Direct Functionalization of Polysaccharide-Based Xylan Phenyl Carbonate Nanoparticles with Tumor Cell Specific Antibodies.

An efficient and easy-to-use approach is presented for obtaining biocompatible polysaccharide-based nanoparticles (NP) that can act as tumor-specific drug delivery agents. Two antibodies are directly immobilized onto reactive xylan phenyl carbonate (XPC) NP; namely Cetuximab (CTX) that binds to human epidermal growth factor receptor (EGFR) and Atezolizumab (ATZ) that binds to programmed death-ligand 1 (PD-L1). High coupling efficiency (up to 100 %) are achieved without any pre-activation and no aggregation occurs during antibody immobilization. By quartz crystal microbalance experiments with dissipation monitoring (QCM-D), flow cytometry assays, and confocal laser scanning microscopy imaging it is demonstrated that the functionalized XPC-NP specifically bind to cells carrying the corresponding antigens. Moreover, the NP retain the antibody specific bioactivities (growth inhibition for CTX and induction of T-cell cytotoxicity for ATZ).

Dually Modified Cellulose as a Non-Viral Vector for the Delivery and Uptake of HDAC3 siRNA.

RNA interference can be applied to different target genes for treating a variety of diseases, but an appropriate delivery system is necessary to ensure the transport of intact siRNAs to the site of action. In this study, cellulose was dually modified to create a non-viral vector for HDAC3 short interfering RNA (siRNA) transfer into cells. A guanidinium group introduced positive charges into the cellulose to allow complexation of negatively charged genetic material. Furthermore, a biotin group fixed by a polyethylene glycol (PEG) spacer was attached to the polymer to allow, if required, the binding of targeting ligands. The resulting polyplexes with HDAC3 siRNA had a size below 200 nm and a positive zeta potential of up to 15 mV. For N/P ratio 2 and higher, the polymer could efficiently complex siRNA. Nanoparticles, based on this dually modified derivative, revealed a low cytotoxicity. Only minor effects on the endothelial barrier integrity and a transfection efficiency in HEK293 cells higher than Lipofectamine 2000TM were found. The uptake and release of the polyplexes were confirmed by immunofluorescence imaging. This study indicates that the modified biopolymer is an auspicious biocompatible non-viral vector with biotin as a promising moiety.

HDACi Delivery Systems Based on Cellulose Valproate Nanoparticles.

The ability of histone deacetylase inhibitors (HDACi) like valproic acid (VPA) as a therapeutic for inflammatory diseases or cancer has increased the interest in HDACi and their targeted transport to diseased tissues. Administration of VPA immobilized on polymeric carriers was found to be a suitable approach to circumvent drawbacks such as rapid metabolization, short serum half-life, or side effects. Polysaccharides are convenient biopolymeric carriers due to their biocompatibility and biodegradability. Furthermore, the hydroxy-, amino-, or carboxylic groups are predestinated for functionalization. The esterification of three hydroxy groups of cellulose with VPA leads to products having a high amount of VPA loading. Subsequent shaping yielded uniform nanoparticles (NPs) of around 150 nm in size capable of releasing VPA in a controlled way under physiological conditions.

Evaluating Release Kinetics from Alginate Beads Coated with Polyelectrolyte Layers for Sustained Drug Delivery.

Current approaches in stem cell-based bone tissue engineering require a release of bioactive compounds over up to 2 weeks. This study presents a polyelectrolyte-layered system featuring sustained release of water-soluble drugs with decreased burst release. The bioactive compounds adenosine 5'-triphosphate (ATP), suramin, and A740003 (a less water-soluble purinergic receptor ligand) were incorporated into alginate hydrogel beads subsequently layered with different polyelectrolytes (chitosan, poly(allyl amine), alginate, or lignosulfonate). Drug release into aqueous medium was monitored over 14 days and evaluated using Korsmeyer-Peppas, Peppas-Sahlin, Weibull models, and a Langmuir-like "Two-Stage" model. Release kinetics strongly depended on both the drug and the polyelectrolyte system. For ATP, five alternating layers of poly(allyl amine) and alginate proved to be most effective in sustaining the release. Release of suramin could be prolonged best with lignosulfonate as polyanion. A740003 showed prolonged release even without layering. Applying polyelectrolyte layers significantly slowed down the burst release. Release curves could be best described with the Langmuir-like model.

Biocompatible sulfated valproic acid-coupled polysaccharide-based nanocarriers with HDAC inhibitory activity.

The development of bio-based nanoparticles (NPs) as drug containers is of increasing interest to circumvent several obstacles in drug therapy such as rapid drug metabolization, short serum half-life, and unspecific side effects. The histone deacetylase inhibitor valproic acid (VPA) is known for its anti-inflammatory as well as for its anti-cancer activity. Here, recently developed VPA-loaded NPs based on cellulose- and dextran VPA esters were modified with sulfuric acid half ester moieties to improve intracellular drug release. The NPs show rapid cellular uptake, are non-toxic in vitro and in vivo, and able to induce histone H3 hyperacetylation. Thus, they represent a potent drug delivery system for the application in a variety of treatment settings, such as inflammation, sepsis and defined cancer types. In addition, the flexible NP-system offers a broad range of further options for modification, e.g. for targeting strategies and multi-drug approaches.

Green Fabrication of Highly Conductive Paper Electrodes via Interface Engineering with Aminocellulose.

Herein, a novel, facile, and versatile approach to fabricate highly flexible and conductive paper is proposed by electroless deposition (ELD) with the assistance of aminocellulose as the interface layer. The obtained Cu nanoparticles (NPs)-coated cellulose paper is highly conductive with a significant low resistance of 0.38 Ω sq-1 after only 10 min of ELD treatment. This conductive cellulose paper shows excellent stability when it suffers from bending, folding, and tape adhesion cycles. With the same method, the Cu NPs can also be successfully deposited on the polypropylene (PP)-filled hybrid paper. The conductive paper exhibits very smooth and hydrophobic surface with high reflection, which can be used for special electronic devices. In a word, the fabrication of aminocellulose interface permits a controlled ELD of metal nanoparticles on paper substrate, and this mild and low-cost method opens up new opportunities for large-scale production of flexible and wearable electronics.

Polysaccharide Nanoparticles Bearing HDAC Inhibitor as Nontoxic Nanocarrier for Drug Delivery.

The histone deacetylase inhibitors (HDACi) are potent drugs in the treatment of inflammatory diseases and defined cancer types. However, major drawbacks of HDACi, such as valproic acid (VPA), are limited serum half-life, side effects and the short circulation time. Thus, the immobilization of VPA in a polysaccharide matrix is used to circumvent these problems and to design a suitable nanocarrier system. Therefore, VPA is covalently attached to cellulose and dextran via esterification with degree of substitution (DS) values of up to 2.20. The resulting hydrophobic polymers are shaped to spherical nanoparticles (NPs) with hydrodynamic diameter between 138 to 221 nm and polydispersity indices from 0.064 to 0.094 by nanoprecipitation and emulsification technique. Lipase treatment of the NPs leads to in vitro release of VPA and hence to an inhibition of HDAC2 activity in a HDAC2 assay. NPs are rapidly taken up by HeLa cells and mainly localize in the cytoplasm. The NPs are hemocompatible and nontoxic as revealed by the shell-less hen's egg model.

Non-Cytotoxic Agarose/Hydroxyapatite Composite Scaffolds for Drug Release.

Healing of large bone defects requires implants or scaffolds that provide structural guidance for cell growth, differentiation, and vascularization. In the present work, an agarose-hydroxyapatite composite scaffold was developed that acts not only as a 3D matrix, but also as a release system. Hydroxyapatite (HA) was incorporated into the agarose gels in situ in various ratios by a simple procedure consisting of precipitation, cooling, washing, and drying. The resulting gels were characterized regarding composition, porosity, mechanical properties, and biocompatibility. A pure phase of carbonated HA was identified in the scaffolds, which had pore sizes of up to several hundred micrometers. Mechanical testing revealed elastic moduli of up to 2.8 MPa for lyophilized composites. MTT testing on Lw35human mesenchymal stem cells (hMSCs) and osteosarcoma MG-63 cells proved the biocompatibility of the scaffolds. Furthermore, scaffolds were loaded with model drug compounds for guided hMSC differentiation. Different release kinetic models were evaluated for adenosine 5'-triphosphate (ATP) and suramin, and data showed a sustained release behavior over four days.

Functional dextran amino acid ester particles derived from N-protected S-trityl-L-cysteine.

This work describes the derivatization of dextran using N-(tert-butyloxycarbonyl)-S-(trityl)-L-cysteine in the presence of N,N'-carbonyldiimidazole (CDI) as a coupling agent. Homogeneous reactions in dimethyl sulfoxide allowed for an efficient coupling of the amino acid derivative to the polymer backbone. Derivatization was confirmed by infrared and 13C NMR spectroscopy, size exclusion chromatography and elemental analysis. The presence of hydrophobic protecting groups resulted in a product that can be shaped into water-insoluble particles stable in an aqueous environment and non-toxic for lung epithelial cells. It is suggested that materials composed of ester bonds between amino acids and polysaccharides are useful for targeted drug delivery, bio-imaging or surface functionalization.

Structural elucidation of a heteropolysaccharide from the wild mushroom Marasmiellus palmivorus and its immune-assisted anticancer activity.

The biological activity of macrofungal polysaccharides (MFPS) depends on their structural features and is a topic of keen interest for researchers since long time. In this communication, we report a water soluble macrofungal heteropolysaccharide (MFPS1) with a molar weight of ˜145,000 g/mol, obtained through alkali extraction, of the wild mushroom, Marasmiellus palmivorus, with significant immunomodulatory properties. In cancer, after the induction of metastasis, the anticancer immune system becomes unresponsive. By studying cytokine secretion and immune phenotyping, it was observed that MFPS1 reactivated the anticancer immune surveillance system. MFPS1 executed T-cell maturation and activation via M1Φ; and also stimulated natural killer (NK) cell and B-cell population. The entire immune activation pathway corroborates its anticancer activity. The RP-HPLC analysis of hydrolyzed MFPS1 showed arabinose, glucose, galactose and mannose as monosaccharide units. The proposed structure of repeating unit was established from methylation analysis, 1D- and 2D NMR study, HR-MS and MALDI-TOF MS analysis.

Chitosan-Cellulose Multifunctional Hydrogel Beads: Design, Characterization and Evaluation of Cytocompatibility with Breast Adenocarcinoma and Osteoblast Cells.

Cytocompatible polysaccharide-based functional scaffolds are potential extracellular matrix candidates for soft and hard tissue engineering. This paper describes a facile approach to design cytocompatible, non-toxic, and multifunctional chitosan-cellulose based hydrogel beads utilising polysaccharide dissolution in sodium hydroxide-urea-water solvent system and coagulation under three different acidic conditions, namely 2 M acetic acid, 2 M hydrochloric acid, and 2 M sulfuric acid. The effect of coagulating medium on the final chemical composition of the hydrogel beads is investigated by spectroscopic techniques (ATR-FTIR, Raman, NMR), and elemental analysis. The beads coagulated in 2 M acetic acid displayed an unchanged chitosan composition with free amino groups, while the beads coagulated in 2 M hydrochloric and sulfuric acid showed protonation of amino groups and ionic interaction with the counterions. The ultrastructural morphological study of lyophilized beads showed that increased chitosan content enhanced the porosity of the hydrogel beads. Furthermore, cytocompatibility evaluation of the hydrogel beads with human breast adenocarcinoma cells (soft tissue) showed that the beads coagulated in 2 M acetic acid are the most suitable for this type of cells in comparison to other coagulating systems. The acetic acid fabricated hydrogel beads also support osteoblast growth and adhesion over 192 h. Thus, in future, these hydrogel beads can be tested in the in vitro studies related to breast cancer and for bone regeneration.

Protein-like fully reversible tetramerisation and super-association of an aminocellulose.

Unusual protein-like, partially reversible associative behaviour has recently been observed in solutions of the water soluble carbohydrates known as 6-deoxy-6-(ω-aminoalkyl)aminocelluloses, which produce controllable self-assembling films for enzyme immobilisation and other biotechnological applications. Now, for the first time, we have found a fully reversible self-association (tetramerisation) within this family of polysaccharides. Remarkably these carbohydrate tetramers are then seen to associate further in a regular way into supra-molecular complexes. Fully reversible oligomerisation has been hitherto completely unknown for carbohydrates and instead resembles in some respects the assembly of polypeptides and proteins like haemoglobin and its sickle cell mutation. Our traditional perceptions as to what might be considered "protein-like" and what might be considered as "carbohydrate-like" behaviour may need to be rendered more flexible, at least as far as interaction phenomena are concerned.

Biocompatible multishell architecture for iron oxide nanoparticles.

The coating of super-paramagnetic iron oxide nanoparticles (SPIONs) with multiple shells is demonstrated by building a layer assembled from carboxymethyldextran and poly(diallydimethylammonium chloride). Three shells are produced stepwise around aggregates of SPIONs by the formation of a polyelectrolyte complex. A growing particle size from 96 to 327 nm and a zeta potential in the range of +39 to -51 mV are measured. Microscopic techniques such as TEM, SEM, and AFM exemplify the core-shell structures. Magnetic force microscopy and vibrating sample magnetometer measurements confirm the architecture of the multishell particles. Cell culture experiments show that even nanoparticles with three shells are still taken up by cells.

Amino-functionalized cellulose nanoparticles: preparation, characterization, and interactions with living cells.

Spherical nanoparticles with sizes from 80 to 200 nm are obtained by self-assembly of highly functionalized 6-deoxy-6-(ω-aminoalkyl)aminocellulosecarbamates. The particles are very stable, nontoxic, and possess primary amino groups that are accessible to further modifications in aqueous suspension. The particles can be labeled with rhodamine B isothiocyanate without changing their size, stability, and shape. The nanoparticles obtained are investigated by means of photo correlation spectroscopy, zeta potential measurements, SEM and fluorescence spectroscopy. Incorporation of the nanoparticles in human foreskin fibroblasts BJ-1-htert and breast carcinoma MCF-7 cells without any transfection reagent is proved by means of confocal laser scanning microscopy.

Decolorization of water and oil-soluble azo dyes by Lactobacillus acidophilus and Lactobacillus fermentum.

The capability of Lactobacillus acidophilus and Lactobacillus fermentum to degrade azo dyes was investigated. The bacteria were incubated under anaerobic conditions in the presence of 6 microg/ml Methyl Red, Ponceau BS, Orange G, Amaranth, Orange II, and Direct Blue 15; 5 microg/ml Sudan I and II; or 1.5 microg/ml Sudan III and IV in deMann-Rogosa-Sharpe broth at 37 degrees C for 36 h, and reduction of the dyes was monitored. Both bacteria were capable of degrading all of the water-soluble azo dyes to some extent. They were also able to completely reduce the oil-soluble diazo dyes Sudan III and IV but were unable to reduce the oil-soluble monoazo dyes Sudan I and II to any significant degree in the concentrations studied. Growth of the bacteria was not significantly affected by the presence of the Sudan azo dyes. Metabolites of the bacterial degradation of Sudan III and IV were isolated and identified by liquid chromatography electrospray ionization tandem mass spectrometry analyses and compared with authentic standards. Aniline and o-toluidine (2-methylaniline), both potentially carcinogenic aromatic amines, were metabolites of Sudan III and IV, respectively.

Acrylamide formed at physiological temperature as a result of asparagine oxidation.

Acrylamide is a probable human carcinogen that is neurotoxic to both humans and animals. It is known to be formed during cooking of foods at temperatures higher than 120 degrees C. The present study demonstrates that acrylamide can also be formed at physiological conditions (37 degrees C, pH 7.4) when asparagine is incubated in the presence of hydrogen peroxide (H(2)O(2)). The formation of acrylamide under these conditions is dependent on the incubation time and the concentration of H(2)O(2). Thus, the results raise the question of the possible endogenous formation of acrylamide in pathological conditions that are associated with long-term oxidative stress. Further studies are therefore warranted to clarify the possible endogenous formation of acrylamide and its significance in chronic conditions that are known to be associated with oxidative stress.

Comparative in vitro study on cytotoxicity, antimicrobial activity, and binding capacity for pathophysiological factors in chronic wounds of alginate and silver-containing alginate.

Chronic wounds contain elevated levels of proteases, proinflammatory cytokines, and free radicals. The presence of bacteria further exaggerates the tissue-damaging processes. For successful treatment, the wound dressing needs to manage wound exudates, create a moist environment, inhibit infection, bind pathophysiological factors that are detrimental to wound healing, and provide thermal isolation. Furthermore, it has to relieve pain, be easy to use, show no allergic potency, and not release toxic residues. The present study suggests a comprehensive in vitro approach to enable the assessment of wound dressings to support optimal conditions for wound healing. Three alginate-based wound dressings: alginate alone, alginate containing ionic silver, and alginate with nanocrystalline silver, were tested for biocompatibility, antimicrobial activity, and influence on chronic wound parameters such as elastase, matrix metalloproteases-2, tumor necrosis factor-alpha, interleukin-8, and free radical formation. Alginate was found to bind considerable amounts of elastase, reduce the concentration of proinflammatory cytokines and inhibit the formation of free radicals. Furthermore, alginate showed antibacterial activity and high biocompatibility. Incorporation of silver into alginate fibers increased antimicrobial activity and improved the binding affinity for elastase, matrix metalloproteases-2, and the proinflammatory cytokines tested. Addition of silver also enhanced the antioxidant capacity. However, a distinct negative effect of silver-containing alginates on human HaCaT keratinocytes was noted in vitro.

Synthesis and characterization of DNA adducts from the HIV reverse transcriptase inhibitor nevirapine.

Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor used against the human immunodeficiency virus type 1 (HIV-1), mostly to prevent mother-to-child HIV transmission in developing countries. One of the limitations of nevirapine use is severe hepatotoxicity, which raises concerns about its administration, particularly in the perinatal and pediatric settings. Nevirapine metabolism involves oxidation of the 4-methyl substituent to 12-hydroxy-NVP and the formation of phenolic derivatives. Further metabolism of 12-hydroxy-NVP by phase II esterification may produce electrophilic derivatives capable of reacting with DNA to yield covalent adducts, which could potentially be involved in the initiation of mutagenic and carcinogenic events. In the present study, we have investigated the reactivity of the synthetic model electrophile, 12-mesyloxy-NVP, toward 2'-deoxynucleosides and DNA. Parallel synthetic studies were conducted with 12-bromo-NVP and 3',5'- O-bis( tert-butyldimethylsilyl)-2'-deoxynucleosides, using palladium(0) catalysis. Multiple adducts from deoxyguanosine, deoxyadenosine, and deoxycytidine were isolated in the reactions with 12-mesyloxy-NVP and characterized by NMR and MS. The adduct structures consistently involved binding through C12 of NVP and N7 or N9 of deoxyguanosine; N1, N3, or N9 of deoxyadenosine; and N3 of deoxycytidine. Reactions conducted under palladium(0) catalysis yielded adducts through O (6) and N1 of deoxyguanosine, N1 of deoxyadenosine, and N3 of deoxycytidine. At least seven deoxynucleoside-NVP adducts were present in DNA reacted with 12-mesyloxy-NVP and enzymatically hydrolyzed. Four of these adducts were identified as 12-(deoxyadenosin-N1-yl)nevirapine, 12-(deoxycytidin-N3-yl)nevirapine, 12-(deoxyguanosin- O(6)-yl)nevirapine, and 12-(deoxyadenosin- N(6)-yl)nevirapine. One depurinating adduct, 12-(guanin-N7-yl)nevirapine, was identified in the thermal neutral DNA hydrolysate. If formed in vivo, some of these adducts would have considerable mutagenic potential. Our results thus suggest that NVP metabolism to 12-hydroxy-NVP may be a factor in NVP hepatocarcinogenicity.

Anaerobic metabolism of 1-amino-2-naphthol-based azo dyes (Sudan dyes) by human intestinal microflora.

The rates of metabolism of Sudan I and II and Para Red by human intestinal microflora were high compared to those of Sudan III and IV under anaerobic conditions. Metabolites of the dyes were identified as aniline, 2,4-dimethylaniline, o-toluidine, and 4-nitroaniline through high-performance liquid chromatography and liquid chromatography electrospray ionization tandem mass spectrometry analyses. These data indicate that human intestinal bacteria are able to reduce Sudan dyes to form potentially carcinogenic aromatic amines.

Transformation of the antibacterial agent norfloxacin by environmental mycobacteria.

Because fluoroquinolone antimicrobial agents may be released into the environment, the potential for environmental bacteria to biotransform these drugs was investigated. Eight Mycobacterium sp. cultures in a sorbitol-yeast extract medium were dosed with 100 microg ml(-1) of norfloxacin and incubated for 7 days. The MICs of norfloxacin for these strains, tested by an agar dilution method, were 1.6 to 25 microg ml(-1). Cultures were extracted with ethyl acetate, and potential metabolites in the extracts were purified by high-performance liquid chromatography. The metabolites were identified using mass spectrometry and nuclear magnetic resonance spectroscopy. N-Acetylnorfloxacin (5 to 50% of the total absorbance at 280 nm) was produced by the eight Mycobacterium strains. N-Nitrosonorfloxacin (5 to 30% of the total absorbance) was also produced by Mycobacterium sp. strain PYR100 and Mycobacterium gilvum PYR-GCK. The MICs of N-nitrosonorfloxacin and N-acetylnorfloxacin were 2- to 38- and 4- to 1,000-fold higher, respectively, than those of norfloxacin for several different bacteria, including the two strains that produced both metabolites. Although N-nitrosonorfloxacin had less antibacterial activity, nitrosamines are potentially carcinogenic. The biotransformation of fluoroquinolones by mycobacteria may serve as a resistance mechanism.