Igor Perevyazko

Polyelectrolyte Complexes of DNA and Linear PEI: Formation, Composition and Properties

In the present study, the complexation between linear 13.4 kDa poly(ethylene imine) (LPEI) and plasmid DNA was investigated. Analytical ultracentrifugation (AUC) was used for size and molar mass determination. Additionally, the morphology was studied by scanning force microscopy. The polyplex formation was investigated in a wide range of PEI nitrogen to DNA phosphate ratios (N/P). At N/P ratios below 1, the PEI/DNA complex formation is characterized by an incomplete DNA condensation and the formation of the primary DNA/PEI complexes. The merging of the initially formed polyplexes occurs at N/P ~2, resulting in the formation of polyplexes with much larger size and high aggregation rate. Stable and uniform polyplexes were formed at N/P > 10, with average sizes of the polyplexes of about 170 ± 65 nm. The content of uncomplexed PEI chains in the polyplex dispersion was estimated at four different N/P ratios, 6.2, 11.6, 28.6, and 57.8, by combining preparative centrifugation with a copper complex assay and by sedimentation velocity analysis as an alternative method. It is demonstrated that virtually all added PEI binds to the DNA at N/P < 2.5; further addition of PEI results in the appearance of a large amount of free PEI in solution. Nevertheless, PEI is able to bind in the whole range of N/P ratios tested. According to the data collected by sedimentation velocity analysis and scanning force microscopy, the single PEI/DNA complexes are composed on average of 8 to 32 single condensed DNA plasmids and 70 ± 25 PEI molecules.

Preparation, Cellular Internalization, and Biocompatibility of Highly Fluorescent PMMA Nanoparticles

Methacrylate monomers were functionalized with a 4-hydroxythiazole chromophore and copolymerized with methyl methacrylate via RAFT. Nanoparticles of 120 and 500 nm in size were prepared without using stabilizers/surfactants. For comparative studies, preparative ultracentrifugation was applied for the separation into small and large particle fractions. All suspensions were characterized by DLS, AUC, and SEM and tested regarding their stability during centrifugation and re-suspension, autoclavation, and incubation in cell culture media. In vitro studies with mouse fibroblast cell line and differently sized NP showed a particle uptake into cells. Biocompatibility, non-toxicity, and hemocompatibility were demonstrated using a XTT assay, a live/dead staining, and an erythrocyte aggregation and hemolysis assay.

Conformation parameters of linear macromolecules from velocity sedimentation and other hydrodynamic methods

Linear macromolecules constitute a broad class of synthetic and natural polymers which are highly useful in various technologies and represent the key molecular systems in living nature. The study of the molecular characteristics of these polymers represents an important problem in fundamental and applied science. The methods of molecular hydrodynamics have been and remain an important way of studying the molar mass, molar mass distribution, size and conformation of linear polymers. This paper discusses the approaches to the problems of hydrodynamic methods, in particular analytical velocity ultracentrifugation, in the study of various types of linear macromolecule. The velocity sedimentation data were processed with three different methods: Sedanal and Sedfit software, and the classical approach of evaluating the rate at which the sedimentation boundary moves. The Sedfit program also allows an evaluation of the frictional ratio values, i.e., the coefficient of translational diffusion. It will be discussed for which systems the estimation of the frictional ratio obtained by Sedfit is adequate and for which it is not. The applications of other hydrodynamic methods (intrinsic viscosity, translational diffusion) are also discussed with a view to obtaining the conformational characteristics of linear macromolecules.

Examination and optimization of the self-assembly of biocompatible, polymeric nanoparticles by high-throughput nanoprecipitation

In recent years, the development of polymer nanoparticle suspensions by nanoprecipitation has gained increased attention both by industry and academia. However, the process by which such formulations are prepared is a highly empirically driven enterprise, whereby developing optimized formulations remains an iterative process. In this contribution, a new approach towards exploration of the materials space for these systems is reported, based on systematically varying processing and formulation to understand their influence on the characteristics of the resulting materials. Taking advantage of the tools and techniques that have already been standardized by informatics-driven life sciences disciplines, we have prepared libraries of nanoparticle formulations of poly(methyl methacrylate-stat-acrylate), poly(lactic-co-glycolic acid), and acetal-derivatized dextran by using a pipetting robot. They were subsequently characterized using a dynamic light scattering plate reader, analytical ultracentrifugation, and scanning electron microscopy. With this high-throughput nanoprecipitation approach, large numbers of materials can be prepared, screened, and the formulation rationally optimized.

Microwave-assisted synthesis of imidazolium ionenes and their application as humidity absorbers†

4,4-Imidazolium ionenes were synthesized under microwave irradiation for the first time and their application as humidity absorbers (water uptake up to 97 wt%) was investigated.

Model system for multifunctional delivery nanoplatforms based on DNA-Polymer complexes containing silver nanoparticles and fluorescent dye

Creation of multifunctional nanoplatforms is one of the new approaches to complex treatment and diagnosis with the monitoring of the curative process. Inclusion of various components into the drug delivery system may reduce toxicity and enhance or modify the therapeutic effects of medicines. In particular, some properties of metal nanoparticles and nanoclusters provide the ability to create new systems for treatment and diagnosis of diseases, biocatalysis and imaging of objects. For example, the ability of metal nanoparticles to enhance the quantum yield of luminescence can be used in bioimaging and therapy. The aim of the research was to construct and examine a multicomponent system based on DNA-polycation compact structure with the inclusion of silver nanoparticles and luminescent dye as a model system for delivery of genes and drugs with the possibility of modification and enhancement of their action.

Hydrodynamic Analysis Resolves the Pharmaceutically-Relevant Absolute Molar Mass and Solution Properties of Synthetic Poly(ethylene glycol)s Created by Varying Initiation Sites

The solution behavior originating from molecular characteristics of synthetic macromolecules plays a pivotal role in many areas, in particular the life sciences. This situation necessitates the use of complementary hydrodynamic analytical methods as the only means for a complete structural understanding of any macromolecule in solution. To this end, we present a combined hydrodynamic approach for studying in-house prepared, low dispersity poly(ethylene glycols)s (PEGs), also known as poly(ethylene oxide)s (PEOs) depending on the classification used, synthesized from varying initiation sites by the living anionic ring opening polymerization. The series of linear PEGs in the molar mass range of only a few thousand to 50 000 g mol-1 have been studied in detail via viscometry and sedimentation-diffusion analysis by analytical ultracentrifugation. The obtained estimations for intrinsic viscosity, diffusion coefficients, and sedimentation coefficients of the macromolecules in the solution-based analysis clearly showed self-consistency of the followed hydrodynamic approach. This self-consistency is underpinned by appropriate and physically sound values of hydrodynamic invariants, indicating adequate values of derived absolute molar masses. The classical scaling relations of Kuhn-Mark-Houwink-Sakurada of all molar-mass dependent hydrodynamic estimates show linear trends, allowing for interrelation of all parametric macromolecular characteristics. Differences among these are ascribed to the observation of α-end and chain-length dependent solvation of the macromolecules, identified from viscometric studies. This important information allows for analytical tracing of variations of scaling relationships and a physically sound estimation of hydrodynamic characteristics. The demonstrated self-sufficient methodology paves an important way for a complete structural understanding and potential replacement of pharmaceutically relevant PEGs by alternative macromolecules offering a suite of similar or tractably distinct physicochemical properties.

Incorporation of core–shell particles into methacrylate based composites for improvement of the mechanical properties

The fracture toughness of polymeric materials and composites can be enhanced by the incorporation of polymer nanoparticles. The combination of a soft core and a hard shell leads to an improvement of the fracture toughness of the polymeric composites. Thereby, the mechanical resistance of the materials is commonly decreased. In our approach, core–shell nanoparticles consisting of an ethylene glycol dimethacrylate (EGDMA) crosslinked poly(butyl acrylate) (PBA) core and a poly(methyl methacrylate) (PMMA) shell were synthesized. The polymer particles were incorporated into triethylene glycol dimethacrylate (TEGDMA)/urethane dimethacrylate (UDMA) based composites in order to tune the mechanical properties. Different core–shell ratios were applied to study the influence on the fracture toughness and E-modulus. An examination of shell-crosslinking with a TEGDMA content of up to 8% was performed to improve particle stability and dispersibility. The particle sizes and morphologies were characterized by dynamic light scattering (DLS), cryogenic transmission electron microscopy (cryo-TEM) and analytical ultracentrifugation (AUC). Latex particle sizes of 70 to 220 nm were obtained. The mechanical properties (flexural strength, E-modulus and K1c) of polymer composites were investigated in three-point bending tests. Core/shell ratios of 50/50 showed a decreasing effect on flexural strength, E-modulus and K1c. Polymer particles with core/shell ratios of 30/70 led to a significant increase of the mechanical properties with maxima of 1.206 MPa m1/2 (K1c) (increase of 65%), E-modulus of 1.90 GPa (increase of 18%) and flexural strength of 79 MPa (increase of 18%). This study represents the first report of a simultaneous improvement of fracture toughness and E-modulus (at the same time) of additive filled polymer composites. The improvement of mechanical properties makes these materials interesting as tougheners for hard tissue applications like bone cements or dental replacement materials.

Unexpected radical polymerization behavior of oligo(2-ethyl-2-oxazoline) macromonomers

A well-defined oligo(2-ethyl-2-oxazoline)acrylate (OEtOxA) macromonomer was obtained by direct end functionalization of the living cationic oxazolinium species from the cationic ring-opening polymerization of EtOx with in situ deprotonated acrylic acid. Kinetic studies during subsequent reversible addition–fragmentation chain transfer (RAFT) polymerization as well as nitroxide mediated polymerization (NMP) experiments revealed proceeding monomer consumption but no increase of the molar mass of the resulting comb polymers. The chain transfer during the radical polymerizations is proposed to result from backbiting and subsequent β-scission of the formed mid-chain radical and took place in a well-defined manner, so that POEtOxA could also be obtained by free radical polymerization with a PDI value below 1.2. A series of POEtOxA was synthesized by RAFT polymerization with varying [monomer]/[chain transfer agent] (M/CTA) ratios and analyzed in detail by means of analytical ultracentrifugation and small angle neutron scattering, indicating that the backbone DP does not exceed 25, which is in accordance with the thermal polymer properties in bulk and in aqueous solution (Tg = 32 °C, Tcp ≈ 73 °C).

Linear poly(ethylene imine)s: true molar masses, solution properties and conformation

In-depth characterization of pharmaceutically relevant polymers plays a pivotal role in many areas, including nanoscience, gene therapy, analytical and polymer chemistry etc. Notwithstanding substantial efforts spent in this area, there are still unresolved challenges and one of the most demanding problems is the determination of absolute molar masses of a broad range of biocompatible cationic polymers. Hereby, we present a combined analytical approach for a distinct and self-consistent characterization of a series of linear poly(ethylene imine)s (PEIs) consisting of six in-house prepared PEIs (0.9 kDa < Mtheor < 250 kDa) and three commercially available linear PEIs (Polyscience labeled as 2.5 kDa, 25 kDa, and 250 kDa). The polymers were studied, in 0.2 M NaBr methanol, by the methods of molecular hydrodynamics: analytical ultracentrifugation, intrinsic viscosity and translational diffusion measurements. Absolute values of the molar masses were evaluated by the classical sedimentation-diffusion analysis resulting in the following range: 1.1 kDa < M < 13.9 kDa. It was demonstrated that the molar masses reported by the manufacturer, as well as theoretical and/or molar masses evaluated by common SEC analysis, are significantly overestimated. The complete set of Kuhn–Mark–Houwink–Sakurada scaling relations shows linear trends over the whole range of the molar masses, whilst the determined scaling indices virtually correspond to the homologous series characterized by a coil conformation ([η] = 0.255 × M0.56; s0 = 0.015 × M0.48; D0 = 994 × M−0.52). The conformational characteristics of LPEI, i.e. equilibrium rigidity (the Kuhn segment length) and the diameter of the PEI chains, were evaluated for the first time and constitute A = 1.9 ± 0.6 nm and d = 0.4 ± 0.2 nm, respectively. The presented self-sufficient analytical approach covers an important area of thorough polymer characterization by nowadays alternative but fundamental hydrodynamic methods allowing for complete structural and molecular analysis of almost any macromolecules in solution.