Nanocarrier Encapsulated Recombinant IkBa as Potential Therapeutic Agent

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Protein therapeutics has been established as a potential weapon in anticancer armamentarium. The limitations of conventional chemo and radiotherapy, including poor response and undesired toxicity, have led researchers to contrive new ways to combat cancer. This quest has generated a whole array of new generation therapy, starting from gene therapy and encompassing protein as well as nanoparticle mediated therapy. Every approach has its own shortcoming, e.g. proteins have a very limited half-life and lose their active conformations if added from outside by conventional routes, thus confining their medical applications. Thus, a new strategy has been devised to deliver the therapeutic protein through nanocarrier based systems to protect its native conformation and burst or sustained release or both as per the requirement. Different types of nanocarriers have been tested as a suitable mode for delivery of therapeutic proteins. The current thesis explores the potential of recombinant I B as a therapeutic protein when delivered via polymeric nanocarriers. This thesis contains an Introduction about the importance of nanocarrier mediated recombinant I B delivery to cancer cells and the relevance of using recombinant I B as a therapeutic molecule. The Review of Literature contains the scholastic insights into the role of I B and previous work done in the field of therapeutic protein delivery and combination therapy, primarily focusing on 5- fluorouracil (5-FU) and curcumin. In Materials and Methods section, the chemicals, enzymes, bacterial strains, cancer cell lines, primers, antibodies etc. and the detailed protocol for each experiment have been elaborated. The Results and Discussions section starts with cloning of I B using cDNA obtained from mammalian cells, followed by purification as a bacterially expressed GST tagged recombinant protein and functional delivery via biodegradable polymeric hydrogel nanocarriers to the cervical carcinoma (HeLa) and glioblastoma (U87MG) cells. Then based on the result, the efficacy of recombinant I B has further been augmented by using it with poly L Lysine coated curcumin nanoparticles as a protein curcumin nanoformulation. Next, to understand molecular effects, I B transfected stable glioblastoma (U87-I B ) cell line was established and its response to common chemotherapeutic agent 5-Fluorouracil (5-FU) was enumerated through cell viability assay, cell cycle and doubling time analysis by flow cytometry and real time PCR for the expression of cyclins.
Supervisor: Siddhartha Sankar Ghosh