Background: Cryosurgery is a minimally invasive cancer treatment using cryogenic temperatures. Intraoperative monitoring of iceball growth is an advantage of the treatment. Unfortunately, the edge of injury or cryolesion develops somewhere inside the edge of the iceball, and cannot be easily monitored. Recently we have shown the ability of TNF-α to enhance destruction within an iceball. This work focuses on identifying the dose and delivery of a novel TNF-α tagged gold nanoparticle (Aurimune^TM) which avoids systemic toxicity vs native TNF-α and show the ability of the nanotherapeutic to extend the edge of injury with the edge of the iceball for improved intraoperative monitoring.

Materials and Methods: Normal skin and human prostate carcinoma (LNCaP Pro 5) grown in the dorsal skin fold chamber (DSFC) in nude mice were pre treated with various doses of TNF-α and frozen after 4 hours by a 1 mm cryoprobe with a tip temperature of -100 °C. LNCaP Pro 5 was transfected with a fluorescent protein (DsRed express (Clontech, CA)) to monitor tumor growth throughout the chamber. The temperature profile in the DSFC was carefully monitored by the use of thermocouples and infrared thermography and injury assessed after 3 days by matching histology with perfusion defects under intravital microscopy. Tumors grown in the rear limb were frozen with a tip temperature of -100 °C and volume changes monitored with time. TNF-α was applied topically (in DSFC only) or injected i.v in to the tail vein. Aurimune^TM consists of a 33 nm size gold nanoparticle coated with PEG-Thiol and TNF-α and was administered i.v.

Results: The growth of tumor was verified by the red fluorescent protein and the extent of necrosis corresponded with the extent of stasis, proven by the use of a dye DiOC-7. The threshold of injury increases to higher temperatures albeit differentially, for both normal skin and tumor with an increase in TNF-α dosage. For a topically treated dose of 200 ng in the DSFC, the threshold of injury increased from -17 ± 3 °C to 2.5 ± 2.8 °C for tumor and from -27.7 ± 5 °C to -2.6 ± 2.7 °C for normal skin. A 5 μg dose of nTNF-α and Aurimune^TM increased the injury extent in tumors to temperatures of 0.9 ± 1.5 °C and -1.6 ± 3.5 °C tumor respectively. The hind limb tumors also witnessed a dramatic shrinkage in all TNF-α treated animals with complete remission observed in 3/7 for Aurimune^TM and 2/7 for nTNF-α injected animals. Systemic toxicity was noted in all nTNF-α groups but none with Aurimune^TM. These results suggest that it is possible to destroy all of a tumor within an iceball by pre-incubation with TNF-α and systemic toxicity can be avoided by Aurimune^TM.

Conclusion: We have shown that TNF-α injection 4 hours before cryosurgery augments cryoinjury in both tumor and normal tissue. Further, the appropriate dose of TNF-α was shown to extend the cryolesion to encompass the entire iceball. While both nTNF-α and Aurimune^TM were effective in achieving destruction inside the entire iceball, only Aurimune^TM accomplished this without toxicity.