso. Chitosan nanoparticles as delivery systems for doxorubicin. J. Control. Rel. 73:255-267 (2001).Google Scholar
  2. 2.
    H.-Q. Mao, K. Roy, V. L. Troung-Le, K. A. Janes, K. Y. Lin, Y. Wang, J. T. August, and K. W. Leong. Chitosan-DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency. J. Control. Rel. 70:399-421 (2001).Google Scholar
  3. 3.
    L. Illum, I. Jabbal-Gill, M. Hinchcliffe, A. N. Fisher, and S. S. Davis. Chitosan as a novel nasal delivery system for vaccines. Adv. Drug Deliv. Rev. 51:81-96 (2001).Google Scholar
  4. 4.
    M. Huang, Z. S. Ma, E. Khor, and L.-Y. Lim. Uptake of FITC-chitosan nanoparticles by A549 cells. Pharm. Res. 19:1488-1494 (2002).Google Scholar
  5. 5.
    Z. S. Ma and L.-Y. Lim. Uptake of chitosan and associated insulin in the Caco-2 cell monolayers: a comparison between chitosan molecules and chitosan nanoparticles. Pharm. Res. 20:1812-1819 (2003).Google Scholar
  6. 6.
    H. S. Blair, J. Guthrie, T. K. Law, and P. Turkington. Chitosan and modified chitosan membranes. I. Preparation and characterization. J. Appl. Polym. Sci. 33:641-656 (1987).Google Scholar
  7. 7.
    K. M. Varum, M. M. Mhyr, R. J. N. Hjerde, and O. Smidsrod. In vitro degradation rates of partially N-acetylated chitosans in human serum. Carbohydr. Res. 299:99-101 (1997).Google Scholar
  8. 8.
    E. Khor. Chitin: Fulfilling a Biomaterials Promise. Elsevier Science, Oxford, UK (2001).Google Scholar
  9. 9.
    D. F. Williams. Progress in biomedical engineering. 4. Definitions in biomaterials. In: Williams, D.F. Editor. Proceedings of a Consensus Conference of the European Society for Materials, Chester, UK. Elsevier, Amsterdam. (1987).Google Scholar
  10. 10.
    K. Y. Lee, W. S. Ha, and H. L. Park. Blood compatibility and biodegradability of partially N-acetylated chitosan derivatives. Biomaterials 16:1211-1216 (1995).Google Scholar
  11. 11.
    K. Tomihata and Y. Ikada. In vitro and in vivo degradation of films of chitin and its deacetylated derivatives. Biomaterials 18:567-575 (1997).Google Scholar
  12. 12.
    H. Zhang and S. H. Neau. In vitro degradation of chitosan by a commercial enzyme preparation: effect of molecular weight and degree of deacetylation. Biomaterials 22:1653-1658 (2001).Google Scholar
  13. 13.
    B. Carreño-Gómez and R. Duncan. Evaluation of the biological properties of soluble chitosan and chitosan microspheres. Int. J. Pharm. 148:231-240 (1997).Google Scholar
  14. 14.
    N. G. M. Schipper, K. M. Varum, and P. Artursson. Chitosans as absorption enhancers for poorly absorbable drugs.1: influence of molecular weight and degree of acetylation on drug transport across human intestinal epithelial (Caco-2) cells. Pharm. Res. 13:1686-1691 (1996).Google Scholar
  15. 15.
    Q.P. Peniston and E. L. Johnson. Process for depolymerization of chitosan. US Patent 3,922,260, November 25, 1975.Google Scholar
  16. 16.
    S. Hirano, Y. Kondo, and K. Fujii. Preparation of acetylation derivatives of modified chito-oligosaccharides by the depolymerisation of partially N-acetylated chitosan with nitrous acid. Carbohydr. Res. 144:338-341 (1985).Google Scholar
  17. 17.
    P. Calvo, C. Remuñn-López, J. L. Vila-Jato, and M. J. Alonso. Novel hydrophilic chitosan-polyethylene oxide nanoparticles as protein carriers. J. Appl. Polym. Sci. 63:125-132 (1997).Google Scholar
  18. 18.
    D. J. Giard. In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J. Natl. Cancer Inst. 51:1417-1423 (1973).Google Scholar
  19. 19.
    T. Mosmann. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity. J. Immunol. Methods 65:55-63 (1983).Google Scholar
  20. 20.
    E. Borenfreund and J. A. Puerner. Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol. Lett. 24:119-124 (1985).Google Scholar
  21. 21.
    S. C. Tan. A study on the isolation and characterization of fungal chitosan. Thesis (M.Sc.), School of Biological Sciences, Faculty of Science, National University of Singapore (1998).Google Scholar
  22. 22.
    S. C. Tan, E. Khor, T. K. Tan, and S. M. Wong. The degree of deacetylation of chitosan: advocating the first derivative UV-spectrophotometry method of determination. Talanta 45:713-719 (1998).Google Scholar
  23. 23.
    C. P. Wan, C. S. Park, and B. H. S. Lau. A rapid and simple microfluorometric phagocytosis assay. J. Immunol. Methods 162:1-7 (1993).Google Scholar
  24. 24.
    S. Sahlin, J. Hed, and I. Rundquist. Differentiation between attached and ingested immune complexes by a fluorescence quenching cytofluorometric assay. J. Immunol. Methods 60:115-124 (1983).Google Scholar
  25. 25.
    T. Jung, W. Kamm, A. Breitenbach, E. Kaiserling, J. X. Xiao, and T. Kissel. Biodegradable nanoparticles for oral delivery of peptides: is there a role for polymers to affect mucosal uptake? Eur. J. Pharm. Biopharm. 50:147-160 (2000).Google Scholar
  26. 26.
    P. Jani, D. E. McCarthy, and A. T. Florence. Nanosphere and microsphere uptake via Peyer's patches: observation of the rate of uptake in the rat after a single oral dose. Int. J. Pharm. 86:239-246 (1992).Google Scholar
  27. 27.
    M. D. Hughes, M. Hussain, Q. Nawaz, P. Sayyed, and S. Akhtar. The cellular delivery of antisense oligonucleotides and ribozymes. Drug Discov. Today 6:303-315 (2001).Google Scholar
  28. 28.
    Y. Yasuomi, S. Kazuya, N Makiya, Y. Fumiyoshi, Y. Kiyoshi, H. Mitsuru, and T. Yoshinobu. Pharmacokinetic analysis of in vivo disposition of succinylated proteins targeted to liver nonparenchymal cells via scavenger receptors: Importance of molecular size and negative charge density for in vivo recognition by receptors. J. Pharmacol. Exp. Ther. 301:467-477 (2002).Google Scholar
  29. 29.
    S. W. Chang, J. Y. Westcott, J. E. Henson, and N. F. Voelkel. Pulmonary vascular injury by polycations in perfused rat lungs. J. Appl. Physiol. 62:1932-1943 (1987).Google Scholar
  30. 30.
    H. M. Ekrami and W. C. Shen. Carbamylation decreases the cytotoxicity but not the drug-carrier properties of polylysines. J. Drug Target. 2:469-475 (1995).Google Scholar
  31. 31.
    L. Dekie, V. Toncheva, P. Dubruel, E. H. Schacht, L. Barrett, and L. W. Seymour. Poly-L-glutamic acid derivatives as vectors for gene therapy. J. Control. Rel. 65:187-202 (2000).Google Scholar
  32. 32.
    P. Ferruti, S. Knobloch, E. Ranucci, R. Duncan, and E. Gianasi. A novel modification of poly(L-lysine) leading to a soluble cationic polymer with reduced toxicity and with potential as a transfection agent. Macromol. Chem. Phys. 199:2565-2575 (1998).Google Scholar

Copyright information

© Plenum Publishing Corporation 2004

Authors and Affiliations

  1. 1.Department of PharmacyNational University of SingaporeSingapore

Personalised recommendations