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The effects of novel polymeric therapeutics based on water-soluble N-(2-hydroxypropyl)methacrylamide copolymers (P(HPMA)) bearing the anticancer drug doxorubicin (Dox), an inhibitor of ABC transporters, or both, on the viability and the proliferation of the murine monocytic leukemia cell line P388 (parental cell line) and its doxorubicin-resistant subline P388/MDR were studied in vitro. The inhibitor derivatives 5-methyl-4-oxohexanoyl reversin 121 (MeOHe-R121) and 5-methyl-4-oxohexanoyl ritonavir ester (MeOHe-RIT), showing the highest inhibitory activities, were conjugated to the P(HPMA) via the biodegradable pH-sensitive hydrazone bond, and the ability of these conjugates to block the ATP driven P-glycoprotein (P-gp) efflux pump was tested. The P(HPMA) conjugate P-Ahx-NH-N═MeOHe-R121 showed a dose-dependent increase in the ability to sensitize the P388/MDR cells to Dox from 1.5 to 24 μM, and achieved an approximately 50-fold increase in sensitization at 24 μM. The P(HPMA) conjugate P-Ahx-NH-N═MeOHe-RIT showed moderate activity at 6 μM (∼10 times higher sensitization) and increased sensitization by 50-fold at 12 μM. The cytostatic activity of the P(HPMA) conjugate P-Ahx-NH-N═MeOHe-R121(Dox) containing Dox and the P-gp inhibitor MeOHe-R121, both bound via hydrazone bonds to the P(HPMA) carrier, was almost 30 times higher than that of the conjugate P-Ahx-NH-N═Dox toward the P388/MDR cells in vitro. A similar result was observed for P-Ahx-NH-N═MeOHe-RIT(Dox), which exhibited almost 10 times higher cytostatic activity than P-Ahx-NH-N═Dox.

A series of model linear copolymers of 2-hydroxyethyl methacrylate (HEMA) and a sterically hindered amine derivative [N-(2,2,6,6-tetramethyl-piperidin-4-yl)methacrylamide (HAS)] were synthesized and characterized. Scavenging activities of the copolymers against reactive oxygen species (peroxyl and hydroxyl radicals) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals were determined. It was found that copolymers with medium HAS content (3.5-4.0 mol %) were better scavengers than copolymers with lower and higher HAS content and also than polyHEMA and polyHAS homopolymers and the HAS monomer. Importantly, these copolymers compared favorably even to established low-molecular weight antioxidant standards (BHA and dexpanthenol). Monomer reactivity ratios were determined, and the microstructure of the copolymers was assessed. Subsequently, cross-linked copolymers in the powder and film forms with optimal HAS content were synthesized. Their scavenging activities against the three types of radicals were determined, revealing that these hydrogels are potent scavengers of reactive oxygen species.

Coiled coils are a common structural motif in many natural proteins that can also be utilized in the design and preparation of drug delivery systems for the noncovalent connection of two macromolecules. In this work, two different pairs of peptides forming coiled coil hetero-oligomers were designed, synthesized, and characterized. While the peptide sequences (VAALEKE)4 and (VAALKEK)4 predominantly form coiled coil heterodimers with randomly orientated peptide chains, (IAALESE)2-IAALESKIAALESE and IAALKSKIAALKSE-(IAALKSK)2 tend to form higher hetero-oligomers with an antiparallel orientation of their peptide chains. The associative behavior of these peptides was studied in aqueous solutions using circular dichroism spectroscopy, size-exclusion chromatography, isothermal titration calorimetry and sedimentation analyses. The orientation of the peptide chains in the coiled coil heterodimers was assessed using fluorescence spectroscopy with fluorescence resonance energy transfer labels attached to the ends of the peptides. The formation of the heterodimer can be used as a general method for the selective noncovalent conjugation of a specific targeting moiety with various drug carrier systems; this process involves simple self-assembly in a physiological solution before drug administration. The preparation of targeted macromolecular therapeutics consisting of a synthetic polymer drug carrier and a recombinant protein targeting ligand is discussed.