Sergey K. Filippov

Poly(2-Oxazoline)s – Are They More Advantageous for Biomedical Applications Than Other Polymers?

Poly(2-alkyl-2-oxazoline)s are biocompatible polymers with polypeptide-isomeric structures that are attracting increasing interest as biomaterials for drug, gene, protein, and radionuclide delivery. They are, however, still relatively new in comparison to other classes of hydrophilic water-soluble polymers already established for such use, including poly(ethylene oxide), polyvinylpyrrolidone, and polymethacrylamides such as poly[N-(2-hydroxypropyl)methacrylamide]. This feature article critically compares the synthetic aspects and physicochemical and biological properties of poly(2-alkyl-2-oxazoline)s and these commonly studied polymers in terms of their suitability for biomedical applications.

Multicompartment lipid cubic nanoparticles with high protein upload: millisecond dynamics of formation.

Membrane shapes, produced by dynamically assembled lipid/protein architectures, are crucial for both physiological functions and the design of therapeutic nanotechnologies. Here we investigate the dynamics of lipid membrane-neurotrophic BDNF protein complexes formation and ordering in nanoparticles, with the purpose of innovation in nanostructure-based neuroprotection and biomimetic nanoarchitectonics. The kinetic pathway of membrane states associated with rapidly occurring nonequilibrium self-assembled lipid/protein nanoarchitectures was determined by millisecond time-resolved small-angle X-ray scattering (SAXS) at high resolution. The neurotrophin binding and millisecond trafficking along the flexible membranes induced an unusual overlay of channel-network architectures including two coexisting cubic lattices epitaxially connected to lamellar membrane stacks. These time-resolved membrane processes, involving intercalation of discrete stiff proteins in continuous soft membranes, evidence stepwise curvature control mechanisms. The obtained three-phase liquid-crystalline nanoparticles of neurotrophic composition put forward important advancements in multicompartment soft-matter nanostructure design.

Polyoxazoline Thermoresponsive Micelles as Radionuclide Delivery Systems

Thermoresponsive polymer micelles are promising drug and radionuclide carriers with a strong passive targeting effect into solid tumors. We have synthesized ABA triblock copolymers poly[2-methyl-2-oxazoline-block-(2-isopropyl-2-oxazoline-co-2-butyl-2-oxazoline)-block-2-methyl-2-oxazoline]. These polymers are molecularly dissolved in aqueous millieu below the cloud point temperature (CPT) of the thermoresponsive central block and above CPT form polymer micelles at CMC 5-10 × 10(-5) g · mL(-1) with diameter ≈200 nm. The phenolic moiety introduced into the copolymer allowed radionuclide labeling with iodine-125 ongoing in good yield with sufficient in vitro stability under model conditions.

DNA/Fusogenic Lipid Nanocarrier Assembly: Millisecond Structural Dynamics

Structural changes occurring on a millisecond time scale during uptake of DNA by cationic lipid nanocarriers are monitored by time-resolved small-angle X-ray scattering (SAXS) coupled to a rapid-mixing stopped-flow technique. Nanoparticles (NPs) of nanochannel organization are formed by PEGylation, hydration, and dispersion of a lipid film of the fusogenic lipid monoolein in a mixture with positively charged (DOMA) and PEGylated (DOPE-PEG2000) amphiphiles and are characterized by the inner cubic structure of very large nanochannels favorable for DNA upload. Ultrafast structural dynamics of complexation and assembly of these cubosome particles with neurotrophic plasmid DNA (pDNA) is revealed thanks to the high brightness of the employed synchrotron X-ray beam. The rate constant of the pDNA/lipid NP complexation is estimated from dynamic roentgenograms recorded at 4 ms time resolution. pDNA upload into the vastly hydrated channels of the cubosome carriers leads to a fast nanoparticle-nanoparticle structural transition and lipoplex formation involving tightly packed pDNA.

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.

Thermoresponsive, Hydrolytically Degradable Polymer Micelles Intended for Radionuclide Delivery

Novel polymer micelles, prepared by self-assembling thermoresponsive poly(N-isopropylacrylamide)-graft-poly[N-(2-hydroxypropyl)methacrylamide] copolymers with hydrolytically degradable N-glycosylamine groups between the polymer blocks are proposed for delivery of diagnostic and therapeutic radionuclides into solid tumors. The micelles are formed by fast heating of an aqueous solution of the copolymer to 37 degrees C. They have a hydrodynamic diameter of 128 nm (measured using dynamic light scattering) and slowly degrade during incubation in aqueous buffer at pH = 7.4. Labeling with both (131)I and (90)Y proceeds with high yields (>85%). The unlabeled polymers are not cytotoxic for any of the tested murine and human cell lines.

Macromolecular HPMA-based nanoparticles with cholesterol for solid-tumor targeting: detailed study of the inner structure of a highly efficient drug delivery system.

We report a rigorous investigation into the detailed structure of nanoparticles already shown to be successful drug delivery nanocarriers. The basic structure of the drug conjugates consists of an N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer bearing the anticancer drug doxorubicin (Dox) bound via a pH-sensitive hydrazone bond and a defined amount of cholesterol moieties that vary in hydrophobicity. The results show that size, anisotropy, and aggregation number N(aggr) of the nanoparticles grows with increasing cholesterol content. From ab initio calculations, we conclude that the most probable structure of HPMA copolymer-cholesterol nanoparticles is a pearl necklace structure, where ellipsoidal pearls mainly composed of cholesterol are covered by a HPMA shell; pearls are connected by bridges composed of hydrophilic HPMA copolymer chains. Using a combination of techniques, we unambiguously show that the Dox moieties are not impregnated inside a cholesterol core but are instead uniformly distributed across the whole nanoparticle, including the hydrophilic HPMA shell surface.

Effect of hydrophobic interactions on properties and stability of DNA-polyelectrolyte complexes.

Polyplexes are polyelectrolyte complexes of DNA and polycations, designed for potential gene delivery. We investigated the properties of new polyplexes formed from cholesterol-modified polycations and DNA. Three complexes were tested; their cholesterol contents were 1.4, 6.3, and 8.7 mol %. UV spectroscopy and fluorescence assay using ethidium bromide proved the formation of polyplexes. The kinetics of turbidity of polyplexes solutions in physiological solution showed that the colloid stability of polyplexes increases with increasing content of cholesterol in polycations. Dynamic, static, and electrophoretic light scattering, small-angle X-ray scattering, and atomic force microscopy were used for characterization of polyplexes. The observed hydrodynamic radii of polyplexes were in the range of 30-60 nm; they were related to the polycation/DNA ratio and hydrophobicity of the used polycations (the cholesterol content). The properties of polyplex particles depend, in addition to polycation structure, on the rate of polycation addition to DNA solutions.

Polyelectrolyte-surfactant complexes formed by poly[3,5-bis(trimethylammoniummethyl)4-hydroxystyrene iodide]-block-poly(ethylene oxide) and sodium dodecyl sulfate in aqueous solutions.

Formation of polyelectrolyte-surfactant (PE-S) complexes of poly[3,5-bis(trimethylammoniummethyl)-4-hydroxystyrene iodide]-block-poly(ethylene oxide) (QNPHOS-PEO) and sodium dodecyl sulfate (SDS) in aqueous solution was studied by dynamic and electrophoretic light scattering, small-angle X-ray scattering (SAXS), atomic force microscopy, and fluorometry, using pyrene as a fluorescent probe. SAXS data from the QNPHOS-PEO/SDS solutions were fitted assuming contributions from free copolymer, PE-S aggregates described by a mass fractal model, and densely packed surfactant micelles inside the aggregates. It was found that, unlike other systems of a double hydrophilic block polyelectrolyte and an oppositely charged surfactant, PE-S aggregates of the QNPHOS-PEO/SDS system do not form core-shell particles and the PE-S complex precipitates before reaching the charge equivalence between dodecyl sulfate anions and QNPHOS polycationic blocks, most likely because of conformational rigidity of the QNPHOS blocks, which prevents the system from the corresponding rearrangement.

Coiled Coil Peptides and Polymer–Peptide Conjugates: Synthesis, Self-Assembly, Characterization and Potential in Drug Delivery Systems

Coiled coils are a common structural motif in many natural proteins that can also be utilized in the design and preparation of drug delivery systems for the noncovalent connection of two macromolecules. In this work, two different pairs of peptides forming coiled coil hetero-oligomers were designed, synthesized, and characterized. While the peptide sequences (VAALEKE)4 and (VAALKEK)4 predominantly form coiled coil heterodimers with randomly orientated peptide chains, (IAALESE)2-IAALESKIAALESE and IAALKSKIAALKSE-(IAALKSK)2 tend to form higher hetero-oligomers with an antiparallel orientation of their peptide chains. The associative behavior of these peptides was studied in aqueous solutions using circular dichroism spectroscopy, size-exclusion chromatography, isothermal titration calorimetry and sedimentation analyses. The orientation of the peptide chains in the coiled coil heterodimers was assessed using fluorescence spectroscopy with fluorescence resonance energy transfer labels attached to the ends of the peptides. The formation of the heterodimer can be used as a general method for the selective noncovalent conjugation of a specific targeting moiety with various drug carrier systems; this process involves simple self-assembly in a physiological solution before drug administration. The preparation of targeted macromolecular therapeutics consisting of a synthetic polymer drug carrier and a recombinant protein targeting ligand is discussed.