Bioengineering functional copolymers. XXI. Synthesis of a novel end carboxyl-trithiocarbonate functionalized poly(maleic anhydride) and its interaction with cancer cells.

A novel carboxyl-trithiocarbonate functionalized polymer with a highly selective antitumor activity was synthesized by a reversible addition-fragmentation chain transfer (RAFT) polymerization of maleic anhydride (MA) with benzoyl peroxide as an initiator and S-1-dodecyl-S-(α,α'-dimethyl-α"-acetic acid)trithiocarbonate as a RAFT agent with the aim to design and synthesize an effective anticancer agent with minimum side effects. The structure, molecular weights and composition of synthesized polymers were investigated by (1)H ((13)C) NMR, MALDI-TOF-MS and GPC analyzes. It was demonstrated that RAFT polymerization of MA was accompanied by a partially controlled decarboxylation of anhydride units and the formation of conjugated double bond fragments in backbone macromolecular chains. The mechanism of interaction of pristine RAFT agent and PMA-RAFT polymer with cancer (HeLa human cervix carcinoma) and normal (L929 Fibroblast) cells was investigated by using a combination of chemical, biochemical, statistical, spectroscopic (SEM and fluorescence inverted microscope) and real-time analysis (RTCA) methods. PMA-RAFT exhibited higher and selective cytotoxicity, apoptotic and necrotic effects toward HeLa cells at relatively low concentrations (around 7.5-75 µg mL(-1), IC(50) = 11.183 µg mL(-1)) and toward Fibroblast cells at high concentrations (IC(50) > 100 µg mL(-1)). The observed highly selective antitumor activity render PMA-RAFT polymers as promising candidates for the utilization in cancer chemotherapy.

[Effects of maleic anhydride-modified poly(D,L-lactic acid) on the adhesion, proliferation and differentiation of osteoblasts].

The main objective of this study was to observe the adhesion, proliferation and differentiation of mouse osteblast-like MC3T3-E1 cells cultured on maleic anhydride-modified poly(D,L-lactic acid) (MPLA) and poly(D,L-lactic acid) (PDLLA) polymers, and to evaluate the cytocompatibility of MPLA polymer. The effects of MPLA and PDLLA polymers on the morphology, adhesion, proliferation, the content of total cellular protein, alkaline phosphatase (ALP) activity and the content of Ca of MC3T3-E1 cells were explored. These results indicated that MC3T3-E1 cells on MPLA polymer adhered and spread more fully. On MPLA polymer, the proliferation, total protein content, ALP activity, Ca content of the cells were significantly higher than those of the cells on PDLLA polymer (P < 0.01). It was concluded that MPLA polymer could promote the adhesion, spreading, proliferation and the synthesis of protein of osteoblasts, and also induced the differentiation and mineralization of osteoblasts, suggesting that MPLA polymer might have the better cytocompatibility than PDLLA.

Sperm characteristics and teratology in rats following vas deferens occlusion with RISUG and its reversal.

The functional success of the reversal of vas occlusion by styrene maleic anhydride (RISUG), using the solvent vehicle, Dimethyl Sulphoxide (DMSO), has been investigated. Reversal with DMSO was carried out in Wistar albino rats 90 days after bilateral vas occlusion. The body weight, organ weight, sperm characteristics, fertility test and teratology, including skeletal morphology were evaluated in vas occlusion and reversal animals and in F(1) progenies to assess the functional success of the occlusion and reversal. Body weight, organ weight and the cauda epididymal sperm characteristics of vas occlusion and reversal animals and of F(1) progenies were comparable to control. Ejaculated spermatozoa in the vaginal smear showed detached head/tail, acrosomal damage, bent midpiece, bent tail and morphological aberrations in sperm head after vas occlusion, which returned to normal, 90 days after reversal. Monthly fertility test, post-injection showed 0% fertility, which improved gradually and 100% fertility was achieved 90 days after reversal. The fertility/pregnancy/implantation record and skeletal morphology of the offspring were comparable to control. The results suggest functional success and safety of vas occlusion reversal by DMSO.

Epoxy Coatings Based on Modified Vegetable Oils for Wood Surface Protection against Fungal Degradation.

The study describes the curing and thermal behavior of a new castor oil maleic anhydride adduct/epoxy oils/5-Bromosalicylic acid coatings and their composites with wood. The epoxidized oils were flax and hemp. The kinetic parameters of the curing and thermal degradation processes were calculated. The resistance of the coated wood surfaces against Cladosporium cladosporioides, Aspergillus brasiliensis, and Penicillium chrysogenum was tested. Color changes, FT-IR and SEM were conducted before and after fungal attack. The decay resistance and color change of raw wood and wood treated samples against fungi was tested. Based on the color changes and according to ASTM D 2017, the decay resistance rating for covered samples was considered as "highly resistant". Chemical resistance and coating performance tests were also undertaken. The obtained results recommend the described materials for applications in wood protective coatings.

A review on various maleic anhydride antimicrobial polymers.

The basic requirement of human beings is better health but the serious health effects and numerous infections caused by rapid growth of harmful pathogens resulting in a large number of deaths and is a significant challenge to modern science. Microbes infecting humans can be stopped in two ways: disinfectants and antimicrobial agents. There is considerable interest from both academics and industry in antimicrobial polymers due to their favorable properties. Maleic anhydride incredibly bears diverse commercial applications due to its versatile chemical structure. Maleic anhydride is an electron-acceptor monomer where the property comes from reactive double bonds and also reactive anhydride groups. This review presents the development of antimicrobial polymers involving maleic anhydride in the macromolecular structure. This article also addresses the applications of antimicrobial polymers with maleic anhydride in numerous sectors.

Encapsulation of tDodSNO generates a photoactivated nitric oxide releasing nanoparticle for localized control of vasodilation and vascular hyperpermeability.

We report the synthesis and characterization of a photoactive nitric oxide (NO) releasing nanoparticle (NP) by encapsulation of the NO donor tert-dodecane S-nitrosothiol (tDodSNO) into a co-polymer of styrene and maleic anhydride (SMA) to afford SMA-tDodSNO. Encapsulation did not affect tDodSNO's stability or NO release profile, but imparted water solubility and protection from degradation reactions with glutathione. Under photoactivation the NP acted as a potent NO donor, with photoactivation acting as a switch to induce localized vasodilation in aortic rings (EC50* 660 nM at 2700 W/m2) and cause vascular hyperpermeability in mesenteric beds (8-fold increase in dye uptake at 1 µM SMA-tDodSNO with 460 W/m2 photoactivation). The NP was markedly superior as a photoactive NO donor in comparison to the S-nitrosothiols GSNO and SNAP, which are commonly used in experimental studies, as well as sodium nitroprusside, a clinically used vasodilator. Future development of this NP may find wide ranging therapeutic applications for treating cardiovascular disease and other disorders related to NO signaling, as well as enhancing macromolecular drug delivery to target organs through selective hyperpermeability. Supporting information describing the biophysical characterization of SMA-tDodSNO is supplied in an accompanying Data in Brief article (Alimoradi et al., doi: 10.1016/j.dib.2018.10.149).

Impact of styrene maleic anhydride (SMA) based hydrogel on rat fallopian tube as contraceptive implant with selective antimicrobial property.

Development of non-hormonal female contraception is a need to combat against increasing population growth. The presently available short term or long term female contraceptives and sterilization methods have their own restrictions and side effects. With this objective, herein, we describe an innovative insight about the use of hydrogel formulation consisting of Styrene Maleic Anhydride (SMA) dissolved in Dimethyl Sulfoxide (DMSO) as non-hormonal fallopian tube contraceptive implant. Firstly, in vitro behavior of SMA hydrogel was evaluated by in vitro swelling and rheological properties to comprehend the polymeric hydrogel property post implantation inside the fallopian tube. Simulated Uterine Fluid (SUF) was used to simulate female reproductive tract environment in this study. Mechanical strength of the hydrogel when subjected to dynamic environment post implantation in the fallopian tube was estimated by the G' values demonstrated. SMA hydrogel expressed selective antimicrobial activity against opportunistic pathogens (Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) while having limited consequence over the growth of Lactobacillus spp. After confirmation of cytocompatibility against primary rat endometrial cell lines, the polymeric hydrogel was implanted inside the uterine horns of Sprague-Dawley rats. In vivo biocompatibility of the hydrogel was confirmed by histological and immunohistochemical evaluation of uterine tissue sections. Hematology, blood biochemistry and organ toxicity (kidney, liver, spleen, lungs and heart) also revealed biocompatibility of SMA hydrogel. The results of the current study indicated that the SMA copolymer dissolved in DMSO to form hydrogel has excellent biocompatibility for application as female contraceptive gel which can be implanted in the fallopian tube.

Charge reversible and biodegradable nanocarriers showing dual pH-/reduction-sensitive disintegration for rapid site-specific drug delivery.

Dual pH-/reduction-sensitive biodegradable poly(methacrylic acid-co-N,N-bis(acryloyl)cystamine)/chitosan/dimethylmaleic anhydride-modified chitosan (PMAABACy/CS/CS-DMMA) nanoparticles with PMAABACy cores as carriers and dimethylmaleic anhydride-modified chitosan as charge reversible shells were rationally designed. PMAABACy cores using N,N-Bis(acryloyl)cystamine (BACy) as a crosslinker and methacrylic acid (MAA) as a monomer were fabricated via a mild and facile one-pot distillation-precipitation polymerization. After that, CS and CS-DMMA were alternately adsorbed on the surface of PMAABACy cores through a mild self-assembly. The results from TEM and DLS reveal that the PMAABACy/CS/CS-DMMA nanoparticles with desired size and hydrodynamic diameter. And then the nanoparticles exhibit the excellent drug-loading capacity and encapsulation efficiency toward anti-cancer agent doxorubicin (DOX), whereas be rapidly triggered to realize the GSH-sensitive site-specific release via the destruction of sulfide cross-linked structure in response to the intracellular environment of tumor cells. Furthermore, their surface charges could transfer from negative in neutral or basic medium to positive in acidic medium to enhance cellular uptake. Most importantly, the excellent anticancer activity has been also revealed using confocal laser scanning microscope (CLSM) analysis, namely successfully delivering DOX molecules to the cell nucleus. These experimental results indicate that such the novel dual pH-/reduction-sensitive biodegradable PMAABACy/CS/CS-DMMA with surface charge reversal have great potential as a desired anticancer drug carrier for cancer therapy.

Role of natural killer cells and macrophages in the nonspecific resistance to tumors in mice stimulated with SMANCS, a polymer-conjugated derivative of neocarzinostatin.

Copoly(styrene-maleic acid)-conjugated neocarzinostatin (SMANCS), a lipophilic derivative of the proteinaceous antitumor antibiotic neocarzinostatin, has been reported to stimulate a nonspecific resistance to tumors (NSRT) in solid tumor-bearing mice, in addition to its chemotherapeutic antitumor effect through the arrest of DNA synthesis by direct DNA strand scission. In the present study, splenic or peritoneal effector cells were used to investigate the ability of SMANCS to augment natural killer (NK) cell activity and to generate cytostatic macrophages (A-M phi). Splenic NK cell activity augmented by SMANCS was characterized by cytotoxicity to various target cells, nylon wool nonadherence, and sensitivity to treatment with anti-asialo-GM1 antiserum or monoclonal anti-Thy-1.2 antibody followed by complement. The A-M phi generated by SMANCS stimulation were characterized by their adherence to a plastic surface coated with fetal calf serum and their ability to phagocytize carbonyl-iron. The maximum level of NK cell activity in the spleens of mice was detected 3 days after i.v. injection of SMANCS, and the highest activity of the peritoneal A-M phi was demonstrated in mice 4 days after SMANCS treatment. On the other hand, the NSRT of mice stimulated with SMANCS was not detectable in mice treated with carrageenan or trypan blue, whereas SMANCS-stimulated NSRT was observed in mice treated with anti-asialo-GM1 antiserum. The NSRT that was stimulated with SMANCS was also demonstrated in mice homozygous for the beige mutation and their non-beige littermates, when NK cell-resistant EL-4 thymoma was used as a tumor target. These results suggest that the expression of NSRT of mice stimulated with SMANCS may require the function of A-M phi, although NK cell activity was also augmented in spleens of mice by administration of SMANCS.

Tumor Acidity-Induced Sheddable Polyethylenimine-Poly(trimethylene carbonate)/DNA/Polyethylene Glycol-2,3-Dimethylmaleicanhydride Ternary Complex for Efficient and Safe Gene Delivery.

Amphiphilic PEI derivatives/DNA complexes are widely used for DNA delivery, but they are unstable in vivo and have cytotoxicity due to the excess cationic charge. PEGylation of cationic complexes can improve sterical stability and biocompatibility. However, PEGylation significantly inhibits cellular uptake and endosomal escape. In this work, sheddable ternary complexes were developed by coating a tumor acidity-sensitive ß-carboxylic amide functionalized PEG layer on the binary complexes of amphiphilic cationic polyethylenimine-poly(trimethylene carbonate) nanoparticles/DNA (PEI-PTMC/DNA). Such sheddable ternary complexes markedly reduced their nonspecific interactions with serum protein in the bloodstream and obtained minimal cytotoxicity due to the protection of the PEG shell. At the tumor site, the PEG layer was deshielded by responding to the tumor acidic microenvironment and the positively charged complexes re-exposed that had higher affinity with negatively charged cell membranes. Meanwhile the positively charged complexes facilitated endosomal escape. Accordingly, this delivery system improved the biocompatibility of gene-loaded complexes and enhanced the gene transfection efficiency. Such PEGylated complexes with the ability to deshield the PEG layer at the target tissues hold great promise for efficient and safe gene delivery in vivo.

Styrene maleic acid neocarzinostatin treatment for hepatocellular carcinoma.

A variety of treatments have recently been introduced to improve the prognosis of hepatocellular carcinoma (HCC). These anticancer therapies include the oily carcinostatic agent styrene maleic acid neocarzinostatin (SMANCS). SMANCS is a chemical conjugate of a synthetic copolymer of styrene maleic acid (SMA) and the proteinaceous anti-cancer agent neocarzinostatin (NCS), which dissolves in organic solvents such as pyridine and acetone, and particularly in Lipiodol. NCS is a simple protein capable of inhibiting DNA synthesis and inducing DNA degradation. Lipiodol is an ethyl ester of iodinated poppy seed oil in which most of the unsaturated double bonds in oleic, linoleic and linolenic acid are almost completely iodinated. When a homogeneous suspension of SMANCS with Lipiodol (SMANCS/Lipiodol) is administered intra-arterially, Lipiodol acts as a carrier of SMANCS. Many studies have demonstrated the clinical efficacy of SMANCS/Lipiodol in the treatment of HCC. We have shown that transcatheter arterial infusion (TAI) with SMANCS/Lipiodol has a more favorable focal therapeutic effect than does epirubicin in Lipiodol in the initial treatment of HCC. However, recent clinical studies have indicated that SMANCS causes severe adverse reactions and complications. We have also reported a case of HCC in which multifocal hepatic infarction developed after TAI with SMANCS/Lipiodol. Arterial administration of SMANCS/Lipiodol, therefore, should be given as peripherally as possible via the tumor feeding arteries, to enhance the efficacy of the agent and to reduce the adverse effects.

Effect of anionic polymeric hydrogels on spermatozoa motility.

The effects of a few synthetic polymers on the motility of human spermatozoa in vitro have been studied. An alternate copolymer of styrene and maleic anhydride, poly(S-MA), poly (styrene-maleic acid), poly(S-MC), poly(hydroxy-ethyl methacrylate-methacrylic acid) copolymer, poly(HEMA-MAC), poly(HEMA) homopolymer and poly(MAC) homopolymer were chosen for this purpose. It was found that all the carboxylic acid containing polymers are strong inhibitors of the motility of spermatozoa. Poly(HEMA) did not have any inhibitory effect on the motility of spermatozoa.

PIP: This paper describes the effects of selected synthetic polymers on the motility of human spermatozoa in vitro. The authors have developed a new mehtod of male fertility control that involves the use of an anionic hydrophilic polymer which when infected into the vas deferens, is precipitated and lowers the pH in the vicinity, thus killing the spermatozoa passing through the vas deferens. The results of this experiment indicate that carboxylic acid-containing polymers are strong inhibitors of sperm motility. Almost all sperm became immotile within 15 minutes after treatment with polymer (styrene-maleic acid) (S-MC) and polymer methacrylic acid (MAC). Polymer (HEMA-MAC), a copolymer containing roughly equal ratios of hydroxyethyl methacrylate and methacrylic acid, showed inhibitory action, but it took 30 minutes for 100% immotility to be achieved. The stronger inhibition of motility observed with poly (S-MC) compared with poly (HEMA-MAC) is attributed to the presence in the former of 2 carboxylic groups on adjacent carbon atoms. It is possible that low pH environment created by ionization of carboxyl groups fixed in the polymer matrix is responsible for killing the spermatozoa. Hydrogen ion concentration is considered to be a key factor in influencing the motility, viability, and metabolism of spermatozoa. Poly (HEMA), containing only hydroxyl groups, did not have any inhibitory effect.

Mechanism of tumor-targeted delivery of macromolecular drugs, including the EPR effect in solid tumor and clinical overview of the prototype polymeric drug SMANCS.

This review article describes three aspects of polymeric drugs. The general mechanism of the EPR (enhanced permeability and retention) effect and factors involved in the effect are discussed, in view of the advantages of macromolecular therapeutics for cancer treatment, which are based on the highly selective EPR-related delivery of drug to tumor. Also described are advantages of more general water-soluble polymeric drugs as primary anticancer agents, using SMANCS as an example. Last, SMANCS/Lipiodol is discussed with reference to the type of formulation for arterial injection with most pronounced tumor selective delivery, as well as its advantages, precautions, and side effects from the clinical standpoint.

Antiproliferative effect of YM881 (SMANCS) on glioma cells in vitro.

Growth inhibitory activity of YM881 (SMANCS) against 3 human cultured cell lines and one rat cultured cell line derived from glioma cells was assessed quantitatively. YM881 showed potent cytotoxicity against all glioma cell lines tested; the IC50 of this drug was 1.9-8.4 micrograms/ml. YM881 may be classified as a concentration-dependent drug but it has also a time-dependent effect. Flow cytometric studies of the DNA histogram showed accumulation in the G2-M phase with YM881. These findings suggest that YM881 may be useful in the treatment of glioma.

Male mediated teratogenic potential evaluation of new antifertility compound SMA in rabbit (Oryctolagus cuniculus).

The male mediated teratogenic potential of a new antifertility compound, styrene maleic anhydride (SMA), was evaluated in rabbits. No teratogenic potential was observed at the doses of 1.25 mg, 2.5 mg and 5.0 mg used in our experiment.

Ultrastructural changes in the vas deferens of langur monkeys Presbytis entellus entellus after vas occlusion with styrene maleic anhydride and after its reversal.

Ultrastructural changes in the vas deferens of langur monkeys after 150 days of vas occlusion with styrene maleic anhydride (SMA) and after 150 days of noninvasive reversal are reported. The vas deferens of the sham-operated control animals revealed active secretory and absorptive functions. The basal cells showed prominent nucleus and sparse cytoplasmic organelles, and the principal cells characterized by oval or irregular nucleus, well developed mitochondria, Golgi bodies, rough endoplasmic reticulum, secretory granules in the Golgi area, free ribosomes, and glycogen granules in the supranuclear region suggesting secretory function. Vesicles and stereocilia in the apex region suggested absorptive functions of the vas deferens. Vas occlusion by SMA resulted in exfoliation of epithelial cells, pyknotic nuclei, and vacuolated cytoplasm virtually devoid of cytoplasmic organelles and stereocilia. After noninvasive reversal, the vas epithelium regained a state of normalcy as evidenced by prominent plasma membrane, nucleus, cytoplasmic organelles, and stereocilia. The results suggest that the exfoliation of the epithelium due to vas occlusion by SMA regains normalcy after 150 days of noninvasive reversal.

Study on the thermal stability of alpha-amylase modified by maleic anhydride copolymer.

The thermal inactivation of mesophilic Bacillus subtilis alpha-amylase modified by maleic anhydride/vinyl acetate copolymer has been studied at different polymer/enzyme ratios in the pH range of relevance to enzymatic catalysis. Enzymatic activity measurements combined with circular dichroism measurements were used to determine the enzyme thermostability. The apparent first-order rate constants and activation energies of thermo-inactivation affected by addition of Ca2+ ions as well as by modification have been calculated. The modified alpha-amylase exhibited sufficiently high catalytic activity with enhanced resistance to the thermal unfolding process.

Factors and mechanism of "EPR" effect and the enhanced antitumor effects of macromolecular drugs including SMANCS.

Both enhanced vascular permeability and angiogenesis of tumor sustain rapid growth of tumor involving many vascular mediators and high vascular density. On the contrary, however, they can be utilized for macromolecular drug delivery to tumor. Impaired reticuloendothelial/lymphatic clearance of macromolecules from the tumor, or lack of such clearance, is another unique characteristic of tumor tissue, which results intratumor retention of macromolecular drugs thus delivered (Figure 1). Consequently, enhanced permeability and retention (EPR) effect is the basis for the selective targeting of macromolecular drugs to tumor, and the EPR concept is now utilized for selective delivery of many macromolecular anticancer agents in aqueous formation for i.v. or i.a. as well as oily formation for i.a. dosing, which is not possible for low-molecular-weight drugs because of rapid washout by capillary vascular blood flow. This EPR concept has been validated in clinical settings with hepatoma and other solid tumors. In our laboratories, several promising macromolecular anticancer drugs after SMANCS, such as PEG-XO, PEG-DAO, PEG-ZnPP, were developed, warranting further investigation for clinical application. More efficient drug delivery to tumor, especially of macromolecular drugs, may be possible by enhancing the EPR effect with the use of various vascular permeability mediators or potentiators. Suppression of the EPR effect by the use of appropriate inhibitors or antidotes, such as the bradykinin antagonist HOE 140 and protease inhibitors or NOS inhibitors, may also be possible. Thus, one may be able to suppress or retard tumor growth and tumor metastasis. Also, by suppressing vascular permeability with antidotes such as the bradykinin antagonist HOE 140, pleural fluid in lung cancer and ascitic fluid in abdominal carcinomatosis may be controlled and the clinical course of cancer patients may be improved. In summary, tumor vasculature can be an excellent target for delivery of macromolecular anticancer drugs; the most beneficial class of drugs in view of tumor-selective targeting based on the EPR effect in solid tumor as well as compliance of patients and ultimate therapeutic efficacy.

Long-term toxicity studies of styrene maleic anhydride in rats.

In male Charles Foster rats, polymer styrene maleic anhydride was injected into the lumen of the vas deferens at dose levels of 1.0, 2.5, and 5.0 mg in each vas deferens of Groups II, III, and IV, respectively, while controls (Group I) received 0.03 ml dimethyl sulfoxide in each vas deferens. The rats were observed for 6 months for toxicity. No change in any of the toxicity parameters in test animals as compared to controls was revealed. Hence, the polymer is safe at the doses used within 6 months of injection.

[Action mechanisms of zinostatin stimalamer (YM881)].

The mechanism of action of zinostatin stimalamer (YM881) was studied. YM881 suppressed colony formation of HeLa cells dose-dependently, and showed cytocidal activity. The compound inhibited DNA synthesis, but neither RNA nor protein synthesis in L1210 cells, and caused cellular DNA strand breaks in HeLa cells and DNA cleavage of PM2 phage supercoiled DNA. YM881 was also shown to cause typical G2/M arrest on L1210 cell cycle, and inhibited DNA polymerase alpha of HeLa cells, but only at high concentrations. Present study showed that antitumor and cytotoxic mechanisms of YM881 were identical to neocarzinostatin (NCS) and due to the cellular DNA strand breaks caused by direct DNA-cleaving activity and the subsequent inhibition of DNA synthesis.