Intracellular delivery enhancement of poly(amino acid) drug carriers by oligoarginine conjugation.

Poly(2-hydroxytethyl aspartamide) (PHEA) was effectively translocated in both fixed and unfixed HeLa cells, when oligoarginine (Arg(8)) known as one of the cell-penetrating peptides was conjugated via a thioether linkage. The internalization of PHEA-Arg(8) into cells was a temperature-dependent process, and the studies at endocytosis inhibition conditions suggested that an endocytosis was a key mechanism. The fluorescence spectra of PHEA-Arg(8) in liposome solutions showed that PHEA-Arg(8) was collectively adsorbed in the negative liposome membrane due to the high cationic property of a conjugated Arg(8), representing that a surface adsorption was a first step in the internalization of PHEA-Arg(8). The membrane leakage activity of PHEA-Arg(8) was much lower than that of Arg(8) own, meaning that PHEA-Arg(8) does not effectively disrupt the cell membrane integrity. The uptake of polymer conjugates increased with the incubation time and reached saturation after several hours. The increase in the number of peptide conjugated to one polymer chain could increase the collective adsorption of polymer conjugates and enhance the cellular uptake. Thus, it is believed that PHEA-Arg(8) could be internalized by an adsorptive-endocytosis. A model conjugate of PHEA-Arg(8) with methotrexate (PHEA-MTX-Arg(8)) inhibited the cell proliferation about several orders of magnitude more active than PHEA-MTX.

Histidine-conjugated poly(amino acid) derivatives for the novel endosomolytic delivery carrier of doxorubicin.

Direct conjugation of histidine to poly(2-hydroxyethyl aspartamide) (PHEA-His) and C18-grafted PHEA (PHEA-g-C18-His) was achieved via an ester linkage using N(alpha)-Boc-L-histidine, followed by the deprotection of Boc groups. PHEA-His series would be expected as an endosomolytic synthetic polymer because of the buffering capacity at physiological and endosomal pH regulated by alpha-amine and imidazole groups in side chains. PHEA-g-C18-His series formed stable self-aggregates due to the hydrophobic interaction between grafted alkyl chains. The size, zeta potential, and micropolarity of PHEA-g-C18-His series greatly increased at pH 5.0, because aggregates swelled by a positive surface charge and the electrostatic repulsion of ionized histidine moieties in the aggregate surface. In the confocal microscopy, it was revealed that PHEA-g-C18-His was more uniformly distributed than PHEA-g-C18 in HeLa cells after 8 h of incubation and was attributed to the endosomolytic ability of conjugated histidine moieties. In doxorubicin-loaded self-aggregate systems, the histidine conjugation improved the cell cytotoxicity by a fast release of loaded doxorubicin at low pH and the endosomolytic ability of conjugated histidine, resulting in the easy nuclear access of doxorubicin.

Self-aggregates of oligoarginine-conjugated poly(amino acid) derivatives as a carrier for intracellular drug delivery.

N(omega)-2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl (N(omega)-Pbf)-protected oligoarginine was directly conjugated to poly(amino acid) derivatives modified with a long alkyl chain. The final concentration of conjugated peptides was easily controlled by the feed ratio of oligoarginine to polymer backbone and a final soluble polymeric system was obtained by the deprotection of N(omega)-Pbf groups. The polymeric conjugates formed stable self-aggregates of size range of 8-40 nm in aqueous solution and effectively internalized into HeLa cells by adsorptive endocytosis.