Design and Synthesis of Xanthone Analogues Conjugated with Aza-aromatic Substituents as Promising G-Quadruplex Stabilizing Ligands and their Selective Cancer Cell Cytotoxic Action.

We have examined the stabilization of higher-order noncanonical G-quadruplex (G4) DNA structures formed by the G-rich sequences in the promoter region of oncogenes such as c-MYC, c-KIT, VEGF and BCl2 by newly synthesized, novel nitrogen-containing aromatics conjugated to xanthone moiety. Compounds with N-heterocyclic substituents such as pyridine (XNiso), benzimidazole (XBIm), quinoxaline (XQX) and fluorophore dansyl (XDan) showed greater effectiveness in stabilizing the G4 DNA as well as selective cytotoxicity for cancer cells (mainly A549) over normal cells both in terms of UV-Vis spectral titrations and cytotoxicity assay. Both fluorescence spectral titrimetric measurements and circular dichroism (CD) melting experiments further substantiated the G4 stabilization phenomenon by these small-molecular ligands. In addition, these compounds could induce the formation of parallel G4 structures in the absence of any added salt condition in Tris⋅HCl buffer at 25 °C. In a polymerase stop assay, the formation of stable G4 structures in the promoter of oncogenes and halting of DNA synthesis in the presence of the above-mentioned compounds was demonstrated by using oncogene promoter as the DNA synthesis template. Apoptosis-mediated cell death of the cancer cells was proved by Annexin V-PI dual staining assay and cell-cycle arrest occurred in the S phase of the cell cycles. The plausible mode of binding involves the stacking of the xanthone core on the G4 DNA plane with the possibility of interaction with the 5'-overhang as indicated by molecular dynamics simulation studies.

Physical-Chemical Characterization of Bilayer Membranes Derived from (±) α-Tocopherol-Based Gemini Lipids and Their Interaction with Phosphatidylcholine Bilayers and Lipoplex Formation with Plasmid DNA.

Membrane formation and aggregation properties of two series of (±) α-tocopherol-based cationic gemini lipids without and with hydroxyl functionalities at the headgroup region (TnS n = 3, 4, 5, 6, 8, and 12; THnS n = 4, 5, 6, 8, and 12) with varying polymethylene spacer lengths were investigated extensively while comparing with the corresponding properties of the monomeric counterparts (TM and THM). Liposomal suspensions of each cationic lipid were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), zeta potential measurements, and small-angle X-ray diffraction studies. The length of the spacer and the presence of hydroxyl functionalities at the headgroup region strongly contribute to the aggregation behavior of these gemini lipids in water. The interaction of each tocopherol lipid with a model phospholipid, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)-derived vesicles, was thoroughly examined by differential scanning calorimetry (DSC) and 1,6-diphenyl-1,3,5-hexatriene (DPH)-doped fluorescence anisotropy measurements. The binding efficiency of the cationic tocopherol liposomes with plasmid DNA (pDNA) was followed by an ethidium bromide (EB) exclusion assay and zeta potential measurements, whereas negatively charged micellar sodium dodecyl sulfate (SDS)-mediated release of the pDNA from various preformed pDNA-liposomal complexes (lipoplex) was studied by an ethidium bromide (EB) reintercalation assay. The structural transformation of pDNA upon complexation with liposome was characterized using circular dichroism (CD) spectroscopic measurements. Gemini lipid-pDNA interactions depend on both the presence of hydroxyl functionalities at the headgroups and the length of the spacer chain between the headgroups. Succinctly, we performed a detailed physical-chemical characterization of the membranes formed from cationic monomeric and gemini lipids bearing tocopherol as their hydrophobic backbone and describe the role of inserting the -OH group at the headgroup of such lipids.

Tumor Chemosensitization through Oncogene Knockdown Mediated by Unique α-Tocopherylated Cationic Geminis.

Herein, siRNA transfection efficiency of a unique set of α-tocopherylated gemini lipids has been established in vitro and in vivo. High efficacy of oncogene silencing achieved using the biomacromolecular assembly, formed from siRNA complexes of co-liposomes containing an α-tocopherylated gemini lipid, has been utilized for tumor regression via chemosensitization. Delivery studies with the gemini bearing hydroxyethyl headgroup with octamethylene spacer (TH8S) pointed to a higher siRNA transfection efficacy than its analog without hydroxyethyl group (T8S). Owing to p53 upregulation, transfected cells showed enhanced sensitivity to the chemotherapeutic agent, doxorubicin. Studies in murine model revealed significantly low levels of survivin mRNA in xenograft tumors injected with siRNA lipoplexes, leading to effective inhibition of tumor growth and an increase in sensitivity of the tumors toward doxorubicin. These findings enable us to propose the anti-survivin siRNA carrying TH8S co-liposomes as a potent member of cancer management strategies using suicide gene therapy.

Correction: Transfection efficiencies of α-tocopherylated cationic gemini lipids with hydroxyethyl bearing headgroups under high serum conditions.

Correction for 'Transfection efficiencies of α-tocopherylated cationic gemini lipids with hydroxyethyl bearing headgroups under high serum conditions' by Bappa Maiti et al., Org. Biomol. Chem., 2018, 16, 1983-1993.

Reduction Responsive Nanovesicles Derived from Novel α-Tocopheryl-Lipoic Acid Conjugates for Efficacious Drug Delivery to Sensitive and Drug Resistant Cancer Cells.

Two novel α-tocopheryl-lipoic acid conjugates (TL1 and TL2) were synthesized for the anticancer drug, doxorubicin (DOX), delivery. Both conjugates were able to form stable nanovesicles. The critical aggregation concentration (CAC) was determined using 4-(N,N-dimethylamino)cinnamaldehyde (DMACA) as a fluorescence probe. Formation of highly packed nanovesicles was characterized by 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence anisotropy and microviscosity measurements. The morphologies of nanovesicles were visualized by transmission electron microscopy (TEM) and atomic force microscopy (AFM). The response of nanovesicles to reducing environment of cells was probed by the addition of dithiothreitol (DTT), which was followed by the increase in the hydrodynamic diameter under dynamic light scattering (DLS) measurements. The encapsulation efficiency of a commonly used anticancer drug, doxorubicin (DOX), in nanovesicles was found to be ∼60% and ∼55% for TL1 and TL2, respectively (TL1-DOX and TL2-DOX). Also, the cumulative drug (DOX) release from DOX-encapsulated nanovesicles in response to biological reducing agent glutathione (GSH) was ∼50% and ∼40% for TL1-DOX and TL2-DOX, respectively, over a period of 10 h. Both TL1-DOX and TL2-DOX delivered the anticancer drug, doxorubicin (DOX), across the DOX-sensitive and DOX-resistant HeLa (HeLa-DOXR) cells in an efficient manner and significantly more efficaciously than the drug alone treatments, especially in HeLa-DOXR cells. The nanovesicle mediated DOX treatment also showed significantly higher cell death when compared to DOX alone treatment in HeLa-DOXR cells. Blood compatibility of the nanovesicles was supported from clotting time, hemolysis, and red blood cell (RBC) aggregation experiments for their potential in vivo applications. Concisely, we present biocompatible and responsive nanovesicles for efficacious drug delivery to drug-sensitive and drug-resistant cancer cells.

Transfection efficiencies of α-tocopherylated cationic gemini lipids with hydroxyethyl bearing headgroups under high serum conditions.

Herein, five new α-tocopheryl cationic gemini lipids with hydroxyethyl bearing headgroups (THnS, n = 4, 5, 6, 8, 12) have been synthesized for efficient plasmid DNA (pDNA) delivery into cancer cells. Among these gemini lipid formulations, the lipid with an octamethylene [-(CH2)8] spacer (TH8S) showed the highest transfection efficiency (TE) that was comparable to that of the commercial standard lipofectamine 2000 (L2K) in terms of luciferase expression in HepG2 (liver hepatocellular carcinoma) cells. The addition of the helper lipid DOPE (1,2-dioleoyl phosphatidyl ethanolamine) with cationic lipids in mixed liposomes further enhanced the TE and the optimized molar ratio was 2 : 1 (DOPE : cationic lipid). The optimized co-liposomal formulation of TH8S (DOPE : TH8S = 2 : 1) showed a higher TE in HepG2, A549 (human lung carcinoma) and MCF7 (human breast adenocarcinoma) cells than other optimized co-liposomal formulations and was also significantly more potent than L2K. The comparison of the TE of DOPE-TH8S (2 : 1) with the gemini lipid T8T (the headgroup devoid of the hydroxyl group) further demonstrated the importance of the hydroxyethyl functionality at the level of the headgroup. Relatively good binding efficiency and easy release of pDNA (pGL3) were also observed with DOPE-TH8S (2 : 1) in the ethidium bromide (EB)-exclusion and re-intercalation assay, which may be the plausible reason for high TE. The lipoplexes were also characterized by atomic force microscopy (AFM), dynamic light scattering (DLS), zeta potential and small angle X-ray diffraction experiments. Greater cellular internalization of fluorescein tagged pDNA was also observed with DOPE-TH8S (2 : 1) lipoplexes compared to that with L2K. Retention of the TE of DOPE-TH8S (2 : 1) lipoplexes under high serum conditions was conferred by the presence of the tocopherol backbone and also the hydroxyethyl functionalities. The cellular internalization pathway of the lipoplexes was characterized by performing transfection experiment in the presence of inhibitors of different endocytic pathways and it was found to be caveolae mediated. An MTT based cell viability assay indicated that the lipoplex mediated gene delivery vectors exhibited low toxicity in all the three cancer cell lines studied.

Efficacious gene silencing in serum and significant apoptotic activity induction by survivin downregulation mediated by new cationic gemini tocopheryl lipids.

Nonviral gene delivery offers cationic liposomes as promising instruments for the delivery of double-stranded RNA (ds RNA) molecules for successful sequence-specific gene silencing (RNA interference). The efficient delivery of siRNA (small interfering RNA) to cells while avoiding unexpected side effects is an important prerequisite for the exploitation of the power of this excellent tool. We present here six new tocopherol based cationic gemini lipids, which induce substantial gene knockdown without any obvious cytotoxicity. All the efficient coliposomal formulations derived from each of these geminis and a helper lipid, dioleoylphosphatidylethanolamine (DOPE), were well characterized using physical methods such as atomic force microscopy (AFM) and dynamic light scattering (DLS). Zeta potential measurements were conducted to estimate the surface charge of these formulations. Flow cytometric analysis showed that the optimized coliposomal formulations could transfect anti-GFP siRNA efficiently in three different GFP expressing cell lines, viz., HEK 293T, HeLa, and Caco-2, significantly better than a potent commercial standard Lipofectamine 2000 (L2K) both in the absence and in the presence of serum (FBS). Notably, the knockdown activity of coliposomes of gemini lipids was not affected even in the presence of serum (10% and 50% FBS) while it dropped down for L2K significantly. Observations under a fluorescence microscope, RT-PCR, and Western blot analysis substantiated the flow cytometry results. The efficient cellular entry of labeled siRNA in GFP expressing cells as evidenced from confocal microscopy put forward these gemini lipids among the potent lipidic carriers for siRNA. The efficient transfection capabilities were also profiled in a more relevant fashion while performing siRNA transfections against survivin (an anti-apoptotic protein) which induced substantial apoptosis. Furthermore, the survivin downregulation improved the therapeutic efficacy levels of an anticancer drug, doxorubicin, significantly. In short, the new tocopherol based gemini lipids appear to be highly promising for achieving siRNA mediated gene knockdown in various cell lines.

α-Tocopherol derived lipid dimers as efficient gene transfection agents. Mechanistic insights into lipoplex internalization and therapeutic induction of apoptotic activity.

In this report, we present cationic dimeric (gemini) lipids for significant plasmid DNA (pDNA) delivery to different cell lines without any marked toxicity in the presence of serum. Six gemini lipids based on α-tocopherol were synthesized, which differed in their spacer chain lengths. Each of these gemini lipids mixed with a helper lipid, 1,2-dioleoyl phosphatidyl ethanolamine (DOPE), was capable of forming stable aqueous suspensions. These co-liposomal systems were examined for their potential to transfect pEGFP-C3 plasmid DNA into nine cell lines of different origins. The transfection efficacies noticed in terms of EGFP expression levels using flow cytometry were well corroborated using independent fluorescence microscopy studies. Significant EGFP expression levels were reported using the gemini co-liposomes, which counted significantly better than one well known commercial formulation, Lipofectamine 2000 (L2 K). Transfection efficacies were also analyzed in terms of the degree of intracellular delivery of labeled plasmid DNA (pDNA) using confocal microscopy, which revealed an efficient internalization in the presence of serum. The cell viability assays performed using optimized formulations demonstrated no significant toxicity towards any of the cell lines used in the study. We also had a look at the lipoplex internalization pathway to profile the uptake characteristics. A caveolae/lipid raft route was attributed to their excellent gene transfection capabilities. The study was further advanced by using a therapeutic p53-EGFP-C3 plasmid and the apoptotic activity was observed using FACS and growth inhibition assay.

Flaring Inflammation and ER Stress by an Organelle-Specific Fluorescent Cage.

The endoplasmic reticulum (ER) plays an important role in protein synthesis and its disruption can cause protein unfolding and misfolding. Accumulation of such proteins leads to ER stress, which ultimately promotes many diseases. Routine screening of ER activity in immune cells can flag serious conditions at early stages, but the current clinically used bio-probes have limitations. Herein, an ER-specific fluorophore based on a biocompatible benzothiadiazole-imine cage (BTD-cage) with excellent photophysical properties is developed. The cage outperforms commercially available ER stains in long-term live cell imaging with no fading or photobleaching over time. The cage is responsive to different levels of ER stress where its fluorescence increases accordingly. Incorporating the bio-probe into an immune disorder model, a 6-, 21-, and 48-fold increase in intensity is shown in THP-1, Raw 246.7, and Jurkat cells, respectively (within 15 min). These results strongly support that this system can be used for rapid visual and selective detection of ER stress. It is envisaged that tailoring molecular interactions and molecular recognition using supramolecular improved fluorophores can expand the library of biological probes for enhanced selectivity and targetability toward cellular organelles.

New Xanthone Derivatives as Potent G-Quadruplex Binders for Developing Anti-Cancer Therapeutics.

Xanthone is an important scaffold for various medicinally relevant compounds. However, it has received scant attention in the design of agents that are cytotoxic to cancer cells via targeting the stabilization of G-quadruplex (G4) nucleic acids. Specific G4 DNA recognition against double-stranded (ds) DNA is receiving epoch-making interest for the development of G4-mediated anticancer agents. Toward this goal, we have synthesized xanthone-based derivatives with various functionalized side-arm substituents that exhibited significant selectivity for G4 DNA as compared to dsDNA. The specific interaction has been demonstrated by performing various biophysical experiments. Based on the computational study as well as the competitive ligand binding assay, it is inferred that the potent compounds exhibit an end-stacking mode of binding with G4 DNA. Additionally, compound-induced conformational changes in the flanking nucleotides form the binding pocket for effective interaction. Selective action of the compounds on cancer cells suggests their effectiveness as potent anti-cancer agents. This study promotes the importance of structure-based screening approaches to get molecular insights for new scaffolds toward desired specific recognition of non-canonical G4 DNA structures.

Liposomal nanoparticles based on steroids and isoprenoids for nonviral gene delivery.

Natural lipid molecules are an essential part of life as they constitute the membrane of cells and organelle. In most of these cases, the hydrophobicity of natural lipids is contributed by alkyl chains. Although natural lipids with a nonfatty acid hydrophobic backbone are quite rare, steroids and isoprenoids have been strong candidates as part of a lipid. Over the years, these natural molecules (steroid and isoprenoids) have been used to make either lipid-based nanoparticle or functionalize in such a way that it could form nano assembly alone for therapeutic delivery. Here we mainly focus on the synthetic functionalized version of these natural molecules which forms cationic liposomal nanoparticles (LipoNPs). These cationic LipoNPs were further used to deliver various negatively charged genetic materials in the form of pDNA, siRNA, mRNA (nucleic acids), and so on. This article is categorized under: Biology-Inspired Nanomaterials > Lipid-Based Structures.

Novel α-tocopherol-ferrocene conjugates for the specific delivery of transgenes in liver cancer cells under high serum conditions.

The delivery of therapeutic genes to a specific organ has drawn significant research attention. Among the pool of various delivery vectors, cationic liposomes (non-viral) are potential candidates for delivering therapeutic genes due to their low immunogenic response. Here, we have developed novel ferrocene-conjugated cationic tocopheryl aggregates as non-viral vectors. These formulations can transfer a reporter gene (pGL3; encoded for luciferase protein) specifically to liver cancer cells (HepG2 and Huh7) instead of non-hepatic cancer cells, such as Caco-2 (human colon carcinoma) and HeLa (cervical cancer) cells. The transfection efficiency (TE) of the optimum liposomal formulation is more significant than commercially available Lipofectamine 2000 (L2K). Notably, it retains its TE under high serum conditions (up to 50% FBS). A coupled effect from conjugated ferrocene and tocopherol in the cationic liposomal formulation might be responsible for the cell-specific delivery and higher serum compatibility. Therefore, the present proposed delivery system may provide a platform for further progress in terms of developing hepatotropic gene delivery systems.

Antibody-Conjugated Vitamin E-Derived Liposomes for Targeted Gene Transfer.

Construction of a vitamin E-based liposomal biomaterial and its ability to deliver therapeutic genes selectively across liver cancer cells are demonstrated herein. In humans, liver regulates the levels of α-tocopherol, i.e., vitamin E, and hepatic cells carry the machinery for its transport. To exploit the presence of tocopherol transport protein, we have selected an efficient gene transfecting α-tocopherol-based twin lipid bearing a hydroxyethylated headgroup and octamethylene spacer (TH8S) for liposome formation. Also, based on the abundancy of the low-density lipoprotein receptor (LDLr) on the cellular surface in the case of hepatocellular carcinoma, anti-LDLr monoclonal antibody is used to confer the targeting ability to liposomes. A facile thiol-maleimide click chemistry is used for antibody decoration on the liposomal surface. Selective delivery of reporter and therapeutic genes (GFP and p53) to cells of hepatic origin was observed using anti-LDLr-tagged TH8S liposomes. Cellular internalization by receptor-mediated endocytosis renders the bioconjugate highly specific as well as highly efficient. Compatibility of the designed material with human blood points to its safety of use in systemic circulation thereby highlighting its in vivo potential. Thus, we report here a versatile biomaterial derived from an essential vitamin that promises potential for targeted suicidal gene therapy.

Palladium-induced transformation of nematic liquid crystals to robust metallogel comprising self-assembled nanowires.

The formation of a nematic liquid-crystalline phase in the bulk along with gelation of a novel asymmetric bolaamphiphilic NDI scaffold has been demonstrated. Further, a discrete metal NDI complex that is capable of forming a robust metallogel through wrapping of the NDI core with the oxyethylene chains of the neighboring molecules has been synthesized.

Engaging Dynamic Surfactant Assemblies in Improving Metal Ion Sensitivity of a 1,4,7-Triazacyclononane-Based Receptor: Differential Optical Response for Cysteine and Histidine.

Self-assembly as well as metal ion binding property of an amphiphilic, dansylated 1,4,7-triazacyclononane (TACN) probe have been investigated in the presence of various surfactant assemblies in aqueous media. As expected, the receptor molecule shows highly sensitive, but rather nonspecific, interaction with metal ions in the bulk water medium. Thus, to achieve the good specificity without dampening the sensitivity of the probe, we embedded the sensor in different surfactant assemblies, such as micelles and vesicles, and explored their metal ion sensing ability. Change in microenvironment by restricting conformational mobility and increasing local hydrophobicity renders a drastic improvement in selectivity and also sensitivity toward Cu2+. Further, the preformed Cu2+ complex of the probe was utilized for exclusive "turn-on" detection of both cysteine (green fluorescence) and histidine (blue fluorescence). The diverse complexation mode of interactions with these amino acids caused a distinct change in the monomer to aggregate ratio, which was reflected in different spectral response. Furthermore, the dansylated probe was involved in developing reusable paper strips for rapid on-site detection of both Cu2+ and cysteine.

Perfluoroarene induces a pentapeptidic hydrotrope into a pH-tolerant hydrogel allowing naked eye sensing of Ca2+ ions.

Self-assembly of a novel thermoresponsive, pyrene-appended oligopeptide sequence VPGKP (PyP) leads to the formation of spherical aggregates in water. The sizes of the globular aggregates of the peptide, PyP, strongly depend on the temperature of its suspension in water and decrease with the decrease in temperature showing a lower critical solution temperature (LCST) phenomenon. Furthermore, a pyrene-octafluoronaphthalene (OFN) 'pair' has been used as a supramolecular synthon to induce hydrogelation of PyP in the presence of an equimolar amount of OFN via complementary quadrupole-quadrupole interactions. The gel shows excellent pH tolerance and thixotropic behavior. Detailed studies suggest the existence of lamellar packing of the gelators in a right-handed helical fashion which yields globular aggregates. The globular aggregates are sticky in nature and form a gel via inter-globular interactions. Addition of Ca2+ ions reinforces the mechanical strength and also reduces the critical gelator concentration of the native gel through coordination with the free -COO- group of the gelator. Therefore, the present hydrogel system could further be used as a naked eye sensor of Ca2+ ions.

First report of charge-transfer induced heat-set hydrogel. Structural insights and remarkable properties.

The remarkable ability of a charge-transfer (CT) complex prepared from a pyrene-based donor (Py-D) and a naphthalenediimide-based acceptor (NDI-A) led to the formation of a deep-violet in color, transparent hydrogel at room temperature (RT-gel). Simultaneously, the RT-gel was diluted beyond its critical gelator concentration (CGC) to obtain a transparent sol. Very interestingly, the resultant sol, on heating above 70 °C, transformed into a heat-set gel instantaneously with a hitherto unknown CGC value. Detailed studies revealed the smaller globular aggregates of the RT-gels fuse to form giant globules upon heating, which, in turn, resulted in heat-set gelation through further aggregation. The thermoresponsive property of Py-D alone and 1 : 1 Py-D : NDI-A CT complex was investigated in detail which revealed the hydrophobic collapse of the oxyethylene chains of the CT complex upon heating was mainly responsible for heat-set gelation. Thixotropy, injectability, as well as stimuli responsiveness of the RT-gels were also addressed. In contrast, heat-set gel did not show thixotropic behavior. The X-ray diffraction (XRD) patterns of the xerogel depicted lamellar packing of the CT stacks in the gel phase. Single crystal XRD studies further evidenced the 1 : 1 mixed CT stack formation in the lamellae and also ruled out orthogonal hydrogen bonding possibilities among the hydrazide unit in the CT gel although such interaction was observed in a single crystal of NDI-A alone. In addition, a Ag(+)-ion triggered metallogelation of NDI-A and nematic liquid-crystalline property of Py-D were also observed.