A. A. Zubareva

Characterization of Protein and Peptide Binding to Nanogels Formed by Differently Charged Chitosan Derivatives

Chitosan (Chi) is a natural biodegradable cationic polymer with remarkable potency as a vehicle for drug or vaccine delivery. Chi possesses multiple groups, which can be used both for Chi derivatization and for particle formation. The aim of this work was to produce stable nanosized range Chi gels (nanogels, NGs) with different charge and to study the driving forces of complex formation between Chi NGs and proteins or peptides. Positively charged NGs of 150 nm in diameter were prepared from hexanoyl chitosan (HC) by the ionotropic gelation method while negatively charged NGs of 190 nm were obtained from succinoyl Chi (SC) by a Ca2+ coacervation approach. NGs were loaded with a panel of proteins or peptides with different weights and charges. We show that NGs preferentially formed complexes with oppositely charged molecules, especially peptides, as was demonstrated by gel-electrophoresis, confocal microscopy and HPLC. Complex formation was accompanied by a change in zeta-potential and decrease in size. We concluded that complex formation between Chi NGs and peptide/proteins is mediated mostly by electrostatic interactions.

Nanoparticles based on succinylchitosan with doxorubicin: Preparation and properties

The conditions of succinylchitosan-based nanostructures with doxorubicin preparation were optimized. Nanoparticles were obtained using a precipitation conservation method for the first time, and the particles were further used to prepare noncovalent complexes with doxorubicin. The physicochemical and biological properties of the studied structures were investigated. It was shown that the average size of the obtained nanostructures is 200–250 nm and their ζ-potential was determined as negative (−20 to −25 mV). Nanoparticles loaded with doxorubicin were shown to demonstrate a positive cytotoxic effect in vitro. Doxorubicin sorbtion on nanoparticles resulted in no changes in the properties of the drug, which may make it possible to lower its toxicity in vivo.

Analysis of toxicity and biocompatibility of chitosan derivatives with different physico-chemical properties

A comparative study of the toxicity and hemocompatibility of chitosan and its derivatives with different acetylation degrees, molecular masses, charges, and hydrophobicity has been performed. It has been shown that only positively charged chitosan derivatives activate platelets and suppress cell proliferation, regardless of the acetylation degree, molecular mass, and hydrophobicity. Chitosan quaternization decreases toxicity at a low degree of substitution and abruptly increases it at a high one. Negatively charged chitosan derivatives were nontoxic and compatible with blood components. It was concluded that the toxicity of chitosan and its derivatives is defined by their charge and solubility at a neutral pH.

CELL BINDING AND PENETRATION OF QUATERNIZED CHITOSAN DERIVATIVES

Chitosan (Ch) is an attractive biopolymer with multiple reactive groups. However it is poorly soluble at neutral pH. Quaternization improves its solubility and permits the development of various positively charged drug delivery systems. The aim of this work was to study the solubility, toxicity, cell binding, and penetration of 20 kDa chitosan with 9, 40, 58 and 98% of quaternary ammonium group substitution (ChQ1 to ChQ4 accordingly). We showed that ChQ with substitution degree >40% was soluble in a wide pH range. Unexpectedly ChQ2 and ChQ3 were more toxic to cells than Ch, ChQ1 and ChQ4. Higher toxicity of ChQ was found against macrophage like cell line RAW264.7 than against epithelial cells MiaPaCa-2. All ChQ, in contrast to unmodified Ch, easily bound and penetrated the cells with the highest uptake by ChQ4. Thus, quaternized chitosan derivatives can be used for biomedical applications.

Antitumor Activity of Furanoallocolchicinoid-Chitosan Conjugate

Colchicine irreversibly binds to tubulin, blocks microtubule formation, and inhibits cell division. However, its usage as an antitumor agent is limited due to its distribution to many tissues and low accumulation in the tumor. The increase in molecule weight can change colchicine biodistribution and decrease side effects. The aim of this work was to study in vivo and in vitro antitumor activity of colchicine-chitosan conjugate. A new allocolchicine derivative – furanoallocolchicinoid 3 was synthesized and conjugated to chitosan (4). Both 3 and 4 induced in vitro tubulin reorganization, cell cycle arrest, and inhibition of cell proliferation in 2D and 3D cultures. Antitumor effect of chitosan, 3, and 4 was studied in Wnt-1 breast tumor bearing mice. Conjugate 4 demonstrated significantly better tumor growth inhibition than 3 possibly as a result of a better accumulation in the tumor.

Interactions of chitosan and its derivatives with cells (review)

Mechanisms of interaction between chitosan, various macromolecules or drug delivery systems and mammalian cells are reviewed. Modernly the role of different physicochemical properties of chitosan and chitosan nanoparticles on the mechanisms of cell bunding, endocytosis and redistribution are poorly understood.

Characterization of physicochemical parameters of nanoparticles formed from modified chitosan

Chitosan is a biodegradable and biocompatible polysaccharide widely used in different applications in medicine. The polycationic structure of the polymer provides many possibilities for its modification. One of the most interesting applications of chitosan is the development of nanosized delivery systems for biologically active molecules. The functional properties of these delivery systems depend severely on the structure and properties of nanoparticles. Using dynamic light scattering, atomic force microscopy, and to confocal microscopy, we have shown that both hexanoyl chitosan (HC) and succinoyl chitosan (SC) formed nanoparticles of comparable diameters from 120 to 250 nm. Using several methods of analysis helped us identify a minor fraction with a larger size of 600 to 700 nm formed by nanoparticle aggregates. The determined ζ potential was from −20 to −25 mV for SC nanoparticles and from 30 to 35 mV for HC nanoparticles.

Antimicrobial Peptides in Health and Disease (Review)

The review describes the latest data on the role of antimicrobial peptides (AMPs) in health and disease, as well as their structure and mechanisms of action. AMPs mediate protection by both direct lysis of bacteria and also by regulation of inflammation and chemotaxis, thus demonstrating immunomodulatory properties. A large amount of data shows that AMPs play an important role in the pathogenesis of multiple chronic diseases with genetic predisposition, such as atopic dermatitis, rosacea, and scleroderma.

Cross-presentation of lactoferrin encapsulated into chitosan-based nanoparticles

Induction of CD8+ cytotoxic T-cell response is essential for the protection from intracellular pathogens. It requires major histocompatibility complex class I processing of newly synthesized proteins transported from the cytosolic pathway. Presentation of mature soluble proteins occurs via a cross-presentation (CP) pathway that is much less efficient in the activation of cytotoxic response. Encapsulation of proteins into polymeric nanoparticles (NPs) can modulate the efficacy of antigen CP. In this article, a model antigen lactoferrin (L) was encapsulated into polysaccharide NPs with different physicochemical properties (size, charge, and hydrophobicity) and used as an immunogen. CD8+ or CD4+ associated IgG2a or IgG1 subclasses of L-specific antibodies, respectively, served as a measure of CD8+ versus CD4+ T-cell activation. Among five types of NPs produced, only succinylchitosan–galactomannan (LSG) and succinylchitosan–PEG-chitosan (LSPC) NPs induced a significant IgG2a response. IgG1 production was comparable in all but hydrophobic succinyl-dodecyl-chitosan (LSD) NPs, where it was only marginal. Confocal studies demonstrated that galactomannan-equipped LSG-NPs induced vacuolar type of CP, while positively charged LSPC-NPs were transported mostly via the cytosolic CP pathway.