Bimal K. Bachhawat

The effect of surface-coupled antigen of liposomes in immunopotentiation

The effect of surface coupled antigens of liposomes on the immunological response has been investigated. Lysozyme was covalently coupled to neutral and positively charged liposomes using glutaraldehyde. Subcutaneous administration of these preparations stimulated a significant antibody response higher than that elicited by the antigen entrapped in neutral liposomes. Immunization by liposomal antigens together with complete Freunds adjuvant resulted in strong immune responses, highest with the antigen coupled to neutral and positively charged liposomes followed by the antigen entrapped in neutral liposomes. Primary and secondary immunization with lysozyme, both entrapped and coupled to liposomes, evoked an IgG response.

Design of liposomes to improve delivery of amphotericin-B in the treatment of aspergillosis

The efficacy and toxicity of free and liposome intercalated amphotericin-B (Amp-B) in controlling Aspergillosis, caused byAspergillus fumigatus in BALB/c mice were studied. Liposomal Amp-B had higher LD50 (8.1 mg/kg) as compared to that of the free drug (1.2 mg/kg). An improvement in the therapeutic index of the drug was observed with liposomal formulation of the drug. We also focussed on the effect of lipid composition and surface sugar in modulating the therapeutic potency of Amp-B. The most effective liposomal preparation was composed of egg phosphatidylcholine (EPC) : L-α-phosphatidylethanolamine, dipalmitoyl (DPPE): cholesterol (Chol) in the molar ratio of 6:1:3. Amp-B intercalated into mannose grafted liposomes (LD50 = 9.3 mg/kg) was more effective as compared to the other formulation tested.

Enhanced intracellular stability and efficacy of PEG modified dextranase in the treatment of a model storage disorder

A model for storage disorders was produced in the livers of mice by the administration of liposomally encapsulated FITC-dextran. Liposomally delivered dextranase was found to be more efficient in degrading the accumulated substrate as compared to the free enzyme. Dextranase was covalently modified with PEG, and liposomes were used as carriers for delivering the free and the modified enzyme to the liver at similar rates. The PEG-dextranase conjugate showed greater intracellular stability as compared to the native enzyme. Liposomally delivered PEG-dextranase, by virtue of its enhanced intracellular stability, could not only degrade the accumulated FITC-dextran, but could also prevent its further accumulation over a period of time. This enhanced intracellular stability of enzymes would be of importance in extending the catalytic life of therapeutically active enzymes and thereby improve their therapeutic potential for the treatment of intracellular storage disorders.

Liposome immune lysis assay (LILA) for gelonin

A complement-mediated liposome immune lysis assay using entrapped calcein was developed for a plant toxin gelonin. Gelonin was covalently coupled to DPPE, and then adsorbed on to the surface of liposomes. Such antigen-bearing liposomes when incubated with anti-gelonin antibody in the presence of guinea pig complement undergo lysis. The detection range is from 3 ng to 60 ng. The method was used to monitor isolation of gelonin by affinity chromatography. It was observed that a minor peak in addition to the major one comes with gelonin, shared common epitopes/epitope with gelonin in immunological reaction. This was further confirmed by SDS-gel electrophoresis indicating the former being an isoform of gelonin. A comparative study of the immunocross-reactivity of ricin and ricin A chain with anti-gelonin antibody was carried out. It was found that while ricin A chain cross-reacted extensively with gelonin antibody and intact ricin elicited little or no cross-reactivity. It is suggested that the present LILA may be employed for the detection and quantitation of ricin A chain by this LILA method.

Mannosylated Liposomes as Carriers for Hamycin in the Treatment of Experimental Aspergillosis in Balb/C Mice

Hamycin incorporated into mannosylated liposomes produced less toxicity and enhanced antifungal activity in experimental aspergillosis in balb/c mice in vivo. Incorporation of cholesterol into mannosylated liposomes led to a decrease in hamycin toxicity. The LD50 (mg/kg) values of hamycin contained in SPC/Chol/DPPE-Man (molar ratio 4:5:1) lipsomes was 2.8 whereas that in SPC/DPPE-Man liposomes (molar ratio 9:1) was 1.4. Incorporation of cholesterol into mannosylated liposomes increased the survival rates of infected animals: 70% survival was recorded after 7 days therapy as well as reduced fungal load in lung, liver, spleen and kidney. HPLC studies of distribution of hamycin in various tissues showed a reduction in the concentration of the liposomal drug in circulation compared to that observed for free drug and neutral liposomes after 1 hour.

Role of Surface Glycolipids - Natural or Synthetic Origin on the Biodistribution of Liposomes

Various sugar residues were incorporated on the surface of liposomes and its effect on the biodistribution and therapeutic importance were discussed here. Galactosylated liposomes were preferentially taken up by liver parenchymal cells whereas mannosylated liposomes were mainly localized in non-parenchyma1 cells. On the other hand, incorporation of dextran on the surface results in increased circulatory half-life of liposomes. The potential application of such liposomes as a carrier of drugs in the diseased condition is also discussed.

Enhanced in vivo catalytic activity of PEG-modified cellulase complex from Trichoderma reesei.

The cellulase complex from Trichoderma reesei was polyethylene glycol (PEG)‐modified with considerable retention of endo‐β‐1,4‐glucanase activity, as evaluated by the carboxymethylcellutase (CMCase) assay. While resistance towards heat denaturation was the same for either form, susceptibility towards proteolysis was slightly greater for the PEG‐conjugate, in contrast to most reports using other enzymes. The circulatory life of endoglucanase activity associated with the complex was enhanced upon PEG‐modification. This was confirmed by demonstrating degradation of chromogen‐tabelled substrate injected (i.v.) after 24hr of administration of the PEG‐ylated enzymes; in contrast the substrate remained undegraded in mice pretreated with the native complex. The PEG‐modified complex could be a good candidate for the treatment of pneumoconioses of textile workers exposed to cotton dust.