David Oupický

Physical properties and in vitro transfection efficiency of gene delivery vectors based on complexes of DNA with synthetic polycations

Biophysical properties of polycation/DNA complexes designed for gene delivery were studied with respect to the conditions of their preparation, chemical structure and molecular weight of the polycations involved. The polycations used included a variety of cationic polymers and copolymers containing primary and tertiary amino or quaternary ammonium groups. It was found that the molecular weight and the size of these polyelectrolyte complexes (PECs) increase with increasing temperature and pH of the buffer. By decreasing the molecular weight of polycations used for PEC formation, the complexes become unstable towards coagulation in aqueous solution at lower pH. The self-assembly of DNA with low-molecular-weight polycations in water provides PECs with the lowest molecular weight, smallest size and the lowest density but their stability in NaCl solutions is very poor. Despite the complexity of the multistep transfection process, a direct correlation between the transfection efficiency in vitro and the stability of the complexes in NaCl solutions and coagulation in 0.15 M NaCl solution was found. DNA complexes with polycations containing primary amino groups showed the best stability in saline solutions and also the best transfection activity. PECs formed by polycations with quaternary ammonium groups were the least resistant to destruction by the added salt and provided the lowest activity in transfection assays. The highest transfection activity was found for DNA complexes formed with a statistical copolymer containing primary and tertiary amines.

Vectors based on reducible polycations facilitate intracellular release of nucleic acids.

Inefficient intracellular delivery of nucleic acids limits the therapeutic usefulness of synthetic vectors such as poly(L‐lysine) (PLL)/DNA polyplexes. This article reports on the characterisation of a new type of synthetic vector based on a linear reducible polycation (RPC) that can be cleaved by the intracellular environment to facilitate release of nucleic acids.

Reducible poly(2-dimethylaminoethyl methacrylate): Synthesis, cytotoxicity, and gene delivery activity

Reducible polycations represent promising carriers of therapeutic nucleic acids. Oligomers of 2-dimethylaminoethyl methacrylate (DMAEMA) containing terminal thiol groups were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization using difunctional chain transfer agent. Reducible poly(DMAEMA) (rPDMAEMA) was synthesized by oxidation of the terminal thiol groups, forming a polymer with disulfide bonds in the backbone. Physico-chemical properties of DNA polyplexes of rPDMAEMA were evaluated by dynamic and static light scattering methods, revealing lower structural density and DNA content than control PDMAEMA polyplexes. Cytotoxicity and transfection activity of rPDMAEMA-based DNA polyplexes were evaluated in vitro. In comparison with control PDMAEMA, only minimum toxic effects of rPDMAEMA were observed in a panel of cell lines. Transfection activity was tested in B16F10 mouse melanoma and six human pancreatic cancer cell lines. rPDMAEMA polyplexes showed a comparable or better activity than control PDMAEMA polyplexes.

Recent advances in delivery of drug-nucleic acid combinations for cancer treatment.

Cancer treatment that uses a combination of approaches with the ability to affect multiple disease pathways has been proven highly effective in the treatment of many cancers. Combination therapy can include multiple chemotherapeutics or combinations of chemotherapeutics with other treatment modalities like surgery or radiation. However, despite the widespread clinical use of combination therapies, relatively little attention has been given to the potential of modern nanocarrier delivery methods, like liposomes, micelles, and nanoparticles, to enhance the efficacy of combination treatments. This lack of knowledge is particularly notable in the limited success of vectors for the delivery of combinations of nucleic acids with traditional small molecule drugs. The delivery of drug-nucleic acid combinations is particularly challenging due to differences in the physicochemical properties of the two types of agents. This review discusses recent advances in the development of delivery methods using combinations of small molecule drugs and nucleic acid therapeutics to treat cancer. This review primarily focuses on the rationale used for selecting appropriate drug-nucleic acid combinations as well as progress in the development of nanocarriers suitable for simultaneous delivery of drug-nucleic acid combinations.

Gene delivery in vitro and in vivo from bioreducible multilayered polyelectrolyte films of plasmid DNA

Layer-by-layer (LbL) films were assembled on flexible stainless steel substrate using plasmid DNA and reducible hyperbranched poly(amido amine) (RHB) polycation. The films were characterized by XPS and their disassembly in reducing conditions confirmed by ellipsometry. Fibroblast and smooth muscle cell attachment and proliferation on DNA/RHB films were indistinguishable from those on control DNA/poly(ethylenimine) (PEI) films. In vitro transfection activity was evaluated using reporter plasmids encoding for secreted alkaline phosphatase (SEAP) and green fluorescent protein (GFP). DNA/RHB films showed higher and longer lasting transfection activity than control DNA/PEI films using SEAP plasmid. It was revealed through the use of GFP plasmid that DNA/RHB films transfected almost the entire cell population growing on the films. In vivo transfection activity was evaluated by subcutaneously implanting a stainless steel substrate coated with the DNA/RHB films containing SEAP plasmid DNA and measuring the levels of SEAP secreted into the blood circulation of rats. It was found that the plasma levels of SEAP peaked at approximately 160 ng SEAP/mL five days post-implantation.

Effect of albumin and polyanion on the structure of DNA complexes with polycation containing hydrophilic nonionic block.

Self-assembling systems based on ionic complexes of DNA with block copolymer of N-(2-hydroxypropyl)methacrylamide with 2-(trimethylammonio)ethyl methacrylate were studied as systems suitable for gene delivery. In this study, the influence of albumin and polyanion on parameters of the DNA polyelectrolyte complexes in aqueous solutions was investigated. Static and dynamic light-scattering methods were used as a main tool for characterizing these interactions. It was found that albumin is not able to release free DNA, but it can rather bind to the complexes forming ternary DNA-polycation-albumin complexes with increased hydrodynamic radii of about 10 nm. Polyanion tested, sodium poly(styrenesulfonate), was able to release free DNA in the presence of a low-molecular-weight electrolyte. In the absence of a low-molecular-weight electrolyte, only formation of ternary complexes and no DNA release was observed. The in vivo biodistribution analysis of DNA complexes showed no effect of the presence of hydrophilic nonionic poly(HPMA) on the circulatory time or organ distribution. The interaction of DNA complexes with albumin and other plasma proteins was suggested to be a major reason for the short circulatory times.

Effect of innate glutathione levels on activity of redox-responsive gene delivery vectors

Redox-responsive polyplexes represent a promising class of non-viral gene delivery vectors. The reducible disulfide bonds in the polyplexes undergo intracellular reduction owing to the presence of high concentrations of reduced glutathione (GSH). Available evidence suggests improved transfection activity of redox-sensitive polyplexes upon artificial modulation of intracellular GSH. This study investigates the effect of innate differences in GSH concentration in a panel of human pancreatic cancer cell lines on activity of reducible polyplexes of the four major classes of nucleic acid therapeutics: plasmid DNA (pDNA), messenger RNA (mRNA), antisense oligodeoxynucleotides (AON) and siRNA. In general, reducible polyplexes of linear poly(amido amines) (PAA) show improved activity compared to non-reducible polyplexes of PAA. Results demonstrate that increased GSH levels are associated with improved transfection of mRNA polyplexes but no clear trend is observed for pDNA, AON and siRNA polyplexes.

Tumor-Penetrating Nanoparticles for Enhanced Anticancer Activity of Combined Photodynamic and Hypoxia-Activated Therapy.

Poor tumor penetration is a major challenge for the use of nanoparticles in anticancer therapy. Moreover, the inability to reach hypoxic tumor cells that are distant from blood vessels results in inadequate exposure to antitumor therapeutics and contributes to development of chemoresistance and increased metastasis. In the present study, we developed iRGD-modified nanoparticles for simultaneous tumor delivery of a photosensitizer indocyanine green (ICG) and hypoxia-activated prodrug tirapazamine (TPZ). The iRGD-modified nanoparticles loaded with ICG and TPZ showed significantly improved penetration in both 3D tumor spheroids in vitro and orthotopic breast tumors in vivo. ICG-mediated photodynamic therapy upon irradiation with a near-IR laser induced hypoxia, which activated antitumor activity of the codelivered TPZ for synergistic cell-killing effect. In vivo studies demonstrated that the nanoparticles could efficiently deliver the drug combination in 4T1 orthotopic tumors. Primary tumor growth and metastasis were effectively inhibited by the iRGD-modified combination nanoparticles with minimal side effects. The results also showed the anticancer benefits of codelivering ICG and TPZ in a single nanoparticle formulation in contrast to a mixture of nanoparticles containing individual drugs. The study demonstrates the benefits of combining tumor-penetrating nanoparticles with hypoxia-activated drug treatment and establishes a delivery platform for PDT and hypoxia-activated chemotherapy.

DNA COMPLEXES WITH BLOCK AND GRAFT COPOLYMERS OF N-(2-HYDROXYPROPYL)METHACRYLAMIDE AND 2-(TRIMETHYLAMMONIO)ETHYL METHACRYLATE

Block and graft copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA) with 2-(trimethylammonio)ethyl methacrylate (TMAEM) were synthesized for the preparation of polyelectrolyte complexes with calf thymus DNA intended for targeted delivery of genes in vivo. In this study, the effects of the poly(HPMA) content of copolymers on the parameters of the interpolyelectrolyte complexes is investigated. Static and dynamic light scattering methods were used as a main tool for characterization. The ability of the copolymers to condense DNA was studied by the ethidium bromide displacement method. The stability of the complexes against precipitation in 0.15 M NaCl and the resistance of the complexed DNA to the action of nucleases was also studied. It was found that the presence of poly(HPMA) in the copolymers has not significantly affected the ability of poly(TMAEM) parts of the copolymers to form complexes with DNA, but has an effect on molecular parameters and aggregation (precipitation) of the complexes. The size of the complexes increases with increasing poly(HPMA) content while their apparent molecular weight decreases. The complex stability against precipitation in 0.15 M NaCl strongly depends on the amount of poly(HPMA) in the copolymer structure. The presence of a sufficiently high content of poly(HPMA) is a prerequisite for achieving good stability. The structure of the complexes changes with increasing poly(HPMA) content from soft balls to the polymer coil. The density of the complexes decreases with increasing poly(HPMA) content independently of the copolymer structure. The DNA complexes of all copolymers showed very good nuclease stability.

Dual‐Function CXCR4 Antagonist Polyplexes To Deliver Gene Therapy and Inhibit Cancer Cell Invasion

A bicyclam-based biodegradable polycation with CXCR4 antagonistic activity was developed with potential for combined drug/gene cancer therapies. The dual-function polycation prevents cancer cell invasion by inhibiting CXCL12 stimulated CXCR4 activation, while at the same time efficiently and safely delivers plasmid DNA into cancer cells.