(737f) Photothermal Therapy of Bladder Cancer and Melanoma Via Targeted Single-Walled Carbon Nanotubes

Single-walled carbon nanotubes (SWNTs) are unique in that they strongly absorb near-infrared (NIR) light, while biological systems have very low levels of absorption of NIR light. This project focuses on targeting the SWNTs using conjugation with the human annexin A5 protein. Annexin A5 (molecular weight of 36,000) is a monomeric protein that binds with high affinity to phophatidylserine (PS) in phospholipid bilayers. PS is the most abundant anionic phospholipid of the plasma membrane and is tightly segregated to the internal side of the plasma membrane in most cell types. Anionic phospholipids are largely absent from the external surfaces of resting mammalian cells under normal conditions. PS is expressed on the external surface of tumor cells and also of endothelial cells that line the blood vessels in tumors, but it is not expressed on the outside surface of the vascular endothelium in normal organs. The SWNTs serve as an NIR target resulting in tumor heating and ablation.

This project focuses on the photothermal therapy treatment of two types of cancer that initially occur on surfacesâ??bladder cancer and melanoma. Approximately 80% of bladder cancer patients have recurrent tumors starting with stage I superficial and quickly progressing towards more muscle invasive and metastatic malignancies with each recurrence. The high incidence of reappearance is believed to be due to the residual tumor left behind in a significant number of patients. The current form of treatment is a transurethral resection for lower grade tumors and cystectomy for higher grade masses. Novel treatments are needed for eliminating all bladder tumor cells with a minimum number of treatments, ideally one. One type of skin cancer, malignant metastatic melanoma (CMM), has a high mortality rate. When CCM becomes metastatic, patient survival rates fall as low as 5%. Over the last half-decade rapid advances in biologic therapeutics and nanotechnology have spawned a new generation of treatment options proving a rich opportunity to investigate combinatorial therapies.

In the study of this therapy for treating bladder cancer, in vitro binding tests confirmed a strong binding affinity of SWNT-annexin A5 to MB49 mouse and J82 human bladder cancer cells. Combining SWNT-annexin A5 heating via NIR light confirmed significant cell death as compared to untreated controls for both cell lines. The in vitro tests provided statistically significant validation for the potential of this targeted ablation therapy. In vivo testing on immune competent mice was conducted to confirm the efficacy of this treatment even further. A biodistribution study followed by FT-Raman analysis confirmed no non-specific accumulation of SWNT-annexin A5 in any organs. Using a 360^o diffuser to deliver NIR light to the inside of mouse bladders, NIR power tolerance tests confirmed that no healthy tissue damage occurred at 50 J/cm². In vivo therapy testing of MB49 cells implanted in the bladder of immune competent mice confirmed a promising treatment modality which is currently being further investigated with more testing using bioluminescence imaging of the cells in combination with fluorescence microscopy.

The work on treating melanoma used B16F10-luciferase mouse melanoma cells injected into immune competent mice. Plasma levels of SWNT-annexin A5 were monitored for up 24 hours after i.v. injection in B6 immune competent albino mice; plasma levels of the conjugate were negligible 1 hour after injection. The minimum injected dose of SWNT-annexin A5 used in combination with photothermal therapy using NIR irradiation was found for obtaining complete eradication of primary tumors (implanted subcutaneously into the flank). Having established the necessary photothermal therapy treatment conditions, we are currently undertaking an evaluation of the synergism between this photothermal therapy and the immunostimulants anti-PD1, anti-CTLA-4, anti-CD73, and cyclophosphamide to ascertain possible mechanisms of harnessing photothermal antigen production to eradicate distant metastases following primary tumor ablation.