Nanoaggregates based on new poly-hydroxyethyl-aspartamide copolymers for oral insulin absorption

The aim of this work was to produce copolymers with an appropriate hydrophilic/hydrophobic balance able to form nanoaggregates with protein molecules and to be used as ideal materials in the field of oral peptide/protein delivery. New anionic polymers obtained by the conjugation of carboxy-bearing ligands, like succinic anhydride and/or cysteine, to hydrophobized α,β-poly(N-2-hydroxyethyl)-dl-aspartamide (PHEA) copolymers have been synthesized and characterized. Starting copolymer was synthesized by the partial derivatization of hydroxyl groups on the PHEA backbone with butylamine (C4) (obtaining the PHEA-C4 copolymer, bearing a butyl moiety). The consecutive reaction of PHEA-C4 with succinic anhydride permitted the PHEA-C4-S copolymer to be obtained, bearing pendant carboxylic groups as well. Finally, part of the pendant carboxylic groups were conjugated to cysteine via an amidic bond, obtaining PHEA-C4-S-Cyst. All synthesized copolymers, PHEA-C4, PHEA-C4-S, and PHEA-C4-S-Cyst, exhibit the ability to interact with insulin in aqueous medium forming nanoaggregates. Physical characterization of prepared insulin/copolymer nanoaggregates was carried out by means of turbidimetric measurements and DLS analysis. These studies demonstrated that synthesized copolymers form colloidal aggregates in the presence of insulin, with size ranging between 62 and 216 nm. Stability studies in the presence of the peptidase α-chymotrypsin showed also the ability of synthesized copolymers to increase insulin stability against enzymatic degradation in the order PHEA-C4-S-Cyst > PHEA-C4-S > PHEA-C4. Moreover, in dosage form such as tablets, the synthesized copolymers displayed the properties to prolong disintegration time and control release of the embedded peptide drug into media mimicking intestinal fluids. The administration of insulin in the presence of PHEA-C4-S-Cyst and PHEA-C4-S copolymers resulted in the ability to provoke a certain absorption of insulin and consequently to induce in vivo hypoglycemic effects on rats after oral administration with respect to free insulin. In particular, the hypoglycemic effect shown by PHEA-C4-S/insulin nanoaggregates was equal to almost 30% of the effect observed after the administration of insulin by conventional subcutaneous administration and about 20% in the case of PHEA-C4-S-Cyst/insulin nanoaggregates. These copolymers are good starting materials for the preparation of an oral dosage form of proteins.