Čestmír Koňák

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.

New HPMA copolymer-based drug carriers with covalently bound hydrophobic substituents for solid tumour targeting

Various conjugates of anticancer drug doxorubicin (Dox) covalently bound by the hydrolytically degradable hydrazone bond to the drug carrier based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers were synthesised. Structure of the conjugates differed in the type and the content of hydrophobic substituent (dodecyl, oleic acid and cholesterol moieties) introduced into the polymer structure. In aqueous solutions the conjugates self-assembled into high-molecular-weight supramolecular structures, such as polymeric micelles or stable hydrophilic nanoparticles 13-37 nm in diameter, depending on the type and the content of hydrophobic substituents. Treatment of mice bearing EL-4 T cell lymphoma with the conjugates in the therapeutic regime of drug administration (i.v.) resulted in significant tumour regression with up to 100% of long-term survivors, depending on the dose and the detailed structure of the carrier. The nanoparticles formed by the conjugate bearing cholesterol moiety exhibited prolonged blood circulation and enhanced tumour accumulation indicating an important role of the EPR effect in excellent anticancer activity of the conjugate.

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.

Semidilute solutions of poly(methacrylic acid) in the absence of salt: Dynamic light-scattering study

Abstract Water solutions of poly(methacrylic acid) of molecular weight Mw = 3.0 × 104 and Mw = 4.0 × 105, neutralized with NaOH, were investigated by photon correlation spectroscopy. At a low degree of neutralization α, the short-time decay only is observed. When α > 0, a second, much slower, process becomes noticeable. It gains quickly in influence when α increases. The reciprocal values of characteristic relaxation times for both dynamic processes were found to decrease linearly with the square of the scattering vector, showing that both processes are diffusive. The following interpretation of these processes founded on the concentration and angle dependences of the corresponding diffusion coefficients and scattering amplitudes is given: (1) for the low-molecular-weight sample (Mw = 3.0 × 104), D∫ (fast process) and be attributed to the Nernst-Hartley diffusion coefficient of polyions, and Ds (slow process) to a diffusion of interchain domains (clusters) with a radius of gyration RG ≈ 50 nm; (2) for the high-molecular-weight sample (Mw = 4.0 × 105), where an overlap of polymer chains occurs, Df can be attributed to a cooperative diffusion coefficient reflecting the concentration fluctuations due to the polyions and counterions, and Ds to slow concentration fluctuations having a large correlation length (≈ 100 nm). The existence of two diffusive modes in salt-free solutions of polyelectrolytes cannot be explained within the framework of the scaling approach as proposed by de Gennes and Odijk.

Novel pH-Responsive Nanoparticles

In this work we report a new type of pH-responsive micelle-like nanoparticle. Reversible nanoscale structures are formed in solutions of a pH-sensitive hydrophobic polyelectrolyte, poly( N-methacryloyl- l-valine) or poly( N-methacryloyl- l-phenylalanine), and nonionic surfactant (Brij 98) in the presence of hydrochloric acid. The influence of composition and pH on particles size and shape was investigated by a variety of methods. An entitys size and polydispersity could be varied in a broad range making them a perspective candidate as a drug carrier. Unlike the case of typical micelles, our results indicate the presence of cavities in the formed particles. A hypothetical model of a nanoparticle and mechanism of formation are proposed.

Dilute and semidilute solutions of ABA block copolymer in solvents selective for A or B blocks: 2. Light scattering and sedimentation study

Abstract Micellar solutions of a triblock copolymer polystyrene- block -poly(hydrogenated butadiene)- block -polystyrene, in heptane and in 1,4- dioxane 25 vol % heptane have been studied by light scattering and sedimentation velocity methods. In dilute solutions, micellar molar mass, hydrodynamic radius of micelles, average volume fraction of polymer segments in a micellar core and in the whole micelle, and the unimer/micelles ratio have been determined. While in semidilute mixed solvent solutions only one diffusion mode, corresponding to collective diffusion, has been determined, at least two modes, diffusion and relaxation ones, have been observed in spectra of decay times in semidilute heptane solutions. Micellar models have been proposed for both systems under study, accommodating satisfactorily all experimental results.

Swelling equilibria of ionized poly(methacrylic acid) gels in the absence of salt

Abstract The kinetic theory of rubber elasticity for polyelectrolytes has been modified by including an effect of the electrostatic persistence length. The relations for swelling equilibria as a function of the degree of neutralization in polyelectrolyte gels are derived. We find that the volume fraction of the polymer in the swollen gel does not decrease monotonically with increasing α, but instead exhibits a minimum near α ∼ 0.1. Beyond this value of α the extent of swelling decreases with increasing degree of neutralization. The theory has been verified by measurements of the swelling behaviour of weakly crosslinked polymethacrylic acid gels as a function of varying degree of neutralization.

Structural parameters of spherical particles prepared by dispersion polymerization of methyl methacrylate

Abstract Poly(methyl methacrylate) (PMMA) dispersions in decane stabilized by polystyrene- block -poly(ethylene- co -propylene) copolymer were prepared with various monomer and steric stabilizer concentrations. The number of PMMA macromolecules formed in the dispersion polymerization does not depend on monomer and stabilizer concentrations. The kinetics of polymerization seems to be only slightly affected by the colloidal character of the system. Correlations have been established between the parameters of the whole system, of dispersion particles and of the individual PMMA macromolecules.

Preparation of DNA complexes with diblock copolymers of poly[N(2-hydroxypropyl)methacrylamide] and polycations

Abstract This study investigated the effects of the speed of component mixing, total concentration of polymers and ionic strength of solvents on formation of interpolyelectrolyte complexes (IPECs) of calf thymus DNA with diblock copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA) and 2-(trimethylammonio)ethyl methacrylate (TMAEMCl). The diblock copolymer consisting of a poly(HPMA) block (A) and poly(TMAEMCl) polycation block (B) with pendant quaternary ammonium groups and with molecular weight of A block higher than that of B was used in the study. Poly(TMAEMCl) polycation were used for comparison. Static and dynamic light scattering methods were used for characterisation of complexes. The apparent mass-average molecular weights, Mwa, and hydrodynamic radii, RHa, of slightly overcompensated IPECs (ϕ=[cation units]/[DNA negative charges]=1.1) prepared by fast mixing of components were found to be only slightly smaller than those prepared by slow mixing. Both Mwa and RHa of IPECs prepared under stoichiometric conditions (ϕ=1) decreased with decreasing total polymer concentration, cT, approaching Mw and RH of single DNA complexes which are the most suitable ones for DNA uptake into cells. The presence of NaCl in solutions resulted in a significant increase in Mwa and RHa of IPECS.

Block copolymer micelles near critical conditions

A three-block copolymer poly(styrene-block-hydrogenated butadiene-block-styrene) forms in 1,4-dioxane and 1,4-dioxane/30 vol% n-heptane mixture spherical micelles with aliphatic cores and polystyrene shells. Properties of these micelles near critical conditions, i.e., above the critical micelle concentration (CMC) and below the critical micelle temperature (CMT) have been studied by light scattering. Integral light scattering is proportional to the weighted contributions of the molar masses of two species present in the solutions under study. Quasielastic light scattering, on the other hand, can provide in principle, the values of diffusion coefficient pertaining predominantly to micelles. Experimental data, namely concentration dependences of the diffusion coefficient and of the apparent mass-average molar mass, strongly deviate from the expected pattern predicted by the model of closed association. The deviation consists in an increase of both molar mass and hydrodynamic radius of micelles in a particular concentration region above CMC.