Improvement of in vitro antioxidant activity of kaempferol by encapsulation in copolymer micelles

Antioxidant capacity of poorly soluble natural antioxidant kaempferol, in particular free or loaded in two types of cationic micelles, was studied on non-enzyme induced lipid peroxidation (LPO) in vitro. The micelles were based on triblock copolymers - poly(2-(dimethylamino)ethyl methacrylate-b-poly(propylene oxide)-b-poly(2-(dimethylamino)ethyl methacrylate (PDMAEMA-PPO-PDMAEMA) and poly(2-(dimethylamino)ethyl methacrylate-b-poly(ε-caprolactone)-b-poly(2-(dimethylamino)ethyl methacrylate (PDMAEMA-PCL-PDMAEMA). The lipid peroxidation was induced by incubating of rat liver microsomes with iron sulphate and ascorbic acid (Fe2+/AA). The effect of free and micellar kaempferol (at concentrations 25, 50 and 75 μg/ml) was assessed after 20 min incubation time. In the non-enzyme lipid peroxidation model, the kaempferol-loaded micelles significantly decreased the formation of malondialdehyde (MDA). The effect of kaempferol loaded in PDMAEMA-PCL-PDMAEMA micelles was more pronounced, showing an improved antioxidant activity in the conditions of oxidative stress and lipid peroxidation in vitro.

Improved gene delivery to K-562 leukemia cells by lipoic acid modified block copolymer micelles

Although there has been substantial progress in the research field of gene delivery, there are some challenges remaining, e.g. there are still cell types such as primary cells and suspension cells (immune cells) known to be difficult to transfect. Cationic polymers have gained increasing attention due to their ability to bind, condense and mask genetic material, being amenable to scale up and highly variable in their composition. In addition, they can be combined with further monomers exhibiting desired biological and chemical properties, such as antioxidative, pH- and redox-responsive or biocompatible features. By introduction of hydrophobic monomers, in particular as block copolymers, cationic micelles can be formed possessing an improved chance of transfection in otherwise challenging cells. In this study, the antioxidant biomolecule lipoic acid, which can also be used as crosslinker, was incorporated into the hydrophobic block of a diblock copolymer, poly{[2-(dimethylamino)ethyl methacrylate]101-b-[n-(butyl methacrylate)124-co-(lipoic acid methacrylate)22]} (P(DMAEMA101-b-[nBMA124-co-LAMA22])), synthesized by RAFT polymerization and assembled into micelles (LAMA-mic). These micelles were investigated regarding their pDNA binding, cytotoxicity mechanisms and transfection efficiency in K-562 and HEK293T cells, the former representing a difficult to transfect, suspension leukemia cell line. The LAMA-mic exhibited low cytotoxicity at applied concentrations but demonstrated superior transfection efficiency in HEK293T and especially K-562 cells. In-depth studies on the transfection mechanism revealed that transfection efficiency in K-562 cells does not depend on the specific oncogenic fusion gene BCR-ABL alone. It is independent of the cellular uptake of polymer-pDNA complexes but correlates with the endosomal escape of the LAMA-mic. A comparison of the transfection efficiency of the LAMA-mic with structurally comparable micelles without lipoic acid showed that lipoic acid is not solely responsible for the superior transfection efficiency of the LAMA-mic. More likely, a synergistic effect of the antioxidative lipoic acid and the micellar architecture was identified. Therefore, the incorporation of lipoic acid into the core of hydrophobic-cationic micelles represents a promising tailor-made transfer strategy, which can potentially be beneficial for other difficult to transfect cell types.

Physicochemical Evaluation of Insulin Complexes with QPDMAEMA-b-PLMA-b-POEGMA Cationic Amphiphlic Triblock Terpolymer Micelles

Herein, poly[quaternized 2-(dimethylamino)ethyl methacrylate-b-lauryl methacrylate-b-(oligo ethylene glycol)methacrylate] (QPDMAEMA-b-PLMA-b-POEGMA) cationic amphiphilic triblock terpolymers were used as vehicles for the complexation/encapsulation of insulin (INS). The terpolymers self-assemble in spherical micelles with PLMA cores and mixed QPDMAEMA/POEGMA coronas in aqueous solutions. The cationic micelles were complexed via electrostatic interactions with INS, which contains anionic charges at pH 7. The solutions were colloidally stable in all INS ratios used. Light-scattering techniques were used for investigation of the complexation ability and the size and surface charge of the terpolymer/INS complexes. The results showed that the size of the complexes increases as INS ratio increases, while at the same time the surface charge remains positive, indicating the formation of clusters of micelles/INS complexes in the solution. Fluorescence spectroscopy measurements revealed that the conformation of the protein is not affected after the complexation with the terpolymer micellar aggregates. It was observed that as the solution ionic strength increases, the size of the QPDMAEMA-b-PLMA-b-POEGMA/INS complexes initially decreases and then remains practically constant at higher ionic strength, indicating further aggregation of the complexes. atomic force microscopy (AFM) measurements showed the existence of both clusters and isolated nanoparticulate terpolymer/protein complexes.