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Development of site-specific drug delivery systems |
Ph
D Students The
programme 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
objectives of the project 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
the reporting year, we have cloned and expressed the full length hemoglobin
receptor from Leishmania including the extreme 3/
end using Poly A tail based primer. Briefly, 893 bp fragment of the respective
gene from 3/
end was amplified using a forward primer
against peptide no.15 and reverse primer against Poly A tail by 3‘RACE.
Subsequently, putative start and stop codons were predicted from 5/
and 3/
end fragments, respectively. Appropriate
forward and reverse primers were designed and used to amplify a full length
(1416 bp) gene from L.donovani cDNA by PCR. The PCR product was cloned,
sequenced and hypothetically translated (471amino acids). The BLAST search
revealed that the cloned protein (Ld-HbR) has -79% homology with Trypanosoma,
52% with yeast, 49% with Drosophila and 46 % with Entamoeba hexokinase.
These results suggested that hemoglobin receptor in Leishmania is
possibly a hexokinase. Subsequently, we have shown that cloned protein retain
hemoglobin binding activity and the binding of the hemoglobin the recombinant
protein is inhibited in the presence of unlabelled hemoglobin and yeast
hexokinase. Moreover, we have prepared several truncated versions of Hb-receptor
protein and showed that the N-terminal end of the protein is the extracellular
hemoglobin binding domain and C-terminal is the cytoplasmic domain of the
receptor. Modulation
of macrophage metabolism through receptor-mediated delivery of biological
response modifiers Earlier,
we have shown that MBSA-MDP treated macrophages enhanced the secretion of a
variety of cytokines including IL-12, IL-6, TNF-a
etc. Moreover, we also have shown that treatment of macrophages with MBSA-MDP
resulted in an increase in the level of Rab7 and decrease in the content of
Rab5 in the macrophages. Accordingly, studies were initiated to determine the
role of respective cytokines in the modulation of particular Rab. Our
preliminary results suggest that IL-12 specifically upregulate the expression
of Rab7 and IL-6 induced the overexpression of Rab5.
Publications
Original
peer-reviewed articles 1.
Singh SB, Tandon R, Krishnamurthy G, Vikram R, Sharma N, Basu SK and
Mukhopadhyay A (2003) Rab5 mediated endosome-endosome fusion regulates
hemoglobin endocytosis in Leishmania donovani. EMBO J 22:5712-5722.
Reviews/
Proceedings 2. Mukhopadhyay A and Basu SK (2003) Intracellular delivery of drugs to macrophages. Adv Biochem Eng Biotechnol 84:183-209. |