Chidinma O. Ibie
Novel polyelectrolyte complexes for oral insulin delivery.
Ibie, Chidinma O.
Colin J. Thompson
Rachel M. Knott
Oral delivery of insulin used for the management of Type 1 diabetes could be referred to as one of the major long-term goals of diabetes research. However, the bioavailability of orally-administrered insulin is significantly compromised by enzymatic degradation in the GI tract, and poor enteral absorption of the protein due to its macromolecular size and hydrophilicity. Nano-sized polymer-protein polyelectrolyte complexes (PECS) formed by electrostatic interactions between insulin and Polyallylamine-based polymers at pH 7.4 have been adapted to facilitate oral insulin delivery. Polyallylamine (15kDa) was quaternised by methylation of its primary amines using methyl iodide, to yield quaternised Paa (QPaa). The average level of polymer quaternisation was determined by elemental analysis and was found to be 72 plus-minus 2 mol%. Subsequent thiolation of Paa and QPaa involved carbodiimide mediated conjugation to N-acetylcysteine (NAC) and modification of the polymers using 2-iminothiolane hydrochloride. These two different thiolation procedures yielded their respective NAC and 4-thibutylamidine (TBA) conjugates: Paa-NAC/QPaa-NAC and Paa-TBA/QPaa-TBA. Estimation of the free thiol content of these thiomers by iodometric titration showed that both Paa-NAC and QPaa-NAC displayed 60 plus-minus 1.2, and 60 plus-mines 4.3 micro-mol free thiol groups per gram polymer, while Paa-TBA and QPaa-TBA conjugates displayed 490 plus-minus 18 and 440 plus-minus 21 micro-mol free thiol groups per gram polymer respectively. Mixing optimal mass ratios of each polymer and insulin in Tris buffer at pH 7.4 resulted in the formation of soluble nanocomplexes. Complexes were characterised by tansmittance measurements, particle size analysis, zeta potential, complexation efficiency and transmission electron microscopy (TEM). Stable polymer-insulin complexes were observed to have hydrodynamic sizes between 50-200nm, positively-charged zeta potential values ranging between 20-40 mV and high insulin complexation efficiency (greater than 90%). Complexation of insulin with TBA conjugates, however, appeared to alter insulin conformation affecting the detection of complexed insulin by HPLC. TEM analysis revealed the formation of bilayered nanovessicles as well as conventional single-layered nanoparticles on complexation of insulin with QPaa and thiolated Paa/QPaa derivatives. In-vitro assessments of enzyme-protective effect of QPaa, Paa-NAC and QPaa-NAC insulin complexes showed that, when compared to a free insulin control, all the aforementioned complexes could protect insulin from degradation by trypsin and alpha-chymotrypsin, but not from pepsin. In-vitro mucin adsorption assays showed that all polymers exhibited a similar mucoadhesive profile with their corresponding insulin PEC, with thiolated Paa derivatives adsorbing greater than 20% more mucin than Paa. Thiolation of QPaa did not result in a noticeable improvement in its mucoadhesive capacity, indicating that polymer-mucin thioldisulphide interactions may be hindered by the presence of quaternary groups. The IC50 of each polymer was determined by MTT assays carried out on Caco-2 cells with or without the inclusion of a 24-hour cell recovery period. An MTT assay conducted without a recovery period indicated that quaternisation of Paa was associated with a six-fold improvement in its IC50. Additionally, cells subjected to a 24-hour recovery period following treatment with QPaa (0.001-4 mgml-1) showed no signs of toxicity. Thiolation of Paa resulted in slight (less than or equal to two-fold) improvements in IC50, while thiolation of QPaa resulted in a decrease in IC50 values obtained both with and without a cell recovery period. Each polymer was subsequently labelled with rhodamine B isothiocyanate (RBITC) and complexed with fluorescein isothiocyanate (FITC)-insulin. Monitoring uptake of these complexes by Caco-2 cells using fluorescence microscopy with DAPI staining indicated that uptake of QPaa and QPaa-TBA complexes was mainly intracellular being localised within the perinuclear area of cells highlighted by DAPI. Hence, intracellular uptake of PECS by Caco-2 cells was enhanced by Paa quaternisation and TBA-based thiolation of QPaa.
IBIE, C.O. 2013. Novel polyelectrolyte complexes for oral insulin delivery. Robert Gordon University, PhD thesis.
|Publication Date||Nov 1, 2013|
|Deposit Date||Mar 17, 2014|
|Publicly Available Date||Mar 17, 2014|
|Keywords||Polyelectrolyte complexation; Insulin; Polyallylamine; Mucoadhesion; Transmucosal transport; Thiolation; Quaternisation; Nanoparticles|
IBIE 2013 Novel polyelectrolyte complexes
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Copyright: the author and Robert Gordon University