title: Uptake and transport of PEG-graft-trimethyl-chitosan copolymer-insulin nanocomplexes by epithelial cells.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Pharmaceutics and Biopharmacy, Philipps-University of Marburg, Ketzerbach 63, D-35032, Marburg, Germany.
authors.forename: Shirui
authors.initials: S
authors.lastname: Mao
authors.forename: Oliver
authors.initials: O
authors.lastname: Germershaus
authors.forename: Dagmar
authors.initials: D
authors.lastname: Fischer
authors.forename: Thomas
authors.initials: T
authors.lastname: Linn
authors.forename: Robert
authors.initials: R
authors.lastname: Schnepf
authors.forename: Thomas
authors.initials: T
authors.lastname: Kissel
mesh_terms: D000465:Algorithms; D000818:Animals; D001692:Biological Transport; D018938:Caco-2 Cells; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D048271:Chitosan / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D007004:Hypoglycemic Agents / Q000008:administration & dosage* / Q000493:pharmacokinetics*; D007328:Insulin / Q000008:administration & dosage* / Q000493:pharmacokinetics*; D008027:Light; D008297:Male; D018613:Microscopy, Confocal; D008970:Molecular Weight; D049329:Nanostructures; D010316:Particle Size; D011092:Polyethylene Glycols / Q000737:chemistry*; D011237:Predictive Value of Tests; D051381:Rats; D012542:Scattering, Radiation; D013778:Tetrazolium Salts; D013844:Thiazoles; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism
chemical_list: D007004:Hypoglycemic Agents; D007328:Insulin; C508427:PEG-graft-trimethyl-chitosan copolymer; D013778:Tetrazolium Salts; D013844:Thiazoles; D011092:Polyethylene Glycols; D048271:Chitosan; C022616:thiazolyl blue