Vivek Dhawan

Lecithin-Based Novel Cationic Nanocarriers (Leciplex) II: Improving Therapeutic Efficacy of Quercetin on Oral Administration†

The objective of the present investigation was to evaluate ability of the novel self-assembled phospholipid- based cationic nanocarriers (LeciPlex) in improving the therapeutic efficacy of a poorly water-soluble natural polyphenolic agent, quercetin (QR), on oral administration. Quercetin loaded LeciPlex (QR-LeciPlex) were successfully fabricated using a biocompatible solvent Transcutol HP. The QR-LeciPlex were characterized for particle size, encapsulation efficiency, zeta potential, and particle morphology by cryo-TEM. UV and fluorescence spectral characterization was carried out to find out the association of QR with LeciPlex. Small angle neutron scattering studies (SANS) were carried out to understand the internal structure of Leciplex and to evaluate the influence of the incorporation of QR in the LeciPlex. Anti-inflammatory and antitumorigenic activity of QR-LeciPlex was determined in comparison to QR suspension to evaluate the potential of LeciPlex in improving oral delivery of QR. QR-LeciPlex exhibited a particle size of ∼400 nm and had excellent colloidal stability. The QR-LeciPlex had a zeta potential greater than +30 mV and exhibited very high encapsulation efficiency of QR (>90%). UV and fluorescence spectral characterization indicated the interaction/association of QR with LeciPlex components. Cryo-TEM studies showed that LeciPlex and QR-LeciPlex have a unilamellar structure. SANS confirmed the unilamellar structure of LeciPlex and indicated that the incorporation of QR does not have any effect on the internal structure of the LeciPlex. QR-LeciPlex exhibited significantly higher anti-inflammatory and antitumorigenic activity (p < 0.01) as compared to that of QR suspension on oral administration.

Cholesterol level affects surface charge of lipid membranes in saline solution

Cholesterol is an important component of all biological membranes as well as drug delivery liposomes. We show here that increasing the level of cholesterol in a phospholipid membrane decreases surface charge in the physiological environment. Through molecular dynamics simulation we have shown that increasing the level of cholesterol decreases Na+ ion binding. Complementary experimental ζ – potential measurements have shown a decreased ζ – potential with increasing cholesterol content, indicative of reduced surface charge. Both experiments and simulations have been carried out on both saturated 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and monounsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. This result is particularly important because membrane surface charge plays an important role in the interactions of biomembranes with peripheral membrane proteins and drug delivery liposomes with the immune system.

Tamoxifen guided liposomes for targeting encapsulated anticancer agent to estrogen receptor positive breast cancer cells: In vitro and in vivo evaluation

Tamoxifen (TMX), an estrogen receptor (ER) antagonist, incorporated at surface of liposomes loaded with Doxorubicin (DOX), was hypothesized to serve as ligand for targeting overexpressed ERs on surface and cytosol of breast cancer cells, in addition to its synergism with DOX in killing MCF-7 cells. The TMX-DOX liposomes demonstrated mean size of 188.8±2.2nm and positive potential of+47mV, both suitable for better cellular interaction. TMX-DOX liposomes sustained DOX release in vitro (25.9%) in pH 7.4 at 48h, in comparison with 64.5% DOX release at pH 5.5. In vitro cell line studies demonstrated that TMX-DOX liposomes were more cytotoxic to ER+ve MCF-7 cells as compared to DOX liposomes, DOX solution and TMX-DOX solution (P<0.05). However, there was no statistical difference in cyto-toxicity of TMX-DOX liposomes and DOX liposomes towards ER-ve MDA-MB-231 cells. Flow cytometry and confocal studies in MCF-7 cells revealed greater cell and nuclear uptake of DOX, with TMX guided liposomes as compared to DOX liposomes and DOX solution. TMX-DOX liposomes demonstrated significantly increased inhibition of MCF-7 cell based tumor growth in nude mice (P<0.05) in comparison to DOX solution and DOX liposomes, indicative of target specificity and higher DOX accumulation at tumor site.

Design of cholesterol arabinogalactan anchored liposomes for asialoglycoprotein receptor mediated targeting to hepatocellular carcinoma: In silico modeling, in vitro and in vivo evaluation

We have developed active targeting liposomes to deliver anticancer agents to ASGPR which will contribute to effective treatment of hepatocellular carcinoma. Active targeting is achieved through polymeric ligands on the liposome surface. The liposomes were prepared using reverse phase evaporation method and doxorubicin hydrocholoride, a model drug, was loaded using the ammonium sulphate gradient method. Liposomes loaded with DOX were found to have a particle size of 200nm with more than 90% entrapment efficiency. Systems were observed to release the drug in a sustained manner in acidic pH in vitro. Liposomes containing targeting ligands possessed greater and selective toxicity to ASGPR positive HepG2 cell lines due to specific ligand receptor interaction. Bio-distribution studies revealed that liposomes were concentrated in the liver even after 3h of administration, thus providing conclusive evidence of targeting potential for formulated nanosystems. Tumor regression studies indicated greater tumor suppression with targeted liposomes thereby establishing superiority of the liposomal system. In this work, we used a novel methodology to guide the determination of the optimal composition of the targeting liposomes: molecular dynamics (MD) simulation that aided our understanding of the behaviour of the ligand within the bilayer. This can be seen as a demonstration of the utility of this methodology as a rational design tool for active targeting liposome formulation.

Cholesterol anchored arabinogalactan for asialoglycoprotein receptor targeting: synthesis, characterization, and proof of concept of hepatospecific delivery

Asialoglycoprotein receptors (ASGPR) are hepatocyte bound receptors, which exhibit receptor mediated endocytosis (RME) for galactose specific moieties. Arabinogalactan (AG), a liver specific high galactose containing branched polysaccharide was hydrophobized using cholesterol (CHOL) as a lipid anchor via a two step reaction process to yield the novel polysaccharide lipid conjugated ligand (CHOL-AL-AG). CHOL-AL-AG was characterized by Fourier transform infra red (FTIR) spectroscopy, (1)H and (13)C nuclear magnetic spectroscopy (NMR), size exclusion chromatography (SEC) and differential scanning calorimetry (DSC). Conventional liposomes (CL) and surface modified liposomes (SML) containing CHOL-AL-AG were prepared using reverse phase evaporation technique. Effect of CHOL-AL-AG concentration on particle size and zeta potential of SML was evaluated. Surface morphology of CL and SML was studied using cryo-transmission electron microscopy (cryo-TEM). In vitro binding affinity of SML and CL was evaluated using Ricinus communis agglutinin (RCA) assay. Cellular uptake of SML and CL was determined on ASGPR expressing HepG2 cell lines by confocal laser scanning microscopy technique (CLSM). FTIR spectra revealed bands at 1736 cm(-1) and 1664 cm(-1) corresponding to ester and carbamate functional groups, respectively. Signals at δ 0.5-2.5 corresponding to the cholestene ring and δ 3-5.5 corresponding to the carbohydrate backbone were observed in (1)H NMR spectrum of the product. CHOL-AL-AG possessed a mean average molecular weight of 27 KDa as determined by size exclusion chromatography. An endothermic peak at 207 °C was observed in the DSC thermogram of CHOL-AL-AG, which was not observed in thermograms of reactants and intermediate product. Synthesized CHOL-AL-AG was successfully incorporated in liposomes to yield SML. Both CL and SML possessed a mean particle size of ∼ 200 nm with polydispersity index of ∼ 0.25. The zeta potential of CLs was observed to be -17 mV whereas zeta potential of SMLs varied from -18 to -22 mV. RCA assay revealed enhanced binding of SML compared to CL confirming presence of galactose on surface of SML. CLSM studies demonstrated enhanced cellular uptake of SMLs compared to CL by HepG2 cells post 3 h administration indicating enhanced uptake by the ASGPR. Thus surface modified liposomes specific to target heptocytes demonstrate a promising approach for targeted drug delivery in liver cancer therapeutics.

Apoptosis induction and anti-cancer activity of LeciPlex formulations.

PurposeCationic agents have been reported to possess anti-neoplastic properties against various cancer cell types. However, their complexes with lipids appear to interact differently with different cancer cells. The purpose of this study was to (i) design and generate novel cationic lecithin nanoparticles, (ii) assess and understand the mechanism underlying their putative cytotoxicity and (iii) test their effect on cell cycle progression in various cancer-derived cell lines. In addition, we aimed to evaluate the in vivo potential of these newly developed nanoparticles in oral anti-cancer delivery.MethodsCationic lecithin nanoparticles were generated using a single step nanoprecipitation method and they were characterized for particle size, zeta potential, stability and in vitro release. Their cytotoxic potential was assessed using a sulforhodamine B assay, and their effect on cell cycle progression was evaluated using flow cytometry. The nanoparticle systems were also tested in vivo for their anti-tumorigenic potential.ResultsIn contrast to cationic agents alone, the newly developed nanoformulations showed a specific toxicity against cancer cells. The mechanism of toxic cell death included apoptosis, S and G2/M cell cycle phase arrest, depending on the type of cationic agent and the cancer-derived cell line used. Both blank and drug-loaded systems exhibited significant anti-cancer activity, suggesting a synergistic anti-tumorigenic effect of the drug and its delivery system.ConclusionsBoth in vitro and in vivo data indicate that cationic agents themselves exhibit broad anti-neoplastic activities. Complex formation of the cationic agents with phospholipids was found to provide specificity to the anti-cancer activity. These formulations thus possess potential for the design of effective anti-cancer delivery systems.

In Vitro and Ex Vivo Evaluations of Lipid Anti-Cancer Nanoformulations: Insights and Assessment of Bioavailability Enhancement

Lipid-based nanoformulations have been extensively investigated for improving oral efficacy of plethora of drugs. Chemotherapeutic agents remain a preferred option for effective management of cancer; however, most chemotherapeutic agents suffer from limitation of poor oral bioavailability that is associated with their physicochemical properties. Drug delivery via lipid-based nanosystems possesses strong rational and potential for improving oral bioavailability of such anti-cancer molecules through various mechanisms, viz. improving their gut solubilisation owing to micellization, improving mucosal permeation, improving lymphatic uptake, inhibiting intestinal metabolism and/or inhibiting P-glycoprotein efflux of molecules in the gastrointestinal tract. Various in vitro characterization techniques have been reported in literature that aid in getting insights into mechanisms of lipid-based nanodevices in improving oral efficacy of anti-cancer drugs. The review focuses on different characterization techniques that can be employed for evaluation of lipid-based nanosystems and their role in effective anti-cancer drug delivery.

Catanionic systems in nanotherapeutics – Biophysical aspects and novel trends in drug delivery applications

ABSTRACT Mixtures of surfactants can result in formation of various structures like micelles, vesicles and inverted micelles. Catanionic vesicular systems are preferred on account of their ease of formation and thermodynamic stability. Furthermore, their charge and surfactant properties render them as useful vehicles for DNA delivery and cytotoxic compounds. They suffer from disadvantages of being leaky and yielding low encapsulation efficiencies which are averse to drug delivery purposes. Extensive efforts are being undertaken to overcome these barriers and render these vesicles amenable to spatial placement and temporal delivery of drugs. This manuscript addresses diverse aspects of catanionic vesicles including their formation, fabrication and stability. The manuscript focuses further on applications of catanionic vesicles in nanodrug delivery. Novel trends in the field of catanionics with respect to bio‐compatibility and novel technologies developed using these systems have also been reviewed. An attempt has been made to compile catanionic systems reported in literature detailing surfactants and therapeutic agents employed to aid understanding and yield information of various facets that drive fabrication and potential utility of these systems in therapeutics. Graphical abstract Figure. No caption available.

Stearylated cycloarginine nanosystems for intracellular delivery – simulations, formulation and proof of concept

Cationization of nanocarriers has been sought after as a viable strategy to surmount cellular barriers that impede intracellular drug and gene delivery. A plethora of cationic compounds including both lipids and polymers have been designed and developed for efficient cellular penetration. The toxicity of these cationic agents, however, precludes their use in drug delivery. Conjugation with biomolecules like sugars and amino acids to produce novel cationic agents is being investigated to generate biocompatible molecules to achieve safe drug delivery. In this study, we propose to mitigate the toxicity of stearylamine, a cationic lipid, by tethering it to arginine, to yield a novel cationic ligand. Ligand loaded liposomes and nanoparticles were fabricated and evaluated for their surface charge and cellular uptake. Furthermore, molecular dynamics simulations were utilized as a tool to understand the accessibility of the novel ligand and stearylamine loaded liposomal systems. This paper presents the one pot synthesis of a novel stearylated arginine dipeptide and its incorporation in delivery systems along with its in vitro and in vivo toxicity evaluation.

Lipid Architectonics for Superior Oral Bioavailability of Nelfinavir Mesylate: Comparative in vitro and in vivo Assessment

AbstractNelfinavir mesylate (NFV), a human immunodeficiency virus (HIV) protease inhibitor, is an integral component of highly active anti retro viral therapy (HAART) for management of AIDS. NFV possesses pH-dependent solubility and has low and variable bioavailability hampering its use in therapeutics. Lipid-based particulates have shown to improve solubility of poorly water soluble drugs and oral absorption, thereby aiding in improved bioavailability. The current study compares potential of vesicular and solid lipid nanocarriers of NFV with drug nanocrystallites and microvesicular systems like cochleates in improving bioavailability of NFV. The paper outlines investigation of systems using in vitro models like in vitro lipolysis, in vitro release, and permeation through cell lines to predict the in vivo potential of nanocarriers. Finally, in vivo pharmacokinetic study is reported which provided proof of concept in sync with results from in vitro studies. Graphical Abstractᅟ