Drug Delivery and Translational Research

, Volume 6, Issue 4, pp 365–379 | Cite as

Improved mucoadhesion and cell uptake of chitosan and chitosan oligosaccharide surface-modified polymer nanoparticles for mucosal delivery of proteins

  • Sathish Dyawanapelly
  • Uday Koli
  • Vimisha Dharamdasani
  • Ratnesh Jain
  • Prajakta Dandekar
Research Article


The main aim of the present study was to compare mucoadhesion and cellular uptake efficiency of chitosan (CS) and chitosan oligosaccharide (COS) surface-modified polymer nanoparticles (NPs) for mucosal delivery of proteins. We have developed poly (d, l-lactide-co-glycolide) (PLGA) NPs, surface-modified COS-PLGA NPs and CS-PLGA NPs, by using double emulsion solvent evaporation method, for encapsulating bovine serum albumin (BSA) as a model protein. Surface modification of NPs was confirmed using physicochemical characterization methods such as particle size and zeta potential, SEM, TEM and FTIR analysis. Both surface-modified PLGA NPs displayed a slow release of protein compared to PLGA NPs. Furthermore, we have explored the mucoadhesive property of COS as a material for modifying the surface of polymeric NPs. During in vitro mucoadhesion test, positively charged COS-PLGA NPs and CS-PLGA NPs exhibited enhanced mucoadhesion, compared to negatively charged PLGA NPs. This interaction was anticipated to improve the cell interaction and uptake of NPs, which is an important requirement for mucosal delivery of proteins. All nanoformulations were found to be safe for cellular delivery when evaluated in A549 cells. Moreover, intracellular uptake behaviour of FITC-BSA loaded NPs was extensively investigated by confocal laser scanning microscopy and flow cytometry. As we hypothesized, positively charged COS-PLGA NPs and CS-PLGA NPs displayed enhanced intracellular uptake compared to negatively charged PLGA NPs. Our results demonstrated that CS- and COS-modified polymer NPs could be promising carriers for proteins, drugs and nucleic acids via nasal, oral, buccal, ocular and vaginal mucosal routes.


Chitosan Chitosan oligosaccharide Poly (d, l-lactide-co-glycolide) Nanoparticle Protein Mucosal delivery 



Mr. Sathish Dyawanapelly would like to thank Department of Biotechnology (BT/PR5372/MED/29/489/2012), Govt. of India for the fellowship. Dr. Prajakta Dandekar is thankful to Ramanujan Fellowship Grant (SR/S2/RJN-139/2011), DST, Govt. of India. Dr. Ratnesh Jain is thankful to Ramalingaswami Fellowship (BT/RLF/RE-ENTRY/51/2011), DBT, Govt. of India. The authors are grateful for the technical assistance of Maruthi Prasanna, Nishant Jain and Kireeti Kumar Kota. PURAC Biopolymers, Netherlands  and Evonik, India is acknowledged for the generous gift of PLGA. Authors are thankful to DST Nanomission (SR/NM/NS 1145/2012) for Imaging Flow Cytometer facility. Dr. Ratnesh Jain is thankful to DST-FIST (SR/FST/ETII-058/2013) for Confocal Microscopy facility.

Compliance with ethical standards

Statement of human and animal rights

‘No animal or human studies were carried out by the authors for this article.’

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

13346_2016_295_MOESM1_ESM.docx (281 kb)
ESM 1 (DOCX 280 kb)


  1. 1.
    Yadav SC, Kumari A, Yadav R. Development of peptide and protein nanotherapeutics by nanoencapsulation and nanobioconjugation. Peptides. 2011;32(1):173–87.CrossRefPubMedGoogle Scholar
  2. 2.
    Gu W-X, Zhu M, Song N, Du X, Yang Y-W, Gao H. Reverse micelles based on biocompatible β-cyclodextrin conjugated polyethylene glycol block polylactide for protein delivery. J Mater Chem B. 2015;3(2):316–22.CrossRefGoogle Scholar
  3. 3.
    Irie T, Uekama K. Cyclodextrins in peptide and protein delivery. Adv Drug Deliv Rev. 1999;36(1):101–23.CrossRefPubMedGoogle Scholar
  4. 4.
    Park K, Kwon IC, Park K. Oral protein delivery: current status and future prospect. React Funct Polym. 2011;71(3):280–7.CrossRefGoogle Scholar
  5. 5.
    Lu Y, Yang J, Sega E. Issues related to targeted delivery of proteins and peptides. AAPS J. 2006;8(3):E466–78.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Stolnik S, Shakesheff K. Formulations for delivery of therapeutic proteins. Biotechnol Lett. 2009;31(1):1–11.CrossRefPubMedGoogle Scholar
  7. 7.
    Mundargi RC, Babu VR, Rangaswamy V, Patel P, Aminabhavi TM. Nano/micro technologies for delivering macromolecular therapeutics using poly (D, L-lactide-co-glycolide) and its derivatives. J Control Release. 2008;125(3):193–209.CrossRefPubMedGoogle Scholar
  8. 8.
    Dong Y, Feng S-S. Poly (d, l-lactide-co-glycolide)/montmorillonite nanoparticles for oral delivery of anticancer drugs. Biomaterials. 2005;26(30):6068–76.CrossRefPubMedGoogle Scholar
  9. 9.
    Blanco MD, Alonso MJ. Development and characterization of protein-loaded poly (lactide-co-glycolide) nanospheres. Eur J Pharm Biopharm. 1997;43(3):287–94.CrossRefGoogle Scholar
  10. 10.
    Cun D, Foged C, Yang M, Frøkjær S, Nielsen HM. Preparation and characterization of poly (DL-lactide-co-glycolide) nanoparticles for siRNA delivery. Int J Pharm. 2010;390(1):70–5.CrossRefPubMedGoogle Scholar
  11. 11.
    Tahara K, Yamamoto H, Hirashima N, Kawashima Y. Chitosan-modified poly (D, L-lactide-co-glycolide) nanospheres for improving siRNA delivery and gene-silencing effects. Eur J Pharm Biopharm. 2010;74(3):421–6.CrossRefPubMedGoogle Scholar
  12. 12.
    Kumar MNVR, Bakowsky U, Lehr CM. Preparation and characterization of cationic PLGA nanospheres as DNA carriers. Biomaterials. 2004;25(10):1771–7.CrossRefGoogle Scholar
  13. 13.
    Verma A, Stellacci F. Effect of surface properties on nanoparticle-cell interactions. Small. 2010;6(1):12–21.CrossRefPubMedGoogle Scholar
  14. 14.
    Faraji AH, Wipf P. Nanoparticles in cellular drug delivery. Bioorg Med Chem. 2009;17(8):2950–62.CrossRefPubMedGoogle Scholar
  15. 15.
    Labhasetwar V. Nanotechnology for drug and gene therapy: the importance of understanding molecular mechanisms of delivery. Curr Opin Biotechnol. 2005;16(6):674–80.CrossRefPubMedGoogle Scholar
  16. 16.
    RaviKumar MNV, Mohapatra SS, Kong X, Jena PK, Bakowsky U, Lehrd CM. Cationic poly (lactide-co-glycolide) nanoparticles as efficient in vivo gene transfection agents. J Nanosci Nanotechnol. 2004;4(8):990–4.CrossRefGoogle Scholar
  17. 17.
    Peniche C, Argüelles-Monal WW, Peniche H, Acosta N. Chitosan: an attractive biocompatible polymer for microencapsulation. Macromol Biosci. 2003;3(10):511–20.CrossRefGoogle Scholar
  18. 18.
    Yang R, Shim W-S, Cui F-D, Cheng G, Han X, Jin Q-R, et al. Enhanced electrostatic interaction between chitosan-modified PLGA nanoparticle and tumor. Int J Pharm. 2009;371(1):142–7.CrossRefPubMedGoogle Scholar
  19. 19.
    Mao S, Sun W, Kissel T. Chitosan-based formulations for delivery of DNA and siRNA. Adv Drug Deliv Rev. 2010;62(1):12–27.CrossRefPubMedGoogle Scholar
  20. 20.
    Ragelle H, Vandermeulen G, Véronique P. Chitosan-based siRNA delivery systems. J Control Release. 2013;172(1):207–18.CrossRefPubMedGoogle Scholar
  21. 21.
    Köping-Höggård M, Vårum KM, Issa M, Danielsen S, Christensen BE, Stokke BT, et al. Improved chitosan-mediated gene delivery based on easily dissociated chitosan polyplexes of highly defined chitosan oligomers. Gene Ther. 2004;11(19):1441–52.CrossRefPubMedGoogle Scholar
  22. 22.
    Richardson SW, Kolbe HJ, Duncan R. Potential of low molecular mass chitosan as a DNA delivery system: biocompatibility, body distribution and ability to complex and protect DNA. Int J Pharm. 1999;178(2):231–43.CrossRefPubMedGoogle Scholar
  23. 23.
    Hu F-Q, Liu L-N, Du Y-Z, Yuan H. Synthesis and antitumor activity of doxorubicin conjugated stearic acid-g-chitosan oligosaccharide polymeric micelles. Biomaterials. 2009;30(36):6955–63.CrossRefPubMedGoogle Scholar
  24. 24.
    Huang X, Du Y-Z, Yuan H, Hu F-Q. Preparation and pharmacodynamics of low-molecular-weight chitosan nanoparticles containing insulin. Carbohydr Polym. 2009;76(3):368–73.CrossRefGoogle Scholar
  25. 25.
    Biswas S, Chattopadhyay M, Sen KK, Saha MK. Development and characterization of alginate coated low molecular weight chitosan nanoparticles as new carriers for oral vaccine delivery in mice. Carbohydr Polym. 2015;121:403–10.CrossRefPubMedGoogle Scholar
  26. 26.
    Hu F-Q, Zhao M-D, Yuan H, You J, Du Y-Z, Zeng S. A novel chitosan oligosaccharide-stearic acid micelles for gene delivery: Properties and in vitro transfection studies. Int J Pharm. 2006;315(1):158–66.CrossRefPubMedGoogle Scholar
  27. 27.
    Amoozgar Z, Park J, Lin Q, Yeo Y. Low molecular-weight chitosan as a pH-sensitive stealth coating for tumor-specific drug delivery. Mol Pharm. 2012;9(5):1262–70.PubMedPubMedCentralGoogle Scholar
  28. 28.
    Murata M, Nakano K, Tahara K, Tozuka Y, Takeuchi H. Pulmonary delivery of elcatonin using surface-modified liposomes to improve systemic absorption: polyvinyl alcohol with a hydrophobic anchor and chitosan oligosaccharide as effective surface modifiers. Eur J Pharm Biopharm. 2012;80(2):340–6.CrossRefPubMedGoogle Scholar
  29. 29.
    Ying X-Y, Cui D, Yu L, Du Y-Z. Solid lipid nanoparticles modified with chitosan oligosaccharides for the controlled release of doxorubicin. Carbohydr Polym. 2011;84(4):1357–64.CrossRefGoogle Scholar
  30. 30.
    Liu X, Huang H, Liu G, Zhou W, Chen Y, Jin Q, et al. Multidentate zwitterionic chitosan oligosaccharide modified gold nanoparticles: stability, biocompatibility and cell interactions. Nanoscale. 2013;5(9):3982–91.CrossRefPubMedGoogle Scholar
  31. 31.
    Bae KH, Park M, Do MJ, Lee N, Ryu JH, Kim GW, et al. Chitosan oligosaccharide-stabilized ferrimagnetic iron oxide nanocubes for magnetically modulated cancer hyperthermia. ACS Nano. 2012;6(6):5266–73.CrossRefPubMedGoogle Scholar
  32. 32.
    Shukla S, Jadaun A, Arora V, Sinha RK, Biyani N, Jain VK. In vitro toxicity assessment of chitosan oligosaccharide coated iron oxide nanoparticles. Toxic Rep. 2015;2:27–39.CrossRefGoogle Scholar
  33. 33.
    Jain S, Datta M. Montmorillonite-PLGA nanocomposites as an oral extended drug delivery vehicle for venlafaxine hydrochloride. Appl Clay Sci. 2014;99:42–7.CrossRefGoogle Scholar
  34. 34.
    Gan Q, Wang T. Chitosan nanoparticle as protein delivery carrier-systematic examination of fabrication conditions for efficient loading and release. Colloids Surf B: Biointerfaces. 2007;59(1):24–34.CrossRefPubMedGoogle Scholar
  35. 35.
    Danhier F, Lecouturier N, Vroman B, Jérôme C, Marchand-Brynaert J, Feron O, et al. Paclitaxel-loaded PEGylated PLGA-based nanoparticles: in vitro and in vivo evaluation. J Control Release. 2009;133(1):11–7.CrossRefPubMedGoogle Scholar
  36. 36.
    Wang G, Yu B, Wu Y, Huang B, Yuan Y, Liu CS. Controlled preparation and antitumor efficacy of vitamin E TPGS-functionalized PLGA nanoparticles for delivery of paclitaxel. Int J Pharm. 2013;446(1):24–33.CrossRefPubMedGoogle Scholar
  37. 37.
    Md S, Khan RA, Mustafa G, Chuttani K, Baboota S, Sahni JK, et al. Bromocriptine loaded chitosan nanoparticles intended for direct nose to brain delivery: pharmacodynamic, pharmacokinetic and scintigraphy study in mice model. Eur J Pharm Sci. 2013;48(3):393–405.CrossRefPubMedGoogle Scholar
  38. 38.
    Parveen S, Sahoo SK. Long circulating chitosan/PEG blended PLGA nanoparticle for tumor drug delivery. Eur J Pharmacol. 2011;670(2):372–83.CrossRefPubMedGoogle Scholar
  39. 39.
    Philippova OE, Korchagina EV, Volkov EV, Smirnov VA, Khokhlov AR, Rinaudo M. Aggregation of some water-soluble derivatives of chitin in aqueous solutions: role of the degree of acetylation and effect of hydrogen bond breaker. Carbohydr Polym. 2012;87(1):687–94.CrossRefGoogle Scholar
  40. 40.
    He C, Hu Y, Yin L, Tang C, Yin C. Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles. Biomaterials. 2010;31(13):3657–66.CrossRefPubMedGoogle Scholar
  41. 41.
    Huang P, Li Z, Hu H, Cui D. Synthesis and characterization of bovine serum albumin-conjugated copper sulfide nanocomposites. J Nanomater. 2010;2010:33.Google Scholar
  42. 42.
    Kaewsaneha C, Opaprakasit P, Polpanich D, Smanmoo S, Tangboriboonrat P. Immobilization of fluorescein isothiocyanate on magnetic polymeric nanoparticle using chitosan as spacer. J Colloid Interface Sci. 2012;377(1):145–52.CrossRefPubMedGoogle Scholar
  43. 43.
    Dandekar P, Jain R, Stauner T, Loretz B, Koch M, Wenz G, et al. A hydrophobic starch polymer for nanoparticle-mediated delivery of docetaxel. Macromol Biosci. 2012;12(2):184–94.CrossRefPubMedGoogle Scholar
  44. 44.
    Paolicelli P, Prego C, Sanchez A, Alonso MJ. Surface-modified PLGA-based nanoparticles that can efficiently associate and deliver virus-like particles. Nanomedicine. 2010;5(6):843–53.CrossRefPubMedGoogle Scholar
  45. 45.
    Cohen-Sela E, Chorny M, Koroukhov N, Danenberg HD, Golomb G. A new double emulsion solvent diffusion technique for encapsulating hydrophilic molecules in PLGA nanoparticles. J Control Release. 2009;133(2):90–5.CrossRefPubMedGoogle Scholar
  46. 46.
    Fonte P, Soares S, Sousa F, Costa A, Seabra V, Reis S, et al. Stability study perspective of the effect of freeze-drying using cryoprotectants on the structure of insulin loaded into PLGA nanoparticles. Biomacromolecules. 2014;15(10):3753–65.CrossRefPubMedGoogle Scholar
  47. 47.
    Abdelwahed W, Degobert G, Stainmesse S, Fessi H. Freeze-drying of nanoparticles: formulation, process and storage considerations. Adv Drug Deliv Rev. 2006;58(15):1688–713.CrossRefPubMedGoogle Scholar
  48. 48.
    Sameti M, Bohr G, Kumar MNVR, Kneuer C, Bakowsky U, Nacken M, et al. Stabilisation by freeze-drying of cationically modified silica nanoparticles for gene delivery. Int J Pharm. 2003;266(1):51–60.CrossRefPubMedGoogle Scholar
  49. 49.
    De Jaeghere F, Allémann E, Feijen J, Kissel T, Doelker E, Gurny R. Freeze-drying and lyopreservation of diblock and triblock poly (lactic acid)-poly (ethylene oxide)(PLA-PEO) copolymer nanoparticles. Pharm Dev Technol. 2000;5(4):473–83.CrossRefPubMedGoogle Scholar
  50. 50.
    Holzer M, Vogel V, Mäntele W, Schwartz D, Haase W, Langer K. Physico-chemical characterisation of PLGA nanoparticles after freeze-drying and storage. Eur J Pharm Biopharm. 2009;72(2):428–37.CrossRefPubMedGoogle Scholar
  51. 51.
    Prajakta D, Ratnesh J, Chandan K, Suresh S, Grace S, Meera V, et al. Curcumin loaded pH-sensitive nanoparticles for the treatment of colon cancer. J Biomed Nanotechnol. 2009;5(5):445–55.CrossRefPubMedGoogle Scholar
  52. 52.
    Thasneem YM, Rekha MR, Sajeesh S, Sharma CP. Biomimetic mucin modified PLGA nanoparticles for enhanced blood compatibility. J Colloid Interface Sci. 2013;409:237–44.CrossRefPubMedGoogle Scholar
  53. 53.
    Ma FK, Li J, Kong M, Liu Y, An Y, Chen XG. Preparation and hydrolytic erosion of differently structured PLGA nanoparticles with chitosan modification. Int J Biol Macromol. 2013;54:174–9.CrossRefPubMedGoogle Scholar
  54. 54.
    Guo M, Rong W-T, Hou J, Wang D-F, Lu Y, Wang Y, et al. Mechanisms of chitosan-coated poly (lactic-co-glycolic acid) nanoparticles for improving oral absorption of 7-ethyl-10-hydroxycamptothecin. Nanotechnology. 2013;24(24):245101.CrossRefPubMedGoogle Scholar
  55. 55.
    Pawar D, Mangal S, Goswami R, Jaganathan KS. Development and characterization of surface modified PLGA nanoparticles for nasal vaccine delivery: effect of mucoadhesive coating on antigen uptake and immune adjuvant activity. Eur J Pharm Biopharm. 2013;85(3):550–9.CrossRefPubMedGoogle Scholar
  56. 56.
    Jagani HV, Josyula VR, Palanimuthu VR, Hariharapura RC, Gang SS. Improvement of therapeutic efficacy of PLGA nanoformulation of siRNA targeting anti-apoptotic Bcl-2 through chitosan coating. Eur J Pharm Sci. 2013;48(4):611–8.CrossRefPubMedGoogle Scholar
  57. 57.
    Slütter B, Bal S, Keijzer C, Mallants R, Hagenaars N, Que I, et al. Nasal vaccination with N-trimethyl chitosan and PLGA based nanoparticles: nanoparticle characteristics determine quality and strength of the antibody response in mice against the encapsulated antigen. Vaccine. 2010;28(38):6282–91.CrossRefPubMedGoogle Scholar
  58. 58.
    He P, Davis SS, Illum L. In vitro evaluation of the mucoadhesive properties of chitosan microspheres. Int J Pharm. 1998;166(1):75–88.CrossRefGoogle Scholar
  59. 59.
    Burton JD. The MTT assay to evaluate chemosensitivity. Chemosensitivity. Springer. 2005. p. 69–78.Google Scholar
  60. 60.
    Grabowski N, Hillaireau H, Vergnaud J, Santiago LA, Kerdine-Romer S, Pallardy M, et al. Toxicity of surface-modified PLGA nanoparticles toward lung alveolar epithelial cells. Int J Pharm. 2013;454(2):686–94.CrossRefPubMedGoogle Scholar
  61. 61.
    Tahara K, Sakai T, Yamamoto H, Takeuchi H, Hirashima N, Kawashima Y. Improved cellular uptake of chitosan-modified PLGA nanospheres by A549 cells. Int J Pharm. 2009;382(1):198–204.CrossRefPubMedGoogle Scholar
  62. 62.
    Foster S, Duvall CL, Crownover EF, Hoffman AS, Stayton PS. Intracellular delivery of a protein antigen with an endosomal-releasing polymer enhances CD8 T-cell production and prophylactic vaccine efficacy. Bioconjug Chem. 2010;21(12):2205–12.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Controlled Release Society 2016

Authors and Affiliations

  1. 1.Department of Pharmaceutical Sciences and TechnologyInstitute of Chemical TechnologyMumbaiIndia
  2. 2.Departments of Chemical EngineeringInstitute of Chemical TechnologyMumbaiIndia

Personalised recommendations