pH-Sensitive Chitosan–Heparin Nanoparticles for Effective Delivery of Genetic Drugs into Epithelial Cells

Chitosan has been extensively studied as a genetic drug delivery platform. However, its efficiency is limited by the strength of DNA and RNA binding. Expecting a reduced binding strength of cargo with chitosan, we proposed including heparin as a competing polyanion in the polyplexes. We developed chitosan–heparin nanoparticles by a one-step process for the local delivery of oligonucleotides. The size of the polyplexes was dependent on the mass ratio of polycation to polyanion. The mechanism of oligonucleotide release was pH-dependent and associated with polyplex swelling and collapse of the polysaccharide network. Inclusion of heparin enhanced the oligonucleotide release from the chitosan-based polyplexes. Furthermore, heparin reduced the toxicity of polyplexes in the cultured cells. The cell uptake of chitosan–heparin polyplexes was equal to that of chitosan polyplexes, but heparin increased the transfection efficiency of the polyplexes two-fold. The application of chitosan–heparin small interfering RNA (siRNA) targeted to vascular endothelial growth factor (VEGF) silencing of ARPE-19 cells was 25% higher. Overall, chitosan–heparin polyplexes showed a significant improvement of gene release inside the cells, transfection, and gene silencing efficiency in vitro, suggesting that this fundamental strategy can further improve the transfection efficiency with application of non-viral vectors

Bio-Inspired Amphiphilic Block-Copolymers Based on Synthetic Glycopolymer and Poly(Amino Acid) as Potential Drug Delivery Systems

In this work, a method to prepare hybrid amphiphilic block copolymers consisting of biocompatible synthetic glycopolymer with non-degradable backbone and biodegradable poly(amino acid) (PAA) was developed. The glycopolymer, poly(2-deoxy-2-methacrylamido-D-glucose) (PMAG), was synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Two methods for modifying the terminal dithiobenzoate-group of PMAG was investigated to obtain the macroinitiator bearing a primary aliphatic amino group, which is required for ring-opening polymerization of N-carboxyanhydrides of hydrophobic α-amino acids. The synthesized amphiphilic block copolymers were carefully analyzed using a set of different physico-chemical methods to establish their composition and molecular weight. The developed amphiphilic copolymers tended to self-assemble in nanoparticles of different morphology that depended on the nature of the hydrophobic amino acid present in the copolymer. The hydrodynamic diameter, morphology, and cytotoxicity of polymer particles based on PMAG-b-PAA were evaluated using dynamic light scattering (DLS) and transmission electron microscopy (TEM), as well as CellTiter-Blue (CTB) assay, respectively. The redox-responsive properties of nanoparticles were evaluated in the presence of glutathione taken at different concentrations. Moreover, the encapsulation of paclitaxel into PMAG-b-PAA particles and their cytotoxicity on human lung carcinoma cells (A549) and human breast adenocarcinoma cells (MCF-7) were studied

Peptide Inhibitors of Vascular Endothelial Growth Factor A : Current Situation and Perspectives

Vascular endothelial growth factors (VEGFs) are the family of extracellular signaling proteins involved in the processes of angiogenesis. VEGFA overexpression and altered regulation of VEGFA signaling pathways lead to pathological angiogenesis, which contributes to the progression of various diseases, such as age-related macular degeneration and cancer. Monoclonal antibodies and decoy receptors have been extensively used in the anti-angiogenic therapies for the neutralization of VEGFA. However, multiple side effects, solubility and aggregation issues, and the involvement of compensatory VEGFA-independent pro-angiogenic mechanisms limit the use of the existing VEGFA inhibitors. Short chemically synthesized VEGFA binding peptides are a promising alternative to these full-length proteins. In this review, we summarize anti-VEGFA peptides identified so far and discuss the molecular basis of their inhibitory activity to highlight their pharmacological potential as anti-angiogenic drugs.Peer reviewe

Poly(lactic acid) and Nanocrystalline Cellulose Methacrylated Particles for Preparation of Cryogelated and 3D-Printed Scaffolds for Tissue Engineering

Different parts of bones possess different properties, such as the capacity for remodeling cell content, porosity, and protein composition. For various traumatic or surgical tissue defects, the application of tissue-engineered constructs seems to be a promising strategy. Despite significant research efforts, such constructs are still rarely available in the clinic. One of the reasons is the lack of resorbable materials, whose properties can be adjusted according to the intended tissue or tissue contacts. Here, we present our first results on the development of a toolbox, by which the scaffolds with easily tunable mechanical and biological properties could be prepared. Biodegradable poly(lactic acid) and nanocrystalline cellulose methacrylated particles were obtained, characterized, and used for preparation of three-dimensional scaffolds via cryogelation and 3D printing approaches. The composition of particles-based ink for 3D printing was optimized in order to allow formation of stable materials. Both the modified-particle cytotoxicity and the matrix-supported cell adhesion were evaluated and visualized in order to confirm the perspectives of materials application

Biocompatible Nanoparticles Based on Amphiphilic Random Polypeptides and Glycopolymers as Drug Delivery Systems

In this research, the development and investigation of novel nanoobjects based on biodegradable random polypeptides and synthetic non-degradable glycopolymer poly(2-deoxy-2-meth-acrylamido-D-glucose) were proposed as drug delivery systems. Two different approaches have been applied for preparation of such nanomaterials. The first one includes the synthesis of block-random copolymers consisting of polypeptide and glycopolymer and capable of self-assembly into polymer particles. The synthesis of copolymers was performed using sequential reversible addition-fragmentation chain transfer (RAFT) and ring-opening polymerization (ROP) techniques. Amphiphilic poly(2-deoxy-2-methacrylamido-D-glucose)-b-poly(L-lysine-co-L-phenylalanine) (PMAG-b-P(Lys-co-Phe)) copolymers were then used for preparation of self-assembled nanoparticles. Another approach for the formation of polypeptide-glycopolymer particles was based on the post-modification of preformed polypeptide particles with an oxidized glycopolymer. The conjugation of the polysaccharide on the surface of the particles was achieved by the interaction of the aldehyde groups of the oxidized glycopolymer with the amino groups of the polymer on particle surface, followed by the reduction of the formed Schiff base with sodium borohydride. A comparative study of polymer nanoparticles developed with its cationic analogues based on random P(Lysco-D-Phe), as well as an anionic one––P(Lys-co-D-Phe) covered with heparin––was carried out. In vitro antitumor activity of novel paclitaxel-loaded PMAG-b-P(Lys-co-Phe)-based particles towards A549 (human lung carcinoma) and MCF-7 (human breast adenocarcinoma) cells was comparable to the commercially available Paclitaxel-LANS

Amphiphilic Polypeptides for VEGF siRNA Delivery into Retinal Epithelial Cells

Polyethyleneimine, poly-L-lysine, chitosan and some others cationic polymers have been thoroughly studied as nucleic acid delivery systems in gene therapy. However, the drug release from these systems proceeds at a very low rate due to extremely high binding between a carrier and gene material. To reduce these interactions and to enhance drug release, we developed a set of amphiphilic polypeptides containing positively and negatively charged amino acids as well as a hydrophobic one. The copolymers obtained were characterized by size-exclusion chromatography, static light scattering, HPLC amino acid analysis and (HNMR)-H-1 spectroscopy. All copolymers formed particles due to a self-assembly in aqueous media. Depending on polypeptide composition, the formation of particles with hydrodynamic diameters from 180 to 900 nm was observed. Stability of polymer particles, loading and release efficiency were carefully studied. Cellular uptake of the particles was efficient and their cytotoxicity was negligible. The application of polymer carriers, containing siRNA, to vascular endothelial growth factor (VEGF-A165) silencing of ARPE-19 cells was successful. The gene silencing was confirmed by suppression of both messenger RNA and protein expression.Peer reviewe

Conformationally Constrained Peptides with High Affinity to the Vascular Endothelial Growth Factor

The design of efficient vascular endothelial growth factor (VEGF) inhibitors is a high-priority research area aimed at the treatment of pathological angiogenesis. Among other compounds, v114* has been identified as a potent VEGF-binding peptide. In order to improve the affinity to VEGF, we built a conformational constrain in its structure. To this aim, C-alpha-tetrasubstituted amino acid Aib was introduced into the N-terminal tail, peptide loop, or C-terminal helix. NMR studies confirmed the stabilization of the helical conformation in proximity to the Aib residue. We found that the induction of the N-terminal helical structure or stabilization of the C-terminal helix can noticeably increase the peptide affinity to the VEGF. These peptides efficiently inhibited VEGF-stimulated cell proliferation as well. The insertion of the non-proteinogenic Aib residue significantly enhanced the stability of the peptides in the vitreous environment. Thus, these Aib-containing peptides are promising candidates for the design of VEGF inhibitors with improved properties.Peer reviewe

Photosensitive poly-l-lysine/heparin interpolyelectrolyte complexes for delivery of genetic drugs

Photo-triggered release of biopharmaceutical drugs inside the cells is a challenging direction of modern science, which requires obtaining new polymeric systems. The interpolyelectrolyte complexes (IPECs) of poly-l-lysine with heparin capable of encapsulation of genetic constructions-such as model oligonucleotide, siRNA, and pDNA-were obtained. Poly-l-lysine to heparin ratios were optimized to provide the appropriate release kinetics of genetic material from the polyplex. In order to impart the obtained IPEC with photosensitive properties, the linker was synthesized as based on 4-brommethyl-3-nitrobenzoic acid. The conditions and kinetics of photosensitive linker destruction were carefully studied. The colloid particles of IPEC were modified with Cy3 probe and their cellular internalization was investigated by flow cytometry method. The efficacy of photosensitive IPECs as siRNA and pDNA delivery system was evaluated. © 2020 by the authors