PLGA-based microspheres loaded with metformin hydrochloride: Modified double emulsion method preparation, optimization, characterization, and in vitro evaluation.

The modified solvent removal method was used to encapsulate metformin hydrochloride (MH) within poly(lactic-co-glycolic acid) (PLGA) microspheres. The study investigated the effect of varying polymer concentrations on the loading and release of the drug from the microspheres. The encapsulation process involved using a double emulsion method, resulting in microspheres with particle diameters ranging from approximately 4.4µm to 2.7µm. The study achieved high encapsulation efficiencies, ranging from 81% to 90%, with drug loadings ranging from 18% to 11%. The release of the drug from the microspheres followed a biphasic pattern over 24 days, with nearly complete release by the end of the study period. Fourier transform infrared spectroscopy (FTIR) analysis indicated that there were no notable differences between PLGA and MH-loaded microspheres, suggesting minimal interactions between MH and PLGA. Differential scanning calorimetry (DSC) and X-ray diffraction (XRD) techniques were used to investigate the state of the MH within the microspheres. The results suggested that the MH was dispersed at a molecular level within the spheres and existed in an amorphous state. This amorphous state of the drug may explain the slow and prolonged release observed in the study.

(+)-Cyanidan-3-ol inhibits epidermoid squamous cell carcinoma growth via inhibiting AKT/mTOR signaling through modulating CIP2A-PP2A axis.

BACKGROUND: Despite recent advances in the treatment of squamous cell skin cancer (SCSC), the disease persists, and treatment resistance develops. Thus, identifying new targets and developing new therapeutic approaches showing low vulnerability to drug resistance is highly needed. PURPOSE: This study aimed to reveal a novel targeted phytotherapeutic strategy for SCSC treatment alone or in combination with standard targeted anticancer molecules. STUDY DESIGN: A library of natural products was utilized to identify molecules that inhibit the growth of skin cancer cells. The anticancer potential of the selected compound was evaluated in human skin squamous carcinoma models, in vitro and in vivo. A comprehensive ingenuity pathway analysis (IPA) strategy and molecular biology technology was adopted to investigate the therapeutic mechanisms in human SCSC. METHODS: The Matrigel invasion chamber, foci formation and soft agar colony formation assays were employed to study the cells invasion and migration potential in vitro. In vivo antitumor effects were evaluated in DMBA/TPA-induced skin papilloma and A431 human skin squamous carcinoma xenograft tumor models. An integrative IPA was employed to identify mechanisms and protein targets in human SCSC.Compounds synergies were determined by the bliss model and evaluated using human SCSC cell lines and xenograft tumors. Histological staining, immunofluorescence imaging, real-time PCR, Western blots, and flow cytometric analyses were employed to analyze apoptosis and cell signaling mechanisms. RESULTS: We identified (+)-cyanidan-3-ol (CD-3) as a selective compound for inhibiting the growth of SCSC cell lines. CD-3 inhibited tumor growth and burden without apparent toxicity and prolonged the survival of tumor-bearing mice. CD-3 inhibitory effects on SCSC growth are mediated via cell cycle arrest and caspase-dependent apoptosis induction. Mechanistic studies showed that CD-3 activates PP2A via inhibiting CIP2A and produces tumor growth inhibitory effects via promoting dephosphorylation of oncogenic AKT/mTOR signaling proteins in SCSC cells and xenograft tumors in a PP2A dependent manner. Furthermore, the combination of CD-3 and mTOR inhibitors (mTORi) synergistically reduced oncogenic phenotypes. CONCLUSIONS: Our study suggests that PP2A activation is an effective strategy for SCSC treatment and the CD-3 and mTORi combination may serve as a promising treatment for SCSC.

Diversity-oriented sustainable synthesis of antimicrobial spiropyrrolidine/thiapyrrolizidine oxindole derivatives: New ligands for a metallo-ß-lactamase from Klebsiella pneumonia.

A simple, environmentally benign and highly proficient microwave assisted one-pot approach for the synthesis of antimicrobial spiropyrrolidine/thiapyrrolizidine oxindole derivatives is reported assembling two pharmacophoric moieties (1,3-indanedione and pyrrolidine/thiapyrrolizidine) in a single molecular framework via three-component 1,3-dipolar cycloaddition reaction of substituted isatin, sarcosine/1,3-thiazoles-4-carboxylic acid and Knoevenagel adduct (2-Cyano-3-phenyl-acrylic acid ethyl ester or 2-Benzylidene-malononitrile) in 2,2,2-trifluoroethanol as a reusable green solvent. Good functional group tolerance and broad scope of usable substrates are other prominent features of the present methodology with high degree of chemo-, regio- and stereoselectivity. The stereochemistry of synthesized compounds was confirmed by single crystal X-ray analysis. All the synthetic compounds were examined for their antimicrobial potential. The synthesized compounds having pyrrolothiazole moiety showed excellent activity against K. pneumoniae as compared to others and even more inhibitory activity than the mentioned drugs, i.e. compounds 6a (MIC=0.09µg/mL), 6b (MIC=0.045µg/mL), 6c (MIC=0.005µg/mL), 6d (MIC=0.19µg/mL). Additionally, compound 6c has shown better binding affinity against New Delhi Metallo-beta-Lactamase-1 (NDM-1) protein in computational docking studies.

In Vitro and In Vivo Evaluation of Small Cationic Abiotic Lipopeptides as Novel Antifungal Agents.

We investigated the antifungal potential of short lipopeptides against clinical fungal isolates with an objective to evaluate their clinical feasibility. All tested lipopeptides exhibit good antifungal activity with negligible difference between the MICs against susceptible and drug-resistant clinical fungal isolates. The MTT assay results revealed the lower cytotoxicity of lipopeptides toward mammalian cells (NRK-52E). In particular, LP24 displayed highest potency against most of the tested fungal isolates with MICs in the range of 1.5-4.5 µg/mL. Calcein dye leakage experiments with model membrane suggested the membrane-active mode of action for LP24. Extending our work from model membranes to intact Aspergillus fumigatus in scanning electron micrographs, we could visualize surface perturbation caused by LP24. LP24 (5 mg/kg) significantly reduces the A. fumigatus burden among the various organs of infected animals, and 70% of the infected mice survived when observed for 28 days. This study underscores the potential of small cationic abiotic lipopeptides to develop into the next-generation antimicrobial therapy.

Growth inhibition and apoptosis induction by (+)-Cyanidan-3-ol in hepatocellular carcinoma.

The objective of this study was to evaluate the cytotoxicity of (+)-cyanidan-3-ol (CD-3) in human hepatocellular carcinoma cell line (HepG2) and chemopreventive potential against hepatocellular carcinoma (HCC) in Balb/c mice. The HepG2 cell line was treated with CD-3 at various concentrations and the proliferation of the HepG2 cells was measure by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), sulforhodamine B (SRB) and lactate dehydrogenase (LDH) assays. Cell apoptosis was detected by Hoechst 33258 (HO), Acridine orange/ethylene dibromide (AO/EB) staining, DNA fragmentation analysis and the apoptosis rate was detected by flow cytometry. The HCC tumor model was established in mice by injecting N-nitrosodiethylamine/carbon tetrachloride (NDEA/CCl4) and the effect of CD-3 on tumor growth in-vivo was studied. The levels of liver injury markers, tumor markers, and oxidative stress were measured. The expression levels of apoptosis-related genes in in-vitro and in vivo models were determined by RT-PCR and ELISA. The CD-3 induced cell death was considered to be apoptotic by observing the typical apoptotic morphological changes under fluorescent microscopy and DNA fragmentation analysis. Annexin V/PI assay demonstrated that apoptosis increased with increase in the concentration of CD-3. The expression levels of apoptosis-related genes that belong to bcl-2 and caspase family were increased and AP-1 and NF-κB activities were significantly suppressed by CD-3. Immunohistochemistry data revealed less localization of p53, p65 and c-jun in CD-3 treated tumors as compared to localization in NDEA/CCl4 treated tumors. Taken together, our data demonstrated that CD-3 could significantly inhibit the proliferation of HepG2 cells in-vitro and suppress HCC tumor growth in-vivo by apoptosis induction.

Cytotoxicity and apoptosis induction in human breast adenocarcinoma MCF-7 cells by (+)-cyanidan-3-ol.

The objective of this study was to evaluate the cytotoxicity and possible signalling pathway implicated in (+)-cyanidan-3-ol (CD-3) induced apoptosis in the human breast adenocarcinoma cell line (MCF-7). The effects of CD-3 on cell proliferation of MCF-7 cells were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), sulforhodamine B (SRB) and lactate dehydrogenase (LDH) assays. Cell apoptosis was detected by Hoechst 33258 (HO) and acridine orange/ethylene dibromide (AO/EB) staining and DNA fragmentation analysis. The expressions of apoptosis-related genes were assessed by RT-PCR and ELISA. Our data revealed that CD-3 induced MCF-7 cell death in a dose-dependent manner. Marked changes in apoptotic morphology was clearly observed after CD-3 treatment. CD-3 induced cell death was considered to be apoptotic by observing the typical apoptotic morphological change under fluorescent microscopy and DNA fragmentation assays. The induction of apoptosis is correlated with the increased mRNA expressions of p53, Bax, and caspase-3, -7, -8 and -9 and decreased mRNA expressions of bcl-2. Subsequently, CD-3 decreased the mRNA expressions of mdm2, p65, c-jun, c-fos in MCF-7 cells. The protein levels of p53, Bax, and caspase-3 were increased, whereas, that of p65, c-jun and Bcl-2 were decreased in MCF-7 cells on CD-3 treatment. These results clearly demonstrated that CD-3 effectively induced growth inhibition and apoptosis in MCF-7 cells.

Human breast adenocarcinoma cytotoxicity and modulation of 7,12-dimethylbenz[a]anthracene-induced mammary carcinoma in Balb/c mice by Acacia catechu (L.f.) Wild heartwood.

OBJECTIVE: The chemopreventive potential of (+)-catechin-rich extract of Acacia catechu (L.f.) Willd. heartwood (AQCE) was evaluated against human breast adenocarcinoma cell line (MCF-7) and 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinoma in Balb/c mice. METHODS: Cell cytotoxicity was investigated using different colorimetric assays. Apoptosis was observed using diphenylamine assay and fluorescent microscopy. AQCE was further evaluated for antitumor activity against DMBA-induced mammary carcinoma. The levels of tumor markers and oxidative stress were measured. Furthermore, level of transcription factors was measured by enzyme-linked immunosorbent assay. RESULTS: The results showed that administration of AQCE showed a dose-dependent growth inhibition response and DNA fragmentation in MCF-7 cells. Tumor multiplicity was significantly decreased to 42.91% with AQCE when compared with DMBA-treated animals. The levels of tumor markers such as total sialic acid and lipid-associated sialic acid were substantially increased after DMBA treatment. However, AQCE treatment restored tumor markers level. AQCE also significantly reduced elevated levels of nitrite and malondialdehyde in DMBA-treated animals. Additionally, AQCE also increased the activities of antioxidant enzymes, viz., catalase, superoxide dismutase, total thiol, reduced glutathione, protein thiol, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase in the mammary tissue and liver mitochondria of DMBA-administered animals. Significant increase in the protein levels of p53, c-jun, and p65 were observed in DMBA-treated mice, whereas less expression was observed in AQCE-treated animals. Eventually, AQCE also significantly improved body weight and maintained the mammary tissue architecture in normal range. CONCLUSIONS: The present data strongly suggest that anticancer potentiality of (+)-catechin-rich AQCE may be attributable to its ability to positively modulate tumor markers as well as the antioxidant system that could decompose the peroxides and, thereby, offer a protection against lipid peroxidation and linked to the expression of transcription factors during DMBA-induced mammary carcinoma.

Chemopreventive efficacy of (+)-catechin-rich aqueous extract of Acacia catechu Willd. Heartwood against 7,12-dimethylbenz[a]anthracene-induced hepatocarcinoma in Balb/c mice.

The objective of this study was to investigate the chemopreventive potential of (+)-catechin-rich extract of Acacia catechu heartwood (AQCE) against 7,12-dimethylbenz[a]anthracene (DMBA)-induced hepatocellular carcinoma in Balb/c mice. The levels of liver injury markers, tumor markers, and oxidative stress were measured in serum and liver tissues. Furthermore, the levels of transcription factors were measured by ELISA. Tumor incidence was found to be 100% in DMBA-treated animals (group 2), whereas, in AQCE-treated animals (group 3), it was 37.5%. AQCE treatment reduced liver injury and restored tumor-marker levels. AQCE also significantly reduced elevated levels of nitrite and hepatic malondialdehyde (MDA) in DMBA-treated animals. Additionally, AQCE modulated the activity of different antioxidant enzymes in liver tissues. Eventually, AQCE also significantly improved body weight, prevented the increase of relative liver weight, and maintained the liver cellular architecture within the normal range of the control. A significant increase in the protein levels of p53, c-jun, and NF-κB (p65) were observed in DMBA-treated mice, whereas low levels of these markers were observed in DMBA+AQCE-treated animals. These findings strongly suggest (1) that (+)-catechin-rich AQCE exerts a chemopreventive effect by modulating the levels of lipid peroxidation and by promoting the enzymatic and non-enzymatic antioxidant defense system and (2) that this effect is linked to the expression of transcription factors during hepatocarcinogenesis.

Human epithelial carcinoma cytotoxicity and inhibition of DMBA/TPA induced squamous cell carcinoma in Balb/c mice by Acacia catechu heartwood.

OBJECTIVES: Acacia catechu heartwood contains significant amounts of polyphenolic compounds that exhibit powerful antioxidant activity. The purpose of this study was to evaluate the cytotoxicity of A. catechu heartwood extracts in a human epithelial carcinoma cell line (A431) and antitumour activity against DMBA/TPA induced squamous cell carcinoma in Balb/c mice. METHODS: Various extracts, including aqueous, ethyl acetate, chloroform and n-hexane, were tested for cytotoxic properties on a human epithelial carcinoma cell line (A431) by using MTT, sulforhodamine B and lactate dehydrogenase leakage assays. The standardized A. catechu heartwood aqueous extract (AQCE) was further evaluated for antitumour activity against 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) induced skin carcinoma in Balb/c mice. KEY FINDINGS: The results showed that administration of AQCE showed a dose-dependent growth inhibition response, with an IC50 value of 78.56 µg/ml. Tumour incidence was significantly decreased (P < 0.001) to 30% with AQCE compared with 100% in the DMBA/TPA group. The AQCE was also found to significantly upregulate different antioxidant enzymes in skin and liver tissue. CONCLUSIONS: The results suggest that AQCE may exert its chemopreventive activity by acting as an antioxidant.

Development and evaluation of carvedilol transdermal patches.

Transdermal patches of carvedilol with a HPMC-drug reservoir were prepared by the solvent evaporation technique. In this investigation, the membranes of Eudragit RL100 and Eudragit RS100 were cast to achieve controlled release of the drug. The prepared patches possessed satisfactory physicochemical characteristics. Thickness, mass and drug content were uniform in prepared batches. Moisture vapour transmission through the patches followed zero-order kinetics. In vitro permeation studies were performed using a K-C diffusion cell across hairless guinea pig skin and followed the super case II transport mechanism. The effects of non-ionic surfactants Tween 80 and Span 80 on drug permeation were studied. The nonionic surfactants in the patches increased the permeation rate, Span 80 exhibiting better enhancement relative to Tween 80. The patches were seemingly free of potentially hazardous skin irritation.