1. Field-Induced Magnetic Nanoparticle Drug Delivery April 9, 2003 BME 273 Group 15 Team Leader : Ashwath Jayagopal (BME, EE, MATH) Members : Sanjay Athavale (BME) and Amit Parikh (BME) Advisor : Dr. Dennis Hallahan, Chairman of Radiation Oncology and Professor of Biomedical Engineering and Radiation Oncology, Vanderbilt University Course Instructor : Dr. Paul King, PE, Associate Professor of Biomedical Engineering, Mechanical Engineering and Anesthesiology, Vanderbilt University

2. Rationale for New Cancer Treatments Since 1990 16 million diagnosed with cancer, 5-year survival rate is 62%, estimated 1,400,000 new cases this year (American Cancer Society) Current treatments such as chemotherapy, surgery, and radiation therapy have disadvantages of extremely harmful side effects, high cost, and long duration Side effects include lower blood counts, flu-like symptoms, hair loss, swelling, scars and wounds, weight fluctuation, nausea, diarrhea, healthy cell death, general weakness of all systems All treatment methods have a range of costs from $1000-$100,000+ depending on the specific case and treatment (ACS) Multiple visits may be required for surgical procedures, chemotherapy, and radiation treatment. There is often a long wait associated with getting treatment

3. A Cancerous Tumor Matrix Matrix -- fibrous internal structure of a tumor, characterized by low permeability; contains cells in various stages of development, proliferation of blood vessels, connective tissue (collagen, laminin) The matrix contains an interstitial basement membrane that forms the framework to which cells are attached The basement membrane separates cells from mesenchymal connective tissue and provides spatial orientation and stability Important for the growth and differentiation of cells and angiogenesis within a tumor Tumors depend on blood vessels for nutrients and oxygen, therefore inhibition of angiogenesis is a goal VUMC Radiation Oncology, using laser scanning microscopy of a squamous cell carcinoma tumor. Highlighted strands indicate blood vessels, connective tissue fibers, and small tumor cells.

4. Project Objectives Develop and test an effective process for facilitating site-specific drug delivery to a tumor using the properties of paramagnetic iron oxide nanoparticles Use an externally applied magnetic field to precisely guide nanoparticle movement through a tumor matrix Enhance nanoparticle delivery using local irradiation and biological factors (an enzyme library which enhances tumor permeability to nanoparticles) Reduce problems associated with current treatment methods dramatically, especially harm to healthy tissue

5. Background Using recently developed methods, medications can be encased in magnetic nanoparticles Given antibody coating, avoids immune reaction, yet lasts in circulation Superparamagnetic iron oxide nanoparticles exhibit strong magnetic properties given an externally applied field, can be produced in uniform sizes and properties (Georgia Tech consortium, Dr. John Zhang, lead investigator) Guided missiles that can deliver tumor -killing drug to affected area without harming healthy tissue Shown here is a ring trap designed to control magnetic nanoparticles (iron oxide) by applying a current. Figure a.) shows the result in a nanoparticle-filled medium when no current is applied. In b.), a 0.35 A current is applied, and the nanoparticles accumulate at the center of the ring. This is analogous to what would be desirable in using a nanoparticle-based tumor drug delivery mechanismLee et. Al, Harvard Univ., J. Appl Phys 79/20, Nov. 2001.

6. Background Matrigel (BD Clontech) is a reconstituted basement membrane -- derived from Engelbroth-Holm-Swarm (EHS) mouse sarcoma, contains laminin, proteoglycan, collagen IV, enactin, and other basement membrane proteins responsible for transport in/out tumor Excellent tumor model, a rich source of ECM proteins, facilitates angiogenesis, nurtures tumor endothelial cells, can induce endothelial cell differentiation in the same pattern as a live tumor Is a cost-effective, safer alternative to in vivo, allows for observation of nanoparticles without imaging methods In order to enhance nanoparticle delivery, enzymes can be used (e.g. collagenase, amylase) to digest these basement membrane proteins to increase permeability Tumor irradiation enhances permeability of the membrane

7. Light micrograph showing angiogenesis in Matrigel solution. Endothelial cells have aligned in a 3-D spatial orientation to create blood vessels in a pattern similar to a tumor matrix. (BD Clontech 2003)

8. Methods Primary design is chemical in nature and involves degradation of the tumor matrix Nanoparticle-enzyme interaction is based on the “sticky” nature of the carbon coating Enzymes should specifically target the constituents of the tumor matrix Irradiation of the tumor weakens critical bonds found in the connective tissue of the tumor matrix

9. Methods Experimental Setup Washing of nanoparticles with PBS solution to eliminate impurities Coating of nanoparticles with specific enzymes including trypsin, collagenase, and amylase Irradiation of tumor Injection of nanoparticles into tumor matrix Application of magnetic field

10. Results Nanoparticles successfully moved through minimally resistant fluid environment. Uncoated Nanoparticles failed to move through Matrigel matrix after 4 hours. Nanoparticle-enzyme combination showed significant movement through the Matrigel matrix.

11. Interpretation of Results Coated nanoparticles show promise in allowing controlled, widespread movement throughout tumor matrix. In a clinical setting these Nanoparticle-enzyme combinations could potentially take the place of previously used nanoparticles. Design is simple, easy to construct, and requires no expensive electromagnets. Our design solves problems associated with clumping, homogenous distribution, and biocompatibility.

12. Future Considerations New Nanoparticle coatings -- Amylase/Collagenase Tumor irradiation by way of X-rays Higher Concentrations of degradation enzymes Incorporation of endothelial cells into Matrigel matrix Try to use different drugs as nanoparticle enclosures Use of cell-receptor mechanisms in conjunction with enzymes to enhance acceptance of nanoparticles into tumor matrix

13. Market Potential With further development of this technology and understanding of drug- nanoparticle relationships, could be applied to numerous tumor varieties, benefiting all cancer patients Drug delivery industry estimated worth $24 billion Implementation in any hospital Cost effective process is appealing at around $1000-2000 depending on drug used; would lower cost of cancer treatment in addition to reducing risks and side effects, if proven effective

14. Conclusions We have covered much ground in developing a process that could potentially be used to treat tumors Our process has been demonstrated to enhance drug-containing nanoparticle mobility through a tumor matrix (i.e. mobility with our enzyme mix, no movement without it); has been called “promising” by several researchers Market potential is strong Experimental procedures used are reproducible Amount of research that could be done within this area has no bounds

15. Acknowledgements Dr. Dennis Hallahan, VUMC Radiation Oncology, VU BME Dr. Ling Geng, VUMC Radiation Oncology Dr. Paul King, PE VU BME Chris Iversen, VUMC Radiation Oncology Sam Kuhn, VU BME Dr. John Zhang, Georgia Tech Biochemistry Dr. Jayaramn Rao, LSUMC (New Orleans)