K M Muraleedharan

Innovative developments in drug delivery technologies rely on our ability to tune the properties of supramolecular and macromolecular carriers through the chemical characteristics of individual components or building-blocks. In this regard, oxanorbornane-based synthetic lipids offer great promise as novel drug delivery systems (NDDS). As part of our efforts to develop them as vehicles for anticancer drugs, we have designed and synthesized a new derivative with a fluorescent tag (NBD) on the head group, and investigated its uptake and distribution in A549 cells. Addition of its DMSO solution to aqueous phase followed by extrusion generated solid lipid particles (SLPs), which were characterized by DLS, AFM and TEM techniques. Vesicles of this lipid in a co-assembled state with phosphatidylcholine (PC) and cholesterol were also prepared by thin-film hydration method. DLS data obtained from samples suspended in PBS showed that average size of SLPs is relatively smaller (∼56 nm) than that of vesicles (∼262 nm). The zeta potential of these particles was between −45 and −51 mV, which favor stable formulations. Confocal microscopic analysis of these aggregates after incubation with A549 cells showed that they get distributed predominantly in the cytosolic side. Concentration- and time-dependent flow cytometry analysis revealed that the uptake commences in the initial 5 min itself, and almost 90% of cells become NBD-positive in 2 h. There was an increase in uptake at higher concentration, indicative of passive diffusion. At the same time, a reduction in uptake at lower temperature (4 °C) compared to that at 37 °C pointed towards some contribution from active transport as well. Variation in uptake after pre-treatment with endocytosis inhibitors such as chlorpromazine and methyl-β-cyclodextrin suggested involvement of clathrin- and caveolae-mediated endocytic pathways. Cell viability and hemolytic assays further indicated that these nanocarriers have good safety profile.

Background: The authors investigated the NF-ҡB inhibitory role of three Benzisothiazolone (BIT) derivatives (1, 2 and 3) in Hodgkin’s Lymphoma cells (L428) which constitutively express activated NF-κB. All three compounds showed dose-dependent NF-κB inhibition (78.3, 70.7 and 34.6%) in the luciferase reporter gene assay and were found cytotoxic at IC50 values of 3.3µg/ml, 4.35µg/ml and 13.8µg/ml, respectively by the XTT assay. BIT 1and BIT 2 (but not BIT 3) suppressed both NF-κB subunits p50 and p65 in cytoplasmic and nuclear extracts in a concentration-dependent manner. Furthermore, BIT 1 showed a moderate synergistic effect with the standard chemotherapy drugs etoposide and doxorubicin, whereas BIT 2 and 3 showed a moderate additive effect to antagonistic effect. Cisplatin exhibited an antagonist effect on all the compounds tested under various concentrations, except in the case of 1.56µg/ml of BIT 3 with 0.156µg/ml of cisplatin. The compounds also inhibited the migration of adherent human lung adenocarcinoma cells (A549) in vitro. We conclude that especially BIT 1 and BIT 2 have in vitro anti-inflammatory and anti-cancer activities, which can be further investigated for future potential therapeutic use. Methods: Inspired by the electrophilic sulfur in Nuphar alkaloids, monomeric and dimeric benzisothiazolones were synthesized from dithiodibenzoic acid and their NF-ҡB inhibitory role was explored. NF-κB inhibition and cytotoxicity of the synthesized derivatives were studied using luciferase reporter gene assay and XTTassay. Immunocytochemistry studies were performed using L428 cells. Cell migration assay was conducted using the A549 cell line. L428 cells were used to conduct combination studies and the results were plotted using CompuSyn software. Results: Benzisothiazolone derivatives exhibited cytotoxicity in Hodgkin’s Lymphoma cells through NF-ҡB inhibition. Potent compounds showed suppression of both NF-κB subunits p50 and p65 in a concentration-dependent manner, both in cytoplasmic and nuclear extracts. Combination studies suggest that benzisothia-zolone derivatives possess a synergistic effect with etoposide and doxorubicin. Furthermore, the compounds also inhibited the migration of A549 cells. Conclusion: Benzisothiazolones bearing one or two electrophilic sulfur atoms as part of the heterocyclic framework exhibited cytotoxicity in Hodgkin’s Lymphoma cells through NF-κB inhibition. In addition, these derivatives also exhibited a synergistic effect with etoposide and doxorubicin along with the ability to inhibit the migration of A549 cells. Our study suggests that BIT-based new chemical entities could lead to potential anti-cancer agents.