title: In-vitro evaluation of the effect of polymer structure on uptake of novel polymer-insulin polyelectrolyte complexes by human epithelial cells.
abstract: The biocompatibility and cellular uptake of polymer, insulin polyelectrolyte complexes (PECs) prepared using polyallylamine-based polymers was evaluated in-vitro using Caco-2 cell monolayers as a predictive model for human small intestinal epithelial cells. Poly(allyl amine) (PAA) and Quaternised PAA (QPAA) were thiolated using either carbodiimide mediated conjugation to N-acetylcysteine (NAC) or reaction with 2-iminothiolane hydrochloride yielding their NAC and 4-thiobutylamidine (TBA) conjugates, respectively. The effect of polymer quaternisation and/or thiolation on the IC50 of PAA was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay carried out on Caco-2 cells (with and without a 24 h recovery period after samples were removed). Uptake of PECs by Caco-2 cells was monitored by microscopy using fluorescein isothiocyanate (FITC) labelled insulin and rhodamine-labelled polymers at polymer:insulin ratios (4:5) after 0.5, 1, 2 and 4 h incubation in growth media (±calcium) and following pre-incubation with insulin. MTT results indicated that quaternisation of PAA was associated with an improvement in IC50 values; cells treated with QPAA (0.001-4 mg mL(-1)) showed no signs of toxicity following a 24 h cell recovery period, while thiolation of QPAA resulted in a decrease in the IC50. Cellular uptake studies showed that within 2-4 h, QPAA and QPAA-TBA insulin PECs were taken up intracellularly, with PECs being localised within the perinuclear area of cells. Further investigation showed that uptake of PECs was unaffected when calcium-free media was used, while presaturating insulin receptors affected the uptake of QPAA, insulin PECs, but not QPAA-TBA PECs. The biocompatibility of PAA and uptake of insulin was improved by both thiol and quaternary substitution.
authors.affiliation: Institute for Heath and Wellbeing Research, Robert Gordon University, Aberdeen, AB10 7GJ, UK.
authors.forename: C
authors.initials: C
authors.lastname: Ibie
authors.affiliation: Institute for Heath and Wellbeing Research, Robert Gordon University, Aberdeen, AB10 7GJ, UK.
authors.forename: R
authors.initials: R
authors.lastname: Knott
authors.affiliation: Institute for Heath and Wellbeing Research, Robert Gordon University, Aberdeen, AB10 7GJ, UK.
authors.forename: C J
authors.initials: CJ
authors.lastname: Thompson
mesh_terms: D000111:Acetylcysteine / Q000737:chemistry / Q000494:pharmacology; D000578:Amidines / Q000737:chemistry / Q000494:pharmacology; D001692:Biological Transport; D018938:Caco-2 Cells; D002470:Cell Survival / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects; D006801:Humans; D007328:Insulin / Q000737:chemistry / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D011073:Polyamines / Q000737:chemistry / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000737:chemistry / Q000494:pharmacology
chemical_list: D000578:Amidines; D007328:Insulin; D011073:Polyamines; D000644:Quaternary Ammonium Compounds; C063994:polyallylamine; D000111:Acetylcysteine
keywords: Fluorescence microscopy; Insulin; MTT assay; Polyelectrolyte complex; Quaternization; Thiolation