Veska Toncheva

Novel vectors for gene delivery formed by self-assembly of DNA with poly(l-lysine) grafted with hydrophilic polymers

Complexes formed between DNA and cationic polymers are attracting increasing attention as novel synthetic vectors for delivery of genes. We are trying to improve biological properties of such complexes by oriented self-assembly of DNA with cationic-hydrophilic block copolymers, designed to enshroud the complex within a protective hydrophilic polymer corona. Poly(L-lysine) (pLL) grafted with range of hydrophilic polymer blocks, including poly(ethylene glycol) (pEG), dextran and poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA), shows efficient binding to DNA and mediates particle self-assembly and inhibition of ethidium bromide/DNA fluorescence. The complexes formed are discrete and typically about 100 nm diameter, viewed by atomic force microscopy. Surface charges are slightly shielded by the presence of the hydrophilic polymer, and complexes generally show decreased cytotoxicity compared with simple pLL/DNA complexes. pEG-containing complexes show increased transfection activity against cells in vitro. Complexes formed with all polymer conjugates showed greater aqueous solubility than simple pLL/DNA complexes, particularly at charge neutrality. These materials appear to have the ability to regulate the physicochemical and biological properties of polycation/DNA complexes, and should find important applications in packaging of nucleic acids for specific biological applications.

Structure-activity relationships of poly(L-lysines): effects of pegylation and molecular shape on physicochemical and biological properties in gene delivery.

The influence of shape, molecular weight and pegylation of linear, grafted, dendritic and branched poly-L-lysines on their DNA delivery properties were investigated. DNA binding, condensation, complex size and morphology, cell uptake and transfection efficiency were determined. Most polylysines condense DNA, linear polymers being more efficient than most dendritic ones. At low molecular weights of PLL DNA binding and condensation were less efficient, particularly with dendrimers. Pegylation did not decrease DNA condensation of PLLs at less than 60% (fraction of M(w)) of PEG. Pegylation stabilized the complexes sterically, but did not protect them from interaction with polyanionic chondroitin sulfate. Cell uptake of polylysine/DNA complexes was high and pegylation increased the transfection efficacy. However, overall transfection level of polylysines is low possibly due to inadequate escape of the complexes from endosomes or poor release of DNA from the complexes. Physicochemical and biological structure-property relationships of poly-L-lysines were demonstrated, but no clear correlations between the tested physicochemical determinants (size of complexes, zeta-potentials, condensation of DNA and the shape of complexes) and biological activities were seen. Transfection activity may be ultimately determined by intracellular factors and/or still unknown features of DNA complexation with the carriers.

Poly-l-glutamic acid derivatives as vectors for gene therapy

This paper describes the synthesis and evaluation of biodegradable derivatives of poly-L-glutamic acid as suitable vectors for gene therapy. When mixed with DNA the new polymers self assemble and form polyelectrolyte complexes. The formation of the complexes and determination of their stability towards disruption by serum albumin was monitored by Ethidium bromide (EtBr) fluorescence spectroscopy. All polymers were able to form complexes and their size, determined by photon correlation spectroscopy, was between 84.5+/-2 nm and 96. 7+/-1.6 nm, depending on the type of polymer and the charge ratio. All complexes were stable towards serum albumin. The results from the biodegradability tests, using tritosomes, show that the polymers are biodegradable and the rate of degradation is influenced by the number of charged groups in the side chains. Haemolysis and red blood cell (RBC) agglutination were assessed and compared to poly(L-lysine) (pLL) and polyethyleneimine (pEI). RBC agglutination was monitored with optical microscopy. Results show that the new polymers are less toxic than pLL and pEI. Preliminary transfection studies show that the polymers are suitable vectors for gene delivery.

Biodegradable polymeric microparticles for drug delivery and vaccine formulation: the surface attachment of hydrophilic species using the concept of poly(ethylene glycol) anchoring segments.

Poly(ethylene glycol)-dextran (PEG-DEX) conjugates have been used as a combined stabilizer and surface modifier to produce resorbable poly(DL-lactide-co-glycolide) (PLG) microparticles by an emulsification/solvent evaporation technique. The use of PEG or dextran polymers alone was incapable of producing microparticles. Particle size measurements revealed smaller mean particle sizes (480 nm) and improved polydispersity when using a 1.2% PEG substituted conjugate relative to a 9% substituted material (680 nm). PLG microparticles modified by post-adsorbed PEG-DEX conjugates flocculated in 0.01 M salt solutions, whereas PLG microparticles prepared using PEG-DEX as a surfactant were stable in at least 0.5 M NaCl solutions. Surface modification of PLG microparticles was confirmed by zeta potential measurements and surface analysis using X-ray photoelectron spectroscopy. The presence of surface exposed dextran was confirmed by an immunological detection method using a dextran-specific antiserum in an enzyme-linked immunosorbent assay. The findings support a model in which the PEG component of the PEG-DEX conjugate provides an anchor to the microparticle surface while the dextran component extends from the particle surface to contribute a steric stabilization function. This approach offers opportunities for attaching hydrophilic species such as targeting moieties to biodegradable microparticles to improve the interaction of drug carriers and vaccines with specific tissue sites.

Synthetic polymers for vectorial delivery of DNA: characterisation of polymer-DNA complexes by photon correlation spectroscopy and stability to nuclease degradation and disruption by polyanions in vitro

Abstract Synthetic polymers or multifunctional block copolymers can be used as gene delivery vectors, designed for self assembly with expression vector DNA. Here we have characterised the interaction between DNA and polylysine of varying molecular weight average (4, 24, 54, 224 k) using ethidium bromide fluorescence to monitor formation of complexes and their disruption by poly( l )aspartic acid (PAA). All poly( l )lysines were able to form complexes with DNA, and sizes measured by photon correlation spectroscopy (PCS) were greater for the larger poly( l )lysine molecular weight fractions (ranging from 37 to 207 nm average diameter). Complexes based on larger poly( l )lysines showed a sigmoidal destabilisation by PAA while complexes based on smaller pLLs showed more linear disruption. Complexes formed between DNA and a linear A-B poly(ethylene glycol)-poly( l )lysine showed an average diameter approximately 53 nm determined by PCS. The block copolymer did not improve the stability of complexes to destabilisation by PAA but did increase resistance of the complexes to nucleolytic degradation.

Physical properties of poly(ester-urethanes) prepared from different molar mass polycaprolactone-diols

Abstract Segmented poly(ester-urethanes) (PEUs) based on poly(ϵ-caprolactone) (PCL) as a soft segment and a non-aromatic diisocyanate in the hard segment were synthesized. The soft segment crystallinity and other physical properties of the PEUs were studied. It was found that the crystallinity and rate of crystallization of the PCL continuous phase in the PEUs decreases and the glass transition temperature of PEUs increases in comparison with the PCL prepolymers. The restriction of the crystallization of the PCL soft segment depends on the hard segment concentration, length of the soft segment, and total molecular weight of the PEUs.

Use of Block Copolymers of Poly(Ortho Esters) and Poly (Ethylene Glycol) Micellar Carriers as Potential Tumour Targeting Systems

Amphiphilic AB and ABA block copolymers have been prepared from poly (ortho esters) and poly (ethylene glycol). Such block copolymers readily form micellar dispersions in water, or buffers. The CMC is in the range of 3 × 10-4–5 × 10-4 g/l which is a value low enough to assure retention of micelle integrity upon intravenous injection. The size, as determined by dynamic light scattering was in the 40–70 nm range. The micelles can be stored in lyophilized form for at lest 8 months and easily reconstituted to the original properties. The micelles are stable in PBS at pH 7.4 and 37°C for 3 days and in a citrate buffer at pH 5.5 and 37°C for 2 h. Stability in the presence of bovine serum albumin depends on the structure of the block copolymer and especially the length of the POE block.

Physicochemical and biological characterisation of an antisense oligonucleotide targeted against the bcl-2 mRNA complexed with cationic-hydrophilic copolymers.

The aim of this study was to evaluate the use of cationic-hydrophilic copolymers for self-assembly with antisense oligonucleotides targeted to the bcl-2 mRNA in order to improve their biocompatibility and modulation of their pharmacokinetics for greater therapeutic usefulness. Examination of the ability of poly(trimethylammonioethyl methacrylate chloride)-poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA-b-pTMAEM) block copolymers to condense the oligonucleotide by fluorescence and electrophoresis techniques showed that complexes were formed more efficiently than with copolymers containing poly(ethylene glycol) blocks grafted onto the backbone of poly(L-lysine) (pLL-g-pEG). In addition, the copolymer pTMAEM-b-pHPMA produced oligonucleotide complexes with the most favourable physicochemical properties appropriate for in vivo applications. The complexes were small (approximately 36 nm in diameter), with low surface charge as measured by zeta potential, relatively stable to physiological salt conditions and could be formed at a DNA concentration of 500 microg/ml. Complex formation with the copolymer pTMAEM-b-pHPMA or pLL-g-pEG reduced the urinary clearance of the oligonucleotide after intravenous injection into mice. However after 30 min, the oligonucleotide complexes were cleared from the bloodstream. These results indicate that for the systemic delivery of oligonucleotides the polymer-derived complexes are not stable enough for prolonged circulation. Instead, these complexes may be more suitable for localised in vivo applications.

Synthesis and Evaluation of Poly(Ethylene Glycol)-Polylysine Block Copolymers as Carriers for Gene Delivery:

Different types of poly(ethylene glycol)-poly(l-lysine) PEG-PLL block copolymers were examined for their ability to form polyelectrolyte complexes with DNA, their toxicity toward red blood cells and their in vitro transfection efficiency. The complexation of the polymers with DNA was studied using the ethidium bromide fluorescence technique. All polymers complexed DNA to form particles with sizes ranging from 80 nm to 150 nm. In most cases, smaller particles were also observed, and sometimes populations of even larger particles could be detected. In vitro toxicity toward red blood cells was low. Agglutination of red blood cells with some of the noncomplexed block copolymers was observed, but the aggregates were less dense than with polylysine. Transfection efficiency of 293 cells in vitro in the presence of chloroquine was dependent upon the charge ratio of polymer/DNA. Efficient transfection was achieved for the PEG-PLL block copolymers with linear PLL blocks. On the other hand, very low transfection efficiency was obtained from the PEG-PLL with a dendritic PLL block.

Thermal Properties and Morphology of Poly(Ester-Urethanes) Prepared from Polycaprolactone-Diol

The soft segment crystallinity and morphology of poly(ester-urethanes) (PEUs) based on poly(ε-caprolactone) (PCL) as a soft segment and an aliphatic diisocyanate in the hard segment were studied. It was found that the restriction of the crystallization of the PCL soft segment depends on the hard segment concentration, the length of the soft segment, and the total molecular mass of the PEUs. The PEU based on a low molecular mass PCL (M=2000) is an amorphous elastic material during a long time after casting from solution or after melt crystallization. A soft-hard segment endothermal mixing transition (Tmix) of about 70-80°C is observed in the DSC curves of this PEU sample.