Cholesterol Sequestration from Caveolae/Lipid Rafts Enhances Cationic Liposome-Mediated Nucleic Acid Delivery into Endothelial Cells.

Delivering nucleic acids into the endothelium has great potential in treating vascular diseases. However, endothelial cells, which line the vasculature, are considered as sensitive in nature and hard to transfect. Low transfection efficacies in endothelial cells limit their potential therapeutic applications. Towards improving the transfection efficiency, we made an effort to understand the internalization of lipoplexes into the cells, which is the first and most critical step in nucleic acid transfections. In this study, we demonstrated that the transient modulation of caveolae/lipid rafts mediated endocytosis with the cholesterol-sequestrating agents, nystatin, filipin III, and siRNA against Cav-1, which significantly increased the transfection properties of cationic lipid-(2-hydroxy-N-methyl-N,N-bis(2-tetradecanamidoethyl)ethanaminium chloride), namely, amide liposomes in combination with 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (AD Liposomes) in liver sinusoidal endothelial cells (SK-Hep1). In particular, nystatin was found to be highly effective with 2-3-fold enhanced transfection efficacy when compared with amide liposomes in combination with Cholesterol (AC), by switching lipoplex internalization predominantly through clathrin-mediated endocytosis and macropinocytosis.

Evaluation of the in vivo genotoxicity of liposomal formulation for delivering anticancer estrogenic derivative (ESC8) in a mouse model.

The genotoxic potential of glucocorticoid receptor (GR)-targeted liposomal formulations of the anticancer drug molecule ESC8 was studied in vivo. A methodical literature review discovered no previous studies on the genotoxicity of ESC8. Genotoxicity was assessed in both male and female mice by various assay systems, such as comet assay, chromosomal aberrations and micronuclei assay, which detect different abnormalities. Eleven groups of male mice and eleven groups of female mice, containing six animals per group, were used in the present study: group I served as vehicle control; group II received the positive control (cyclophosphamide 40 mg/kg; CYP); and animals in group III to XI received free drug (ESC8), DX liposome and drug-associated DX liposomal formulation (DXE), respectively, dissolved in 5% solution of glucose at a drug-dose of 1.83, 3.67 and 7.34 mg/kg, respectively. Same drug treatments were followed for the female mice groups. The obtained data revealed the safety of DXE, which did not show substantial genotoxic effects at different dose levels. In contrast, the positive control, CYP, exhibited highly substantial irregular cytogenetic variations in comparison with the control group in different assays.

Identification and characterization of forced degradation products of vortioxetine by LC/MS/MS and NMR.

Vortioxetine (VTX) is a novel multimodal antidepressant drug that affects the serotoninergic and noradrenergic systems. In this work, the forced degradation of VTX was studied according to (ICH) Q1A (R2) guidelines. The study revealed that VTX was stable under thermal stress conditions and hydrolytic stress conditions i.e., acidic, basic and neutral conditions. In contrast, six degradation products (DPs) were formed under photolytic and oxidative stress conditions. The DPs were identified and characterized by high-resolution LC/MS and LC/MS/MS. The structures of major DPs were further confirmed by the synthesis and characterization by 1H and 13C NMR data. A possible mechanism for the formation of the VTX DPs via photolytic/oxidative stress degradation pathway was proposed.

Chumacin-1 and Chumacin-2 from Pseudomonas aeruginosa strain CGK-KS-1 as novel quorum sensing signaling inhibitors for biocontrol of bacterial blight of rice.

The in vitro inhibition of quorum sensing signal, xanthan gum secretion, biofilm formation in different Xanthomonas pathovars and biological control of bacterial blight of rice by the two bioactive extrolites produced by Pseudomonas aeruginosa strain CGK-KS-1 were explored. These extrolites were extracted from Diaion HP-20 resin with methanol and purified by preparative-thin layer chromatography. Further, spectroscopic structural elucidation revealed the tentative identity of these extrolites to be (R,3E,5E,9Z,11E)-13-((3S,5R)-5-acetyl-2,6-dimethylheptan-3-yl)-10-hydroxy-4-methyl-1,8-diazabicyclo[9.3.1]pentadeca-3,5,9,11(15),13-pentaen-2-one and (R,3E,5E,8E,11E)-13-((3S,5R)-5-acetyl-2,6-dimethylheptan-3-yl)-4-methyl-1,8-diazabicyclo[9.3.1]pentadeca-3,5,8,11(15),13-pentaene-2,10-dione, named as Chumacin-1 and Chumacin-2, respectively. Antimicrobial assay showed Chumacin-1 and Chumacin-2 exhibited a strong in vitro growth inhibition against various Xanthomonas pathovars. Quorum sensing overlay assay using a reporter strain Chromobacterium violaceum strain CV026 showed that Chumacin-1 and Chumacin-2 inhibited quorum sensing signaling. The mechanistic studies revealed that these extrolites inhibited the production of quorum sensing signaling factor, cis-11-methyl-2-dodecenoic acid; suppressed the xanthan gum secretion and also inhibited the biofilms formed by various Xanthomonas pathovars. Both Chumacin-1 and Chumacin-2 showed ROS generation in the test Xanthomonas strains, resulting in in vitro cell membrane damage was revealed through CSLM and FE-SEM micrographs. Further, greenhouse experiments using Samba Mashuri (BPT-5204) revealed that seed treatment with Chumacin-1 and Chumacin-2 along with foliar spray groups showed up to ˜80% reduction in bacterial blight disease in rice. To the best of our knowledge, this is the first report on new quorum sensing inhibitors, Chumacin-1 and Chumacin-2 produced by Pseudomonas aeruginosa strain CGK-KS-1 exhibiting DSF inhibition activity in Xanthomonas oryzae pv. oryzae.

Oestrogen receptor-mediated liposomal drug delivery for treating melanoma.

Function of steroid hormone oestrogen that transactivates oestrogen receptor (ER) is expressed in multiple organs. Except for malignancies of gynaecological organs, ER remains largely unutilised as a target to treat cancers of ER-expressing brain, prostate, skin etc. We have previously developed oestrogen targeting cationic lipid molecule (ES-C10), which showed targeted killing of ER + breast and skin cancer cells. In this study, we explored the targeting ability of ES-C10 as a ligand as well as its additive killing effect (if any), when incorporated in two different liposomes (DCME and DCDE), carrying two anticancer molecules MCIS3 and Docetaxel™, respectively. DCME and DCDE exhibited higher cytotoxicity in ER + cancer cells than in ER - cancer or in non-cancer cells. Both liposomes induced ER-mediated cytotoxicity and caspase 3-induced apoptosis in ER + melanoma cells. Further, decreased levels of pAkt, and increased levels of PTEN and p53 were also observed. Both the targeted liposomes were least haemolytic. These selectively delivered drug-cargoes to tumour mass over other vital organs and induced better anti-tumour effect, which led to increased survivability than their respective controls. In conclusion, we demonstrated the development of two independent liposomal drug-delivery systems associated with an anticancer, oestrogen-structure based ligand for efficient, ER-mediated anti-melanoma effect.