Protease-catalyzed synthesis of α-poly-L-Lysine and amphiphilic poly(L-lysine-co-L-phenylalanine) in a neat non-toxic organic solvent.

Subtilisin Carlsberg (alkaline protease from Bacillus licheniformis) catalyzes the syntheses of high molecular weights (ca. 20 KDa) cationic α-poly-L-lysine and amphiphilic poly(α-L-lysine-co-L-phenylalanine) in neat organic solvent. The synthesis is conducted in liquid 1,1,1,2-tetrafluoroethane solvent, which is a hydrophobic non-toxic gas that does not deplete the ozone layer and approved for pharmaceutical applications. Solubility of substrates and adequate protease activity in this system with low water environment limits the reaction of hydrolysis of the growing peptide chains. The pressurization of this organic compressed fluid to liquid has low-pressure requirements (25 bar, 40 ºC), and its complete evaporation at atmospheric pressure after completing the reaction ensures solvent-free residues in products. The resulting polypeptides present null cytotoxicity according to MTT and NR analyses, as well as Calcein/EthD-1 assay in human cells.

Cross-Linking Chitosan into Hydroxypropylmethylcellulose for the Preparation of Neem Oil Coating for Postharvest Storage of Pitaya (Stenocereus pruinosus).

The market trend for pitaya is increasing, although the preservation of the quality of this fruit after the harvest is challenging due to microbial decay, dehydration, and oxidation. In this work, the application of antimicrobial chitosan-based coatings achieved successful postharvest preservation of pitaya (Stenocereus pruinosus) during storage at 10 ± 2 °C with a relative humidity of 80 ± 5%. The solution of cross-linked chitosan with hydroxypropylmethylcellulose with entrapped Neem oil (16 g·L-1) displayed the best postharvest fruit characteristics. The reduction of physiological weight loss and fungal contamination, with an increased redness index and release of azadirachtin from the microencapsulated oil, resulted in up to a 15 day shelf life for this fruit. This postharvest procedure has the potential to increase commercial exploitation of fresh pitaya, owing to its good taste and high content of antioxidants.

Antiproliferative and Antimigratory Activity of Poly-gallic Acid in Cancer Cell Lines.

BACKGROUND/AIM: Enzyme-mediated grafting of poly (gallic acid) (PGAL) and L-arginine and a-L-lysine onto PGAL produces reactive oxygen species (ROS)-suppressor multiradical molecules with low cytotoxicity, high thermostability and water solubility with cancer treatment potential. This study examined the anticancer effects of these molecules in hepatic (HepG2, ATCC HB-8065), breast (MCF7, ATCC HTB-22), and prostate (PC-3, ATCC CRL-1435 and DU 145, ATCC HTB-81) cancer cell lines, as well as in fibroblasts from healthy human skin as control cells. MATERIALS AND METHODS: PGAL was synthesized by the oxidative polymerization of the naturally abundant GA using laccase from Trametes versicolor. Insertions of amino acids L-arginine and α-L-lysine on the PGAL chain were carried out by microwave. The cells of dermal fibroblast (Fb) were obtained from primary skin cultures and isolated from skin biopsies. The cancer cells lines of hepatic (HepG2), breast (MCF7), and prostate (PC-3, DU 145) were obtained from ATCC. The viability of the cancer cells and the primary culture was obtained by the MTT assay. Proliferation was demonstrated by crystal violet assay. Cell migration was determined by Wound healing assay. Finally, cell cycle analysis was carried out with cells. RESULTS: The results show that 200 µg/ml of PGAL cultured in vitro with prostate cancer cells decreased viability, proliferation, and migration, as well as arrested cells in the G1 and S phases of the cell cycle. In contrast, the dermal fibroblasts and the hepatic line remained unaffected. The random grafting of L-Arg and a-L-Lys onto the PGAL chain also decreased the viability of prostate cancer cells. CONCLUSION: PGAL and PGAL-grafted amino acids are potential adjuvants for prostate cancer treatment, with improved physicochemical characteristics compared to GA.

Antiphagocytic Properties of Polygallic Acid with Implications in Gouty Inflammation.

Polygallic acid (PGAL) has been used in vitro to protect synoviocytes from monosodium urate (MSU) crystals due to its anti-inflammatory properties. However, MSU crystals can also activate other cells of the synovial fluid (SF). We studied the impact of PGAL on the phagocytosis of MSU crystals, inflammation, and oxidative stress using an in vitro model with SF leukocytes and THP-1 monocyte cells. SF leukocytes were stimulated with PGAL and MSU crystals, proinflammatory cytokines and phagocytosis were assessed. In THP-1 cells, the effect of PGAL on the phagocytosis of MSU crystals and the levels of IL-1ß, IL-6, TNF-α, and reactive oxygen species (ROS) was evaluated. PGAL was added to THP-1 cultures 24 h before MSU crystal addition as a pre-treatment, and IL-1ß was measured. One-way ANOVA with Tukey's post hoc test was performed, and a P value < 0.05 was considered statistically significant. PGAL (100 µg/mL) decreased phagocytosis in SF leukocytes by 14% compared to cells exposed to crystals without PGAL. In THP-1 cells, 100 and 200 µg/mL PGAL reduced phagocytosis by 17% and 15%, respectively. In SF cells, there was a tendency to decrease IL-1ß and IL-6. In THP-1 cells, decreases in IL-1ß and TNF-α, as well as a slight decrease in ROS, were identified. PGAL pre-treatment resulted in a reduction of IL-1ß. PGAL inhibits MSU phagocytosis by exerting an anti-inflammatory effect on cells exposed to crystals. The use of PGAL before an acute attack of gout suggests an important protective factor to control the inflammation.

Radiosterilized Pig Skin, Silver Nanoparticles and Skin Cells as an Integral Dressing Treatment for Burns: Development, Pre-Clinical and Clinical Pilot Study.

Radiosterilized pig skin (RPS) has been used as a dressing for burns since the 1980s. Its similarity to human skin in terms of the extracellular matrix (ECM) allows the attachment of mesenchymal stem cells, making it ideal as a scaffold to create cellularized constructs. The use of silver nanoparticles (AgNPs) has been proven to be an appropriate alternative to the use of antibiotics and a potential solution against multidrug-resistant bacteria. RPS can be impregnated with AgNPs to develop nanomaterials capable of preventing wound infections. The main goal of this study was to assess the use of RPS as a scaffold for autologous fibroblasts (Fb), keratinocytes (Kc), and mesenchymal stem cells (MSC) in the treatment of second-degree burns (SDB). Additionally, independent RPS samples were impregnated with AgNPs to enhance their properties and further develop an antibacterial dressing that was initially tested using a burn mouse model. This protocol was approved by the Research and Ethics Committee of the INRLGII (INR 20/19 AC). Transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis of the synthesized AgNPs showed an average size of 10 nm and rounded morphology. Minimum inhibitory concentrations (MIC) and Kirby-Bauer assays indicated that AgNPs (in solution at a concentration of 125 ppm) exhibit antimicrobial activity against the planktonic form of S. aureus isolated from burned patients; moreover, a log reduction of 1.74 ± 0.24 was achieved against biofilm formation. The nanomaterial developed with RPS impregnated with AgNPs solution at 125 ppm (RPS-AgNPs125) facilitated wound healing in a burn mouse model and enhanced extracellular matrix (ECM) deposition, as analyzed by Masson's staining in histological samples. No silver was detected by energy-dispersive X-ray spectroscopy (EDS) in the skin, and neither by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) in different organs of the mouse burn model. Calcein/ethidium homodimer (EthD-1), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and scanning electron microscopy (SEM) analysis demonstrated that Fb, Kc, and MSC could attach to RPS with over 95% cell viability. Kc were capable of releasing FGF at 0.5 pg above control levels, as analyzed by ELISA assays. An autologous RPS-Fb-Kc construct was implanted in a patient with SDB and compared to an autologous skin graft. The patient recovery was assessed seven days post-implantation, and the patient was followed up at one, two, and three months after the implantation, exhibiting favorable recovery compared to the gold standard, as measured by the cutometer. In conclusion, RPS effectively can be used as a scaffold for the culture of Fb, Kc, and MSC, facilitating the development of a cellularized construct that enhances wound healing in burn patients.

Enzymatic poly(gallic acid)-grafted α-l-lysine inhibits Staphylococcus aureus and Escherichia coli strains with no cytotoxicity for human cells.

The α-l-Lysine (LL) grafting onto the enzymatic poly(gallic acid) (PGAL) produces a helicoidal brush-like antimicrobial polymer containing outer positive-charged moieties. Best results are found with ca. 16 mol% α-LL-grafting for the inhibition of gram-positive Staphylococcus aureus and gram-negative Escherichia coli strains. Membrane permeability, confocal and scanning electron microscopy studies suggest a pore-formation and translocation mechanisms by initial electrostatic interaction of positive charged polymer at the negatively charged bacterial membranes. The attained polymer displays high concentration of hemolysis (Hc) in erythrocytes, and no lymphocyte mitochondrial activity. Interestingly, PGAL-LL is not cytotoxic on human dermal fibroblast. The antioxidant activity after the LL hybridization is also demonstrated by DPPH, ORAC, FRAP and hydroxyl radical scavenging, which enhances the preservation of human cells in addition to antimicrobial for this polymer.

Antioxidant and antimicrobial material by grafting of L-arginine onto enzymatic poly(gallic acid).

Microwave-mediated grafting of L-Arg onto naturally derived and stable multiradical poly(gallic acid) (PGAL) in aqueous media has been successfully achieved. This polymeric material has no adverse effect in human cells as there is no hemolytic activity upon MTT and Neutral Red assays. The analytical and computational characterization studies carried out in this study describe a helical molecular structure with random incorporation of L-Arginine pendant groups from PGAL's backbone. The antioxidant properties of the precursor polymer are preserved as proved by the elimination of stable DPPH and hydroxyl radical scavenging, as well as the FRAP and ORAC assays. Regarding the latter, the oxygen radical inhibition is enhanced compared to PGAL, which is attributed to the guanidyl moieties. PGAL-g-L-Arg displays antimicrobial activity against Gram (+) Listeria monocytogenes and Staphylococcus aureus strains with a MIC of 0.8 g/L and a bacteriostatic effect against Gram (-) Escherichia coli. Additionally, scanning electron and confocal fluorescence microscopies as well as crystal violet colorimetric assay demonstrate that the mechanism involved in the bacterial inhibition is related to the formation of porous channels on the membrane, which is discussed according to the helical secondary structure of the polymer and the amino acid guanidyl moieties interacting to bacterial membranes.

The enzymatic poly(gallic acid) reduces pro-inflammatory cytokines in vitro, a potential application in inflammatory diseases.

Cytokines like IL-6, TNF-α, and IL-1ß are important mediators of inflammation in many inflammatory diseases, as well as in cellular processes like cell proliferation and cell adhesion. Finding new molecules that decrease cell proliferation, adhesion (inflammatory infiltrate), and pro-inflammatory cytokine release could help in the treatment of many inflammatory diseases. The naturally derived poly(gallic acid) (PGAL), produced enzymatically from gallic acid in aqueous medium, is a non-toxic, thermostable multiradical polyanion that is antioxidant and has potential biomedical uses. Experimental evidence has demonstrated that PGAL reduces pro-inflammatory cytokines, which are the target of some inflammatory diseases. PGAL decreased IL-6, TNF-α, and IL-1ß production in human monocytes exposed to PMA without affecting cell viability. Additionally, PGAL reduced cell proliferation by affecting the transition from the S phase to the G2 phase of the cell cycle. Cell adhesion experiments showed that PMA-induced cell adhesion was diminished with the presence of PGAL, particularly at a concentration of 200 µg/mL. These properties of PGAL show a potential use for treating inflammatory diseases, such as psoriasis or arthritis.