Raghav Goel

Biodistribution of TNF-α-coated gold nanoparticles in an in vivo model system

AIM In this study, we describe the biodistribution of CYT-6091, a colloidal gold (Au)-based nanomedicine that targets the delivery of TNF-alpha to solid tumors. MATERIALS & METHODS A single intravenous injection of CYT-6091 coated with 5 microg TNF-alpha was given to human prostate tumor-bearing or naive (without tumor) nude mice. Tissues were harvested and analyzed at specific time points for Au nanoparticles by atomic emission spectroscopy and TNF-alpha by ELISA. RESULTS The two constituents of CYT-6091, TNF-alpha and Au, exhibited different behavior in blood, with TNF-alpha showing a faster decay than the Au nanoparticles. Between 0 and 4 h after injection, TNF-alpha showed a preferential accumulation in the tumor. Au was observed to accumulate preferentially in the liver between 4 and 12 h, and showed some clearance over time (4 months). CONCLUSION These data suggest that CYT-6091 delivers TNF-alpha preferentially to the tumor and that upon TNF-alpha degradation, the liver takes up Au, which is cleared slowly over time.

TNF-α–based accentuation in cryoinjury—dose, delivery, and response

Cryosurgery is a minimally invasive cancer treatment using cryogenic temperatures. Intraoperative monitoring of iceball growth is an advantage of the treatment. However, whereas the iceball can be easily visualized, destruction within the iceball is incomplete and the means to monitor the “kill zone” are urgently needed. Recently, we have shown the ability of tumor necrosis factor-α (TNF-α) to enhance destruction within an iceball. To avoid systemic toxicity, we delivered TNF-α selectively to the tumor by a gold nanoparticle of 30-nm diameter (CYT-6091) tagged with TNF-α and thiol-derivatized polyethylene glycol. Using a dorsal skin fold chamber (DSFC) in a nude mouse, both normal skin and human prostate carcinoma (LNCaP Pro 5) were pretreated with soluble TNF-α (topically or i.v.) or CYT-6091 (i.v.) and frozen after 4 h. The cryolesion was assessed after 3 days by comparing histologic necrosis with perfusion defects. Hind limb tumors were also treated by visibly encompassing the tumor with an iceball and assessing gross changes over time. A 5-μg dose of soluble TNF-α or CYT-6091 increased the temperature threshold of necrosis in the tumor in the DSFC from −14.0 ± 1.6°C (n = 6) to 0.9 ± 1.5°C (n = 6) and −1.5 ± 3.7°C (n = 6), respectively. In hind limb tumors, the same dose resulted in significant tumor shrinkage and remission in 2 of 8 (for soluble TNF-α) and in 3 of 8 (for CYT-6091). The nanoparticle alone group without TNF-α increased the temperature threshold of necrosis to −7.0 ± 2.3°C in the tumor in the DSFC and more shrinkage of the tumor in the hind limb when compared with cryo alone treatment. Systemic toxicity was noted in all soluble TNF-α groups but none with CYT-6091. These results suggest that it is possible to destroy all of a tumor within an iceball by preincubation with TNF-α and systemic toxicity can be avoided by CYT-6091. [Mol Cancer Ther 2007;6(7):2039–47]

ADJUVANT APPROACHES TO ENHANCE CRYOSURGERY

Molecular adjuvants can be used to enhance the natural destructive mechanisms of freezing within tissue. This review discusses their use in the growing field of combinatorial or adjuvant enhanced cryosurgery for a variety of disease conditions. Two important motivations for adjuvant use are: (1) increased control of the local disease in the area of freezing (i.e., reduced local recurrence of disease) and (2) reduced complications due to over-freezing into adjacent tissues (i.e., reduced normal functional tissue destruction near the treatment site). This review starts with a brief overview of cryosurgical technology including probes and cryogens and major mechanisms of cellular, vascular injury and possible immunological effects due to freeze-thaw treatment in vivo. The review then focuses on adjuvants to each of these mechanisms that make the tissue more sensitive to freeze-thaw injury. Four broad classes of adjuvants are discussed including: thermophysical agents (eutectic forming salts and amino acids), chemotherapuetics, vascular agents and immunomodulators. The key issues of selection, timing, dose and delivery of these adjuvants are then elaborated. Finally, work with a particularly promising vascular adjuvant, TNF-alpha, that shows the ability to destroy all cancer within a cryosurgical iceball is highlighted.

Tumor necrosis factor-α–induced accentuation in cryoinjury: mechanisms in vitro and in vivo

Cryosurgical treatment of solid cancer can be greatly assisted by further translation of our finding that a cytokine adjuvant tumor necrosis factor-α (TNF-α) can achieve complete cancer destruction out to the intraoperatively imaged iceball edge (-0.5°C) over the current clinical recommendation of reaching temperatures lower than -40°C. The present study investigates the cellular and tissue level dose dependency and molecular mechanisms of TNF-α-induced enhancement in cryosurgical cancer destruction. Microvascular endothelial MVEC and human prostate cancer LNCaP Pro 5 (LNCaP) cells were frozen as monolayers in the presence of TNF-α. Normal skin and LNCaP tumor grown in a nude mouse model were also frozen at different TNF-α doses. Molecular mechanisms were investigated by using specific inhibitors to block nuclear factor-κB–mediated inflammatory or caspase-mediated apoptosis pathways. The amount of cryoinjury increased in a dose-dependent manner with TNF-α both in vitro and in vivo. MVEC were found to be more cryosensitive than LNCaP cells in both the presence and the absence of TNF-α. The augmentation in vivo was significantly greater than that in vitro, with complete cell death up to the iceball edge in tumor tissue at local TNF-α doses greater than 200 ng. The inhibition assays showed contrasting results with caspase-mediated apoptosis as the dominant mechanism in MVEC in vitro and nuclear factor-κB–mediated inflammatory mechanisms within the microvasculatures the dominant mechanism in vivo. These results suggest the involvement of endothelial-mediated injury and inflammation as the critical mechanisms in cryoinjury and the use of vascular-targeting molecules such as TNF-α to enhance tumor killing and achieve the clinical goal of complete cell death within an iceball. [Mol Cancer Ther 2008;7(7):2547–55]

Pre-Conditioning Cryosurgery: Cellular and Molecular Mechanisms and Dynamics of TNF-α Enhanced Cryotherapy in an in vivo Prostate Cancer Model System

Cryosurgery is increasingly being used to treat prostate cancer; however, a major limitation is local recurrence of disease within the previously frozen tissue. We have recently demonstrated that tumor necrosis factor alpha (TNF-α), given 4h prior to cryosurgery can yield complete destruction of prostate cancer within a cryosurgical iceball. The present work continues the investigation of the cellular and molecular mechanisms and dynamics of TNF-α enhancement on cryosurgery. In vivo prostate tumor (LNCaP Pro 5) was grown in a dorsal skin fold chamber (DSFC) on a male nude mouse. Intravital imaging, thermography, and post-sacrifice histology and immunohistochemistry were used to assess iceball location and the ensuing biological effects after cryosurgery with and without TNF-α pre-treatment. Destruction was specifically measured by vascular stasis and by the size of histologic zones of injury (i.e., inflammatory infiltrate and necrosis). TNF-α induced vascular pre-conditioning events that peaked at 4h and diminished over several days. Early events (4-24 h) include upregulation of inflammatory markers (nuclear factor-κB (NFκB) and vascular cell adhesion molecule-1 (VCAM)) and caspase activity in the tumor prior to cryosurgery. TNF-α pre-conditioning resulted in recruitment of an augmented inflammatory infiltrate at day 3 post treatment vs. cryosurgery alone. Finally, pre-conditioning yielded enhanced cryosurgical destruction up to the iceball edge at days 1 and 3 vs. cryosurgery alone. Thus, TNF-α pre-conditioning enhances cryosurgical lesions by vascular mechanisms that lead to tumor cell injury via promotion of inflammation and leukocyte (esp. neutrophil) recruitment.

Use of Tumor Necrosis Factor–alpha-coated Gold Nanoparticles to Enhance Radiofrequency Ablation in a Translational Model of Renal Tumors

OBJECTIVES Radiofrequency ablation (RFA) has been most effective when the tumors are small, exophytic, and away from vital structures. We enlarged the size of the ablation kill zone by infusing a 30-nm tumor necrosis factor-alpha and polyethylene glycol-coated gold nanoparticle (CYT-6091, CytImmune Sciences, Inc.) before ablation in a rabbit kidney tumor model. MATERIALS AND METHODS A total of 37 New Zealand White rabbits had VX-2 tumors implanted into their bilateral kidneys; they were then split into 3 treatment groups of 10 rabbits each and a sham group of 7 rabbits as follows: (1) CYT-6091 only, (2) RFA only, (3) CYT-6091 followed 4 hours later by RFA. Gross and microscopic measurements of the ablation size as well as histologic analysis using hematoxylin and eosin staining were performed to determine the effect of CYT-6091 on the ablation. RESULTS The RFA + CYT-6091 group had a larger zone of complete cell death than the RFA-only group when measured on microscopic examination (0.30 +/- 0.07 vs 0.23 +/- 0.03 mL, P = .03). The zone of partially ablated tissue was smaller in the RFA + CYT-6091 group than in the RFA-only group (0.08 +/- 0.02 vs 0.13 +/- 0.05 mL, P = .01). CONCLUSIONS We have demonstrated the efficacy of CYT-6091 in enhancing RFA in a translational kidney tumor model. The potential usage of CYT-6091 to improve RFA of renal cell carcinoma merits further study.

Tumor necrosis factor-α-induced accentuation in cryoinjury

Cryosurgical treatment of solid cancer can be greatly assisted by further translation of our finding that a cytokine adjuvant tumor necrosis factor-α (TNF-α) can achieve complete cancer destruction out to the intraoperatively imaged iceball edge (-0.5°C) over the current clinical recommendation of reaching temperatures lower than -40°C. The present study investigates the cellular and tissue level dose dependency and molecular mechanisms of TNF-α-induced enhancement in cryosurgical cancer destruction. Microvascular endothelial MVEC and human prostate cancer LNCaP Pro 5 (LNCaP) cells were frozen as monolayers in the presence of TNF-α. Normal skin and LNCaP tumor grown in a nude mouse model were also frozen at different TNF-α doses. Molecular mechanisms were investigated by using specific inhibitors to block nuclear factor-κB–mediated inflammatory or caspase-mediated apoptosis pathways. The amount of cryoinjury increased in a dose-dependent manner with TNF-α both in vitro and in vivo. MVEC were found to be more cryosensitive than LNCaP cells in both the presence and the absence of TNF-α. The augmentation in vivo was significantly greater than that in vitro, with complete cell death up to the iceball edge in tumor tissue at local TNF-α doses greater than 200 ng. The inhibition assays showed contrasting results with caspase-mediated apoptosis as the dominant mechanism in MVEC in vitro and nuclear factor-κB–mediated inflammatory mechanisms within the microvasculatures the dominant mechanism in vivo. These results suggest the involvement of endothelial-mediated injury and inflammation as the critical mechanisms in cryoinjury and the use of vascular-targeting molecules such as TNF-α to enhance tumor killing and achieve the clinical goal of complete cell death within an iceball. [Mol Cancer Ther 2008;7(7):2547–55]

Tumor necrosis factor-alpha induced enhancement of cryosurgery

Local recurrence of cancer after cryosurgery is related to the inability to monitor and predict destruction of cancer (temperatures > -40°C) within an iceball. We previously reported that a cytokine adjuvant TNF-α could be used to achieve complete cancer destruction at the periphery of an iceball (0 to -40°C). This study is a further development of that work in which cryosurgery was performed using cryoprobes operating at temperatures > -40°C. LNCaP Pro 5 tumor grown in a dorsal skin fold chamber (DSFC) was frozen at -6°C after TNF-α incubation for 4 or 24 hours. Tumors grown in the hind limb were frozen with a probe tip temperature of -40°C, 4 or 24 hours after systemic injection with TNF-α. Both cryosurgery alone or TNF-α treatment alone caused only a minimal damage to the tumor tissue at the conditions used in the study. The combination of TNF-α and cryosurgery produced a significant damage to the tumor tissue in both the DSFC and the hind limb model system. This augmentation in cryoinjury was found to be time-dependent with 4-hour time period between the two treatments being more effective than 24-hour. These results suggests the possibility of cryotreatment at temperatures > -40°C with the administration of TNF-α.

Biodistribution of TNF-alpha coated gold nanoparticles in an in vivo cancer model

Over the past several years, there has been an increasing interest in the use of nanoparticles as a tool for treatment of cancer. We have shown tremendous augmentation and control (without toxicity) of both heat and cold-based thermal therapy for cancer treatment with a gold based nanodrug-CYT-6091 (Cytimmune Sciences, Inc.) [1–3]. To reach the full potential of these nanodrugs for both stand-alone solid cancer treatment and as adjuvant to thermal therapy, there is a need to understand the in vivo biodistribution and their short-term and long-term tissue interaction.Copyright