Nanoaggregates of Biphilic Carboxyl-Containing Copolymers as Carriers for Ionically Bound Doxorubicin.

Application of nanocarriers for drug delivery brings numerous advantages, allowing both minimization of side effects common in systemic drug delivery and improvement in targeting, which has made it the focal point of nanoscience for a number of years. While most of the studies are focused on encapsulation of hydrophobic drugs, delivery of hydrophilic compounds is typically performed via covalent attachment, which often requires chemical modification of the drug and limits the release kinetics. In this paper, we report synthesis of biphilic copolymers of various compositions capable of self-assembly in water with the formation of nanoparticles and suitable for ionic binding of the common anticancer drug doxorubicin. The copolymers are synthesized by radical copolymerization of N-vinyl-2-pyrrolidone and acrylic acid using n-octadecyl-mercaptan as a chain transfer agent. With an increase of the carboxyl group's share in the chain, the role of the electrostatic stabilization factor of the nanoparticles increased as well as the ability of doxorubicin as an ion binder. A mathematical description of the kinetics of doxorubicin binding and release is given and thermodynamic functions for the equilibrium ionic binding of doxorubicin are calculated.

In Vitro Assessment of Poly-N-Vinylpyrrolidone/Acrylic Acid Nanoparticles Biocompatibility in a Microvascular Endothelium Model.

An amphiphilic copolymer of N-vinyl-2-pyrrolidone and acrylic acid-namely, p(VP-AA)-OD6000 (p(VP-AA))-was synthesized to prepare p(VP-AA) nanoparticles (NPs). Furthermore, the copolymer was linked with CFSE, and the so-prepared nanoparticles were loaded with the DiI dye to form D nanoparticles (DNPs). In this study, as demonstrated by immunofluorescence microscopy, immunofluorescence, and confocal microscopy, DNPs were readily taken up by human microvascular endothelial cells (HMEC-1) cells in a concentration-dependent manner. Upon uptake, both the CFSE dye (green stain) and the DiI dye (red stain) were localized to the cytoplasm of treated cells. Treatment with p(VP-AA) did not affect the viability of normal and challenged with LPS, HMEC-1 cells at 0.010 mg/mL and induced a dose-dependent decrease of these cells' viability at the higher concentrations of 0.033 and 0.066 mg/mL (p ≤ 0.01; p ≤ 0.001, respectively). Furthermore, we focused on the potential immunological activation of HMEC-1 endothelial cells upon p(VP-AA) NPs treatment by assessing the expression of adhesion molecules (E-Selectin, ICAM-1, and V-CAM). NPs treatments at concentrations utilized (p = NS) did not affect individual adhesion molecules' expression. p(VP-AA) NPs do not activate the endothelium and do not affect its viability at pharmacologically relevant concentrations.

Hybrid (Bovine Serum Albumin)/Poly(N-vinyl-2-pyrrolidone-co-acrylic acid)-Shelled Microbubbles as Advanced Ultrasound Contrast Agents.

Microbubbles are routinely used ultrasound contrast agents in the clinic. While a soft protein shell is commercially preferable for imaging purposes, a rigid polymer shell demonstrates prolonged agent stability. Hence, combining polymers and proteins in one shell composition can advance microbubble properties. We formulated the hybrid "protein-copolymer" microbubble shell with a complex of bovine serum albumin and an amphiphilic copolymer of N-vinyl-2-pyrrolidone and acrylic acid. The resulting microbubbles demonstrated advanced physicochemical and acoustic properties, preserving in vitro biocompatibility. Adjusting the mass ratio between protein and copolymer allowed fine tuning of the microbubble properties of concentration (by two orders, up to 1010 MBs/mL), mean size (from 0.8 to 5 µm), and shell thickness (from 28 to 50 nm). In addition, the minimum air-liquid surface tension for the "protein-copolymer" solution enabled the highest bubble concentration. At the same time, a higher copolymer amount in the bubble shell increased the bubble size and tuned duration and intensity of the contrast during an ultrasound procedure. Demonstrated results exemplify the potential of the hybrid "protein-polymer" microbubble shell, allowing tailoring of microbubble properties for image-guided applications, combining advances of each material involved in the formulation.

Amphiphilic Poly-N-vinylpyrrolidone Nanoparticles as Carriers for Nonsteroidal, Anti-Inflammatory Drugs: Pharmacokinetic, Anti-Inflammatory, and Ulcerogenic Activity Study.

Nanoparticles are increasingly utilized as drug delivery agents. Previously, we have developed a drug delivery system based on amphiphilic derivatives of poly-N-vinylpyrrolidone (PVP-OD4000) with excellent biocompatibility. In the current study, we assessed the pharmacokinetics, anti-inflammatory profile, and ulcerogenic potential of indomethacin (IMC)-loaded PVP-OD4000 nanoparticles compared to the free drug. Wistar male rats were utilized for a pharmacokinetics study and an anti-inflammatory study. Loaded IMC exhibited a slower elimination rate (p < 0.05) and a higher blood plasma concentration at 8 and 24 h after intraperitoneal injection compared with free IMC. In addition, decreased uptake of loaded IMC in the liver and kidney compared to free IMC (p < 0.05) was detected. Furthermore, PVP-OD4000 nanoparticles loaded with IMC showed an enhanced anti-inflammatory effect compared to free IMC (p < 0.05) in carrageenan-induced and complete Freund's adjuvant-induced−(CFA) sub-chronic and chronic paw edema treatment (p < 0.01; p < 0.01). Notably, upon oral administration of loaded IMC, animals had a significantly lower ulcer score and Paul's Index (3.9) compared to the free drug (p < 0.05). The obtained results suggest that IMC loaded to PVP nanoparticles exhibit superior anti-inflammatory activity in vivo and a safe gastrointestinal profile and pose a therapeutic alternative for the currently available NSAIDs' administration.

Synthesis of Amphiphilic Copolymers of N-Vinyl-2-pyrrolidone and Allyl Glycidyl Ether for Co-Delivery of Doxorubicin and Paclitaxel.

Co-delivery of chemotherapeutics in cancer treatment has been proven essential for overcoming multidrug resistance and improving the outcome of therapy. We report the synthesis of amphiphilic copolymers of N-vinyl-2-pyrrolidone and allyl glycidyl ether of various compositions and demonstrate that they can form nanoaggregates capable of simultaneous covalent immobilization of doxorubicin by the epoxy groups in the shell and hydrophobic-driven incorporation of paclitaxel into the core of nanoparticles. The structure of the obtained copolymers was characterized by 13C NMR, IR, and MALDI spectroscopy, as well as adsorption at the water/toluene interface. A linear increase in the number-average molecular weight of amphiphilic copolymers and a decrease in the number-average diameter of macromolecular aggregates with an increase in the ratio N-vinyl-2-pyrrolidone/allyl glycidyl ether were observed. The assembled nanocarriers were characterized by DLS. The reported novel nanocarriers can be of interest for delivery and co-delivery of a wide range of pharmacological preparations and combined therapy for cancer and other deceases.

Synthesis, Self-Assembly and In Vitro Cellular Uptake Kinetics of Nanosized Drug Carriers Based on Aggregates of Amphiphilic Oligomers of N-Vinyl-2-pyrrolidone.

Development of nanocarrier-based drug delivery systems is a major breakthrough in pharmacology, promising targeted delivery and reduction in drug toxicity. On the cellular level, encapsulation of a drug substantially affects the endocytic processes due to nanocarrier-membrane interaction. In this study we synthesized and characterized nanocarriers assembled from amphiphilic oligomers of N-vinyl-2-pyrrolidone with a terminal thiooctadecyl group (PVP-OD). It was found that the dissolution free energy of PVP-OD depends linearly on the molecular mass of its hydrophilic part up to M¯n = 2 × 104, leading to an exponential dependence of critical aggregation concentration (CAC) on the molar mass. A model hydrophobic compound (DiI dye) was loaded into the nanocarriers and exhibited slow release into the aqueous phase on a scale of 18 h. Cellular uptake of the loaded nanocarriers and that of free DiI were compared in vitro using glioblastoma (U87) and fibroblast (CRL2429) cells. While the uptake of both DiI/PVP-OD nanocarriers and free DiI was inhibited by dynasore, indicating a dynamin-dependent endocytic pathway as a major mechanism, a decrease in the uptake rate of free DiI was observed in the presence of wortmannin. This suggests that while macropinocytosis plays a role in the uptake of low-molecular components, this pathway might be circumvented by incorporation of DiI into nanocarriers.

Novel chitosan and bacterial cellulose biocomposites tailored with polymeric nanoparticles for modern wound dressing development.

Dressing biomaterials play a key role in wound management keeping a moisture medium and protecting against external factors. Natural and synthetic materials could be used as dressings where chitosan and bacterial cellulose is one of the most important solutions. These biopolymers have been used for wound dressing based on their non-toxic, biodegradable, and biocompatible features. In this study, biocomposites based on bacterial cellulose and chitosan membranes tailored with antimicrobial loaded poly(N-isopropylacrylamide)/polyvinyl alcohol nanoparticles were prepared. Core-shell polymeric nanoparticles, bacterial cellulose/chitosan membranes, and biocomposites were independently loaded with silver sulfadiazine, a well-known sulfonamide antibacterial agent used in the therapy of mild-to-moderate infections for sensitive organisms. The chemistry, structure, morphology, and size distribution were investigated by Fourier transformed infrared spectroscopy (FTIR-ATR), RAMAN spectroscopy, Scanning electron (SEM) and Transmission electron microscopy (TEM), and Dynamic light scattering (DLS). In vitro release behaviors of silver sulfadiazine from polymeric nanoparticles and biocomposites were investigated. The biological investigations revealed good biocompatibility of both the nanoparticles and the biocomposites in terms of human dermal fibroblasts viability and proliferation potential. Finally, the drug-loaded polymeric biomaterials showed promising characteristics, proving their high potential as an alternative support to develop a biocompatible and antibacterial wound dressing.

Kinetics and Mechanism of Synthesis of Carboxyl-Containing N-Vinyl-2-Pyrrolidone Telehelics for Pharmacological Use.

It was found that sulfanylethanoic and 3-sulfanylpropanoic acids are effective regulators of molecular weight with chain transfer constants of 0.441 and 0.317, respectively, and show an unexpected acceleration effect on the radical polymerization of N-vinyl-2-pyrrolidone, initiated by 2,2'-azobisisobutyronitrile. It was determined for the first time that the thiolate anions of mercapto acids form a high-temperature redox initiating system with 2,2'-azobisisobutyronitrile during the radical polymerization of N-vinyl-2-pyrrolidone in 1,4-dioxane. Considering the peculiarities of initiation, a kinetic model of the polymerization of N-vinyl-2-pyrrolidone is proposed, and it is shown that the theoretical orders of the reaction rate, with respect to the monomer, initiator, and chain transfer agent, are 1, 0.75, 0.25, and are close to their experimentally determined values. Carboxyl-containing techelics of N-vinyl-2-pyrrolidone were synthesized so that it can slow down the release of the anticancer drug, doxorubicin, from aqueous solutions, which can find its application in the pharmacological field.

Chemical Oxidative Polymerization of Methylene Blue: Reaction Mechanism and Aspects of Chain Structure.

The kinetic regularities of the initial stage of chemical oxidative polymerization of methylene blue under the action of ammonium peroxodisulfate in an aqueous medium have been established by the method of potentiometry. It was shown that the methylene blue polymerization mechanism includes the stages of chain initiation and growth. It was found that the rate of the initial stage of the reaction obeys the kinetic equation of the first order with the activation energy 49 kJ × mol-1. Based on the proposed mechanism of oxidative polymerization of methylene blue and the data of MALDI, EPR, and IR spectroscopy methods, the structure of the polymethylene blue chain is proposed. It has been shown that polymethylene blue has a metallic luster, and its electrical conductivity is probably the result of conjugation over extended chain sections and the formation of charge transfer complexes. It was found that polymethylene blue is resistant to heating up to a temperature of 440 K and then enters into exothermic transformations without significant weight loss. When the temperature rises above 480 K, polymethylene blue is subject to endothermic degradation and retains 75% of its mass up to 1000 K.

Live In-Vivo Neuroimaging Reveals the Transport of Lipophilic Cargo Through the Blood-Retina Barrier with Modified Amphiphilic Poly-N-Vinylpyrrolidone Nanoparticles.

The blood-retina barrier (BRB), analogous to the blood-brain barrier, is a major hurdle for the passage of drugs from the blood to the central nervous system. Here, we designed polymeric nanoparticles from amphiphilic poly-/V-vinylpyrrolidone (Amph-PVP NPs) as a new carrier-system and investigated their ability to pass the BRB using a live In-Vivo neuroimaging system for the retina in rats and ex-vivo wholemounted retinae preparation. Amph-PVP NPs were loaded with hydrophobic fluorescent markers as a surrogate for hydrophobic drugs. Linking these NPs with the hydrophobic fluorescence marker Carboxyfluorescein-succinimidyl-ester (CFSE) to the surface, induced the passage of the cargo into the retina tissue. In particular, we observed a substantial internalization of the CFSE-linked NPs into blood cells. We propose surface- modified Amph-PVP NPs as a potential new nano-carrier platform to target posterior eye and potentially brain diseases while camouflaged by blood cells.

Amphiphilic Poly(N-Vinylpyrrolidone) Nanoparticles Loaded with DNA Plasmids Encoding Gn and Gc Glycoproteins of the Rift Valley Fever Virus: Preparation and In Vivo Evaluation.

The aim of the study was to develop amphiphilic poly(N-vinylpyrrolidone) (PVP) nanoparticles (NPs) loaded with DNA plasmids encoding Gn and Gc glycoproteins of the Rift Valley fever virus (RVFV) and to study the humoral response in vivo. DNA plasmids were protected from extracellular nucleases by loading in NPs from PVP derivatives modified with amino acids ß-alanine (Ala7-PVPOD4000) or glycine (Gly7.5-PVP-OD4000) fabricated by the original self-assembly technique. The obtained NPs were administered in mice and the enhancement of humoral response compared to this one in case of immunization with native DNA plasmids was demonstrated. The NPs loaded with DNA plasmids are promising for the fabrication of various DNA particulate vaccines.

Assessment of Amphiphilic Poly-N-vinylpyrrolidone Nanoparticles' Biocompatibility with Endothelial Cells in Vitro and Delivery of an Anti-Inflammatory Drug.

Nanoparticles (NPs) produced from amphiphilic derivatives of poly-N-vinylpyrrolidone (Amph-PVP), composed of various molecular weight polymeric hydrophilic fragments linked into hydrophobic n-alkyl chains of varying lengths, were previously shown to exert excellent biocompatibility. Although routes of administration can be different, finally, most nanosystems enter the blood circulation or lymphatic vessels, and by this, they establish direct contact with endothelial cells. In this study, Amph-PVP NPs and fluorescently labeled Amph-PVP-based NPs, namely "PVP" NPs (Amph-PVP-NPs (6000 Da) unloaded) and "F"-NPs (Amph-PVP-NPs (6000 Da) loaded with fluorescent FITC), were synthesized to study Amph-PVP NPs interactions with HMEC-1 endothelial cells. PVP NPs were readily uptaken by HMEC-1 cells in a concentration-dependent manner, as demonstrated by immunofluorescence imaging. Upon uptake, the FITC dye was localized to the perinuclear region and cytoplasm of treated cells. The generation of lipopolysaccharide (LPS)-induced activated endothelium model revealed an increased uptake of PVPNPs, as shown by confocal microscopy. Both unloaded PVP NPs and F-NPs did not affect EC viability in the 0.01 to 0.066 mg/mL range. Furthermore, we focused on the potential immunological activation of HMEC-1 endothelial cells upon PVPNPs treatment by assessing the expression of their E-Selectin, ICAM-1, and VCAM-1 adhesion receptors. None of the adhesion molecules were affected by NP treatments of both activated by LPS and nonactivated HMEC-1 cells, at the utilized concentrations (p = NS). In this study, PVP (6000 Da) NPs were used to encapsulate indomethacin, a widely used anti-inflammatory drug. The synthesized drug carrier complex did not affect HMEC-1 cell growth and expression of E-selectin, ICAM-1, and VCAM-1 adhesion receptors. In summary, PVP-based NPs are safe for use on both basal and activated endothelium, which more accurately mimics pathological conditions. Amph-PVP NPs are a promising drug delivery system.

Fluorouracil neutrophil extracellular traps formation inhibited by polymer nanoparticle shielding.

Venous thromboembolism is a frequent complication occurring in patients suffering from neoplastic diseases. Since neutrophil extracellular traps (NETs) play an important role both in the development of the tumor growth process and in inducing complications such as thrombosis, indubitably the investigation of the effect of antitumor drugs on the formation of neutrophil extracellular traps and on the ability of such drugs to prevent NETs contribution on carcinogenesis is of great interest. In the present work we studied the effect of 5-fluorouracil (5FU) and its shielded -by amphiphilic poly-N-vinylpyrrolidone (Amph-PVP) nanoparticles-nanoscaled polymeric form on the activation of human neutrophils under ex vivo conditions. Free 5FU at concentrations varying from 0.01 to 10 mg/ml was found to cause a significant (two to three times) and rapid (after 20 min) increase in the total amount of NETs in the blood. Importantly, when 5FU-loaded Amph-PVP nanoparticles were studied under the same conditions, the appearance of NETs in the blood was completely blocked providing strong evidence of their potential as delivery system for 5FU in antitumor therapy.

Reprogramming of CaCo2 colorectal cancer cells after using the complex of poly-(N-vinylpyrrolidone) with small non-coding RNAs.

Small non-coding RNAs control normal development and differentiation in the embryo. These regulatory molecules play a key role in the development of human diseases and are used often today for researching new treatments for different pathologies. In this study, CaCo2 colorectal adenocarcinoma cells were initially epigenetically reprogrammed and transformed into CD4+ cells with nano-sized complexes of amphiphilic poly-(N-vinylpyrrolidone) (PVP) with miRNA-152 and piRNA-30074. The transformation of cells was confirmed by morphological and genetic changes in the dynamic of reprogramming. CD4+ lymphocytes marker was detected using immunofluorescence. Amphiphilic poly-(N-vinylpyrrolidone)/small non-coding RNAs complexes were investigated for transfection efficiency and duration of transfection of CaCo2 colorectal adenocarcinoma cells using fluorescence.

Poly(3-hydroxybutyrate-CO-3-hydroxyvalerate) PHBHV biocompatible nanocarriers for 5-FU delivery targeting colorectal cancer.

Aiming to address the issue of poor bioavailability of most anti-tumor medicines against colorectal cancer, we developed a targeted anticancer nanocarrier using biocarriers able to both bind and easily release their load in a controlled manner. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) carriers were obtained via the emulsification-diffusion method, loaded with 5-fluorouracil and then characterized in terms of particle morphology and size (SEM, DLS), drug uptake and release. The cytotoxic potential of the 5-fluorouracil-loaded polymer nanocarriers on human adenocarcinoma cells (HT-29 cell line) was investigated. The in vitro studies clearly demonstrated that while the nanocarriers themselves slightly alter HT-29 cell viability, when loaded with 5-fluorouracil they significantly decrease cell viability, suggesting that the polymer itself exhibits low cytotoxicity and the drug-loaded carrier acts in an efficient manner to kill HT-29 human adenocarcinoma cells.

Nanosized carriers based on amphiphilic poly-N-vinyl-2-pyrrolidone for intranuclear drug delivery.

AIM: Ability to deliver drugs into the cell nuclei can significantly increase the efficacy of cancer therapies, in particular in the case of multidrug-resistant cancer Results: Polymer nanocarriers based on amphiphilic thiooctadecyl-terminated poly-N-vinyl-2-pyrrolidone were produced and loaded with a model hydrophobic drug, curcumin. Two commonly used loading approaches - emulsification and ultrasonic dispersion - were found to lead to two different size distributions with distinctively different biological effect. While nanocarriers produced via the emulsion method penetrated cells by dynamin-dependent endocytic mechanisms, sub-100 nm dispersion-produced nanocarriers were capable of crossing the membranes via biologically independent mechanisms. CONCLUSION: This finding opens an intriguing possibility of intranuclear delivery by merely tailoring the size of polymeric carriers, thus promising a new approach for cancer therapies.

Mechanistic understanding of nanoparticles' interactions with extracellular matrix: the cell and immune system.

Extracellular matrix (ECM) is an extraordinarily complex and unique meshwork composed of structural proteins and glycosaminoglycans. The ECM provides essential physical scaffolding for the cellular constituents, as well as contributes to crucial biochemical signaling. Importantly, ECM is an indispensable part of all biological barriers and substantially modulates the interchange of the nanotechnology products through these barriers. The interactions of the ECM with nanoparticles (NPs) depend on the morphological characteristics of intercellular matrix and on the physical characteristics of the NPs and may be either deleterious or beneficial. Importantly, an altered expression of ECM molecules ultimately affects all biological processes including inflammation. This review critically discusses the specific behavior of NPs that are within the ECM domain, and passing through the biological barriers. Furthermore, regenerative and toxicological aspects of nanomaterials are debated in terms of the immune cells-NPs interactions.

Amphiphilic poly-N-vinylpyrrolidone nanoparticles as carriers for non-steroidal, anti-inflammatory drugs: In vitro cytotoxicity and in vivo acute toxicity study.

Polymeric nanoparticles were prepared from self-assembled amphiphilic N-vinylpyrrolidone polymers in aqueous media and evaluated as novel carriers of indomethacin, a non-steroidal, anti-inflammatory drug. It was determined that these nanoparticles could be created in spherical morphologies with sizes less than 100nm, narrow size distributions and high indomethacin contents(up to 35%) combined with high drug loading efficiencies(up to 95%). In cytotoxicity tests using the human embryonic stem cell derived fibroblasts (EBF-H9) and hepatocellular carcinoma cells (HepG2), the indomethacin-loaded polymeric nanoparticles showed higher cell viability compared to that of free indomethacin at the same concentration. The median LD50 values, determined by the Litchfield-Wilcoxon method, were 55-70mg/kg body weight depending on the polymer molecular design in both mice and rats. Based on the acquired results, these novel amphiphilic poly-N-vinylpyrrolidone nanoparticles can be considered as potential carriers for new, highly efficient, injectable drug delivery systems for hydrophobic drugs such as indomethacin.

Emerging aspects of nanotoxicology in health and disease: From agriculture and food sector to cancer therapeutics.

Nanotechnology is an evolving scientific field that has allowed the manufacturing of materials with novel physicochemical and biological properties, offering a wide spectrum of potential applications. Properties of nanoparticles that contribute to their usefulness include their markedly increased surface area in relation to mass, surface reactivity and insolubility, ability to agglomerate or change size in different media and enhanced endurance over conventional-scale substance. Here, we review nanoparticle classification and their emerging applications in several fields; from active food packaging to drug delivery and cancer research. Nanotechnology has exciting therapeutic applications, including novel drug delivery for the treatment of cancer. Additionally, we discuss that exposure to nanostructures incorporated to polymer composites, may result in potential human health risks. Therefore, the knowledge of processes, including absorption, distribution, metabolism and excretion, as well as careful toxicological assessment is critical in order to determine the effects of nanomaterials in humans and other biological systems. Expanding the knowledge of nanoparticle toxicity will facilitate designing of safer nanocomposites and their application in a beneficial manner.

Delta-sleep inducing peptide entrapment in the charged macroporous matrices.

Various biomolecules, for example proteins, peptides etc., entrapped in polymer matrices, impact interactions between matrix and cells, including stimulation of cell adhesion and proliferation. Delta-sleep inducing peptide (DSIP) possesses numerous beneficial properties, including its abilities in burn treatment and neuronal protection. DSIP entrapment in two macroporous polymer matrices based on copolymer of dimethylaminoethyl methacrylate and methylen-bis-acrylamide (Co-DMAEMA-MBAA) and copolymer of acrylic acid and methylen-bis-acrylamide (Co-AA-MBAA) has been studied. Quite 100% of DSIP has been entrapped into positively charged Co-DMAEMA-MBAA matrix, while the quantity of DSIP adsorbed on negatively charged Co-AA-MBAA was only 2-6%. DSIP release from Co-DMAEMA-MBAA was observed in saline solutions (0.9% NaCl and PBS) while there was no DSIP release in water or 25% ethanol, thus ionic strength was a reason of this process.