Impact of the Hydrocarbon Chain Length of Biodegradable Ester-Bonded Cationic Gemini Surfactants on Self-Assembly, In Vitro Gene Transfection, Cytotoxicity, and Antimicrobial Activity.

Gemini surfactants, due to their unique structural features and enhanced properties compared to conventional surfactants, are becoming more popular in the domain of colloid and interface science, drug delivery, and gene delivery science. This distinct class of surfactants forms a wide range of self-assembled aggregates depending on their chemical structure and environmental conditions. The present work aims to develop Gemini with three distinct chain lengths linked through the ester group and quaternary nitrogen head groups that can bind DNA molecules and ultimately serve as vectors for DNA transfection. Thus, we synthesized three distinct cationic Gemini with 12, 14, and 16 carbons in their tails and studied the effect of the hydrocarbon chain length on their physicochemical properties and biological applications. The self-assembly of these Geminis in aqueous solution was investigated by a number of techniques, including surface tension, electrical conductivity, fluorescence probe, calorimetry, dynamic light scattering, and atomic force microscopy. All three Gemini were extremely surface active and self-assembled above a very low critical micelle concentration. Calorimetric studies suggested the formation of thermodynamically favorable aggregates in an aqueous medium. Chain length dependence was observed in the size as well as the morphology of the aggregates. These Gemini ions were found to bind DNA strongly, as indicated by the high binding constant values. In vitro gene transfection studies using the RAW 264.7 cell line suggested that all three cationic Gemini had transfection efficiencies comparable to that of commercial standard turbofectamine. MTT assay was also performed for concentration selection while using these Gemini as transfection vectors. Overall, it was observed that Gemini had very little cytotoxicity within the investigated concentration range, highlighting the significance of the ester link within the structure. When compared with known antimicrobials such as kanamycin and ampicillin, all three Gemini furnished excellent antimicrobial activity in both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) microorganisms.

Self-Assembly, In Vitro Gene Transfection, and Antimicrobial Activity of Biodegradable Cationic Bolaamphiphiles.

Bolaamphiphiles or bolaforms have drawn particular interest in drug and gene delivery, and studies of bolaforms have been growing continuously. Bolaforms, due to their unique structure, exhibit specific self-assembly behavior in water. The present work aims to develop biodegradable cationic bolaforms with a better gene transfection ability. In this work, a novel cationic bolaform (Bola-1) with head groups bearing hydroxyl (OH) functionality was designed and synthesized to investigate self-assembly and gene transfection efficiency. The self-assembly behavior of Bola-1 in water was compared with that of the hydrochloride salt (Bola-2) of its precursor molecule to investigate the effect of the -OH functionality on their solution properties. Several techniques, including surface tension, electrical conductivity, fluorescence probe, calorimetry, dynamic light scattering, and atomic force microscopy, were employed for the physicochemical characterization of Bola-1 and Bola-2. Despite the presence of polar urea groups in the spacer chain, both bolaforms were found to form spherical or elongated micelles above a relatively low critical aggregation concentration (CAC). The presence of the OH group was found to significantly affect the CAC value. The results of calorimetric measurements suggested a thermodynamically favorable aggregate formation in salt-free water. Despite stronger binding efficiency with calf thymus DNA, in vitro gene transfection studies performed using adherent cell Hek 293 suggested that both Bola-1 and Bola-2 have gene transfection efficiency comparable to that of turbofectamine standard. Both bolaforms were found to exhibit significant in vitro cytotoxicity at higher concentrations. Also, the bolaforms showed beneficial antibacterial activity at higher concentrations.

Activated group 3 innate lymphoid cells promote T-cell-mediated immune responses.

Group 3 innate lymphoid cells (ILC3s) have emerged as important cellular players in tissue repair and innate immunity. Whether these cells meaningfully regulate adaptive immune responses upon activation has yet to be explored. Here we show that upon IL-1ß stimulation, peripheral ILC3s become activated, secrete cytokines, up-regulate surface MHC class II molecules, and express costimulatory molecules. ILC3s can take up latex beads, process protein antigen, and consequently prime CD4(+) T-cell responses in vitro. The cognate interaction of ILC3s and CD4(+) T cells leads to T-cell proliferation both in vitro and in vivo, whereas its disruption impairs specific T-cell and T-dependent B-cell responses in vivo. In addition, the ILC3-CD4(+) T-cell interaction is bidirectional and leads to the activation of ILC3s. Taken together, our data reveal a novel activation-dependent function of peripheral ILC3s in eliciting cognate CD4(+) T-cell immune responses.

An insight into the mechanism of inhibition of unusual bi-subunit topoisomerase I from Leishmania donovani by 3,3'-di-indolylmethane, a novel DNA topoisomerase I poison with a strong binding affinity to the enzyme.

DIM (3,3'-di-indolylmethane), an abundant dietary component of cruciferous vegetables, exhibits a wide spectrum of pharmacological properties. In the present study, we show that DIM is a potent inhibitor of Leishmania donovani topoisomerase I with an IC50 of 1.2 microM. Equilibrium dialysis shows that DIM binds strongly to the free enzyme with a binding constant of 9.73x10(-9) M. The binding affinity of DIM to the small subunit is 8.6-fold more than that of the large subunit of unusual LdTOP1LS (bi-subunit L. donovani topoisomerase I). DIM stabilizes topoisomerase I-DNA cleavage complexes in vitro and also in vivo. Like CPT (camptothecin), DIM inhibits the religation step when the drug was added to preformed topoisomerase I-DNA binary complex. Hence, DIM is similar to CPT with respect to its ability to form the topoisomerase I-mediated 'cleavable complexes' in vitro and in vivo. But unlike CPT, DIM interacts with both free enzyme and substrate DNA. Therefore DIM is a non-competitive class I inhibitor of topoisomerase I. DIM also inhibits the relaxation activity of the CPT-resistant mutant enzyme LdTOP1Delta39LS (N-terminal deletion of amino acids 1-39 of LdTOP1LS). The IC50 values of DIM in simultaneous and enzyme pre-incubation relaxation assays were 3.6 and 2.9 muM respectively, which are higher than that of wild-type topoisomerase I (LdTOP1LS), indicating that the affinity of DIM to LdTOP1Delta39LS is less than that for LdTOP1LS. This is the first report on DIM as an L. donovani topoisomerase I poison. Our study illuminates a new mode of action of enzyme inhibition by DIM that might be exploited for rational drug design in human leishmaniasis.

Leishmania donovani bisubunit topoisomerase I gene fusion leads to an active enzyme with conserved type IB enzyme function.

All eukaryotic topoisomerase I enzymes are monomeric enzymes, whereas the kinetoplastid family (Trypanosoma and Leishmania) possess an unusual bisubunit topoisomerase I. To determine what happens to the enzyme architecture and catalytic property if the two subunits are fused, and to explore the functional relationship between the two subunits, we describe here in vitro gene fusion of Leishmania bisubunit topoisomerase I into a single ORF encoding a new monomeric topoisomerase I (LdTOPIL-fus-S). It was found that LdTOPIL-fus-S is active. Gene fusion leads to a significant modulation of in vitro topoisomerase I activity compared to the wild-type heterodimeric enzyme (LdTOPILS). Interestingly, an N-terminal truncation mutant (1-210 amino acids) of the small subunit, when fused to the intact large subunit [LdTOPIL-fus-Delta(1-210)S], showed reduced topoisomerase I activity and camptothecin sensitivity in comparison to LdTOPIL-fus-S. Investigation of the reduction in enzyme activity indicated that the nonconserved 1-210 residues of LdTOPIS probably act as a 'pseudolinker' domain between the core and catalytic domain of the fused Leishmania enzyme, whereas mutational analysis of conserved His453 in the core DNA-binding domain (LdTOPIL) strongly suggested that its role is to stabilize the enzyme-DNA transition state through hydrogen bonding to one of the nonbridging oxygens. Taken together, our findings provide an insight into the details of the unusual structure of bisubunit topoisomerase I of Leishmania donovani.