Objectives and results of the work done in the last semester

Inhibition studies of nanoparticle internalization by the cell lines in the presence of Mannose 6-phosphate and antisera to mpr300 in separate experiments

Inhibition of nanoparticle internalization by 20mM mannose 6-phosphate. (A) DAPI, (B) blocking receptor with 20mM mannose 6-phosphate followed by incubation with nanoparticles, (C) merged image of A &B, (D) is the transmission image. Cell line is NIH3T3; Bar in the panel is 50 m. Colocalization of internalized nanoparticles with Lamp1 protein

Lyophilization of phosphomannan core nanoparticles and Coupling of Phosphomannan core (PMC) nanoparticles to DVS activated seralose matrix

The phosphomannan core nanoparticle prepared and characterized in my earlier studies were lyophilized in order to couple them to the DVS activated seralose matrix and were coupled to the same, with a long term objective to use this matrix for the purification of mannose 6phosphate receptor proteins. Isolation of Cathepsin D from Unio homogenate and Beta D Glucuronidase from goat liver tissue. Unio tissue was homogenized and passed through Lactose affinity chromatography column and the lactose specific lectin was isolated, the unbound fraction of the homogenate was processed further and was passed through Pepstatin A affigel column. The bound cathepsin D protein was eluted from the column using elution buffer. The eluted fractions were concentrated by amicon concentrator. The sample will be used for nanoparticle preparation and characterized. Goat liver tissue was homogenized and was processed further to isolate another lysosomal enzyme Beta D Glucuronidase Initially the homogenate was passed through Phenyl sepharose column and eluted fractions were assayed for enzyme activity. Active fractions were pooled and passed through DE-52 (anion exchanger). Unbound sample was removed by extensive washing with column buffer. Gradient elution was performed for enzyme purification, initially 50mM NaCl in column buffer was used for elution and then 100 mM and finally 200 Mm I could find the enzyme activity in 50mM fractions, active fractions were pooled and concentrated. To further purify the protein G-200 sephadex matrix was packed in 100ml column and the partially purified sample was passed through the matrix. The eluate after the void volume were carefully collected and assayed for enzyme activity and analyzed by SDS-PAGE as shown below, the band pattern of the enzyme has to be further confirmed

Preparation and characterization of galactan and galactomannan polysaccharide (from Strachnous seeds) nanoparticles with Mohammad

Programme of work for next semester

Preparation of nanoparticles for these lysosomal enzymes one prepared from the invertebrates and one from the vertebrates, and carrying out similar studies as described in my earlier reports, would further strengthen the role of the receptor in nanoparticle delivery in specific cell types. Preparation of antisera for PMC and PMP-BSA in rabbits to quantify the nanoparticles developed in the study. In vitro blotting techniques for recognition of nanoparticles by receptor/antibody to receptor blots Isolation plant polysaccharides (from Moringa oleifera seeds) and preparation of nanoparticles and characterization of the same.