Research Paper

Pharmaceutical Research

, Volume 22, Issue 12, pp 2058-2068

First online:

Uptake and Transport of PEG-Graft-Trimethyl-Chitosan Copolymer–Insulin Nanocomplexes by Epithelial Cells

  • Shirui MaoAffiliated withDepartment of Pharmaceutics and Biopharmacy, Philipps-University of MarburgCollege of Pharmacy, Shenyang Pharmaceutical University
  • , Oliver GermershausAffiliated withDepartment of Pharmaceutics and Biopharmacy, Philipps-University of Marburg
  • , Dagmar FischerAffiliated withDepartment of Pharmaceutics and Biopharmacy, Philipps-University of Marburg
  • , Thomas LinnAffiliated withMedical Clinic III and Policlinic, Justus Liebig University
  • , Robert SchnepfAffiliated withComplex Biosystems
  • , Thomas KisselAffiliated withDepartment of Pharmaceutics and Biopharmacy, Philipps-University of Marburg Email author 

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Purpose

The effect of chitosan and polyethylene glycol (PEG)ylated trimethyl chitosan copolymer structure on the uptake and transport of insulin nanocomplexes was evaluated and transport mechanisms were investigated.

Methods

Insulin nanocomplexes were prepared from chitosan and its copolymers by self-assembly. Complex uptake in Caco-2 cells was quantified by measuring the cell-associated fluorescence and cellular localization was visualized by confocal laser scanning microscopy (CLSM) using tetra-methyl-rhodamine isothiocyanate-labeled insulin. The transport of selected insulin complexes through Caco-2 monolayers was then investigated and compared with in vivo uptake by nasal epithelium in diabetic rats.

Results

All complexes were 200–400 nm in diameter, positively charged, and displayed an insulin loading efficiency of approximately 90%. In vitro release of insulin from the complexes was dependent on the medium pH. Insulin uptake was enhanced by nanocomplex formation, and was dependent on incubation time, temperature, and concentration. Complex uptake in Caco-2 cells was inhibited by 25.2 ± 1.3%, 13.0 ± 1.0%, and 16.6 ± 0.7% in the presence of cytochalasin D, sodium azide, and 2,4-dinitrophenol, respectively. The uptake mechanism was assumed to be adsorptive endocytosis. Additionally, cell uptake efficiency was shown to be influenced by a combination of polymer molecular weight, viscosity, and positive charge density. However, none of the nanocomplexes displayed improved transport properties when compared to insulin transport data after 2 h incubation with Caco-2 monolayers. This result was further confirmed with animal experiments.

Conclusions

Small, stable insulin nanocomplexes were formed using PEGylated trimethyl chitosan copolymers, which significantly enhanced the uptake of insulin in Caco-2 cells by adsorptive endocytosis. However, nanocomplexation did not seem to enhance transcellular insulin transport across cell monolayers, which is in line with animal data in rats. This implies that PEGylated trimethyl chitosan complexes with insulin need further optimization and the Caco-2 cell line is a predictable in vitro cell culture model for drug absorption.

Key Words

Caco-2 cells insulin nanocomplexes PEGylation trimethyl chitosan uptake