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Multidrug resistance (MDR) is often caused by the overexpression of efflux pumps, such as ABC transporters, in particular, P-glycoprotein (P-gp). Here, we investigate the di- and tri- block amphiphilic polymer systems based on polypropylene glycol (PPO) and copolymers of (N-(2-hydroxypropyl)methacrylamide) (PHPMA) as potential macromolecular inhibitors of P-gp, and concurrently, carriers of drugs, passively targeting solid tumors by the enhanced permeability and retention (EPR) effect. Interestingly, there were significant differences between the effects of di- and tri- block polymer-based micelles, with the former being significantly more thermodynamically stable and showing much higher P-gp inhibition ability. The presence of Boc-protected hydrazide groups or the Boc-deprotection method did not affect the physico-chemical or biological properties of the block copolymers. Moreover, diblock polymer micelles could be loaded with free PPO containing 5&ndash, 40 wt % of free PPO, which showed increased P-gp inhibition in comparison to the unloaded micelles. Loaded polymer micelles containing more than 20 wt % free PPO showed a significant increase in toxicity, thus, loaded diblock polymer micelles containing 5&ndash, 15 wt % free PPO are potential candidates for in vitro and in vivo application as potent MDR inhibitors and drug carriers.

The study compared the physico-chemical and biological properties of a water-soluble star-like polymer nanomedicine with three micellar nanomedicines formed by self-assembly of amphiphilic copolymers differing in their hydrophobic part (statistical, block and thermosensitive block copolymers). All nanomedicines showed a pH-responsive release of the drug, independent on polymer structure. Significant penetration of all polymer nanomedicines into tumor cells in vitro was demonstrated, where the most pronounced effect was observed for statistical- or diblock copolymer-based micellar systems. Tumor accumulation in vivo was dependent on the stability of the nanomedicines in solution, being the highest for the star-like system, followed by the most stable micellar nanomedicines. The star-like polymer nanomedicine showed a superior therapeutic effect. Since the micellar systems exhibited slightly lower systemic toxicity, they may exhibit the same efficacy as the star-like soluble system when administered at equitoxic doses. In conclusion, treatment efficacy of studied nanomedicines was directly controlled by the drug pharmacokinetics, namely by their ability to circulate in the bloodstream for the time needed for effective accumulation in the tumor due to the enhanced permeability and retention (EPR) effect. Easy and scalable synthesis together with the direct reconstitution possibility for nanomedicine application made these nanomedicines excellent candidates for further clinical evaluation.

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.

Various click chemistry azide–alkyne cycloaddition reactions were used to attach azide group-terminated peptides to polymer drug carriers in an effort to conjugate biologically active molecules with polymer drug carriers by directly binding unprotected peptides to these polymers. Three methods using click chemistry to conjugate the azide group-containing molecules with synthetic polymers were compared: (1) click chemistry with a Cu(I) catalyst in aqueous and organic solvents, (2) click reactions using ruthenium complex catalysts in DMF and (3) metal-free click chemistry based on a dibenzocyclooctyne (DBCO) reactive group. The suitability of these reactions was verified for the non-covalent attachment of targeting moieties to these polymer carriers via peptide–peptide interactions. Moreover, RAFT polymerization was suggested for the synthesis of semitelechelic copolymers containing a single DBCO group at the polymer chain end and for the preparation of well-defined diblock copolymer drug carriers consisting of specific peptide and hydrophilic polymer blocks.

New amphiphilic diblock polymer nanotherapeutics serving simultaneously as a drug delivery system and an inhibitor of multidrug resistance were designed, synthesized, and evaluated for their physico-chemical and biological characteristics. The amphiphilic character of the diblock polymer, containing a hydrophilic block based on the N-(2-hydroxypropyl)methacrylamide copolymer and a hydrophobic poly(propylene oxide) block (PPO), caused self-assembly into polymer micelles with an increased hydrodynamic radius (Rh of approximately 15nm) in aqueous solutions. Doxorubicin (Dox), as a cytostatic drug, was bound to the diblock polymer through a pH-sensitive hydrazone bond, enabling prolonged circulation in blood, the delivery of Dox into a solid tumor and the subsequent stimuli-sensitive controlled release within the tumor mass and tumor cells at a decreased pH. The applicability of micellar nanotherapeutics as drug carriers was confirmed by an in vivo evaluation using EL4 lymphoma-bearing C57BL/6 mice. We observed significantly higher accumulation of micellar conjugates in a solid tumor because of the EPR effect compared with similar polymer-drug conjugates that do not form micellar structures or with the parent free drug. In addition, highly increased anti-tumor efficacy of the micellar polymer nanotherapeutics, even at a sub-optimal dose, was observed. The presence of PPO in the structure of the diblock polymer ensured, during in vitro tests on human and mouse drug-sensitive and resistant cancer cell lines, the inhibition of P-glycoprotein, one of the most frequently expressed ATP-dependent efflux pump that causes multidrug resistance. In addition, we observed highly increased rate of the uptake of the diblock polymer nanotherapeutics within the cells. We suppose that combination of unique properties based on MDR inhibition, stimuli sensitiveness (pH sensitive activation of drug), improved pharmacokinetics and increased uptake into the cells made the described polymer micelle a good candidate for investigation as potential drug delivery system.

Temperature-induced micellization of CAE-85, a carboxylic acid end-standing derivative of a triblock copolymer Pluronic P85, was studied by attenuated total reflectance Fourier-transform infrared spectroscopy (ATR FTIR) and density functional theory (DFT) model calculations. It was found that in polymer micelles carboxyl end groups dissociated and it was a two stage process. The first stage of deprotonation appeared with the onset of micellization and it was in agreement with predictions of existing models and theories for ionization processes in micellar corona. In micelles well above the critical micellization temperature, the degree of CAE-85 deprotonation increased further to values significantly higher compared to unimer solution. It is proposed that such deprotonation correlates with the formation of hydrogen bonded carboxyl end groups enabled by sufficient density of chains in the corona of developing micelles. It was demonstrated that the proton dissociation constant, p K m , specific to the micellar form existed and was different from the proton dissociation constant of solution of unimers, p K a .

The synthesis, physico-chemical behavior, and in vitro intracellular degradation of new biodegradable graft, diblock or multiblock polymer carriers that were designed to deliver bioactive compounds by passive tumor targeting were investigated. The graft polymer carriers consisted of the N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer backbone grafted with a semitelechelic HPMA copolymer. The diblock polymer carriers were prepared by condensation of two semitelechelic HPMA copolymers. The multiblock polymer drug carrier was prepared by oxidative polycondensation of PEG-bis-cysteine. In all three carrier systems, the single polymers were linked via biodegradable disulfide bonds forming the graft, diblock or multiblock polymers. These polymers are potential polymer carriers for solid tumor-specific drug delivery with subsequent intracellular degradation to short polymer fragments that can be excreted by glomerular filtration. Prolonged blood circulation, accumulation in solid tumors, and drug release from these carriers have been reported. Here, degradation of the polymers in model buffer solutions mimicking intracellular environment as well as after incubation with EL4 T-cell lymphoma cancer cells were investigated. In both cases, degradation resulted in polymer fragments of molecular weight below the renal threshold.

Reductively degradable multiblock polymers for gene and drug delivery were synthesised by oxidative polycondensation of poly(ethylene glycol)-cysteine derivatives containing ester or urethane bonds. Hydrolysis of the polymers at pH = 5.5, 7.4 and 8.0 and reductive degradation with glutathione and dithiothreitol were studied. The hydrolysis rate of polymer esters increased with increase in pH; the hydrolysis of polymer urethanes was negligible. Chemical substitution of the pendant COOH or NH 2 groups significantly affected the rate of the polymer degradation. Surface modification of poly(L-lysine)-DNA complexes with PEG-cystine multiblocks led to the formation of reductively degradable nanoparticles.

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.

Syntheses and characterization of polymer conjugates of 9-[2-(phosphonomethoxy)ethyl] (PME) derivatives of adenine (PMEA), 2,6-diaminopurine (PMEDAP) and guanine (PMEG) are described. The phosphonate group of these acyclic nucleotide analogues was activated by reaction with triphenylphosphine and di(2-pyridyl) disulfide (TPP-PDS). The activated phosphonate reacted with a random copolymer containing N-(2-hydroxypropyl)methacrylamide (HPMA) and N-(3-methacrylamidopropanoyl)ethane-1,2-diamine (Ma-AP-ED) units. The phosphonamide bond between the nucleotide and polymer carrier proved to be relatively stable at physiological pH 7.4 while at pH 5.0 (corresponding to endosomal or lysosomal compartments of cells) underwent slow hydrolysis. The rate of hydrolysis (drug release) was shown to depend on the detailed structure of the heterocyclic base. The polymer-drug conjugates described in the paper represent a new family of antiviral and cytostatic drugs with potentially improved pharmacokinetics, sustained drug release and diminished non-specific toxicity.

The synthesis and physico-chemical characterisation of a biodegradable multiblock polymer drug carrier based on poly(ethylene glycol) (PEG) is described. The blocks of PEG ( Mw 2,000) are connected by an enzymatically degradable tripeptide derivative consisting of one lysine and two glutamic acid residues. Doxorubicin (Dox), was attached to the polymer carrier via a hydrazone bond susceptible to acid hydrolysis at pH 5.0. Human immunoglobulin (IgG) was covalently linked to the polymer-Dox conjugate by the reaction of 2-pyridyldisulfanyl groups of the polymer with thiol groups of the antibody modified with 2-iminothiolane. The resulting antibody-polymer-drug conjugates were characterised by size-exclusion chromatography, UV/VIS spectrophotometry, electrophoresis and amino acid analysis. All polymers studied (both with and without IgG) showed high anti-proliferative activity against concanavalin A-stimulated murine splenocytes and various cancer cell lines in vitro. The polymer-Dox conjugate (without IgG) exhibited a significant anti-tumor efficacy against murine EL4 T-cell lymphoma and human colorectal carcinoma SW620 in vivo.

The synthesis and characterization of a water-soluble biodegradable multiblock polyurethane based on poly(ethylene glycol) blocks interconnected by a pentapeptide derivative N,N'-bis(aspartylprolyl)lysine is described. The pentapeptide linkages are susceptible both to chemical hydrolysis in neutral and mild acid medium (pH 5) and to enzymatic degradation by lysosomal enzyme cathepsin B. Anti-cancer drug doxorubicin was attached to a hydrazide derivative of the polymer via a hydrolytically labile hydrazone bond. Both the pH-triggered hydrolytic release of the drug and the degradation of the polymer carrier by enzymatic and chemical hydrolysis were studied using liquid chromatography.

Diblock and multiblock polymers of poly(ethylene glycol) containing degradable ester bonds between the blocks were synthesized and characterized. Monofunctional poly(ethylene glycol) (PEG 2000) was modified by aliphatic dicarboxylic acids (malonic, succinic, glutaric, maleic) to obtain monocarboxylic polymers PEG-COOH containing ester bonds. Diblock polymers (4000) were prepared by polycondensation of a diamine (ethane-1,2-diamine, L-lysine) and the semitelechelic PEG-COOH. The relationship between the structure of the linkage connecting two PEG blocks and the rate of its hydrolytic degradation was studied at pH 5.5, 7.4 and 8.0. The rate of hydrolysis of all polymers was significant already under mild alkaline conditions (pH 7.4 and 8.0) and increased with increasing pH. The ester bonds of polymers with saturated dicarboxylic acid moieties were stable at pH 5.5. However, the presence of double bond in the acid moiety substantially decreased the stability of the polymer not only in alkaline but also in acid medium. The results of this model study can be utilized in the design of biodegradable high-molecular-weight drug carriers and polymers for preparation of "stealth" systems intended for therapeutic application.

Thermoresponsive polymeric surfactant CAE85 is a telechelic carboxyl group derivative of Pluronic P85 and its carboxyl end-groups undergo deprotonation into carboxylate groups upon micellization. Micelle formation and disintegration were studied here by means of small angle X-ray scattering, FTIR and Raman spectroscopy, quantum mechanical calculations and dynamical mechanical analysis. The deprotonation was observed in aqueous solutions of CAE85 for concentrations from 5 wt% to 30 wt% at temperatures above the corresponding critical micellization temperature (CMT). The most likely cause is a difference between the proton dissociation constant of the micelle (pK(m)) and the proton dissociation constant of the unimers in solution (pK(a)); our observations indicate that pKmpK(a). For concentrations up to 15 wt%, the presence of carboxylate groups in CAE85 lowered the CMT in comparison with P85. In addition, the behavior of CAE85 was generally not thermo-reversible and reproducible upon cooling. Quantum chemical calculations showed that, in the dense micelle corona, the deprotonated states were more stable than hydrogen-bonded states of neutral molecules, which is likely to affect the equilibrium processes in the micelle. In contrast to the unmodified P85, no gelation was observed in the case of CAE85. By studying the processes at all the levels of organization from nanoscale charge formation through micellization to the macroscale process of gelation, our understanding of polymeric micelle formation may be advanced.

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.

The reductively degradable multiblock polymer designed for use in drug delivery was synthesized by oxidative polycondensation of the PEG-cysteine derivatives containing amide bonds between PEG and cysteine. Polymer degradation and release of doxorubicin linked to the carrier through acido-labile hydrazone bond were studied. The rate of amide bond hydrolysis was negligible compared with that of reduction of disulfide bonds. The hydrazone bond was stable at pH 7.4 but 68% of the drug was released after 24 h at pH 5.