Olga Samsonova

Triazine dendrimers as nonviral vectors for in vitro and in vivo RNAi: the effects of peripheral groups and core structure on biological activity.

A family of triazine dendrimers, differing in their core flexibility, generation number, and surface functionality, was prepared and evaluated for its ability to accomplish RNAi. The dendriplexes were analyzed with respect to their physicochemical and biological properties, including condensation of siRNA, complex size, surface charge, cellular uptake and subcellular distribution, their potential for reporter gene knockdown in HeLa/Luc cells, and ultimately their stability, biodistribution, pharmacokinetics and intracellular uptake in mice after intravenous (iv) administration. The structure of the backbone was found to significantly influence siRNA transfection efficiency, with rigid, second generation dendrimers displaying higher gene knockdown than the flexible analogues while maintaining less off-target effects than Lipofectamine. Additionally, among the rigid, second generation dendrimers, those with either arginine-like exteriors or peripheries containing hydrophobic functionalities mediated the most effective gene knockdown, thus showing that dendrimer surface groups also affect transfection efficiency. Moreover, these two most effective dendriplexes were stable in circulation upon intravenous administration and showed passive targeting to the lung. Both dendriplex formulations were taken up into the alveolar epithelium, making them promising candidates for RNAi in the lung. The ability to correlate the effects of triazine dendrimer core scaffolds, generation number, and surface functionality with siRNA transfection efficiency yields valuable information for further modifying this nonviral delivery system and stresses the importance of only loosely correlating effective gene delivery vectors with siRNA transfection agents.

Biophysical characterization of hyper-branched polyethylenimine-graft- polycaprolactone-block-mono-methoxyl-poly(ethylene glycol) copolymers (hy-PEI-PCL-mPEG) for siRNA delivery

A library of mono-methoxyl-poly(ethylene glycol)-block-poly(ε-caprolactone) (mPEG-PCL) modified hyperbranched PEI copolymers (hy-PEI-PCL-mPEG) was synthesized to establish structure function relationships for siRNA delivery. These amphiphilic block-copolymers were thought to provide improved colloidal stability and endosomal escape of polyplexes containing siRNA. The influence of the mPEG chain length, PCL segment length, hy-PEI molecular weight and the graft density on their biophysical properties was investigated. In particular, buffer capacity, complex formation constants, gene condensation, polyplex stability, polyplex size and zeta-potential were measured. It was found that longer mPEG chains, longer PCL segments and higher graft density beneficially affected the stability and formation of polyplexes and reduced the zeta-potential of siRNA polyplexes. Significant siRNA mediated knockdown was observed for hy-PEI25k-(PCL900-mPEG2k)(1) at N/P 20 and 30, implying that the PCL hydrophobic segment played a very important role in siRNA transfection. These gene delivery systems merit further investigation under in vivo conditions.

Amphiphilic and biodegradable hy-PEI-g-PCL-b-PEG copolymers efficiently mediate transgene expression depending on their graft density

Novel biodegradable amphiphilic copolymers hy-PEI-g-PCL-b-PEG were prepared by grafting PCL-b-PEG chains onto hyper-branched poly(ethylene imine) as non-viral gene delivery vectors. Our investigations focused on the influence of graft densities of PCL-b-PEG chains on physico-chemical properties, DNA complexation and transfection efficiency. We found that the transfection efficiencies of these polymers increased at first towards an optimal graft density (n=3) and then decreased. The buffer-capacity-test showed almost exactly the same tendency as transfection efficiency. Cytotoxicity (MTT-assay) depended on the cooperation of PEG molecular weight and graft density of PCL-b-PEG chains. With increasing the graft density, cytotoxicity, zeta-potential, affinity with DNA, stability of the polyplexes and CMC-values were reduced strongly and regularly. Increasing the excess of polymer over DNA was shown to result in a decrease of the observed particle size to 100-200 nm.

Optimising the self-assembly of siRNA loaded PEG-PCL-lPEI nano-carriers employing different preparation techniques

Amphiphilic cationic block copolymers consisting of poly(ethylene glycol), poly(ε-caprolactone) and poly(ethylene imine) spontaneously assemble to nano-sized particulate carriers, which can be utilised for complexation of nucleic acids (small-interfering RNA), representing a multifunctional vector system, designed for drug and gene delivery. Apart from polymer design and charge ratio, a more homogeneous complexation could lead to a more uniform charge distribution, subsequently increasing colloidal stability, RNA protection and consequently transfection efficiency. Microfluidic mixing techniques, bringing cationic polymer and nucleic acid together at a constant ratio during the entire mixing process, have the potential for a gentler complexation. In the present study carriers were prepared by a solvent displacement technique. In a first step complex size for addition of RNA during (addition to the aqueous or the organic phase) or after (classical pipetting or microfluidic mixing) carrier assembly was determined by dynamic light scattering. Suitable N/P ratios have previously been selected by measuring size and ζ-potential as a function of N/P. Subsequently, for the most promising techniques (loading after assembly), colloidal stability, the ability to protect RNA as well as transfection efficiency in vitro were compared. Finally, parameters for the superior microfluidic mixing process were optimised with the help of a central composite design. Generally, gentler loading leads to more homogeneous complexes. Hence, possibly due to a more consistent surface coating, loading after carrier assembly resulted in less aggregation. In comparison to bulk mixing, microfluidic assembly exhibited smaller diameters (179±11 vs. 230±97nm), less heterogeneity (PDI=0.205±0.028 vs. 0.353±0.161), enhanced RNA protection (RNA recovery=30.6±1.0 vs. 15.4±1.4%) as well as increased transfection performance (34.8±1.5 vs. 24.5±2.2% knockdown). Therefore, microfluidic complexation represents a reproducible alternative for formulating gene delivery carriers with superior colloidal stability, RNA protection and transfection efficiency.

A Chiral, Low-Cytotoxic [Ni15]-Wheel Complex

A new chiral [Ni15] complex with a Schiff-base ligand derived from o-vanillin and L-glutamic acid is presented, emphasizing the properties relevant for biology and materials science. The formation of the complex molecules in solution is confirmed by AFM and dynamic light scattering studies. The compound is weakly antiferromagnetic with considerable admixture of excited states, comprising negligibly interacting [Ni3] units. Studies of the interactions with two cell lines indicate low cytotoxicity.

Functionalized polyglycerol amine nanogels as nanocarriers for DNA.

Polyglycerol based nanogels (nPG) can function as cellular delivery systems. These nPGs are synthesized with different amine densities (nPG amines) by acid-catalyzed epoxide-opening polymerization using a mini-emulsion approach and surface modification. All the synthesized nanogels are characterized by NMR, dynamic light scattering, and ζ-potential, showing slightly positive surface charge and a homogeneous size of ≈100 nm. The use of these systems for delivery applications is demonstrated with regard to polyplex formation, cytotoxicity, and cellular uptake studies. It is depicted that the CE50 value of the high loaded nPG amines is eight times higher than the low loaded ones. The influence of the amine loading percentage on the nanogel and the effects of polyvalency in these architecture is discussed.

The use of isothermal titration calorimetry and molecular dynamics to show variability in DNA transfection performance.

The mechanism causing variability in DNA transfection efficacy for low-molecular-weight pDMAEMA (poly(2-(dimethylamino)ethyl methacrylate) and pDMAEMA-b-pHEMA (poly(2-(dimethyl amino)ethylmethacrylate)-block-poly(2-hydroxyl methacrylate)) has so far remained unclear, apart from the evidence of beneficial effects of the pHEMA grafting. This study has explicitly characterized the electrostatically driven self-assembly process of linear polymethacrylate polymers with DNA-generating nanocarriers for efficient gene transfection. Isothermal titration calorimetry (ITC) showed clear differences in binding-heat profiles of homo-polycationic and pHEMA grafted polymers with DNA. Polyethylene imine, a branched polycationic polymer of 25kDa with high transfection potential that has previously been successfully used in transfection experiments, demonstrated a heat flow profile that was partly identical to pDMAEMA-b-pHEMA. Computational molecular dynamics (MD) simulated the folding process of polymer in water from a linear to a coiled state: homo-pDMAEMA and pHEMA grafts reduced their overall positive charge accessibility upon folding, down to 45% and 63%, respectively. The homo-pDMAEMA formed the globular conformation more preferably than pHEMA grafts, thus impeding electrostatic interaction with DNA. These findings substantiate the known disadvantage of low-molecular-weight linear polymers compared to higher-molecular-weight polymers in transfection performance; here we have disclosed the ability of a non-cationic chain elongation to be beneficial for the self-assembly process. The combination of MD and ITC has proved to be a suitable approach for carrier-payload interaction studies and may be used to predict the efficacy of a polymer as a nanocarrier from the flexibility of its structure.

Influence of amine-modified poly(vinyl alcohol)s on vibrating-membrane nebulizer performance and lung toxicity.

A suitable aerosol droplet size and formulation output rate is essential for the therapy of lung diseases under application of nebulizers. The current study investigated the potential of amine-modified poly(vinyl alcohol)s as excipients for inhalation delivery. A change of conductivity (effective at <0.1mg/ml) and viscosity (effective at >0.1mg/ml) of samples that were supplemented with charge-modified polymers had a significant influence on the generated droplet size (shift from ~8 to ~4 μm) and formulation throughput rate (shift from ~0.2 to ~1.0 g/min), where polymers with a higher amine density (and molecular weight) showed an elevated activity. Biocompatibility assessment of polymers in A549 cells and an isolated lung model resulted in cell lysis and lung edema formation dependent on the type (degree of amine substitution) and dose of polymer applied. Suitable compositions and concentrations of amine-modified poly(vinyl alcohol)s were identified with respect to an optimized nebulizer performance and acceptable biocompatibility. Charge-modified polymers represent novel excipients with potential to improve inhalation therapy.

Evaluation of liposomal carnosine in adjuvant arthritis

Liposomal carnosine could overcome the problems associated with direct application of this peptide. Anti-inflammatory and antioxidant effects of liposomal and non-liposomal carnosine in adjuvant arthritis were compared. The experiments were done on healthy animals, untreated arthritic animals, arthritic animals with oral administration of carnosine, and with subcutaneous administration of liposomal carnosine, both administered in the same daily dose of 150 mg/kg b.w. during 28 days. Carnosine reduced hind paw volume on day 28. Both forms markedly decreased interleukin-1β, matrix metalloproteinase-9 and monocyte chemoattractant protein-1 (MCP-1) in plasma on day 14. Only liposomal carnosine reduced significantly MCP-1. Malondialdehyde, 4-hydroxynonenal, resistance to Fe2+-induced oxidation and protein carbonyls were significantly corrected after administration of any form of carnosine. Liposomal carnosine corrected more effectively the oxidative stress in plasma than did carnosine. In brain tissue, our results showed protective ability of both forms of carnosine against oxidation of proteins and lipids. They also corrected the resistance to Fe2+-induced oxidation in arthritic animals. We found that only liposomal carnosine decreased the mRNA expression of inducible nitric oxide synthase in cartilage tissue. It can be concluded that the liposomal drug-delivery system is improving the pharmacological properties of carnosine administered in arthritis.