Arehalli S. Manjappa

Antibody derivatization and conjugation strategies: Application in preparation of stealth immunoliposome to target chemotherapeutics to tumor

A great deal of effort has been made over the years to develop liposomes that have targeting vectors (oligosaccharides, peptides, proteins and vitamins) attached to the bilayer surface. Most studies have focused on antibody conjugates since procedures for producing highly specific monoclonal antibodies are well established. Antibody conjugated liposomes have recently attracted a great deal of interest, principally because of their potential use as targeted drug delivery systems and in diagnostic applications. A number of methods have been reported for coupling antibodies to the surface of stealth liposomes. The objective of this review is to enumerate various strategies which are employed in the modification and conjugation of antibodies to the surface of stealth liposomes. This review also describes various derivatization techniques of lipids prior and after their use in the preparation of liposomes. The use of single chain variable fragments and affibodies as targeting ligands in the preparation of immunoliposomes is also discussed.

Bone metastasis targeting: A novel approach to reach bone using Zoledronate anchored PLGA nanoparticle as carrier system loaded with Docetaxel

In spite of good research in drug delivery, bone targeting remains largely unexplored. Even some of the bone diseases are seldom cured just because of poor distribution of drug at the bone site. Zoledronate (ZOL) having strong affinity towards bone and its utility in bone metastasis management makes it perfect ligand for bone targeting. Recent studies revealed that ZOL in combination with docetaxel showed significant synergism in the management of bone metastasis. From the results, it is clear that ZOL-conjugated PLGA nanoparticles (NPs) showed more cellular uptake than pegylated PLGA NPs with change in cellular uptake route. In vitro studies on MCF-7 and BO2 cell line revealed that ZOL anchored PLGA-PEG NPs showed enhanced cell cytotoxicity, increase in cell cycle arrest and more apoptotic activity. PLGA-PEG-ZOL NPs found to block mevalonate pathway and increase accumulation of apoptotic metabolites such as ApppI. In vivo animal studies using technetium-99m radiolabeling showed prolong blood circulation half-life, reduced liver uptake and significantly higher retention of ZOL tagged NPs at the bone site with enhanced tumor retention. Here, we can conclude that the targeting ability of ZOL enhanced by strong affinity to bone, enhanced endocytosis of ZOL anchored PLGA-PEG NPs.

Opsonization, Biodistribution, Cellular Uptake and Apoptosis Study of PEGylated PBCA Nanoparticle as Potential Drug Delivery Carrier

ABSTRACTPurposeFor nanocarrier-based targeted delivery systems, preventing phagocytosis for prolong circulation half life is a crucial task. PEGylated poly(n-butylcyano acrylate) (PBCA) NP has proven a promising approach for drug delivery, but an easy and reliable method of PEGylation of PBCA has faced a major bottleneck.MethodsPEGylated PBCA NPs containing docetaxel (DTX) by modified anionic polymerization reaction in aqueous acidic media containing amine functional PEG were made as an single step PEGylation method. In vitro colloidal stability studies using salt aggregation method and antiopsonization property of prepared NPs using mouse macrophage cell line RAW264 were performed. In vitro performance of anticancer activity of prepared formulations was checked on MCF7 cell line. NPs were radiolabeled with 99mTc and intravenously administered to study blood clearance and biodistribution in mice model.ResultsThese formulations very effectively prevented phagocytosis and found excellent carrier for drug delivery purpose. In vivo studies display long circulation half life of PBCA-PEG20 NP in comparison to other formulations tested.ConclusionsThe PEGylated PBCA formulation can work as a novel tool for drug delivery which can prevent RES uptake and prolong circulation half life.

Investigation on design of stable etoposide-loaded PEG-PCL micelles: effect of molecular weight of PEG-PCL diblock copolymer on the in vitro and in vivo performance of micelles.

In the present study, six different molecular weight diblock copolymer of methoxy poly (ethylene glycol)-b-poly (ϵ-caprolactone) (MPEG-PCL) were synthesized and characterized and was used for fabrication of etoposide-loaded micelles by nanoprecipitation technique. The particle size and percentage drug entrapment of prepared micelles were found to be dependent on the molecular weight of PCL block and drug to polymer ratio. The maximum drug loading of 5.32% was found in micellar formulation MPEG5000-PCL10000, while MPEG2000-PCL2000 exhibited 2.73% of maximum drug loading. A variation in the fixed aqueous layer thickness and PEG surface density of micellar formulations was attributed to difference in MPEG molecular weight and interaction of PEG and PCL block of copolymer. The MPEG2000-PCL2000 micelles demonstrated poor in vitro stability among other micellar formulations, due to its interaction with bovine serum albumin and immediate release of drug from micelles. Furthermore, plain etoposide and MPEG2000-PCL2000 micelles exhibited greater extent of hemolysis, due to presence of surfactants and faster release of drug from micelles, respectively. The biodistribution studies carried out on Ehrlich ascites tumor-bearing Balb/C mice confirmed higher accumulation of etoposide-loaded micellar formulation at tumor site compared to plain etoposide due to enhanced permeability and retention effect.

Laminin receptor-targeted etoposide loaded polymeric micelles: a novel approach for the effective treatment of tumor metastasis.

Background: Tumor-targeted delivery is a desirable approach to improve therapeutic outcome of anticancer drug due to enhanced efficacy and reduced toxicity. Purpose: The present study was aimed to target laminin receptor over-expressed tumor cells using YIGSR (Tyr-Ile-Gly-Ser-Arg) conjugated etoposide loaded micelles in the treatment of metastasis. Methods: YIGSR conjugated micelles prepared using synthesized carboxyl and methoxy terminated poly(ethylene glycol)-b-poly(ϵ-caprolactone) block copolymers were evaluated for it efficacy against highly metastatic B16F10 cell lines conducting cytotoxicity, colony formation, cell migration, cellular uptake and flow cytometry studies. The in-vivo antimetastatic effect of micelles was evaluated using experimental metastatic model on C57BL/6 mice. Results: YIGSR conjugated micelles of particle size 45.2 ± 3.77 nm and zeta potential of−5.7 ± 1.3 mV demonstrated enhanced cytotoxicity and cellular uptake with significant reduction in colony formation and cell migration activities compared to non-conjugated micelles. Furthermore, a markedly inhibition in lung colony formation was observed with these micelles. Discussion: An enhanced cellular internalization of YIGSR conjugated micelles due to laminin receptor based endocytosis resulted in to higher cytotoxicity as well as antimetastatic effect against highly metastatic B16F10 cells. Conclusion: These studies indicate that YIGSR conjugated nanocarrier can be a promising approach in the treatment of tumor metastasis.

Anti-neuropilin 1 antibody Fab′ fragment conjugated liposomal docetaxel for active targeting of tumours

Abstract Neuropilin-1, a transmembrane receptor entailed in wide range of human tumour cell lines and diverse neoplasms, mediates the effects of VEGF and Semaphorins during the processes of cellular proliferation, survival and migration. In view of this, we had developed and evaluated in vitro and in vivo efficacy of anti-neuropilin-1 immunoliposomes against neuropilin-1 receptor expressing tumours. The PEGylated liposomes loaded with docetaxel were prepared using thin film hydration method. Functionalised PEGylated liposomes were prepared using post-insertion technique. Anti-neuropilin-1 immunoliposomes were prepared by covalently conjugating Fab′ fragments of neuropilin-1 antibody to functionalised PEGylated liposomes via thioether linkage. In vivo evaluation of Taxotere and liposomal formulations was performed using intradermal tumour model to demonstrate anti-angiogenic and tumour regression ability. The modified Fab′ fragments and immunoliposomes were found to be immunoreactive against A549 cells. Further, docetaxel loaded PEGylated liposomes and PEGylated immunoliposomes demonstrated higher in vitro cytotoxicity than Taxotere formulation at the same drug concentration and exposure time. The live imaging showed distinctive cellular uptake of functional immunoliposomes. Further, significant decrease in micro-blood vessel density and tumour volumes was observed using bio-engineered liposomes. The results clearly highlight the need to seek neuropilin-1 as one of the prime targets in developing an anti-angiogenic therapy.

Acylated chitosan anchored paclitaxel loaded liposomes: Pharmacokinetic and biodistribution study in Ehrlich ascites tumor bearing mice

Acylated chitosan (Myristoyl and Octanoyl) coated paclitaxel-loaded liposomal formulation was developed with an aim to overcome the cremophor EL related toxicities. They were evaluated for drug entrapment, in vitro drug release, and cytotoxicity and cell uptake behavior using A549 cells. The 99mTc radio-labeled formulations were also evaluated in vivo in Ehrlich Ascites Tumor (EAT) bearing mice for biodistribution and tumor uptake. The mean particle size of both coated and uncoated liposomal formulations was found to be in the range of 180-200 nm with high drug entrapment efficiency (>90% in case of uncoated liposomes and 80 ± 5% in case of coated liposomes). The uncoated liposomes displayed negative zeta potential (-10.5 ± 4.9 mV) whereas coated liposomes displayed positive zeta potential in the range of +21 to +27 mV. Slower drug release was observed in case of liposomes coated with acylated chitosans as compared to uncoated and native chitosan coated liposomes. All liposomal formulations were found less cytotoxic than paclitaxel injection (Celtax™, Celon Labs, India). In vitro cell uptake and intracellular distribution studies confirmed the cytosolic delivery of uncoated and coated liposomes. The myristoyl chitosan coated liposomal system (LMC) exhibited improved pharmacokinetic, biodistribution and tumor uptake characteristics over other formulations. These obtained results confirmed the potential application of acylated chitosn coated liposomal delivery systems (LMC) in tumor targeting of paclitaxel and other drugs.

Drug-fortified liposomes as carriers for sustained release of NSAIDs: The concept and its validation in the animal model for the treatment of arthritis

&NA; Drug‐fortified cationic liposomes of 6‐methoxy‐2‐naphthylacetic acid (6‐MNA) were prepared and characterized by various techniques. The residence time of drug‐fortified liposomes in joint cavity was evaluated by intra‐articular (IA) administration of the radio‐labeled (99mTc) liposomal formulation in the inflamed joints in rats. The cationic liposomal formulation composed of 6‐MNA (3) as an active agent, its double salt (4) with the lipid 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine (DSPE), and pharmaceutically acceptable excipients such as hydrogenated soyabean phospatidylcholine (HSPC) and 1,2‐dioleyloxy‐3‐trimethylammoniumpropane chloride (DOTAP) were developed using thin film hydration technique. The cryo‐TEM analysis confirmed that the prepared optimized liposomal formulation (DFL‐2) was a mixture of small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs). In addition, the TEM analysis confirmed that the prepared liposomes were of spherical in shape having liposome size in the range of 500–900 nm and zeta potential of about +30 mV. The developed cationic liposomes exhibited sustained release profile of payload of 6‐MNA for over >12 h and about five times higher retention in the inflamed animal joints after 24 h (by scintigraphy of the joints) as compared to the plain 6‐MNA solution when administered by IA route. The anti‐inflammatory activity of prepared liposomal composition is evaluated by Freunds adjuvant induced arthritic model in rats. The liposomal formulation was well tolerated by all animals indicating good biocompatibility. Further, the cationic liposomal formulation treated group showed decreased erythrocyte sedimentation rate (ESR), and C‐reactive protein (CRP) level in comparison to the control and the standard groups in the in vivo study. The improved efficacy of the drug‐fortified liposomal formulation was due to the coupled effect of longer retention and sustained release of the active drug 6‐MNA in the joints. From the obtained results it could be concluded that the combined effect of the cationic charge on the drug‐fortified liposomes and the inherent affinity of the active agent towards the synovial joint tissues, coupled with slow release of the active drug due to double salt approach at the site of administration could potentially decrease the frequency of IA drug administration. Hence such a formulation could prove to be a therapeutic boon for the management of late stage arthritis.