Dual drug loaded polypeptide delivery systems for cancer therapy.

The present study was aimed to prepare and examine in vitro novel dual-drug loaded delivery systems. Biodegradable nanoparticles based on poly(L-glutamic acid-co-D-phenylalanine) were used as nanocarriers for encapsulation of two drugs from the paclitaxel, irinotecan, and doxorubicin series. The developed delivery systems were characterised with hydrodynamic diameters less than 300 nm (PDI < 0.3). High encapsulation efficiencies (≥75%) were achieved for all single- and dual-drug formulations. The release studies showed faster release at acidic pH, with the release rate decreasing over time. The release patterns of the co-encapsulated forms of substances differed from those of the separately encapsulated drugs, suggesting differences in drug-polymer interactions. The joint action of encapsulated drugs was analysed using the colon cancer cells, both for the dual-drug delivery sytems and a mixture of single-drug formulations. The encapsulated forms of the drug combinations demonstrated comparable efficacy to the free forms, with the encapsulation enhancing solubility of the hydrophobic drug paclitaxel.

Amphiphilic Polypeptides Obtained by Post-Polymerization Modification of Poly-l-Lysine as Systems for Combined Delivery of Paclitaxel and siRNA.

The development of effective anti-cancer therapeutics remains one of the current pharmaceutical challenges. The joint delivery of chemotherapeutic agents and biopharmaceuticals is a cutting-edge approach to creating therapeutic agents of enhanced efficacy. In this study, amphiphilic polypeptide delivery systems capable of loading both hydrophobic drug and small interfering RNA (siRNA) were developed. The synthesis of amphiphilic polypeptides included two steps: (i) synthesis of poly-αl-lysine by ring-opening polymerization and (ii) its post-polymerization modification with hydrophobic l-amino acid and l-arginine/l-histidine. The obtained polymers were used for the preparation of single and dual delivery systems of PTX and short double-stranded nucleic acid. The obtained double component systems were quite compact and had a hydrodynamic diameter in the range of 90-200 nm depending on the polypeptide. The release of PTX from the formulations was studied, and the release profiles were approximated using a number of mathematical dissolution models to establish the most probable release mechanism. A determination of the cytotoxicity in normal (HEK 293T) and cancer (HeLa and A549) cells revealed the higher toxicity of the polypeptide particles to cancer cells. The separate evaluation of the biological activity of PTX and anti-GFP siRNA formulations testified the inhibitory efficiency of PTX formulations based on all polypeptides (IC50 4.5-6.2 ng/mL), while gene silencing was effective only for the Tyr-Arg-containing polypeptide (56-70% GFP knockdown).

Polymyxin B Conjugates with Bio-Inspired Synthetic Polymers of Different Nature.

The emergence and growth of bacterial resistance to antibiotics poses an enormous threat to humanity in the future. In this regard, the discovery of new antibiotics and the improvement of existing ones is a priority task. In this study, we proposed the synthesis of new polymeric conjugates of polymyxin B, which is a clinically approved but limited-use peptide antibiotic. In particular, three carboxylate-bearing polymers and one synthetic glycopolymer were selected for conjugation with polymyxin B (PMX B), namely, poly(α,L-glutamic acid) (PGlu), copolymer of L-glutamic acid and L-phenylalanine (P(Glu-co-Phe)), copolymer of N-vinyl succinamic acid and N-vinylsuccinimide (P(VSAA-co-VSI)), and poly(2-deoxy-2-methacrylamido-D-glucose) (PMAG). Unlike PGlu and PMAG, P(Glu-co-Phe) and P(VSAA-co-VSI) are amphiphilic and form nanoparticles in aqueous media. A number of conjugates with different polymyxin B loading were synthesized and characterized. In addition, the complex conjugates of PGLu or PMAG with polymyxin B and deferoxamine (siderophore) were obtained. A release of PMX B from Schiff base and amide-linked polymer conjugates was studied in model buffer media with pH 7.4 and 5.8. In both cases, a more pronounced release was observed under slightly acidic conditions. The cytotoxicity of free polymers and PMX B as well as their conjugates was examined in human embryonic kidney cells (HEK 293T cell line). All conjugates demonstrated reduced cytotoxicity compared to the free antibiotic. Finally, the antimicrobial efficacy of the conjugates against Pseudomonas aeruginosa was determined and compared. The lowest values of minimum inhibitory concentrations (MIC) were observed for polymyxin B and polymyxin B/deferoxamine conjugated with PMAG. Among the polymers tested, PMAG appears to be the most promising carrier for delivery of PMX B in conjugated form due to the good preservation of the antimicrobial properties of PMX B and the ability of controlled drug release.

Amphiphilic Polypeptides Obtained by the Post-Polymerization Modification of Poly(Glutamic Acid) and Their Evaluation as Delivery Systems for Hydrophobic Drugs.

Synthetic poly(amino acids) are a unique class of macromolecules imitating natural polypeptides and are widely considered as carriers for drug and gene delivery. In this work, we synthesized, characterized and studied the properties of amphiphilic copolymers obtained by the post-polymerization modification of poly(α,L-glutamic acid) with various hydrophobic and basic L-amino acids and D-glucosamine. The resulting glycopolypeptides were capable of forming nanoparticles that exhibited reduced macrophage uptake and were non-toxic to human lung epithelial cells (BEAS-2B). Moreover, the developed nanoparticles were suitable for loading hydrophobic cargo. In particular, paclitaxel nanoformulations had a size of 170-330 nm and demonstrated a high cytostatic efficacy against human lung adenocarcinoma (A549). In general, the obtained nanoparticles were comparable in terms of their characteristics and properties to those based on amphiphilic (glyco)polypeptides obtained by copolymerization methods.

Self-Assembled Nanoparticles Based on Block-Copolymers of Poly(2-Deoxy-2-methacrylamido-d-glucose)/Poly(N-Vinyl Succinamic Acid) with Poly(O-Cholesteryl Methacrylate) for Delivery of Hydrophobic Drugs.

The self-assembly of amphiphilic block-copolymers is a convenient way to obtain soft nanomaterials of different morphology and scale. In turn, the use of a biomimetic approach makes it possible to synthesize polymers with fragments similar to natural macromolecules but more resistant to biodegradation. In this study, we synthesized the novel bio-inspired amphiphilic block-copolymers consisting of poly(N-methacrylamido-d-glucose) or poly(N-vinyl succinamic acid) as a hydrophilic fragment and poly(O-cholesteryl methacrylate) as a hydrophobic fragment. Block-copolymers were synthesized by radical addition-fragmentation chain-transfer (RAFT) polymerization using dithiobenzoate or trithiocarbonate chain-transfer agent depending on the first monomer, further forming the hydrophilic block. Both homopolymers and copolymers were characterized by 1H NMR and Fourier transform infrared spectroscopy, as well as thermogravimetric analysis. The obtained copolymers had low dispersity (1.05-1.37) and molecular weights in the range of ~13,000-32,000. The amphiphilic copolymers demonstrated enhanced thermal stability in comparison with hydrophilic precursors. According to dynamic light scattering and nanoparticle tracking analysis, the obtained amphiphilic copolymers were able to self-assemble in aqueous media into nanoparticles with a hydrodynamic diameter of approximately 200 nm. An investigation of nanoparticles by transmission electron microscopy revealed their spherical shape. The obtained nanoparticles did not demonstrate cytotoxicity against human embryonic kidney (HEK293) and bronchial epithelial (BEAS-2B) cells, and they were characterized by a low uptake by macrophages in vitro. Paclitaxel loaded into the developed polymer nanoparticles retained biological activity against lung adenocarcinoma epithelial cells (A549).

Towards the Development of a 3-D Biochip for the Detection of Hepatitis C Virus.

The early diagnostics of hepatitis C virus (HCV) infections is currently one of the most highly demanded medical tasks. This study is devoted to the development of biochips (microarrays) that can be applied for the detection of HCV. The analytical platforms of suggested devices were based on macroporous poly(glycidyl methacrylate-co-di(ethylene glycol) dimethacrylate) monolithic material. The biochips were obtained by the covalent immobilization of specific probes spotted onto the surface of macroporous monolithic platforms. Using the developed biochips, different variants of bioassay were investigated. This study was carried out using hepatitis C virus-mimetic particles (VMPs) representing polymer nanoparticles with a size close to HCV and bearing surface virus antigen (E2 protein). At the first step, the main parameters of bioassay were optimized. Additionally, the dissociation constants were calculated for the pairs "ligand-receptor" and "antigen-antibody" formed at the surface of biochips. As a result of this study, the analysis of VMPs in model buffer solution and human blood plasma was carried out in a format of direct and "sandwich" approaches. It was found that bioassay efficacy appeared to be similar for both the model medium and real biological fluid. Finally, limit of detection (LOD), limit of quantification (LOQ), spot-to-spot and biochip-to-biochip reproducibility for the developed systems were evaluated.