Decisive role of hydrophobic side groups of polypeptides in thermosensitive gelation.

Thermosensitive hydrogels based on PEG and poly(l-glutamate)s bearing different hydrophobic side groups were separately synthesized by the ring-opening polymerization (ROP) of l-glutamate N-carboxyanhydrides containing different alkyl protected groups, that is, methyl, ethyl, n-propyl, and n-butyl, using mPEG(45)-NH(2) as macroinitiator. The resulting copolymers underwent sol-gel transitions in response to temperature change. Interestingly, the polypeptides containing methyl and ethyl showed significantly lower critical gelation temperatures (CGTs) than those bearing n-propyl and butyl side groups. Based on the analysis of (13)C NMR spectra, DLS, circular dichroism spectra, and ATR-FTIR spectra, the sol-gel transition mechanism was attributed to the dehydration of poly(ethylene glycol) and the increase of ß-sheet conformation content in the polypeptides. The in vivo gelation test indicated that the copolymer solution (6.0 wt %) immediately changed to a gel after subcutaneous injection into rats. The mass loss of the hydrogel in vitro was accelerated in the presence of proteinase K, and the MTT assay revealed that the block copolymers exhibited no detectable cytotoxicity. The present work revealed that subtle variation in the length of a hydrophobic side group displayed the decisive effect on the gelation behavior of the polypeptides. In addition, the thermosensitive hydrogels could be promising materials for biomedical applications due to their good biocompatibility, biodegradability, and the fast in situ gelation behavior.

Toxicity studies of poly(anhydride) nanoparticles as carriers for oral drug delivery.

PURPOSE: To evaluate the acute and subacute toxicity of poly(anhydride) nanoparticles as carriers for oral drug/antigen delivery. METHODS: Three types of poly(anhydride) nanoparticles were assayed: conventional (NP), nanoparticles containing 2-hydroxypropyl-ß-cyclodextrin (NP-HPCD) and nanoparticles coated with poly(ethylene glycol) 6000 (PEG-NP). Nanoparticles were prepared by a desolvation method and characterized in terms of size, zeta potential and morphology. For in vivo oral studies, acute and sub-acute toxicity studies were performed in rats in accordance to the OECD 425 and 407 guidelines respectively. Finally, biodistribution studies were carried out after radiolabelling nanoparticles with (99m)technetium. RESULTS: Nanoparticle formulations displayed a homogeneous size of about 180 nm and a negative zeta potential. The LD(50) for all the nanoparticles tested was established to be higher than 2000 mg/kg bw. In the sub-chronic oral toxicity studies at two different doses (30 and 300 mg/kg bw), no evident signs of toxicity were found. Lastly, biodistribution studies demonstrated that these carriers remained in the gut with no evidences of particle translocation or distribution to other organs. CONCLUSIONS: Poly(anhydride) nanoparticles (either conventional or modified with HPCD or PEG6000) showed no toxic effects, indicating that these carriers might be a safe strategy for oral delivery of therapeutics.

Construction of an environmentally friendly octenylsuccinic anhydride modified pH-sensitive chitosan nanoparticle drug delivery system to alleviate inflammation and oxidative stress.

An environmentally friendly octenylsuccinic anhydride modified pH-sensitive chitosan-octenylsuccinic anhydride (OSA-CS) was synthesized. The critical micelle concentration (CMC) of the modified chitosan was 27 µg/mL, the graft polymers can form solubilized curcumin (CUR) and quercetin (QUE) nanoparticles. The drug-loaded nanoparticles had high encapsulation efficiency and drug loading content, the self-assembly of graft polymers formed spherical uniform nanoparticles with an approximate diameter of 150-180 nm. The nanoparticles were stable under storage conditions and in serum. The results revealed that OSA-CS exhibited excellent biocompatibility, no cytotoxicity. Additionally, the results of pH sensitivity and drug release experiments showed that the nanoparticles were highly sensitive to weakly acidic conditions (pH 6.0) and showed a faster release rate, while they were reasonably stable at physiological conditions (pH 7.4). The drug-loaded nanoparticles exhibited higher cellular uptake in vitro, and exhibited stronger anti-inflammatory and antioxidant efficacy. Therefore, OSA-CS-based nanoparticles are a promising hydrophobic drug delivery system for pH-response targeting therapy.

Nature and kinetics of redox imbalance triggered by respiratory and skin chemical sensitizers on the human monocytic cell line THP-1.

Low molecular weight reactive chemicals causing skin and respiratory allergies are known to activate dendritic cells (DC), an event considered to be a key step in both pathologies. Although generation of reactive oxygen species (ROS) is considered a major danger signal responsible for DC maturation, the mechanisms leading to cellular redox imbalance remain poorly understood. Therefore, the aim of this study was to unveil the origin and kinetics of redox imbalance elicited by 1-fluoro-2,4-dinitrobenzene (DNFB) and trimellitic anhydride chloride (TMAC), two golden standards of skin and chemical respiratory allergy, respectively. To track this goal, we addressed the time course modifications of ROS production and cellular antioxidant defenses as well as the modulation of MAPKs signaling pathways and transcription of pathophysiological relevant genes in THP-1 cells. Our data shows that the thiol-reactive sensitizer DNFB directly reacts with cytoplasmic glutathione (GSH) causing its rapid and marked depletion which results in a general increase in ROS accumulation. In turn, TMAC, which preferentially reacts with amine groups, induces a delayed GSH depletion as a consequence of increased mitochondrial ROS production. These divergences in ROS production seem to be correlated with the different extension of intracellular signaling pathways activation and, by consequence, with distinct transcription kinetics of genes such as HMOX1, IL8, IL1B and CD86. Ultimately, our observations may help explain the distinct DC phenotype and T-cell polarizing profile triggered by skin and respiratory sensitizers.

Preventing false-negatives in the in vitro skin sensitization testing of acid anhydrides using interleukin-8 release assays.

In vitro safety tests may be used as replacements for animal tests owing to their accuracy and high-throughput performance. However, several in vitro skin sensitization tests produce false-negative results such as acid anhydride. Here, we investigated the relationship between false-negative results of acid anhydride and its hydrolysis by aqueous vehicle. Differences in the pattern of hydrolysis for phthalic anhydride (PAH) due to addition of 1 drop of stock solution of PAH in liquid paraffin (LP) dispersion medium and PAH in DMSO were analyzed in a cell-free system. The results showed that use of LP dispersion medium stabilized the concentration of PAH in water over 5min by sustained-release, although almost all PAH converted to phthalic acid in water within 5min using DMSO. Additionally, treatment of THP-1 cells with PAH and phthalic acid using LP dispersion medium for 5min resulted in a 32-fold increase in IL-8 release for PAH as compared with that in the vehicle control. In contrast, for PAH using aqueous vehicle and phthalic acid using LP dispersion medium, there were no significant increases in IL-8 release. Similarly, using LP dispersion medium, trimellitic anhydride significantly increased IL-8 release was observed.

Genotoxic evaluation of poly(anhydride) nanoparticles in the gastrointestinal tract of mice.

Gantrez® AN 119-based NPs have been developed as oral drug carriers due to their strong bioadhesive interaction with components of the gastrointestinal mucosa and to their adaptable surface. The use of mannosamine to coat Gantrez® AN 119-based NPs results in a high mucus-permeable carrier, able to reach the gastrointestinal epithelium. Although their efficacy to transport a therapeutic agent has been demonstrated, their safety has not yet been thoroughly studied. They have proved to be non-cytotoxic, non-genotoxic and non-mutagenic in vitro; however, the in vivo toxicity profile has not yet been determined. In this study, the in vivo genotoxic potential of Gantrez® AN 119 NPs coated with mannosamine (GN-MA-NP) has been assessed using the in vivo comet assay in combination with the enzyme formamidopyrimidine DNA glycosylase in mice, following the OECD test guideline 489. To determine the relevant organs to analyse and the sampling times, an in vivo biodistribution study was also carried out. Results showed a statistically significant induction of DNA strand breaks and oxidized bases in the duodenum of animals exposed to 2000 mg/kg bw. However, this effect was not observed at lower doses (i.e. 500 and 1000 mg/kg which are closer to the potential therapeutic doses) or in other organs. In conclusion, GN-MA-NP are promising nanocarriers as oral drug delivery systems.

Topical application versus intranasal instillation: a qualitative comparison of the effect of the route of sensitization on trimellitic anhydride-induced allergic rhinitis in A/J mice.

Allergic airway diseases caused by low-molecular weight chemicals including trimellitic anhydride (TMA) have been linked to Th2 cytokines and are characterized by mucus hypersecretion and infiltration of lymphocytes and eosinophils into the airways. The most common route of human exposure to chemical respiratory allergens is inhalation. Most murine models, however, use topical exposure to sensitize mice. The present study tests the hypothesis that topical sensitization on the ears of mice with TMA will induce a qualitatively similar immunologic and pathologic response in the nasal airways after intranasal challenge to that induced after intranasal sensitization and challenge. A/J mice were sensitized topically or by intranasal instillation followed by intranasal challenge with TMA in an ethyl acetate/olive oil vehicle. Intranasal challenge with TMA in mice that were either topically or intranasally sensitized with TMA caused a marked allergic rhinitis, of similar severity, characterized by an influx of eosinophils and lymphocytes. Both the topical and intranasal routes of sensitization also caused significant increases in total serum IgE after intranasal challenge with TMA. In addition, both the topical and intranasal routes of sensitization caused significant increases in the mRNA expression of the Th2 cytokines IL-4, IL-5, and IL-13. Collectively, these findings suggest that topical application is effective in sensitizing mice to TMA and induces a nasal airway lesion and associated immune response after intranasal challenge, which is qualitatively similar to that induced by intranasal sensitization and challenge. Skin exposure may be a potential route of sensitization of the respiratory tract to chemical allergens.

Modification of wood with environmentally friendly chemicals to improve decay resistance.

Turkish pine (Pinus brutia Ten.) solid wood was chemically modified to various weight percent gains (WPG) using either acetic, methacrylic, hexanoic and succinic anhydrides. Laboratory soil block decay testing using the brown rot fungus C. puteana and G. trabeum was performed and weight loss calculated. All high degree of modified samples showed good biological resistance and severely reduced degradation. Chemical modification of wood is very important for the prevention of biodeterioration which are environmentally friendly and also not hazardous to human beings.

Toxicity, biodegradation and elimination of polyanhydrides.

Although originally developed for the textile industry, polyanhydrides have found extensive use in biomedical applications due to their biodegradability and excellent biocompatibility. Polyanhydrides are most commonly synthesized from diacid monomers by polycondensation. Efficient control over various physicochemical properties, such as biodegradability and biocompatibility, can be achieved for this class of polymers, due to the availability of a wide variety of diacid monomers as well as by copolymerization of these monomers. Biodegradation of these polymers takes place by the hydrolysis of the anhydride bonds and the polymer undergoes predominantly surface erosion, a desired property to attain near zero-order drug release profile. This review examines the mode of degradation and elimination of these polyanhydrides in vivo as well as the biocompatibility and toxicological aspects of various polyanhydrides.

Immunotoxicology of organic acid anhydrides (OAAs).

Organic acid anhydrides (OAAs) have considerable economic importance due to their extensive use in the production of alkyd, epoxy, and polyester resins. Occupational exposure to OAAs has been associated with a variety of health effects, which may be classified into two major categories of direct toxicity/irritant and hypersensitivity. The hypersensitivity diseases associated with OAA exposure are thought to be related to the reactivity of these chemicals and in particular their ability to form protein conjugates that may be recognized as neo-antigens by the immune system. This review will present a brief discussion of the basic chemistry of these compounds and the environmental and biological monitoring methods used for exposure measurements. The clinical syndromes associated with exposure to these compounds will be discussed along with factors that may affect disease susceptibility. Finally, animal models that have been developed to examine the mechanisms of disease will be discussed.

IgG and IgE antibody responses following exposure of Brown Norway rats to trimellitic anhydride: comparison of inhalation and topical exposure.

A variety of chemicals can cause sensitisation of the respiratory tract and occupational asthma, including certain acid anhydrides, diisocyanates and reactive dyes. As yet, no well-validated methods are available for the toxicological evaluation of the respiratory sensitising potential of chemicals. One approach which has been explored recently is the evaluation of induced IgE responses or cytokine expression patterns in rats or mice following topical exposure to chemical. Thus, it has been demonstrated that topical exposure of rodents to respiratory sensitising chemicals, but not to contact allergens, causes a dose-dependent and time-related increase in the concentration of total IgE. Using the reference respiratory allergen trimellitic anhydride (TMA), we have considered here the influence of route of exposure on the nature of induced immune responses. Specific IgG and IgE antibody responses and changes in total serum concentration of IgE have been measured following exposure of Brown Norway (BN) starin rats to TMA by topical administration or by inhalation. Exposure to TMA by both routes resulted in the stimulation of specific IgG and IgE antibody, although responses were considerably more vigorous after dermal exposure. Topical treatment also provoked marked and sustained increases in total serum IgE levels, whereas exposure via the respiratory tract stimulated a more transient elevation of this immunoglobulin in a minority of animals which reached statistical significance only at the highest dose group. The lesser vigour of the immune response following inhalation exposure is likely to be related to the considerably lower total antigenic dose which is delivered by this route. Nevertheless, these results show that the nature of immune response with respect to antibody isotype profile provoked by topical administration of TMA is qualitatively comparable with that stimulated by inhalation exposure to the same chemical. For the purposes of hazard assessment and identification of potential chemical respiratory allergens as a function of induced changes in serum IgE concentration, however, the evidence is that topical administration of test material is the preferred route of exposure.

Antibody specificity to the chemical structures of organic acid anhydrides studied by in-vitro and in-vivo methods.

The objective of the study was to evaluate the structure-activity relationship for the antigenic activity of different organic acid anhydrides (OAAs). The specificity of guinea pig (GP) IgG1 to different anhydrides was studied by ELISA-inhibition, PCA, and airway provocation tests of cross-reactivity with different OAA conjugates. In the airway provocation tests, lung resistance and plasma extravasation of Evan's Blue dye was measured. The ELISA-inhibition tests showed a wide range in antibody specificity. Modelling of ring configuration, methyl group substitution, double bond position, and cis/trans isomerism of anhydride forming carboxyl groups influenced the specificity. There was a general consistency in cross-reactivity of anti-cis-hexahydrophthalic anhydride IgG1 versus GP serum albumin conjugates of trans-hexahydrophthalic anhydride, phthalic anhydride, and succinic anhydride as shown by ELISA-inhibition, PCA, and airway provocation tests. It is concluded that various modifications of the chemical structures of a hapten are recognized by the hapten-specific antibodies, and that these differences may have clinical relevance. In particular, the ring structure and the positions of double bonds and of methyl groups are important. Further, the in-vitro ELISA-inhibition tests show a good agreement with the in-vivo PCA and bronchial provocation tests.

Toxicology and biocompatibility considerations in the evaluation of polymeric materials for biomedical applications.

It is necessary to determine the safety of polymeric materials through laboratory and animal testing before biomedical use can become more widespread. This article describes many of the methods used in analyzing the toxicology and biocompatibility of potentially important biomedical polymers. The study method used in the development of an important new class of polymeric materials for human use, the poly(anhydrides), is presented from laboratory bench to human clinical trials.

In vitro anti-proliferation/cytotoxic activity of cantharidin (Spanish Fly) and related derivatives.

The anti-cancer therapeutic promise of cantharidin is limited because of its high mammalian toxicity. In order to find new anti-cancer lead compounds with reduced toxicity of the cantharidin prototype, the following seven derivatives were screened against the human SH-SY5Y neuroblastoma and MCF-7 breast cancer cells in vitro: 2,3-dimethyl-7-oxabicylo-[2.2.1]heptane-2,3-dicarboxylic anhydride (cantharidin) [1], 1-cyclohexen-1,2-dicarboxylic anhydride [2], cis-4-cyclohexen-1,2-dicarboxylic anhydride [3], cis-1, 2-cyclohexanedicarboxylic anhydride [4], exo-7-oxabicyclo[2.2.1]hept-5-ene-2-3 dicarboxylic anhydride [5], exo-7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic anhydride (norcantharidin) [6], and (S)-(-)-O-acetylmalic anhydride [7]. Cantharidin, was found to be the most effective anti-proliferative compound on both cell lines. However, on the human neuroblastoma cells cantharidin was of equal toxicity to compound [6]. Mode of action studies revealed that cantharidin inhibited growth factor-mediated activation of mitogen activated protein kinase (MAPkinase) and attenuated the de-phosphorylation of the extracellular regulated kinases 1 and 2 (erk1 and erk2).

[Use of the "exposure level-body condition" relations for evaluation of the effects of complex atmospheric pollution]. / Ispol'zovanie zavisimosti "kontsentratsiia-status organizma" dlia otsenki vozdeistviia kompleksa atmosfernykh zagriaznenii.

Criteria for the organism status arrangement in terms of environmental effects expression from adaptation to its break-down were worked up. The criteria may be used in developing of the hygienic standardization of environmental effects.

Pyridoxylamine reactivity kinetics as an amine based nucleophile for screening electrophilic dermal sensitizers.

Chemical allergens bind directly, or after metabolic or abiotic activation, to endogenous proteins to become allergenic. Assessment of this initial binding has been suggested as a target for development of assays to screen chemicals for their allergenic potential. Recently we reported a nitrobenzenethiol (NBT) based method for screening thiol reactive skin sensitizers, however, amine selective sensitizers are not detected by this assay. In the present study we describe an amine (pyridoxylamine (PDA)) based kinetic assay to complement the NBT assay for identification of amine-selective and non-selective skin sensitizers. UV-Vis spectrophotometry and fluorescence were used to measure PDA reactivity for 57 chemicals including anhydrides, aldehydes, and quinones where reaction rates ranged from 116 to 6.2 × 10(-6) M(-1) s(-1) for extreme to weak sensitizers, respectively. No reactivity towards PDA was observed with the thiol-selective sensitizers, non-sensitizers and prohaptens. The PDA rate constants correlated significantly with their respective murine local lymph node assay (LLNA) threshold EC3 values (R(2) = 0.76). The use of PDA serves as a simple, inexpensive amine based method that shows promise as a preliminary screening tool for electrophilic, amine-selective skin sensitizers.

Naphtho[1,2-b]furan-4,5-dione induces apoptosis and S-phase arrest of MDA-MB-231 cells through JNK and ERK signaling activation.

Naphtho[1,2-b]furan-4,5-dione (NFD), prepared from 2-hydroxy-1,4-naphthoquinone and chloroacetaldehyde in an efficient one-pot reaction, exhibits anti-carcinogenic effect. The results of present study showed that NFD inhibited the proliferation of breast cancer MDA-MB-231 cells through the induction of S-phase arrest and apoptosis. NFD-induced S-phase arrest was associated with a marked decrease in the protein expression of cyclin A, cyclin B, and cyclin-dependent kinase (Cdk)2. NFD-induced apoptosis was characterized by increase of sub-G1 population, phosphatidylserine (PS) externalization, and activation of caspases. Moreover, up-regulation of Bad and down-regulation of Bcl-2, Bcl-X(L), and survivin led to the loss of mitochondrial membrane potential (DeltaPsim), the release of cytochrome c and sequential activation of caspase-9 and caspase-3. NFD activated c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-regulated kinase (ERK) in MDA-MB-231 cells. Inhibitors of JNK (SP600125) and ERK (PD98059), but not p38 MAPK (SB203580) suppressed NFD-induced S-phase arrest and apoptosis in MDA-MB-231 cells. Both SP600125 and PD98059 attenuated Bad up-regulation, and reversed down-regulation of Bcl-2, Bcl-X(L), survivin, cyclin A, cyclin B, and Cdk2 in NFD-treated cells. Taken together, our data show that JNK and ERK-signaling pathways play important roles in NFD-mediated S-phase arrest and apoptosis of MDA-MB-231 cells.