Cannabimimetic N-Stearoylethanolamine as "Double-Edged Sword" in Anticancer Chemotherapy: Proapoptotic Effect on Tumor Cells and Suppression of Tumor Growth versus Its Bio-Protective Actions in Complex with Polymeric Carrier on General Toxicity of Doxorubicin In Vivo.

This study reports a dose-dependent pro-apoptotic action of synthetic cannabimimetic N-stearoylethanolamine (NSE) on diverse cancer cell lines, including multidrug-resistant models. No antioxidant or cytoprotective effects of NSE were found when it was applied together with doxorubicin. A complex of NSE with the polymeric carrier poly(5-(tert-butylperoxy)-5-methyl-1-hexen-3-yn-co-glycidyl methacrylate)-graft-PEG was synthesized. Co-immobilization of NSE and doxorubicin on this carrier led to a 2-10-fold enhancement of the anticancer activity, particularly, against drug-resistant cells overexpressing ABCC1 and ABCB1. This effect might be caused by accelerated nuclear accumulation of doxorubicin in cancer cells, which led to the activation of the caspase cascade, revealed by Western blot analysis. The NSE-containing polymeric carrier was also able to significantly enhance the therapeutic activity of doxorubicin in mice with implanted NK/Ly lymphoma or L1210 leukemia, leading to the complete eradication of these malignancies. Simultaneously, loading to the carrier prevented doxorubicin-induced elevation of AST and ALT as well as leukopenia in healthy Balb/c mice. Thus, a unique bi-functionality of the novel pharmaceutical formulation of NSE was revealed. It enhanced doxorubicin-induced apoptosis in cancer cells in vitro and promoted its anticancer activity against lymphoma and leukemia models in vivo. Simultaneously, it was very well tolerated preventing frequently observed doxorubicin-associated adverse effects.

Covalent Conjugate of Ser-Pro-Cys Tripeptide with PEGylated Comb-Like Polymer as Novel Killer of Human Tumor Cells.

Recently, we detected a previously unknown Ser-Pro-Cys (SPC) tripeptide in the blood serum of multiple sclerosis patients. Its role as a biomarker of the autoimmune disease was suggested, although its origin and real biological activity remained unclear. Here, we created a biocompatible PEGylated comb-like polymer that was used as a platform for covalent immobilization of the SPC, which provided a possibility to explore the biological activity of this tripeptide. This macromolecular conjugate was synthesized via a reaction of the terminal epoxide group of the biocompatible copolymer of dimethyl maleate (DMM) and polyethylene glycol methyl ether methacrylate (PEGMA) with the amino group of the SPC tripeptide. Unexpectedly, the resulting conjugate containing SPC demonstrated anticancer activity in vitro. It possessed pro-apoptotic action toward human tumor cells, while there was no cytotoxic effect of that conjugate toward normal lymphocytes of human peripheral blood. The detected biological effects of the created conjugate inspired us to carry out a thorough study of structural and colloidal-chemical characteristics of this surface-active copolymer containing side PEG chains and a terminal nontoxic synthetic fragment. The copolymer composition, in particular, the content of the peptide fragment, was determined via elemental analysis and NMR spectroscopy. At CMC, it formed polymeric micelle-like structures with a hydrodynamic diameter of 180 ± 60 nm. The conjugation of the peptide fragment to the initial comb-like copolymer caused a change of zeta-potential of the formed micelle-like structures from -0.15 to 0.32 mV. Additional structural modification of the created polymeric nanoplatform was performed via attachment of fluorescein isothiocianate (FITC) dye that permitted monitoring of the behavior of the bioactive SPC-functionalized conjugate in the treated tumor cells. Its penetration into those cells and localization in their cytoplasm were revealed. The principal novelty of this study consists in finding that covalent conjugation of two nontoxic compounds-SPC tripeptide and comb-like PEGylated polymer-led to an unexpected synergy which appeared in the distinct cytotoxic action of the macromolecular complex toward human tumor cells. A potential role of peculiarities of the colloidal-chemical properties of the novel conjugate in its cytotoxic effect are discussed. Thus, synthesized comb-like PEGylated polymers can provide a prospective nanoplatform for drug delivery in anticancer chemotherapy.

Fabrication of 1D-Nanofiber/Fe2O3 Composites with Tailored Magnetic Properties.

A novel flexible electrospun nanofiber/γ-Fe2O3 composite has been obtained from suspension of γ-Fe2O3 nanoparticles in polyvinylpyrolidone solution in dimethylformamide. The impedance spectroscopy of the synthesized nanofiber/γ-Fe2O3 composite was carried out. Negative magnetoresistance and giant magnetocapacitance effects, as well as phenomenon of a "negative capacitance" at room temperature were observed in magnetic field (2.75 κOe) in infra-low frequency range. The polarization properties and volt-ampere characteristics of the nanocomposite in the applied magnetic field indicate the increase in the dielectric permittivity ɛ and the emergence of spin electromotive force, which enables us to accumulate of electric energy at quantum level. A quantum-mechanical model, which explained the non-monotonous behaviour of the volt-ampere characteristic of the novel nanofiber based composite, has been suggested.

Fluorine-containing block/branched polyamphiphiles forming bioinspired complexes with biopolymers.

Colloidal-chemical characteristics of block/branched cationic and non-ionic polyamphiphiles containing poly(fluorine-alkyl methacrylate) (poly(FMA)) block and their intermolecular complexes with biopolymers were studied. The dependences of their surface activity and micelle size on the length of hydrophobic and hydrophilic blocks, as well as the length of side fluorine-alkyl branches were established. Poly(FMA)-block-poly(DMAEMA) was used for formation of interpolyelectrolyte complexes with plasmid DNA (pDNA) via their electrostatic interaction. Novel non-viral polyplexes were tested as gene delivery systems for mammalian cells. The results of DLS, TEM and MALDI-ToF studies demonstrated disaggregation of lysozyme (LYZ) aggregates in the presence of poly(FMA)-block-poly(NVP) and formation of the polyamphiphile…LYS complex possessing antibacterial action.

Comb-like PEG-containing polymeric composition as low toxic drug nanocarrier.

BACKGROUND: Development of biocompatible multifunctional polymeric drug carriers is crucial in modern pharmaceutics aimed to create "smart" drugs. The high potential of the PEGylated comb-like polymeric nanocarrier (PNC) in delivering both traditional and experimental drugs to tumor cells in vitro and in vivo has been demonstrated previously. In the present study, we investigated the general toxicity of polyethylene glycol (PEG) processed with both covalent and non-covalent attachments of PEG to compose a comb-like polymer that behaves like a simple chain of n monomers decorated with swollen side chains. The PNC possesses properties of a water-soluble surfactant containing methyl-terminated PEG side branches in some monomer units attached covalently to the carbon chain backbone. RESULTS: We have demonstrated that the synthesized PNC possesses weak toxic effects toward human leukemia cells (HL-60 and Jurkat lines), as well as toward hepatocellular (HepG2), colon (HCT116) and breast (MCF-7) tumor cell lines. Additionally, after a long period (20 days) of intraperitoneal administration, the PNC had no significant toxic effects in laboratory white mice (470 mg/kg body mass in 1 ml) and Wistar rats (440 mg/kg body mass in 10 ml). CONCLUSION: The developed PNC we studied can be qualified as a compound of grade 4 toxicity (low toxicity substance). The reduced toxicity of this PNC in combination with its improved bioavailability and previously detected capability to enhance cytotoxicity toward tumor cells in vitro and potential tumor treatment effects in vivo suggests its potential as a safe drug delivery platform for treating various diseases, especially cancer.

Hepatic metallothioneins in molecular responses to cobalt, zinc, and their nanoscale polymeric composites in frog Rana ridibunda.

Despite numerous studies suggesting a dramatic decline of amphibians, the biochemical mechanisms of adaptation in these animals to polluted environment are poorly studied. The aim of this study was to elucidate the ability to release cobalt (Co) and zinc (Zn) from their nanoscale complexes (NCs) derived from the polymeric substance of N-vinylpyrrolidone (PS) in the liver of amphibian (Rana ridibunda). Frog males were subjected to 14days exposure to waterborne Co(2+) (50µg/L), Zn(2+) (100µg/L), as well as corresponding concentrations of Co-NC, Zn-NC or PS. Main attention was paid to MT's interrelations with indices of stress and toxicity. Only Co(2+) and Zn(2+) caused elevation of the correspondent metal in MTs. Co(2+) caused down-regulation of cathepsin D activity, while Zn(2+), Zn-NC and the PS up-regulated this activity. Zn(2+) provoked 1.6 times increase of metal-bounded form of the MT (MT-Me), while all other exposures caused the elevation of the ratio of MT total protein concentration (MT-SH) and concentrations of the MT-Me and/or immunoreactive (MTi) form (up to ~10 times) accompanied by a decrease in the levels of oxyradicals. The increased DNA fragmentation and down-regulation of caspase-3 activity in relation to the redox state of glutathione and/or lactate/pyruvate were shown at all exposures. These data indicate the vulnerability of the redox state of cellular thiols and inability to release Co and Zn from NCs in frog's liver.

Responses of hepatic metallothioneins and apoptotic activity in Carassius auratus gibelio witness a release of cobalt and zinc from waterborne nanoscale composites.

The main goal of this study was to evaluate the ability of fish Carassius auratus tissues to release cobalt (Co) and zinc (Zn) cations present in the applied Co- and Zn-containing nanoscale composites (NCs). Male fish was subjected to 14day long action of Co- and Zn-NCs, as well as of Co(2+) and Zn(2+) or polymeric substance (PS) used for the NC preparation and derived from the vinylpyrrolidone. 50µg∙L(-1) of Co and 100µg∙L(-1) of Zn were applied either as a salt or a nanocomposite. Both Co and Co-NC increased (3.1 and 2.3 times, respectively) concentration of total Co, metallothionein-related Co (3.7 and 6.6 times, respectively) and thiols (by 71 and 95%, respectively), and caspase-3 activity (2.2 and 3.7 times, respectively) in the fish liver. At the same time, Co and Co-NC decreased glutathione level (1.8 and 1.9 times, respectively) and activated vitellogenesis (5.1 and 9.9 times, respectively) in the fish liver. Both Zn and Zn-NC increased markedly concentrations of metallothionein-related Zn (2.4 and 2.9 times, respectively) and Cu (2.8 and 3.2 times, respectively), and decreased metallothionein-related thiol (2.5 and 4.2 times, respectively), oxyradical (by 30.4 and 44.2%, respectively), and caspase-3 (3.0 and 5.3 times, respectively) levels in the fish liver. These peculiarities are common for metal and metal-NC and witness a release of metal from NS in fish organism. The differences in the levels of DNA strand breaks, biotransformation enzymes and total Zn levels in the liver were dependent on the kind of exposure.

Biophysical study of novel oligoelectrolyte-based nonviral gene delivery systems for mammalian cells.

BACKGROUND: Gene therapy is an important treatment for genetic and acquired diseases. The success of gene therapy is largely dependent on the development of suitable vectors for gene transfer. Vectors are needed to overcome cellular barriers and to achieve efficient DNA delivery with low cytotoxicity. In the present study, we synthesized and characterized a novel comb-like oligoelectrolyte nanocarrier, BG-2, as a nonviral gene delivery vector. METHODS: A novel surface-active oligoelectrolyte of comb-like structure was synthesized via controlled radical copolymerization using oligoperoxide Cu(+2) coordinating complex as a multisite initiator of graft copolymerization. The critical micellar concentration was determined by Nile Red fluorescence. Complex formation of DNA with BG-2 was determined by YOYO-1 fluorescence. The physicochemical properties of DNA in complex with BG-2 have been investigated by electrophoresis, dynamic light scattering and fluorescence spectroscopy. The BG-2/DNA complex was demonstrated by scanning electron microscopy. Interactions between BG-2/DNA complex and model membranes were also studied. The sensitivity of the DNA molecule, complexed with BG-2, against deoxyribonuclease I and serum nucleases was assessed by agarose gel electrophoresis. BG-2 efficiency in the transfection of HeLa cells was determined by measuring luciferase gene expression using a luminometer and cytotoxicity was also evaluated. RESULTS: BG-2 oligoelectrolyte was successful in overcoming cellular barriers as a result of forming stable and small sized complexes with DNA, interacting with model membranes in a desirable manner and protecting DNA from nuclease. The transfection efficiency was quite high and cytotoxicity was low. CONCLUSIONS: BG-2 appears to be a promising nonviral vector with low cytotoxicity and efficient transfection properties.

A novel method for genetic transformation of yeast cells using oligoelectrolyte polymeric nanoscale carriers.

The genetic transformation of target cells is a key tool in modern biological research, as well as in many gene therapy and biotechnology applications. Here we describe a new method for delivery of DNA into several industrially important species of yeast, including Saccharomyces cerevisiae. Our method is based on the use of a novel nanoscale oligoelectrolyte polymer possessing a comb-like structure as a carrier molecule. Direct comparisons to standard transformation methods clearly show that our approach: (i) yields two times more transformants of Hansenula polymorpha NCYC 495 compared to electroporation approaches and 15 times more transformants compared to lithium acetate protocols, as well as (ii) 5 times more Pichia pastoris GS115 transformants compared to electroporation and 79 times more transformants compared to lithium acetate. Taken together, these results clearly indicate genetic transformation of yeasts using oligoelectrolyte polymer carriers is a highly effective means of gene delivery.

Evaluation of biotargeting and ecotoxicity of Co²âº-containing nanoscale polymeric complex by applying multi-marker approach in bivalve mollusk Anodonta cygnea.

Cobalt (Co(2+)) is present in many nanoscaled materials created for various applications. The key goal of our study was to develop sensitive approaches for assessing the bio-risks associated with using novel Co(2+)-containing nanoscaled polymeric complex (Co-NC). Freshwater bivalve mollusk Anodonta cygnea (Unionidae) was subjected to 14 d action of the developed Co-NC, as well as of Co(2+) applied in the corresponding concentration (50 µg L(-1)) or polymeric substance (PS). All experimental groups under study have demonstrated signs of toxic targeting, notably changes in DNA characteristics, oxidative stress (with particularities in each exposed group) and activation of anaerobiosis (Co(2+) and Co-NC). However, the group exposed to Co-NC showed some advantages that can be related to the activation of metallothionein (MT) function (increase in the level of MT-related SH-groups (MT-SH)): low level of oxyradical formation, no increase in protein carbonylation and vitellogenin-like proteins concentration unlike in Co(2+) and PS exposed groups. On the other hand, Co(2+) increased metal (Co, Cu, Zn and Cd) binding to MT (MT-Me) without changes in MT-SH level jointly with activation of oxyradical formation and apoptosis and decreasing of lysosomal membrane stability. PS per se initiated unbalanced changes in activities of the biotransformation enzymes ethoxyresorufin-O-deethylase and glutathione-S-transferase. Thus, Co(2+) complexing with the developed PS prevented bio-toxic effects of free Co(2+) ions and PS per se, at least in the studied hydrobiont. The MT-SH was the main distinguishing index of Co-NC group selected by classification and regression tree analysis.

The use of oligoperoxide-coated magnetic nanoparticles to label stem cells.

Iron oxide nanoparticles obtained by the coprecipitation of Fe(II) and Fe(III) salts and oxidation were coated with a novel poly(vinyl acetate-co-5-tert-(butylperoxy)-5-methylhex-1-en-3-yne-co-butyl acrylate-co-maleic anhydride) (PVBM) oligomer to ensure colloidal stability. The magnetic nanoparticles were thoroughly characterized by a range of physico-chemical methods, which proved the presence of the coating on the particles. Experiments with rat mesenchymal stem cells (rMSCs) confirmed that PVBM-coated gamma-Fe2O3 nanoparticles were not cytotoxic and that the average efficiency of stem cell labeling was good and comparable to that obtained with commercial agents. The cells labeled with PVBM-coated gamma-Fe2O3 nanoparticles displayed excellent contrast on magnetic resonance (MR) images. Such particles are thus promising for in vivo MR imaging of transplanted cells. Moreover, PVBM offers the possibility of additional modification by grafting compounds that reduce non-specific protein adsorption.

The peculiarities of homogeneous nucleation of reactive Cu0 colloidal particles in the presence of functional oligoperoxides.

A method is proposed that combines the stage of formation of colloidal metal and metal-oxide particles with the stage of their surface modification by functional surface-active oligoperoxides (FSAP), which are sorbed irreversibly. Investigation of copper particle homogeneous nucleation kinetics witnesses the significant influence of supermolecular micelle-like structures formed by FSAP in solution on the reduction rate of Cu2+ cations caused by a phenomenon analogous to micellar catalysis. The rate constants of copper reduction in different local zones of the process have been determined. Particle homogeneous nucleation kinetics in the presence of surface-active oligoperoxides has been found to correspond to the main regularities of the Michaelis-Menten equation describing micellar catalysis. The carrying out of copper particle formation in distinct zones correlates well with the particle size distribution. The presence of reactive ditertiary peroxidic fragments on the particle surface as a result of FSAP sorption confers reliable protection from oxidation, hydrophobicity, and ability to form free radicals and participate in elementary stages of radical processes.