Letter of Research Statement Cancer is a major leading cause of mortality and morbidity worldwide.

Cancer progresses from the uncontrolled growth of cells to the formation of a primary tumour mass, vascularisation and subsequent spread (metastasis) of cancer cells to other parts of the body where secondary tumours may form.1 Gliomas are well known primary brain tumours, which are classified by increasing grades of malignancy. The clinical outcome is very poor even with multimodality treatment, including surgery, radiation, chemotherapy and immunotherapy.2 The currently available anticancer therapeutics has less than optimal usefulness for brain tumour mainly because of delivery problems to the tumour. Several types of nanoparticles have been used to enhance drug delivery to glioma but liposome-based methods are considered to be among the promising methods.3 Development of nanobiotechnology-based innovative delivery shows a scope of innovation for glioma treatment. Prognosis of another major cancer type hepatocellular carcinoma (HCC) remains extremely poor and curative treatment (liver transplantation, surgical resection, and radiofrequency ablation) is also unsuccessful most of the cases.4 Development of selective internal radiotherapy (SIRT) to deliver high tumoricidal doses while limiting the radiation induced liver disease is the challenge for drug delivery to treat HCC. Currently, anti-cancer drugs are toxic to both tumour and normal cells, thus the efficacy of chemotherapy is often limited by the side-effects of the drug. Increased selectivity of drugs towards cancer cells will reduce the toxicity to normal tissue. Nanoscale delivery devices, such as dendrimers (spherical, branched polymers), silica-coated micelles, ceramic nanoparticles, and cross-linked liposomes can be targeted to cancer cells.5 Useful Nanosystems for tumour-targeted drug delivery are yet to be developed for targeted drug delivery. One of the biggest motivations behind nanoparticle-assisted combination chemotherapy is the ability to unify the pharmacokinetics of different drugs by simultaneously delivering multiple therapeutic agents to the target site. This would minimize the gap between in vitro and in vivo studies and enhance the possibility of bench-to-bedside translation. Novel nanoparticles can be preprogrammed to alter their structure and properties during the drug delivery process to make them most effective for the different extra- and intracellular delivery steps. Tumour-targeting principles include systemic passive targeting and active receptor targeting. Physical forces (e.g., electric or magnetic fields, ultrasound, hyperthermia or light) contribute to focusing and triggered activation of nanosystems. Biological drugs delivered with programmed nanosystems include plasmid DNA, siRNA, miRNA and other therapeutic nucleic acids.6

Letter of Research Statement

Evolved as a defense mechanism against RNAvirus, RNA interference (RNAi) is the phenomenon in which small interfering RNA (siRNA) of 2123 nucleotides in length silences the target gene by binding to its complementary mRNA and triggering the degradation of target mRNA.7 The major bottleneck in the development of siRNA therapeutics is the efficient delivery of siRNA to target cells.8 The crucial challenge of in vivo application of RNAi therapy is developing efficient delivery system to transport the siRNA into the tissue and finally into the cytoplasm.9 A series of carriers, including cationic lipids, peptides, synthetic or biodegradable polymers have been developed to enhance the cross membrane capability of siRNA. After entering the cytoplasm, the anticancer efficacy of siRNA will not display unless it is released from the endosome and silence the target mRNA. For this reason, fusogenic agents that can disrupt the endosome are generally required for the delivery system.10 Electrostatic interaction between siRNA and cationic lipid is relatively uncontrolled and the encapsulation of siRNA is therefore incomplete.11 Careful designing and backbone modification is the major challenge to prove it to be the best approaches to address these limitations. miRNA profiling has revealed distinct expression signatures in various human cancers, including glioma.12 Limitations associated with siRNA & miRNA therapy are the difficulty in systemic delivery to tissues and the need for modifications to enhance their stability and cellular uptake & an additional issue with this method is the transient nature of miRNA expression.13 One way of addressing these limitations would be to use a gene therapy approach that results in longer lasting miRNA expression. In this approach, a plasmid or viral vector driven by a pol II or III promoter upstream of short hairpin RNA (shRNA) is used to express a specific miRNA which is then loaded into RISC after being processed into mature miRNA by Dicer. These constructs have long-lasting silencing compared to double stranded miRNA mimetics and their capacity to express multiple miRNAs from one transcript. Examples include the use of adeno-associated virus (AAV) vectors as therapeutics for miRNA delivery to the liver and brain.14,15 However, the eliciting of immune response and the possibility of insertional mutagenesis is still a limiting factor. Radiation therapy (RT) is another successful therapeutic approach for cancer. Besides its cytotoxic effects on tumour cells, RT also have immunomodulatory effects.16 The idea of Targeted Radionuclide Therapy has been recently developed. The major advantage of targeted radionuclide therapy over either modality alone is that a molecule (eg, antibody, antibody fragment, peptide, or anticancer drug) specifically targeted to cancer cells delivers more destructive radiation than to healthy tissue, as opposed to either the chemical alone, or a dose of systemic radiation. The goal is to achieve a favorable benefit-risk profile for the agent(s) that are being used for the management of cancer.17

Letter of Research Statement

Nanotargeted radionuclide therapy is a modality that allows the delivery of a large payload of radiolabeled chemotherapeutics or imaging agents to tumour targets. Radiolabeled nanocarriers have to be developed to image and treat tumour models and these techniques will allow co-delivery of radiochemotherapeutics and simultaneous multifunctional imaging that is an advantageous characteristic of nanotargeted radionuclides for cancer imaging and therapy.18 As example, Ibritumomab tiuxetan is a two-part modality, consisting of the ibritumonab portion, a monoclonal antibody that recognizes a submicroscopic site on the surface of lymphoma cells called CD20, and tiuxetan, a special molecular that is a binding site for radioactive isotope (Yttrium-90 or Indium-111). The monoclonal antibody moiety of ibritumonab tiuxetan recognizes the CD20 protein on the surface of the (lymphoma) cancer cell and binds to that site. The radiation emitted from the isotope (Yttrium-90 or Indium-111) damages the cancer cells DNA/ helps in imaging and kills those malignant cells.19 Liposomes can be employed as lipid carriers of targeted radionuclide therapies. The combined liposome (nuclisome) therapy with both radioisotope and anticancer agent is at least 5 times more effective, at least in vitro, in eradicating cancer cells than the anticancer agent alone.20 Development of nanotargeted delivery system for radionuclide is still a new area to be explored. In all the cases, opportunity lies in the development of appropriate nanotargeted delivery system, assessing the performance of the delivery system, understanding the underlying mechanisms and optimizing the system for desired therapeutics. Through my previous academic & laboratory experience I have objectively prepared myself to explore in the area of targeted drug delivery and cancer therapeutics. In the Masters program, I have studied drug designing & molecular modeling, molecular mechanism of drug action, cancer biology & carcinogenesis, anticancer drugs, macromolecules, biocatalysis, polymer chemistry, biotechnology & drug delivery, combinatorial chemistry, and molecular mechanism of drug resistance in the Advanced Medicinal Chemistry courses. Advanced Pharmaceutical Chemistry courses included advanced analytical techniques like NMR spectroscopy, Mass spectroscopy, IR spectroscopy, X-ray crystallography, chromatography, chirality analysis and biostatistics. In the Masters thesis, I put a lot of effort into developing research skills and learning essential laboratory techniques. While working in laboratory for thesis and extramural projects I learned to isolate chemical compounds, use gel filtration chromatography, analyze by western blot, elucidate structure from Mass and NMR spectrum, apply microbiological assay techniques, culture cell lines, design animal models for laboratory testing, and interpret results applying biostatistics. In my current job as Lecturer in the Department of Pharmacy at Northern University Bangladesh, I have supervised two undergraduate projects - Chemistry and Pharmacology of Opioid Analgesics, Drug of Abuse and the Advanced Literature Study and Pharmacology of Neurodegenerative Diseases, Therapeutics and Advanced Literature Study. I believe these experiences will significantly help me to excel in research.

Letter of Research Statement

My future work will primarily focus on designing nanotargeted drug delivery system for cancer diagnosis and therapeutics using nanopolymeric materials, non-viral vectors and liposomes. To minimize the toxicity of anticancer drugs, effectively diagnose tumour & reach the drug at the target tumour cells and produce desired action, development of nanotargeted drug delivery is necessary for the clinical applications of the available anticancer drugs. The unified process for designing nanotargeted drug delivery for aggressive primacy tumours like glioma is still missing. My approach to filling in this gap in the literature will utilize a diversity of techniques and pharmaceutical engineering knowledge available at this time. Moreover, understanding the basic cellular mechanisms for a particular carcinoma I will develop an in vivo applicable delivery system for siRNA and miRNA.

Md. Rezaul Karim Applicant- Erasmus Mundus Doctorate fellowship for PhD in Nanomedicine

Letter of Research Statement

References: 1. Kakde D, Jain D, Shrivastava V, Kakde R, Patil AT. Cancer Therapeutics-Opportunities, Challenges and Advances in Drug Delivery. J App Pharm Sci 2011; 01(09): 01-10. 2. Osada H, Tokunaga T, Nishi M, Hatanaka H, Abe Y, Tsugu A, Kijima H, Yamazaki H, Ueyama Y, Nakamura M. Overexpression of the Neuropilin 1 (NRP1) Gene Correlated with Poor Prognosis in Human Glioma. Anticancer Research 2004; 24: 547-552. 3. Lu Y, Mahato RI (eds.), Pharmaceutical Perspectives of Cancer Therapeutics, Springer Science Business Media 2009; 258-59. 4. Box CV, Lacoeuille F, Roux J, Aube C, Garcion E, Lepareur N, Oberti F, Bouchet F, Noiret N, Garin E, Benot JP, Couturier O, Hindre F. Lipid Nanocapsules Loaded with Rhenium-188 Reduce Tumor Progression in a Rat Hepatocellular Carcinoma Model. PloseOne 2011; 6:3, e16926. 5. Brigger I, Dubernet C, Couvreur P. Nanoparticles in cancer therapy and diagnosis, Advanced Drug Delivery Reviews 2002; 54:631-651. 6. Lu Y, Mahato RI (eds.), Pharmaceutical Perspectives of Cancer Therapeutics, Springer Science Business Media 2009; 254. 7. Lu Y, Mahato RI (eds.), Pharmaceutical Perspectives of Cancer Therapeutics, Springer Science Business Media 2009; 399 8. Lu Y, Mahato RI (eds.), Pharmaceutical Perspectives of Cancer Therapeutics, Springer Science Business Media 2009; 407. 9. Takeshita F,Ochiya T. Therapeutic potential of RNA interference against cancer. Cancer Sci 2006; 97: 689696. 10. Heidel JD. Linear cyclodextrin-containing polymers and their use as delivery agents. Expert Opin Drug Deliv 2006; 3: 641646. 11. Spagnou S, Miller AD, Keller M. Lipidic carriers of siRNA: differences in the formulation, cellular uptake, and delivery with plasmid DNA. Biochemistry 2004; 43: 1334813356. 12. Godlewski J, Nowicki MO, Bronisz A, Williams S, Otsuki A, Nuovo G, Chaudhury AR, Herbert B, Newton HB,E. Antonio Chiocca EA,and Sean Lawler S. Targeting of the Bmi-1 Oncogene/Stem Cell Renewal Factor by MicroRNA-128 Inhibits Glioma Proliferation and Self-Renewal. Cancer Res 2008; 68:9125-9130. 13. Prakash TP, Allerson CR, Dande P, Vickers TA, Sioufi N, Jarres R, Baker BF, Swayze EE, Griffey RH, Bhat B. Positional effect of chemical modifications on short interference RNA activity in mammalian cells. J Med Chem 2005; 48(13):42474253. 14. Xia H, Mao Q, Paulson HL, Davidson BL. siRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol 2002; 20(10):10061010.

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15. Uprichard SL, Boyd B, Althage A, Chisari FV. Clearance of hepatitis B virus from the liver of transgenic mice by short hairpin RNAs. Proc Natl Acad Sci USA 2005; 102(3):773778. 16. Mozaffari F et al. NK-cell and T-cell functions in patients with breast cancer: effects of surgery and adjuvant chemo- and radiotherapy. Br J Cancer 2007; 97: 105111. 17. Ting G, Chang CH, Wang HE, Lee TW. Nanotargeted radionuclides for cancer nuclear imaging and internal radiotherapy. J Biomed Biotechnol 2010; 1-17. 18. Ting G, Chang CH, Wang H, Lee TW. Nanotargeted Radionuclides for Cancer Nuclear Imaging and Internal Radiotherapy. Journal of Biomedicine and Biotechnology 2010; 17. 19. Song H, Sgouros G. Radioimmunotherapy of solid tumors: searching for the right target. Curr Drug Deliv 2011; 8:26-44. 20. Song H, Sgouros G. Radioimmunotherapy of solid tumors: searching for the right target. Curr Drug Deliv 2011; 8:26-44.