Comparative Evaluation of Nimesulide-Loaded Nanoparticles for Anticancer Activity Against Breast Cancer Cells

Recent clinical and epidemiological researches have declared that non-steroidal anti-inflammatory agents may display as antineoplastic agents and indicate pro-apoptotic and antiproliferative effects on cancer cells. The major purpose of this research was to develop a novel poly(ethyleneglycol)-block-poly(ε-caprolactone) (PEG-b-PCL) nano-sized particles encapsulated with nimesulide (NMS), a selective COX-2 inhibitor, and to evaluate its anticancer activity against MCF-7 breast cancer cells. NMS-encapsulated PEG-b-PCL nanoparticles were fabricated using three different production techniques: (i) by emulsion-solvent evaporation using a high shear homogenizer, (ii) by emulsion-solvent evaporation using an ultrasonicator, and (iii) by nanoprecipitation. Nanoparticles were evaluated with respect to the entrapment efficiency, size characteristics, drug release rates, thermal behavior, cell viability assays, and apoptosis. The resulting nanoparticles were found to be spherical shapes with negative surface charges. The average diameter of all nanoparticles ranged between 148.5 and 307.2 nm. In vitro release profiles showed that all nanoparticles exhibited a biphasic release pattern. NMS-loaded PEG-b-PCL nanoparticles demonstrated significant anticancer activity against MCF-7 breast cancer cells in a dose-dependent manner, and the effects of nanoparticles on cell proliferation were significantly affected by the preparation techniques. The nanoparticles developed in this work displayed higher potential for the NMS delivery against breast cancer treatment for the future.     

Purification and activity characterization of polysaccharides in the medicinal lichen <Emphasis Type="Italic">Umbilicaria tornata</Emphasis> from Taibai Mountain,China

Water-soluble polysaccharides from Umbilicaria tornata (UTP) were purified and preliminarily characterized. The antioxidant and antitumor activities of crude UTP and two purified fractions (UTP-1 and UTP-2) were evaluated using in vitro experiments. The results showed that the molecular weights of UTP-1 and UTP-2 were 84.86 and 28.66 kDa, respectively. Both UTP-1 and UTP-2 were composed of glucose and xylose, with their molar ratios being 1.3:0.9 and 0.9:4.6, respectively. In addition, crude UTP, UTP-1 and UTP-2 showed dose-dependent DPPH and hydroxyl radical scavenging and reducing activities. However, crude UTP exhibited stronger antioxidant activity than UTP-1 and UTP-2, particularly in terms of DPPH radicals. Crude UTP and the two purified fractions inhibited the growth of HeLa, HepG2, A375, MCF-7, SGC7901 and Caco2 cancer cells in vitro. Compared with UTP-1 and UTP-2, crude UTP presented significantly higher antitumor activity in vitro against HeLa and HepG2 cells (p?<?0.05). These findings provide a scientific basis for the deeper exploration and resource development of U. tornata.     

<Emphasis Type="Italic">In Vivo</Emphasis> Anticancer Efficacy and Toxicity Studies of a Novel Polymer Conjugate <Emphasis Type="Italic">N</Emphasis>-Acetyl Glucosamine (NAG)–PEG–Doxorubicin for Targeted Cancer Therapy

A novel polymer–drug conjugate, polyethylene glycol–N-(acetyl)-glucosamine–doxorubicin (PEG-NAG-DOX) was evaluated in this study for its in vivo potential for treatment of tumours demonstrating improved efficacy and reduced toxicity. The proposed polymer–drug conjugate comprised of polyethylene glycol–maleimide (mPEG-MAL, 30000 Da) as a carrier, doxorubicin (DOX) as an anticancer drug and N-acetyl glucosamine (NAG) as a targeting moiety as well as penetration enhancer. Doxorubicin has a potent and promising anticancer activity; however, severe cardiotoxicity limits its application in cancer treatment. By modifying DOX in PEG-NAG-DOX prodrug conjugate, we aimed to eliminate this limitation. In vivo anticancer efficacy of the conjugate was evaluated using BDF mice-induced skin melanoma model by i.v. administration of DOX conjugates. Anticancer efficacy studies were done by comparing tumour volume, body weight, organ index and percent survival rate of the animals. Tumour suppression achieved by PEG-NAG-DOX at the cumulative dose of 7.5 mg/kg was two-fold better than that achieved by DOX solution. Also, the survival rate for PEG-NAG-DOX conjugate was >70% as compared to <50% survival rate for DOX solution. In addition, toxicity studies and histopathological studies revealed that while maintaining its cytotoxicity towards tumour cells, PEG-NAG-DOX conjugate showed no toxicities to major organs. Therefore, PEG-NAG-DOX conjugate can be suggested as a desirable candidate for targeted cancer therapy.     

Co-delivery of Vorinostat and Etoposide <Emphasis Type="Italic">Via</Emphasis> Disulfide Cross-Linked Biodegradable Polymeric Nanogels: Synthesis,Characterization, Biodegradation,and Anticancer Activity

Treatment regimens for cancer patients using single chemotherapeutic agents often lead to undesirable toxicity, drug resistance, reduced uptake etc. Combination of two or more drugs is therefore becoming an imperative strategy to overcome these limitations. A step forward can be taken through delivery of the drugs used in combination via nanoparticles. Co-administration of chemotherapeutic drugs encapsulated in nanoparticles has been shown to result in synergistic effects and enhanced therapeutic efficacy. In present study, we explored the combination treatment of histone deacetylase inhibitor vorinostat (VOR) and topoisomerase II inhibitor etoposide (ETOP). The concurrent combination treatment of VOR and ETOP resulted in synergistic effect on human cervical HeLa cancer cells. VOR and ETOP were encapsulated into poly(ethylene glycol) monomethacrylate (POEOMA)-based disulfide cross-linked nanogels. The nanogels were synthesized using atom transfer radical polymerization (ATRP) via cyclohexane/water inverse mini-emulsion and were degradable in presence of intracellular glutathione (GSH) concentration. Both the drugs were loaded into the nanogels by physical encapsulation method and characterized by Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), X-ray diffraction (XRD), dynamic light scattering (DLS), and differential scanning calorimetry (DSC). Both VOR- and ETOP-loaded nanogels showed sustained release profile. Furthermore, combination treatment drugs encapsulated of POEOMA nanogel demonstrated enhanced synergistic cytotoxic effect compared with combination of free drugs. Enhanced synergistic cell killing efficiency of drug-loaded POEOMA nanogels was due to increased apoptosis via caspase 3/7 activation. Therefore, combination of VOR- and ETOP-loaded PEG-based biodegradable nanogels may provide a promising therapy with enhanced anticancer effect.     

An integrated approach of bioinformatic prediction and in vitro analysis identified that miR-34a targets <Emphasis Type="Italic">MET</Emphasis> and <Emphasis Type="Italic">AXL</Emphasis> in triple-negative breast cancer

Background

Breast cancer is the most prevalent cancer among women, and AXL and MET are the key genes in the PI3K/AKT/mTOR pathway as critical elements in proliferation and invasion of cancer cells. MicroRNAs (miRNAs) are small non-coding RNAs regulating the expression of genes.

Methods

Bioinformatic approaches were used to find a miRNA that simultaneously targets both AXL and MET 3′-UTRs. The expression of target miRNA was evaluated in triple-negative (MDA-MB-231) and HER2-overexpressing (SK-BR-3) breast cancer cell lines as well as normal breast cells, MCF-10A, using quantitative real-time PCR. Then, the miRNA was overexpressed in normal and cancer cell lines using a lentiviral vector system. Afterwards, effects of overexpressed miRNA on the expression of AXL and MET genes were evaluated using quantitative real-time PCR.

Results

By applying bioinformatic software and programs, miRNAs that target the 3′-UTR of both AXL and MET mRNAs were determined, and according to the scores, miR-34a was selected for further analyses. The expression level of miR-34a in MDA-MB-231 and SK-BR-3 was lower than that of MCF-10A. Furthermore, AXL and MET expression in SK-BR-3 and MDA-MB-231 was lower and higher, respectively, than that of MCF-10A. After miR-34a overexpression, MET and AXL were downregulated in MDA-MB-231. In addition, MET was downregulated in SK-BR-3, while AXL was upregulated in this cell line.

Conclusions

These findings may indicate that miR-34a is an oncogenic miRNA, downregulated in the distinct breast cancer subtypes. It also targets MET and AXL 3′-UTRs in triple-negative breast cancer. Therefore, it can be considered as a therapeutic target in this type of breast cancer.

     

<Emphasis Type="Italic">In Vitro</Emphasis> Characterization and Evaluation of the Cytotoxicity Effects of Nisin and Nisin-Loaded PLA-PEG-PLA Nanoparticles on Gastrointestinal (AGS and KYSE-30), Hepatic (HepG2) and Blood (K562) Cancer Cell Lines

The aim of this study was an in vitro evaluation and comparison of the cytotoxic effects of free nisin and nisin-loaded PLA-PEG-PLA nanoparticles on gastrointestinal (AGS and KYSE-30), hepatic (HepG2), and blood (K562) cancer cell lines. To create this novel anti-cancer drug delivery system, the nanoparticles were synthesized and then loaded with nisin. Subsequently, their biocompatibility, ability to enter cells, and physicochemical properties, including formation, size, and shape, were studied using hemolysis, fluorescein isothiocyanate (FITC), Fourier transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), and scanning electron microscopy (SEM), respectively. Then, its loading efficiency and release kinetics were examined to assess the potential impact of this formulation for the nanoparticle carrier candidacy. The cytotoxicities of nisin and nisin-loaded nanoparticles were evaluated by using the MTT and Neutral Red (NR) uptake assays. Detections of the apoptotic cells were done via Ethidium Bromide (EB)/Acridine Orange (AO) staining. The FTIR spectra, SEM images, and DLS graph confirmed the formations of the nanoparticles and nisin-loaded nanoparticles with spherical, distinct, and smooth surfaces and average sizes of 100 and 200 nm, respectively. The loading efficiency of the latter nanoparticles was about 85–90%. The hemolysis test represented their non-cytotoxicities and the FITC images indicated their entrance inside the cells. An increase in the percentage of apoptotic cells was observed through EB/AO staining. These results demonstrated that nisin had a cytotoxic effect on AGS, KYSE-30, HepG2, and K562 cancer cell lines, while the cytotoxicity of nisin-loaded nanoparticles was more than that of the free nisin.     

Lectin-Conjugated Clarithromycin and Acetohydroxamic Acid-Loaded PLGA Nanoparticles: a Novel Approach for Effective Treatment of <Emphasis Type="Italic">H. pylori</Emphasis>

Helicobacter pylori infection remains challenging as it mainly colonized beneath the deep gastric mucosa and adheres to epithelial cells of the stomach. Concanavalin-A (Con-A)-conjugated gastro-retentive poly (lactic-co-glycolic acid) (PLGA) nanoparticles of acetohydroxamic acid (AHA) and clarithromycin (CLR) were prepared and evaluated under in vitro conditions. Solvent evaporation method was employed for preparation of nanoparticles and characterized for particle size distribution, surface morphology, percent drug entrapment, and in vitro drug release in simulated gastric fluid. Optimized nanoparticles were conjugated with Con-A and further characterized for Con-A conjugation efficiency and mucoadhesion and tested for in vitro anti-H. pylori activity. The conjugation with Con-A further sustained the drug release over a period of 8 h when compared to non-conjugated nanoparticles of AHA and CLR. In vitro anti H. pylori study confirmed that Con-A-conjugated nanoparticles containing both drugs, i.e., CLR and AHA, had shown maximum zone of inhibition compared to other formulations. In a nut shell, results suggest that the developed systems could be used for better therapeutic activity against H. pylori infection.     

Targeted cowpea chlorotic mottle virus-based nanoparticles with tumor-homing peptide F3 for photothermal therapy

Our aim was to devise targeted drug delivery systems using genetically modified cowpea chlorotic mottle virus (CCMV) capsids by fusion expression with tumorhoming peptide F3 for efficient delivery of therapeutic substances into tumor cells. The RNA-binding domain at the N terminus (amino acid residues 1–25) of CCMV capsid protein (CP) was selectively deleted, and F3 was inserted for the expression in Pichia pastoris. After chromatographic purification, F3-CCMV capsids were obtained via selfassembly of the F3-CP fusion protein and then analyzed by transmission electron microscopy and dynamic light scattering analysis, which revealed spherical nanoparticles (NPs) ca. 18 nm in diameter with regular monodispersity. Near-infrared fluorescent dye IR780 iodide, which has been applied for cancer imaging, photodynamic therapy, and photothermal therapy, was encapsulated in F3-CCMV NPs. The resultant F3-CCMV-IR780 NPs showed excellent molecular targeting to nucleolin receptor overexpressed on the surface of MCF-7 tumor cells. Furthermore, the in vitro cellular uptake and cell viability assay proved a photothermal effect by a single dose of near-infrared laser irradiation. The present system may offer a programmable nanoscaffoldbased drug delivery system vehicle for fabrication of promising therapeutic substances for cancer therapy.     

Isolation of Natural Naphthoquinones from <Emphasis Type="Italic">Juglans regia</Emphasis> and In Vitro Antioxidant and Cytotoxic Studies of Naphthoquinones and the Synthetic Naphthofuran Derivatives

The naphthoquinones and their derivatives containing hydroxyl group exhibit wide range of pharmacological activities, such as antioxidant, antibacterial, antiviral, anticancer, antimalarial, and antifungal activities. In particular, the antioxidant and anticancer behaviors of these compounds continue to draw attention of researchers. In the present communication, three natural naphthoquinones—juglone, lawsone, and plumbagin—isolated from the chloroform extract of nutshells of Juglans regia Linn. and two 1,4-naphthoquinone derivatives—ethyl-5-hydroxynaphtho[ 1,2-b]furan-3-carboxylate and diethylnaphtho[1,2-b:4,3-b′]difuran-3,4-dicarboxylate—and three 5-hydroxy- 1,4-naphthoquinone derivatives—diethyl-7-hydroxynaphtho[1,2-b:4,3-b']difuran-3,4-dicarboxylate,4-ethoxycarbonyl- 7-hydroxynaphtho[1,2-b:4,3-b']difuran-3-carboxylic acid, and 7-hydroxynaphtho[1,2-b:4,3-b']difuran-3,4- dicarboxylic acid were synthesized and examined for their in vitro antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) bioassays. In addition, the cytotoxicity test using human hepatocellular liver carcinoma cell line (HepG2) was carried out for all the compounds. The 5-hydroxy-1,4-naphthoquinone derivatives displayed almost equivalent scavenging activity in DPPH assay and higher activity in ABTS assay relative to ascorbic acid. On the other hand, naphthoquinones Juglone and Plumbagin showed lesser antioxidant activity, but higher cytotoxic activity than naphthofurans except for diethyl naphtho[1,2-b:4,3-b′]difuran-3,4-dicarboxylate, which showed excellent cytotoxic activity.     

Effects of transgenic expression of <Emphasis Type="Italic">Brevibacterium linens</Emphasis> methionine gamma lyase (MGL) on accumulation of <Emphasis Type="Italic">Tylenchulus semipenetrans and key aminoacid contents</Emphasis> in <Emphasis Type="Italic">Carrizo citrange</Emphasis>

Alpha-dioxygenases (α-DOX) catalyzing the primary oxygenation of fatty acids to oxylipins were recently found in plants. Here, the biological roles of the pepper α-DOX (Ca-DOX) gene, which is strongly induced during non-host pathogen infection in chili pepper, were examined. Virus-induced gene silencing demonstrated that down-regulation of Ca-DOX enhanced susceptibility to bacterial pathogens and suppressed the hypersensitive response via the suppression of pathogenesis-related genes such as PR4, proteinase inhibitor II and lipid transfer protein (PR14). Ca-DOX-silenced pepper plants also exhibited more retarded growth with lower epidermal cell numbers and reduced cell wall thickness than control plants. To better understand regulation of Ca-DOX, transgenic Arabidopsis plants harboring the β-glucuronidase (GUS) reporter gene driven from a putative Ca-DOX promoter were generated. GUS expression was significantly induced upon avirulent pathogen infection in transgenic Arabidopsis leaves, whereas GUS induction was relatively weak upon virulent pathogen treatment. After treatment with plant hormones, early and strong GUS expression was seen after treatment of salicylic acid, whereas ethylene and methyl jasmonate treatments produced relatively weak and late GUS signals. These results will enable us to further understand the role of α-DOX, which is important in lipid metabolism, defense responses, and growth development in plants.     

Imidazole-Bearing Polymeric Micelles for Enhanced Cellular Uptake,Rapid Endosomal Escape,and On-demand Cargo Release

The complex design of multifunctional nanomedicine is beneficial to overcome the multiple biological barriers of drug delivery, but it also presents additional hurdles to clinical translation (e.g., scaling-up and quality control). To address this dilemma, we employed a simple imidazole-bearing polymer micelle for enhanced cellular uptake, facilitated endosomal escape, and on-demand release of a model drug, SN-38. The micelles were crosslinked by the reversible imidazole/Zn²⁺ coordination with a drug loading of ca. 4% (w/w) and a diameter less than 200 nm. Under mimicked tumor microenvironment (pH 6.8), the surface charge of micelles reversed from negative to positive, leading to enhanced micelles uptake by model 4T1 cells. Such effect was verified by fluorescent labelling of micelles. Compared to imidazole-free nanocarriers, the charge-reversal micelles delivered significantly more SN-38 to 4T1 cells. Due to the proton sponge effect, imidazole-bearing micelles could rapidly escape from endosomes compared to the control micelles, as evidenced by the kinetic analysis of micelle/endosome co-localization. The coordination crosslinking also enabled the acid-triggered drug release. This work provides a “three birds with one stone” approach to achieve the multifunctionality of nanocarriers without complicated particle design, and opens new avenues of advancing nanomedicine translation via simple tailored nanocarriers.     

Magnetic Nanoparticles for the Delivery of Dapagliflozin to Hypoxic Tumors: Physicochemical Characterization and Cell Studies

In solid tumors, hypoxia (lack of oxygen) is developed, which leads to the development of resistance of tumor cells to chemotherapy and radiotherapy through various mechanisms. Nevertheless, hypoxic cells are particularly vulnerable when glycolysis is inhibited. For this reason, in this study, the development of magnetically targetable nanocarriers of the sodium-glucose transporter protein (SGLT2) inhibitor dapagliflozin (DAPA) was developed for the selective delivery of DAPA in tumors. This nanomedicine in combination with radiotherapy or chemotherapy should be useful for effective treatment of hypoxic tumors. The magnetic nanoparticles consisted of a magnetic iron oxide core and a poly(methacrylic acid)-graft-poly(ethyleneglycol methacrylate) (PMAA-g-PEGMA) polymeric shell. The drug (dapagliflozin) molecules were conjugated on the surface of these nanoparticles via in vivo hydrolysable ester bonds. The nanoparticles had an average size of ~ 70 nm and exhibited a DAPA loading capacity 10.75% (w/w) for a theoretical loading 21.68% (w/w). The magnetic responsiveness of the nanoparticles was confirmed with magnetophoresis experiments. The dapagliflozin-loaded magnetic nanoparticles exhibited excellent colloidal stability in aqueous and biological media. Minimal (less than 15% in 24 h) drug release from the nanoparticles occurred in physiological pH 7.4; however, drug release was significantly accelerated in pH 5.5. Drug release was also accelerated (triggered) under the influence of an alternating magnetic field. The DAPA-loaded nanoparticles exhibited higher in vitro anticancer activity (cytotoxicity) against A549 human lung cancer cells than free DAPA. The application of an external magnetic field gradient increased the uptake of nanoparticles by cells, leading to increased cytotoxicity. The results justify further in vivo studies of the suitability of DAPA-loaded magnetic nanoparticles for the treatment of hypoxic tumors.     

Fine-scale differences in genetic and census population size ratios between two stream fishes

Comparing the ratio of effective number of breeders (N _b ) to adult population size (N) among closely related coexisting species can provide insights into the role of life history on N _b /N ratios and inform conservation programs towards limiting the loss of evolutionary potential in natural populations. We estimated N _b and N in two coexisting salmonid fishes (Brook trout and Atlantic salmon) for 3–4 consecutive years in two small, adjacent streams in Newfoundland, Canada, using mark-recapture (N), linkage disequilibrium (N _b(LD)), and sibship frequency approaches (N _b(Sib) ). We found that N _b /N ratios were about 20-fold greater in Atlantic salmon than in brook trout (mean 0.20, range 0.06–0.56 vs. mean 0.02, range 0.01–0.05, respectively). This difference was consistent across N _b estimators. In addition, we found that removing migrants reduced N _b : the strength of the effect was weak for N _b(LD) and much stronger for N _b(Sib). Our results highlight the importance of subtle ecological differences and gene flow in shaping N _b /N. They also provide some evidence that the linkage between demographic and evolutionary processes varies between closely related taxa and suggest that a more complete understanding of the N _b /N range across various species is an important component of conservation genetics and management.     

Construction of flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>-overproducing strains of the thermotolerant methylotrophic yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> (<Emphasis Type="Italic">Pichia angusta</Emphasis>)

L-Lactate cytochrome c oxidoreductase (flavocytochrome b ₂, FC b ₂) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker’s yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b ₂ producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b ₂. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b ₂ activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b ₂ producer characterized by a sixfold increased (to 3 μmol min^?1 mg^?1 protein in cell-free extract) activity of the enzyme.     

Rapid antimicrobial susceptibility testing and β-lactam-induced cell morphology changes of Gram-negative biological threat pathogens by optical screening

Background

For Yersinia pestis, Burkholderia pseudomallei, and Burkholderia mallei, conventional broth microdilution (BMD) is considered the gold standard for antimicrobial susceptibility testing (AST) and, depending on the species, requires an incubation period of 16–20?h, or 24–48?h according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. After a diagnosis of plague, melioidosis or glanders during an outbreak or after an exposure event, the timely distribution of appropriate antibiotics for treatment or post-exposure prophylaxis of affected populations could reduce mortality rates.

Results

Herein, we developed and evaluated a rapid, automated susceptibility test for these Gram-negative bacterial pathogens based on time-lapse imaging of cells incubating in BMD microtitre drug panels using an optical screening instrument (oCelloScope). In real-time, the instrument screened each inoculated well containing broth with various concentrations of antibiotics published by CLSI for primary testing: ciprofloxacin (CIP), doxycycline (DOX) and gentamicin (GEN) for Y. pestis; imipenem (IPM), ceftazidime (CAZ) and DOX for B. mallei; and IPM, DOX, CAZ, amoxicillin-clavulanic acid (AMC) and trimethoprim-sulfamethoxazole (SXT) for B. pseudomallei. Based on automated growth kinetic data, the time required to accurately determine susceptibility decreased by ≥70% for Y. pestis and?≥?50% for B. mallei and B. pseudomallei compared to the times required for conventional BMD testing. Susceptibility to GEN, IPM and DOX could be determined in as early as three to six hours. In the presence of CAZ, susceptibility based on instrument-derived growth values could not be determined for the majority of B. pseudomallei and B. mallei strains tested. Time-lapse video imaging of these cultures revealed that the formation of filaments in the presence of this cephalosporin at inhibitory concentrations was detected as growth. Other β-lactam-induced cell morphology changes, such as the formation of spheroplasts and rapid cell lysis, were also observed and appear to be strain- and antibiotic concentration-dependent.

Conclusions

A rapid, functional AST was developed and real-time video footage captured β-lactam-induced morphologies of wild-type B. mallei and B. pseudomallei strains in broth. Optical screening reduced the time to results required for AST of three Gram-negative biothreat pathogens using clinically relevant, first-line antibiotics compared to conventional BMD.

     

Antifungal Activity of Chitosan-Coated Poly(lactic-co-glycolic) Acid Nanoparticles Containing Amphotericin B

Amphotericin B (AmB) is one of the most used drugs for the treatment of systemic fungal infections; however, the treatment causes several toxic manifestations, including nephrotoxicity and hemolytic anemia. Chitosan-coated poly(lactide-co-glycolide) (PLGA) nanoparticles containing AmB were developed with the aim to decrease AmB toxicity and propose the oral route for AmB delivery. In this work, the antifungal efficacy of chitosan-coated PLGA nanoparticles containing AmB was evaluated in 20 strains of fungus isolates from patients with vulvovaginal candidiasis (01 Candida glabrata and 03 Candida albicans), bloodstream infections (04 C. albicans and 01 C. tropicalis) and patients with urinary tract infection (04 Candida albicans, 02 Trichosporon asahii, 01 C. guilhermondii, 03 C. glabrata) and 01 Candida albicans ATCC 90028. Moreover, the cytotoxicity over erythrocytes was evaluated. The single-emulsion solvent evaporation method was suitable for obtaining chitosan-coated PGLA nanoparticles containing AmB. Nanoparticles were spherical in shape, presented mean particle size about 460 nm, positive zeta potential and encapsulation efficiency of 42%. Moreover, nanoparticles prolonged the AmB release. All the strains were susceptible to plain AmB and nanostructured AmB, according to EUCAST breakpoint version 8.1 (resistant > 1 μg/mL), using broth microdilution method. In C. albicans (urine, blood, and vulvovaginal secretion isolates, and 1 ATCC), the MIC value of AmB-loaded nanoparticles varied from 0.25 to 0.5 μg/mL and EUCAST varied from 0.03 to 0.5 μg/mL. In urine and vulvovaginal secretion isolates of C. glabrata, the MIC value of AmB-loaded nanoparticles varied from 0.25 to 0.5 μg/mL and EUCAST varied from 0.03 to 0.015 μg/mL. In urine isolates of C. guilhermondii, the MIC value of AmB-loaded nanoparticles was 0.12 μg/mL and EUCAST was 0.06 μg/mL. In blood isolates of C. tropicalis, the MIC value of AmB-loaded nanoparticles was 0.5 μg/mL and EUCAST was 0.25 μg/mL. Finally, in urine isolates of T asahii, the MIC value of AmB-loaded nanoparticles was 1 μg/mL and EUCAST varied from 0.5 to 1 μg/mL. In the cytotoxicity assay, plain AmB was highly hemolytic (100% in 24 h) while AmB-loaded chitosan/PLGA nanoparticles presented negligible hemolysis.     

Evaluation of cytotoxic activities of snake venoms toward breast (MCF-7) and skin cancer (A-375) cell lines

Snake venoms are mixtures of bioactive proteins and peptides that exhibit diverse biochemical activities. This wide array of pharmacologies associated with snake venoms has made them attractive sources for research into potentially novel therapeutics, and several venom-derived drugs are now in use. In the current study we performed a broad screen of a variety of venoms (61 taxa) from the major venomous snake families (Viperidae, Elapidae and “Colubridae”) in order to examine cytotoxic effects toward MCF-7 breast cancer cells and A-375 melanoma cells. MTT cell viability assays of cancer cells incubated with crude venoms revealed that most venoms showed significant cytotoxicity. We further investigated venom from the Red-bellied Blacksnake (Pseudechis porphyriacus); venom was fractionated by ion exchange fast protein liquid chromatography and several cytotoxic components were isolated. SDS-PAGE and MALDI-TOF mass spectrometry were used to identify the compounds in this venom responsible for the cytotoxic effects. In general, viper venoms were potently cytotoxic, with MCF-7 cells showing greater sensitivity, while elapid and colubrid venoms were much less toxic; notable exceptions included the elapid genera Micrurus, Naja and Pseudechis, which were quite cytotoxic to both cell lines. However, venoms with the most potent cytotoxicity were often not those with low mouse LD₅₀s, including some dangerously venomous viperids and Australian elapids. This study confirmed that many venoms contain cytotoxic compounds, including catalytic PLA₂s, and several venoms also showed significant differential toxicity toward the two cancer cell lines. Our results indicate that several previously uncharacterized venoms could contain promising lead compounds for drug development.     

Characteristics and expression of genes encoding two small heat shock protein genes lacking introns from <Emphasis Type="Italic">Chilo suppressalis</Emphasis>

Small heat shock proteins (sHSPs) constitute a large, diverse, and functionally uncharacterized family of heat shock proteins. To gain insight regarding the function of sHSPs in insects, we identified genes encoding two sHSPs, Cshsp22.9b and Cshsp24.3, from the rice pest Chilo suppressalis. The cDNAs of Cshsp22.9b and Cshsp24.3 encoded proteins of 206 and 216 amino acids with isoelectric points of 5.79 and 9.28, respectively. Further characterization indicated that both Cshsp22.9b and Cshsp24.3 lacked introns. Real-time quantitative PCR indicated that Cshsp22.9b and Cshsp24.3 were expressed at higher levels within the fat body as compared to other tissues (head, epidermis, foregut, midgut, hindgut, Malpighian tubules, and hemocytes). Expression of Cshsp22.9b and Cshsp24.3 was lowest in the hindgut and Malpighian tubules, respectively. Cshsp22.9b and Cshsp24.3 showed identical patterns in response to thermal stress from ?11 to 43 °C, and both genes were up-regulated by hot and cold temperatures. The mRNA (messenger ribonucleic acid) expression levels of Cshsp22.9b (KY701308) and Cshsp24.3 (KY701309) were highest after a 2-h exposure at 39 °C and started to decline at 42 °C. In response to cold temperatures, both Cshsp22.9b and Cshsp24.3 showed maximal expression after a 2-h exposure to ?3 °C. The two Cshsps were more responsive to hot than cold temperature stress and were not induced by mildly cold or warm temperatures. In conclusion, Cshsp22.9b and Cshsp24.3 could play a very important role in the regulation of physiological activities in C. suppressalis that are impacted by environmental stimuli.