Development of site-specific drug delivery systems

Project Assistant
Ganga Krishnamurthy

Ph D Student
Vikram

This program aims at developing systems for intracellular delivery of drugs or pharmacologically active agents selectively to specific cell types by exploiting the efficiency of the process of receptor-mediated endocytosis. The broad objectives are i) exploration of heme acquisition mechanism in Leishmania donovani, ii) drug targeting for overcoming multidrug resistance in cancer chemotherapy, iii) modulation of macrophage metabolism through receptor-mediated delivery of biological response modifiers and iv) therapeutic strategies based on scavenger receptor-mediated delivery of antisense oligonucleotides.

Exploration of heme acquisition mechanism in L.donovani

 In this reporting year, we have further characterized the purified 46 kD hemoglobin-binding protein from Leishmania promastigote. We have raised the polyclonal antibody by immunizing mice with purified protein. Specificity of the polyclonal antibody was determined by ELISA and Western blot analysis. Immunofluorescence studies with this antibody revealed the presence of the 46 kDa protein, the putative hemoglobin receptor, in the flagellar pocket of Leishmania promastigote. Simultaneously, spleen from the immunized mice was dissected out and fusion was carried out with SP2/0 mouse myeloma cells to prepare monoclonal antibodies. Hybrids were assayed for the presence of antibodies from the culture supernatant by ELISA using immobilized purified protein. Cells from the positive hybrids were subsequently cloned by serial dilution. About 50 positive clones were selected. Further characterization of these antibody-secreting clones is under progress.

N-terminal sequence (AHFYFAI) of the purified protein revealed no apparent match in the database. Based on the N-terminal and a tentative internal sequence, we have designed degenerate sense and antisense primers. Subsequently, these primers were used to carry out a PCR reaction using Leishmania genomic DNA as template. Three fragments T1 (~500 bp), T2 (~400 bp) and B (~300 bp) were amplified which were subsequently cloned into pGEM-T vector and sequenced. The sequences were fed into the BLAST program to look for compatible sequences in the Leishmania genome. T2 fragment gave a high scoring match with the genomic sequences. Southern hybridization was done using T2 as well as the other two fragments as probe. T2 hybridized with a fragment of ~1 kb of the genomic DNA digest, while T1 and B did not give any clear signal. Further studies are in progress to screen for the desired clones using T2 as probe.

Modulation of macrophage metabolism through receptor-mediated delivery of biological response modifiers

Previously, we have shown that Salmonella containing phagosomes promotes fusion with early endosomes through enhance recruitment of Rab5, an early acting GTPase, and also deplete Rab7, a targeting signal for the late lysosomal compartment, there by survive in the macrophages. In the reporting year, we explored the mechanism of killing of intracellular Salmonella by scavenger receptor mediated delivery of MDP. We have shown that scavenger receptor-mediated delivery of muramyl dipeptide reduces the content of Rab5 and enhances the content of Rab7 in the cells as well as on Salmonella -containing phagosomes. Reduced content of the Rab5 inhibited the fusion of Salmonella -containing phagosomes with early endosomes and enhanced expression of Rab7 facilitated their targeting to lysosomes. Scavenger receptor-mediated delivery of muramyl dipeptide thus subverted the mechanism that Salmonella uses for its survival by diverting the vesicular transport pathway towards lysosomal killing. This finding offers a general therapeutic strategy against macrophage-associated infections caused by intracellular pathogens that survive in the host cell by resisting transport to lysosomes.

Publications

Original peer-reviewed articles

1.   *Srividya S, Roy RP, Basu SK and Mukhopadhyay A (2000) Scavenger receptor mediated delivery of muramyl dipeptide activates antitumour efficacy of macrophages by enhanced secretion of tumour suppressive cytokines. J Leukocyte Biol 67:683-690.(*in press last year, since published).

2.   *Hashim S, Mukherjee K, Raje M, Basu SK and Mukhopadhyay A (2000) Live Salmonella modulate expression of rab proteins to persist in a specialized compartment and escape transport to lysosomes. J Biol Chem 275:16281-16288 (*in press last year, since published).