Tatiana K. Bronich

Evaluation of polyether-polyethyleneimine graft copolymers as gene transfer agents

Cationic copolymers consisting of polycations linked to non-ionic polymers are evaluated as non-viral gene delivery systems. These copolymers are known to produce soluble complexes with DNA, but only a few studies have characterized the transfection activity of these complexes. This work reports the synthesis and characterization of a series of cationic copolymers obtained by grafting the polyethyleneimine (PEI) with non-ionic polyethers, poly (ethylene oxide) (PEO) or Pluronic 123 (P123). The PEO–PEI conjugates differ in the molecular mass of PEI (2 kDa and 25 kDa) and the degree of modification of PEI with PEO. All of these conjugates form complexes upon mixing with plasmids, which are stable in aqueous dispersion for several days. The sizes of the particles formed in these systems vary from 70 to 200 nm depending on the composition of the complex. However, transfection activity of these systems is much lower than that of PEI (25 kDa) or Superfect as assessed in in vitro transfection experiments utilizing a luciferase reporter expression in Cos-7 cells as a model system. In contrast, conjugate of P123 with PEI (2 kDa) mixed with free P123 (9:1(wt)) forms small and stable complexes with DNA (110 nm) that exhibit high transfection activity in vitro. Furthermore, gene expression is observed in spleen, heart, lungs and liver 24 h after i.v. injection of this complex in mice. Compared to 1,2-bis(oleoyloxy)-(trimethylammonio) propane:cholesterol (DOTAP:Chol) and PEI (25 kDa) transfection systems, the P123-PEI system reveals a more uniform distribution of gene expression between these organs, allowing a significant improvement of gene expression in liver.

Block and Graft Copolymers and Nanogel™ Copolymer Networks for DNA Delivery into Cell

Abstract Self-assembling complexes from nucleic acids and synthetic polymers are evaluated for plasmid and oligonucleotide (oligo) delivery. Polycations having linear, branched, dendritic, block- or graft copolymer architectures are used in these studies. All these molecules bind to nucleic acids due to formation of cooperative systems of salt bonds between the cationic groups of the polycation and phosphate groups of the DNA. To improve solubility of the DNA/polycation complexes, cationic block and graft copolymers containing segments from polycations and non-ionic soluble polymers, for example, poly(ethylene oxide) (PEO) were developed. Binding of these copolymers with short DNA chains, such as oligos, results in formation of species containing hydrophobic sites from neutralized DNA-polycation complex and hydrophilic sites from PEO. These species spontaneously associate into polyion complex micelles with a hydrophobic core from neutralized polyions and a hydrophilic shell from PEO. Such complexes are very small (10-40 nm) and stable in solution despite complete neutralization of charge. They reveal significant activity with oligos in vitro and in vivo. Binding of cationic copolymers to plasmid DNA forms larger (70-200 nm) complexes, which are practically inactive in cell transfection studies. It is likely that PEO prevents binding of these complexes with the cell membranes (“stealth effect”). However attaching specific ligands to the PEO-corona can produce complexes, which are both stable in solution and bind to target cells. The most efficient complexes were obtained when PEO in the cationic copolymer was replaced with membrane-active PEO-b-poly(propylene oxide)-b-PEO molecules (Pluronic 123). Such complexes exhibited elevated levels of transgene expression in liver following systemic administration in mice. To increase stability of the complexes, NanoGel™ carriers were developed that represent small hydrogel particles synthesized by cross-linking of PEI with double end activated PEO using an emulsification/solvent evaporation technique. Oligos are immobilized by mixing with NanoGel™ suspension, which results in the formation of small particles (80 nm). Oligos incorporated in NanoGel are able to reach targets within the cell and suppress gene expression in a sequence-specific fashion. Further, loaded NanoGel particles cross-polarized monolayers of intestinal cells (Caco-2) suggesting potential usefulness of these systems for oral administration of oligos. In conclusion the approaches using polycations for gene delivery for the design of gene transfer complexes that exhibit a very broad range of physicochemical and biological properties, which is essential for design of a new generation of more effective non-viral gene delivery systems.

Core cross-linked block ionomer micelles as pH-responsive carriers for cis-diamminedichloroplatinum(II)

Benefits of the frequently prescribed platinum (II) chemotherapy drugs are compromised by undesirable side effects, poor pharmacokinetics and development of drug resistance. Polymer micelles derived from amphiphilic block copolymers, offer a novel macromolecular platform for carrier based delivery of such compounds. Soft polymeric nanocarriers were synthesized by template-assisted method involving condensation of the poly(ethylene oxide)-b-polymethacrylate anions by metal ions into core-shell block ionomer complex micelles followed by chemical cross-linking of the polyion chains in the micelle cores. The resulting micelles can efficiently incorporate cisplatin with a high loading capacity (up to 42% w/w). Core cross-linking stabilized the micelles against structural disintegration and prevented premature drug release. The reversible cisplatin entrapment involved the carboxylate groups of the micellar core. The drug was released in a pH-responsive manner, without loss of its biological activity. The stable cross-linked polymer micelles can potentially improve platinum (II) drug disposition with improved therapeutic potential.

Cisplatin-loaded core cross-linked micelles: comparative pharmacokinetics, antitumor activity, and toxicity in mice

Polymer micelles with cross-linked ionic cores are shown here to improve the therapeutic performance of the platinum-containing anticancer compound cisplatin. Biodistribution, antitumor efficacy, and toxicity of cisplatin-loaded core cross-linked micelles of poly(ethylene glycol)-b-poly(methacrylic acid) were evaluated in a mouse ovarian cancer xenograft model. Cisplatin-loaded micelles demonstrated prolonged blood circulation, increased tumor accumulation, and reduced renal exposure. Improved antitumor response relative to free drug was seen in a mouse model. Toxicity studies with cisplatin-loaded micelles indicate a significantly improved safety profile and lack of renal abnormalities typical of free cisplatin treatment. Overall, the study supports the fundamental possibility of improving the potential of platinum therapy using polymer micelle-based drug delivery.

A simple and highly effective catalytic nanozyme scavenger for organophosphorus neurotoxins

ABSTRACT A simple and highly efficient catalytic scavenger of poisonous organophosphorus compounds, based on organophosphorus hydrolase (OPH, EC 3.1.8.1), is produced in aqueous solution by electrostatic coupling of the hexahistidine tagged OPH (His6‐OPH) and poly(ethylene glycol)‐b‐poly(l‐glutamic acid) diblock copolymer. The resulting polyion complex, termed nano‐OPH, has a spherical morphology and a diameter from 25 nm to 100 nm. Incorporation of His6‐OPH in nano‐OPH preserves catalytic activity and increases stability of the enzyme allowing its storage in aqueous solution for over a year. It also decreases the immune and inflammatory responses to His6‐OPH in vivo as determined by anti‐OPH IgG and cytokines formation in Sprague Dawley rats and Balb/c mice, respectively. The nano‐OPH pharmacokinetic parameters are improved compared to the naked enzyme suggesting longer blood circulation after intravenous (iv) administrations in rats. Moreover, nano‐OPH is bioavailable after intramuscular (im), intraperitoneal (ip) and even transbuccal (tb) administration, and has shown ability to protect animals from exposure to a pesticide, paraoxon and a warfare agent, VX. In particular, a complete protection against the lethal doses of paraoxon was observed with nano‐OPH administered iv and ip as much as 17 h, im 5.5 h and tb 2 h before the intoxication. Further evaluation of nano‐OPH as a catalytic bioscavenger countermeasure against organophosphorus chemical warfare agents and pesticides is warranted.

Substitution reactions in ternary systems of macromolecules

Abstract Substitution reactions were investigated on a macromolecular system containing two different types of poly-anions, namely sodium polymethacrylate (A), sodium polyethylene sulphonate (B), and the poly-cations of poly-4-vinyl-N-ethylpyridinium bromide (C). The reaction was carried out by adding to the water-soluble non-stoichiometric poly-electrolytic complex (PEC) portions of the competing polymer: PEC(AC) +B⇆PEC(BC)+A (1). Reaction (1) can have two mechanisms depending on the reaction conditions; firstly dissociation of PEC(AC) will cause the C-parts to enter the solution where their bonding of the B macromolecules will yield PEC(BC), and secondly by an electrostatic reaction of the B-parts with the ionic groups of C present in the defects (loops) of PEC(AC) to form mixed ternary PEC which are then transformed to PEC(BC) due to phase-separation of the solutions. Variation of the reaction (1) conditions (addition of a low mol.wt. electrolyte, a change of the length of the polymeric reagents, etc.) can result in an effective control of the substitution route in one or the other direction. The general validity of the process types described is shown.

Specific features of complex interchange reactions between polyelectrolytes

Abstract The reaction of complex interchange has been studied in three-component mixtures of polyelectrolytes containing two different polyanions and a single polycation. The reaction products are associates containing all three polymeric species. The composition and structure of the resulting particles have been determined. A mechanism is proposed for the reaction of complex interchange between polyelectrolytes.