Biophysical Interactions of Novel Oleic Acid Conjugate and its Anticancer Potential in HeLa Cells.

Novel conjugate 2,6-Diisopropylphenol-Oleic acid (2,6P-OLA) has shown potent anticancer activity on various cancer cell lines (Khan et al. Lipids 47:973-986, 2012). In the present study, the protein-or/ and DNA-binding property of 2,6P-OLA was evaluated that could predict its potential toxic effect, in vitro. Preferential structural stability and interaction mechanism of 2,6P-OLA to human serum albumin (HSA) and calf thymus DNA (CT-DNA) was used as model molecules, employing fluorescence spectroscopy (FS) and circular dichroism (CD) methods. The binding and apoptotic activities of conjugate were determined on bacterial recombinant DNA, pBR322 and human cancer cell line, HeLa, respectively. FS studies showed a strong conjugate binding affinity to HSA with overall binding constant of K = 7.66 (±0.03) × 10(2) M(-1). Higher concentration induced detectable changes in the CD spectrum of HSA. The conjugate complexation altered HSA secondary conformation by an increase in α-helices and decrease in ß-sheets. Flourescence quenching studies with CT-DNA exhibited K = 1.215 × 10(2) L mol(-1) where 2,6P-OLA efficiently displaced the ethidium bromide (EtBr) bound DNA indicating its strong competition with EtBr for intercalation. Similarly, 2,6P-OLA was able to partially bind pBR322, resulting in decrease the intensity of EtBr gradually. The conjugate significantly reduced survival of HeLa cells. Morphological studies revealed altered cell morphology, suggesting apoptotic death of HeLa cells. Overall, our data shows that 2,6P-OLA bind well with both HSA and DNA and possessed anticancer activities.

Biophysical interactions, docking studies and cytotoxic potential of a novel propofol-linolenate: a multi-technique approach.

In the present study, we have analyzed the biophysical interactions of alpha-linolenic acid conjugate (2,6P-ALA) with human serum albumin (HSA) and calf thymus DNA (CT-DNA); and also determined its effect on human cancer cell lines. The results of interactions between 2,6P-ALA and HSA intrinsic fluorescence indicated static quenching of HSA by the target conjugate with overall Stern-Volmer quenching constant (Ksv) value of 1.8 × 103 M-1. At high concentrations, 2,6P-ALA caused conformational variations in HSA with evident increase in α-helices. Docking studies also revealed preferential binding of 2,6P-ALA at the hydrophobic cavity of site IB with suggestive involvement of hydrophobic forces. Likewise, the conjugate was also able to quench the fluorescence intensity of CT-DNA with static type of quenching signifying the probability of interaction between them. In case of competitive interaction with ethidium bromide (EB) bound CT-DNA also; the conjugate replaced the EB depicting intercalation to be the main type of binding force. Results of cytotoxic effect of 2,6P-ALA showed significant inhibition of cancer cells growth in a concentration-dependent manner. Conjugate was most potent on MCF-7 cells. Fluorescence microscopic image of MCF-7 cells at IC50 concentration of 24 µM revealed distinct morphological changes that were characteristic of programed cell death. Overall, these results complement with the previous findings of 2,6P-ALA and provide added statistics about the prospect of their transport in blood plasma.Communicated by Ramaswamy H. Sarma.

Therapeutic potential of functionalized siRNA nanoparticles on regression of liver cancer in experimental mice.

Short interfering RNA (siRNA) possesses special ability of silencing specific gene. To increase siRNA stability, transportation and its uptake by tumor cells, effective delivery to the appropriate target cells is a major challenge of siRNA-based therapy. In the present study, an effective, safe and biocompatible survivin siRNA encapsulated, GalNAc decorated PEGylated PLGA nanoconjugates (NCs) viz., GalNAc@PEG@siRNA-PLGA were engineered and their synergistic antitumor efficacy was evaluated for targeted delivery in HCC bearing experimental mice. GalNAc@PEG@siRNA-PLGA NCs were characterized for size, bioavailability, toxicity and biocompatibility. Their antitumor potential was evaluated considering gene silencing, apoptosis, histopathology and survival of treated mice. Exceptional accumulation of hepatocytes, reduction in survivin expression and prominent regression in tumor size confirmed the ASGPR-mediated uptake of ligand-anchored NCs and silencing of survivin gene in a targeted manner. Increased DNA fragmentation and potential modulation of caspase-3, Bax and Bcl-2 factors specified the induction of apoptosis that helped in significant inhibition of HCC progression. The potential synchronous and tumor selective delivery of versatile NCs indicated the effective payloads towards the target site, increased apoptosis in cancer cells and improved survival of treated animals.

Additive potential of combination therapy against cryptococcosis employing a novel amphotericin B and fluconazole loaded dual delivery system.

Cryptococcus neoformans is one of the most lethal fungi causing mortality across the globe. Immuno-competent patients and patients taking immuno-suppressive medications are extremely susceptible to its infection. For effective removal of cryptococcal burden, there is an urgent need for new forms of therapy. In the present study, we have explored the potential effects of amphotericin B (AMB) and fluconazole (FLC) in combination, against cryptococcosis in Swiss albino mice. To enhance the therapeutic potential of the tested drugs, they were entrapped into fibrin microspheres; a dual delivery vehicle comprising of poly-lactide co-glycolide (PLGA) microsphere that was additionally encapsulated into the fibrin cross-linked plasma bead. Dynamics of fibrin microspheres included survival and fungal burden in lung, liver and spleen of treated mice. While each drug was effective in combination or alone, prominent additive potential of AMB and FLC were clearly observed when used in fibrin microsphere. Significant reduction in fungal burden and increase in survival rate of AMB + FLC-fibrin microspheres treated mice shows an extensive accessibility of both tested drugs without any side-effects. A full potential of two-drug combination encapsulated in fibrin microspheres proposes an effective approach of safe delivery to the target site in their intact form and decrease the drug associated toxicities.

Antifungal efficacy of amphotericin B encapsulated fibrin microsphere for treating Cryptococcus neoformans infection in Swiss albino mice.

A natural and biocompatible fibrin microsphere is one of the most promising dual delivery vehicle as compared to other traditionally designed delivery modalities. It represents sustained delivery of encapsulated drug and is easily biodegradable in the blood circulation. In the present study, we evaluated the systemic augmentation of the antifungal activity of amphotericin B loaded in fibrin microsphere (AMB-fibrin microsphere) against cryptococcosis in Swiss albino mice. Mice infected with Cryptococcus neoformans were treated with 0.5mg/kg AMB-fibrin microsphere that was given alternately for 7 days. The antifungal potential of AMB-fibrin microsphere was assessed on the basis of reduction of cfu count in the systemic circulation and various vital organs of infected mice. The formulation was found to be highly effective in reducing intracellular pathogen from the experimental animals where fibrin microsphere significantly controlled the release of amphotericin B for longer time duration. The AMB-fibrin microsphere chemotherapy was significantly more effective than free amphotericin B in reducing the fungal burden and showed better survival efficacy (p<0.05). The current study demonstrating the use of novel amphotericin B loaded fibrin microsphere not only imparts protection to the encapsulated amphotericin B but also offers an effective strategy to decrease the drug associated toxicities.

Targeted nano-delivery of novel omega-3 conjugate against hepatocellular carcinoma: Regulating COX-2/bcl-2 expression in an animal model.

The present approach enumerates the effectiveness of tuftsin tagged nano-liposome for the cytosolic transport of 2,6-di-isopropylphenol-linolenic acid conjugate against liver cancer in mice. Initially, the conjugate in its free form was examined for anticancer potential on HepG2 liver cancer cells. Induction of apoptosis and suppression of migration and adhesion of HepG2 cells confirmed the effectiveness of conjugate as an anticancer agent. After this, role of the conjugate entrapped in a nano-carrier was evaluated in animal model. The nano-formulation comprising of conjugate bearing tuftsin tagged liposome was firsly characterized and then its therapeutic effect was determined. The nano-formulation had 100-130nm size nanoparticles and showed sustained release of the conjugate in the surrounding milieu. The nano-formulation distinctly reduced the expression of COX-2, an important molecule that is vastly expressed in hepatocellular carcinoma. The utilization of in-house engineered nano-formulation was also successful in significantly up-regulating Bax and down-regulating bcl-2 gene expression eventually helping in better survival of treated mice. Histopathological analysis also revealed positive recovery of the general architecture and the violent death of cancer cells by apoptosis at tumor specific site. The site specific delivery of conjugate entrapped in tuftsin tagged liposomes was highly safe as well as efficaceous. Nano-formulation based approach showed a visible chemotherapeutic effect on liver cancer progression in experimental mice thereby making it a potential candidate for treatment of liver cancer in clinical settings.

Biochemical characterization of a protein of albumin multigene family from serum of African catfish Clarias gariepinus Bloch.

A monomorphic albumin-like protein (CfSA) has been purified to homogeneity from the serum of African air-breathing catfish Clarias gariepinus Bloch. It has a molecular mass of approximately =70 kD and shows a lesser electrophoretic mobility than human serum albumin (HSA) in native gels. The protein exhibits cross-reactivity against rabbit anti-HSA serum and shows considerable similarity with HSA in secondary structure, however, with some differences, as indicated by a slight shift in the peaks around 267 nm and 278 nm and the absence of shoulders at 276 and 283. A certain degree of similarity also exists between their tertiary structures with respect to aromatic asymmetric environment as indicated by far-UV CD spectra and the visible range CD spectra of bilirubin complexes. CfSA-bilirubin complex is mainly characterized by bisignate CD Cotton effects (CDCEs), having minima and maxima wavelengths at 406 and 486 nm, respectively and unlike HSA, it shows prominent additional maxima around 426 nm. Based on the number of sulfhydryls, CfSA is in the rank of advanced teleosts. The occurrence of albumin in C. gariepinus in relation to the evolutionary dichotomy of albumin and other members of its multigene family in class Pisces has been discussed.

Anticancer efficacy of a novel propofol-linoleic acid-loaded escheriosomal formulation against murine hepatocellular carcinoma.

AIM: The preparation and characterization of a novel escheriosomal nanoparticle formulation of a potent anticancer conjugate, 2,6-diisopropylphenol-linoleic acid (2,6P-LA), and evaluation of its anticancer efficacy against diethyl nitrosamine-induced hepatocellular carcinoma (HCC) in BALB/c mice. MATERIALS & METHODS: Escheriosomized 2,6P-LA nanoparticles were characterized for size, zeta-potential, entrapment efficiency, release kinetics and in vivo toxicity. Their anticancer potential was evaluated on the basis of survival, DNA fragmentation, caspase-3 activation, western blot analysis of apoptotic factors and histopathological changes in hepatocytes of treated animals. RESULTS: The escheriosomized 2,6P-LA nanoparticles exhibited low toxicity, biocompatibility and bioavailability. As revealed by apoptosis induction, survival rate, expression profiles of Bax, Bcl-2 and caspase-9, escheriosomized 2,6P-LA nanoparticles were more effective in the treatment of HCC than the free form of 2,6P-LA in experimental animals. CONCLUSION: 2,6P-LA-bearing escheriosome nanoparticles are effective in suppressing HCC in mice. Original submitted 17 January 2012; Revised submitted 27 August 2012; Published online 14 January 2013.

Design, synthesis and in vitro anticancer evaluation of a stearic acid-based ester conjugate.

AIM: Chemical synthesis and characterization of a lipophilic ester conjugate, propofol stearate and evaluation of its anticancer efficacy on human breast cancer cell lines MDA-MB-361, MCF-7 and MDA-MB-231. MATERIALS AND METHODS: The chemical structure of the synthesized conjugate was characterized by spectroscopic studies. Its anticancer potential was evaluated on the basis of growth inhibition, cancer cell adhesion and migration and apoptosis induction. RESULTS: Propofol stearate exhibited significant (p<0.05) growth inhibition of breast cancer cells in a concentration-dependent manner. MDA-MB-231 cells showed highest susceptibility towards the inhibitory effect of the conjugate. Moreover, treatment of MDA-MB-231 cancer cells with 25 µM propofol stearate potentially suppressed their adhesion (~34%) and migration (~41%), and induced apoptosis (~25%). CONCLUSION: Exogenously-applied stearic acid as an ester derivative, inhibits the growth of human breast cancer cells and shows a beneficial role in the treatment of breast cancer, in vitro.

Vaccine potential of cytosolic proteins loaded fibrin microspheres of Cryptococcus neoformans in BALB/c mice.

Cryptococcosis is a leading mycological cause of mortality among immunologically compromised individuals. In order to develop an effective vaccine against Cryptococcus neoformans, the cytosolic proteins (Cp) of the pathogen have been used as an antigen in combination with different formulations. In the present study, we have demonstrated that Cp encapsulated poly-lactide co-glycolide (PLGA) microsphere further co-encapsulated into the biocompatible fibrin cross-linked plasma beads (Fib-PLGA-Cp) mediated cytosolic delivery elicited strong immune response in the BALB/c mice. In contrast, other formulations of Cp failed to impart significant level of protection. The immune response, involved with Fib-PLGA-Cp protection, appear to interact with the target cells by both endocytosis as well as membrane fusion mode, thus helping in the activation of both CD4(+) and CD8(+) T-cells. Analysis of cytokine profiles in immunized animals revealed that the protective response was associated with the Th1/Th2 polarization in favor of type-1 cytokine [interferons (IFN)-γ and interleukin (IL)-2] cells. Furthermore, vaccination with Fib-PLGA-Cp elicited high immunoglobulin (Ig) G(l) and IgG(2a) isotype response; successfully cleared fungal burden in vital organs and also increased the survival rate of immunized animals. Altogether the present study is a clear indicative of the possible use of fibrin microsphere-based targeted delivery of cytosolic proteins to induce protective immune responses against experimental murine cryptococcosis.

Synthesis and characterization of novel n-9 fatty acid conjugates possessing antineoplastic properties.

The present study enumerates the synthesis, spectroscopic characterization, and evaluation of anticancer potential of esters of two n-9 fatty acids viz., oleic acid (OLA) and ricinoleic acid (RCA) with 2,4- or 2,6-diisopropylphenol. The synthesis strategy involved esterification of the hydroxyl group of diisopropylphenol (propofol) to the terminal carboxyl group of n-9 fatty acid. The synthesized propofol-n-9 conjugates having greater lipophilic character were tested initially for cytotoxicity in-vitro. The conjugates showed specific growth inhibition of cancer cell lines whereas no effect was observed in normal cells. In general, pronounced growth inhibition was found against the human skin malignant melanoma cell line (SK-MEL-1). The anticancer potential was also determined by testing the effect of these conjugates on cell migration, cell adhesion and induction of apoptosis in SK-MEL-1 cancer cells. Propofol-OLA conjugates significantly induced apoptosis in contrast to propofol-RCA conjugates which showed only weak signals for cytochrome c. Conclusively, the synthesized novel ester conjugates showed considerable moderation of anti-tumor activity. This preliminary study places in-house synthesized conjugates into the new class of anticancer agents that possess selectivity toward cancer cells over normal cells.

Antifungal efficacy of amphotericin B encapsulated fibrin microsphere for treating Cryptococcus neoformans infection in Swiss albino mice

Abstract A natural and biocompatible fibrin microsphere is one of the most promising dual delivery vehicle as compared to other traditionally designed delivery modalities. It represents sustained delivery of encapsulated drug and is easily biodegradable in the blood circulation. In the present study, we evaluated the systemic augmentation of the antifungal activity of amphotericin B loaded in fibrin microsphere (AMB-fibrin microsphere) against cryptococcosis in Swiss albino mice. Mice infected with Cryptococcus neoformans were treated with 0.5 mg/kg AMB-fibrin microsphere that was given alternately for 7 days. The antifungal potential of AMB-fibrin microsphere was assessed on the basis of reduction of cfu count in the systemic circulation and various vital organs of infected mice. The formulation was found to be highly effective in reducing intracellular pathogen from the experimental animals where fibrin microsphere significantly controlled the release of amphotericin B for longer time duration. The AMB-fibrin microsphere chemotherapy was significantly more effective than free amphotericin B in reducing the fungal burden and showed better survival efficacy (p < 0.05). The current study demonstrating the use of novel amphotericin B loaded fibrin microsphere not only imparts protection to the encapsulated amphotericin B but also offers an effective strategy to decrease the drug associated toxicities.