Ivaylo Dimitrov

Polymeric nanoparticle engineering: from temperature-responsive polymer mesoglobules to gene delivery systems.

A novel approach for the preparation of nano- and microcapsules in aqueous solutions by using thermoresponsive polymer (TRP) templates (mesoglobules) is described. The method comprised three steps: formation of mesoglobules, coating the templates by seeded radical copolymerization, followed by core dissolution and core removal upon cooling. When mesoglobule entraps biomacromolecules during the process of their formation, it makes it possible to load a controlled amount of bioactive compounds without covalent attachment. Special attention is paid to the mesoglobule dissolution upon cooling, as well as their loading efficiency. Details on the outer shell formation and the possibilities for targeting ligands incorporation and control of the shell porosity are discussed. Finally, the seeded radical copolymerization was used for covering DNA complexes with cationic copolymers bearing TRP blocks. This Review is an attempt to convince researchers of the promising perspectives for using mesoglobules as potential reservoirs, carriers, and transferring agents for biologically active substances.

Insulin/poly(ethylene glycol)-block-poly(L-lysine) Complexes: Physicochemical Properties and Protein Encapsulation.

Insulin (INS) was encapsulated into complexes with poly(ethylene glycol)-block-poly(L-lysine) (PEG-b-PLys), which is a polypeptide-based block copolymer (a neutral-cationic block polyelectrolyte). The particular cationic-neutral block copolymer can complex INS molecules in aqueous media via electrostatic interactions. Light-scattering techniques are used to study the complexation process and structure of the hybrid nanoparticles in a series of buffers, as a function of protein concentration. The physicochemical and structural characteristics of the complexes depend on the ionic strength of the aqueous medium, while the concentration of PEG-b-PLys was constant through the series of solutions. As INS concentration increased the size distribution of the complexes decreased, especially at the highest ionic strength. The size/structure of complexes diluted in biological medium indicated that the copolymer imparts stealth properties and colloidal and biological stability to the complexes, features that could in turn affect the clearance properties in vivo. Therefore, these studies could be a rational roadmap for designing the optimum complexes/effective nanocarriers for proteins and peptides.

Nucleation of insulin crystals in a wide continuous supersaturation gradient.

Abstract: Modifying the classical double pulse technique, by using a supersaturation gradient along an insulin solution contained in a glass capillary tube, we found conditions appropriate for the direct measurement of nucleation parameters. The nucleation time lag has been measured. Data for the number of crystal nuclei versus the nucleation time were obtained for this hormone. Insulin was chosen as a model protein because of the availability of solubility data in the literature. A comparison with the results for hen‐egg‐white lysozyme, HEWL was performed.

Probabilistic approach to lysozyme crystal nucleation kinetics

Nucleation of lysozyme crystals in quiescent solutions at a regime of progressive nucleation is investigated under an optical microscope at conditions of constant supersaturation. A method based on the stochastic nature of crystal nucleation and using discrete time sampling of small solution volumes for the presence or absence of detectable crystals is developed. It allows probabilities for crystal detection to be experimentally estimated. One hundred single samplings were used for each probability determination for 18 time intervals and six lysozyme concentrations. Fitting of a particular probability function to experimentally obtained data made possible the direct evaluation of stationary rates for lysozyme crystal nucleation, the time for growth of supernuclei to a detectable size and probability distribution of nucleation times. Obtained stationary nucleation rates were then used for the calculation of other nucleation parameters, such as the kinetic nucleation factor, nucleus size, work for nucleus formation and effective specific surface energy of the nucleus. The experimental method itself is simple and adaptable and can be used for crystal nucleation studies of arbitrary soluble substances with known solubility at particular solution conditions.

Hypergravity as a Crystallization Tool

Abstract:  The centrifugal increase of concentration is nondestructive, rapid, and simple technology. Therefore it is used to create a higher supersaturation that is required for crystal nucleation, as the one that is appropriate for the subsequent growth. Crystal nucleation is evoked in glass capillary tubes filled with protein solutions. The couple ferritin/ apoferritin is used as model proteins in the present article. Although differing in their masses the two (quasi) spherical molecules have exactly the same size and surface properties. Together with the temperature‐independent solubility this makes them very convenient for our investigations. Decoupling nucleation and growth, for example, by means of hypergravity makes it possible to grow quasi‐equidimensional crystals. The use of monodisperse crystalline forms of therapeutic agents can ensure constant time‐release of protein‐based medications.

Functional block copolymer nanocarriers for anticancer drug delivery

Polymer-based nanocarriers for anticancer drug delivery bearing “clickable”, biodegradable, pH-sensitive and subcellular targeting functions were designed and successfully obtained. Firstly, well-defined functional amphiphilic diblock copolymers were synthesized applying a multistep controlled polymerization and modification procedure. As a result, copolymers comprising alkyne-end functionalized biodegradable poly(D,L-lactide) and polycationic poly(N,N-dimethylaminoethyl methacrylate) blocks were obtained. The latter blocks were additionally functionalized with subcellular targeting triphenylphosphonium cations. The amphiphilic block copolymers self-associated in aqueous media into nanosized functional micelles that were able to incorporate the natural anticancer drug curcumin into their biodegradable cores. The in vitro cytotoxicity evaluation of block copolymer micelles indicated a low intrinsic inhibitory potential on the proliferation of different human cell lines. More importantly, the drug-loaded nanocarriers demonstrated an obvious ability to induce apoptosis and exhibited more prominent inhibition of the NF-κB transcription factor in cancer cell lines and their drug-resistant analogues, as compared to the free drug. The obtained results are optimistic for potential application of the functional block copolymers in nanomedicine.

A view on the aggregation issue in lysozyme crystallization

The aggregation of lysozyme in crystallizing solutions prior to crystal nucleation and during crystal growth has been the subject of numerous investigations over the past two decades. Nevertheless, it remains a controversial rather than a well-recognized phenomenon. In this study, we investigate the growth of tetragonal lysozyme crystals in quiescent solutions at the early crystal growth stage using seven different protein concentrations ranging from 25 mg ml−1 to 55 mg ml−1, 5% NaCl (w/v), 0.1 M sodium acetate buffer (pH 4.0), and constant temperature (22.0 °C). The growth rates of lysozyme crystals are determined by analyzing the time-lapse images of the evolving visible crystal area, recorded at relatively high magnification (600×). The calculated crystal growth rates are then analyzed according to an equilibrium distribution of lysozyme aggregates, which is based on a model two-body process (monomer ↔ dimer ↔ tetramer ↔ octamer ↔ hexadecamer) developed by M. Li et al. (1995). All types of aggregates are considered separately. We found that both octamers and tetramers are probable candidates for crystal growth units and suggested the most voluminous aggregate fraction as strongly influencing the crystal growth process. We also interpret the lysozyme crystal nucleation in the frame of the classical nucleation theory (CNT) and in accordance with the assumed two-body aggregation reaction set. Our results reveal that the tetramer appears to be the least achievable nucleus size across the whole protein concentration range, where classical nucleation could still be an observable phenomenon.