Abstract/Summary

Linear polyethyleneimine (L-PEI) has been extensively used in various fields, such as pharmaceutical formulations, gene delivery, and water treatment. Though L-PEI is considered as a potential gene delivery vector or as a pharmaceutical excipient, the applications of L-PEI are limited as L-PEI displays relatively high toxicity and low biocompatibility. The secondary amine groups within L-PEI can interact with cell membranes and the extracellular matrix, and these interactions are predominantly electrostatically driven. Herein, we selected succinic anhydride, phthalic anhydride, methacrylic anhydride, crotonic anhydride and maleic anhydride to modify L-PEI to improve functionality and lower its toxicity. Firstly, L-PEI was prepared by fully hydrolyzing commercially available poly(2-ethyl-2-oxazoline) (PEOZ, 50 kDa) and then reacted with different anhydrides. The obtained succinylated L-PEI, phthaylated L-PEI, methacrylated L-PEI, crotonylated L-PEI and maleylated L-PEI were fully characterized using 1 H-NMR and FTIR spectroscopies, turbidity-pH measurements and electrophoretic mobility. The resultant polymers (succinylated L-PEI, phthaylated L-PEI and maleylated L-PEI) were the polyampholytes which each have an isoelectric point (pHIEP), and two cationic polyelectrolytes methacrylated L-PEI, crotonylated L-PEI according to their structures. Water-soluble polymers generally exhibit mucoadhesive activity, interacting with mucin via electrostatic effects or hydrogen bonding or/and formation of interpenetrating layer between polymers and mucus gel. Mucoadhesion of polymers can provide significant opportunities when designing pharmaceutical formulations, such as tablets, films, patches and gels. However, the mucoadhesive properties of polyampholytes are rarely reported in the literature. This work thus explored the factors affecting the mucoadhesive properties of both synthetic and natural polyampholytes. Turbidimetric titrations and isothermal titration calorimetry (ITC) were conducted to investigate the interactions between polyampholytes and porcine gastric mucin in solutions. Both synthetic and natural polyampholyte demonstrated more pronounced interactions with mucin at pH<pHIEP than at pH≥pHIEP, where the polyampholytes are positively charged and mucin remains negatively charged. Electrostatic effects are predominantly responsible for their mucoadhesion whilst hydrogen bonding and hydrophobic effects have synergistic effects. A system of polyampholyte and fluorescein isothiocyanate (FITC) coated tablets were used to assess adhesion to porcine gastric mucosa at different pHs in essentially “static” systems. In addition, to reflecting fluid dynamics encountered on clinical application, the polyampholytes were labelled fluorescently and prepared in solutions to determine their retention using a fluorescence microscopy-based flow-through assay. These ex vivo assays confirmed that the polyampholytes exhibited superior mucoadhesive properties at pH<pHIEP. All these studies demonstrated solution pH and pHIEP of polyampholytes are primary factors affect mucoadhesive properties of polyampholytes, and hydrogen bonding, hydrophobic effects, water transport, capillary forces, penetration also contribute to mucoadhesion. To test the generalisability of these findings, the retention of methacrylated L-PEI, crotonylated L-PEI, maleylated L-PEI and succinylated L-PEI on bovine palpebral conjunctiva at physiological pH (pH=7.4) was assessed using a fluorescence microscopy-based flow-through assay. Methacrylated-L-PEI and crotonylated L-PEI exhibited strong mucoadhesive properties at pH 7.4, due to the formation of covalent bonding between unsaturated C=C moieties within these synthetic cationic polyelectrolytes and mucin thiol groups. Conversely, maleylated L-PEI and succinylated L-PEI were poorly-mucoadhesive since physiological pH was above their isoelectric point, leading to electrostatic repulsion between the polyampholytes and mucin. In addition, the contribution of amine groups within these polyelectrolytes to adhesion are minimal at pH=7.4, where these polyelectrolytes are either non-charged or negatively charged. Toxicological evaluation and irritation studies of the modified L-PEI derivatives were undertaken. In vivo assays with planaria and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cell viability assay, and slug mucosa irritation assay suggested anhydride modified L-PEIs alleviated the adverse toxicity effects seen for the parent L-PEI. In summary, modification of L-PEI with organic anhydrides enhanced mucoadhesive properties and biocompatibility of L-PEI, and reduced its toxicity, displaying great potential of modified L-PEI derivates as novel water-soluble functional excipients for mucoadhesive delivery systems.