Inducing Targeted Mild Hyperthermia in Murine Tumor Models through Photothermal Conversion of Near-infrared Light by Intratumoral Gold Nanorods

Targeted Hyperthermic therapy is an emerging modality in cancer treatment designed to selectively elevate tumor temperatures while minimizing damage to surrounding healthy tissue. This method describes a highly efficient, low resource intensive and reproducible way of inducing gold nanorod mediated hyperthermia in solid tumors. Employing direct intratumoral injection of gold nanorods followed by exposure to near-infrared light allows for precise, controlled, and highly localized heating of tumors.

The success of this protocol depends on efficient deep tissue activation of gold nanorods using the appropriate wavelength of near-infrared light, as well as accurate real-time monitoring of internal tumor temperature throughout the procedure. Begin daily tumor measurements starting around day seven, post-cancer cell injection, or whenever tumors become palpable. Prior to beginning the procedure, measure the width, length, and height of the tumor to calculate total tumor volume.

Aim to perform the procedure once tumors have reached an approximate volume of 50 millimeters cubed. For our model, this tumor volume was achieved between day 10 and 12 post-cancer cell injection. In a sterile biological safety cabinet, use a one mil syringe with a 31 gauge needle to draw up the appropriate volume of gold nanorods based off of each tumor volume.

Prior to gold nanorod injection, anesthetize the mouse using a nose cone delivering 2%isoflurane at a flow rate of 0.8 liters per minute. Apply eye lubricant to both eyes to prevent drying and minimize any potential laser damage. If necessary, shave excess fur around the tumor to ensure skin surface exposure and use ethanol to disinfect the tumor surface, wiping off any fur or debris.

Prior to laser exposure, inject gold nanorods intratumorally at a concentration of one microgram per millimeter cubed of tumor volume. If necessary, split up the injection between two intratumoral sites to ensure efficient distribution of golden nanorods inside the tumor. Ensure to wipe off any excess golden nanorods on the skin surface after injection.

For control group mice, administer an intratumoral injection of sterile PBS of the equivalent volume used for the golden nanorod treatment groups. Clean the intratumoral temperature probe with ethanol prior to insertion. Gently insert the temperature probe into the middle of the tumor mass.

Apply a uniform layer of aloe vera gel approximately five millimeters thick over the tumor surface to prevent skin ulceration and minimize the risk of thermal injury during treatment. Adjust the laser to line up directly above the middle of the tumor. Use the irradiance lookup table to determine the best laser height based on the required laser beam diameter to cover the tumor surface area.

Switch on the laser and thermoelectric cooling source. Then plug in the laser system to the power source. Turn on the thermoelectric cooling source.

The blue LED light will turn on and after 30 seconds, the temperature will stabilize at 25 degrees Celsius. Based off the irradiance lookup table, adjust the laser current output to achieve a target irradiance of approximately one watts per centimeter squared. In our experiment, a laser height of 1.5 centimeters, a beam diameter of 1.3 centimeters, and a laser current of 1800 milliamps resulted in an irradiance value of 0.82 watts per centimeter squared, which was optimized for our tumor model.

From this point onwards, ensure all individuals in the laser operation room wear certified laser safety glasses for the wavelength of light used. Set up a thermo camera adjacent to the laser platform directed at the mouse to monitor skin surface temperature during laser procedure. Begin recording on the thermo-coupled data logging software and ensure internal tumor temperature versus time graph is being displayed in real time.

Turn the laser safety key to enable source output. Begin laser administration by pushing down on the foot pedal At laser start time, the internal tumor temperature will start to increase, taking approximately 15 seconds to two minutes to enter target hyperthermic range of 42 to 48 degrees. If the internal tumor temperature approaches 48 degrees, take your foot off the pedal, allowing the temperature to drop back down.

This drop in temperature should become visible in real time on the temperature versus time display graph. Once the internal tumor temperature has dropped to around 43 degrees, re-enable laser administration using the foot pedal. The temperature should begin to rise again without dropping out of hyperthermic range.

Continue this on off cycling of the laser to ensure the tumor remains in hyperthermic range. The temperature versus time display graph should begin to reflect this on off cycling pattern of the laser. Throughout the maintenance of tumor hyperthermia, continually monitor skin surface temperature to ensure it stays below 50 degrees Celsius.

After five minutes of maintaining hyperthermia, release the foot pedal to stop laser administration and allow the internal tumor temperature to drop back down to around 37 degrees Celsius. Once the tumor has returned to a normal temperature, remove the temperature monitoring probe and wipe off any excess aloe vera gel on the tumor surface. Remove the mouse from nose cone anesthetic and monitor animal arousal prior to returning to cage For recovery post procedure, place the cage partially on a heat pad as shown here.

Ensure mice have returned to normal behavior before returning them to their housing facility. This table provides irradiance values in watts per centimeter squared for different laser current output settings in milliamps and working distances in centimeters. This table is used to determine the appropriate laser height based on the required beam diameter to cover the tumor surface and to achieve a target irradiance of approximately one watts per centimeter square.

Green highlighted values in this table indicate optimal irradiance levels near one watts per centimeter square. This figure shows representative internal tumor temperature profiles over time in milliseconds recorded by the internal tumor temperature probe throughout tumor exposure to near-infrared light. Figure A shows the on off laser cycling pattern in a gold nanorod injected mouse required to maintain the internal tumor temperature in hyperthermic range.

It highlights the importance of close monitoring of internal tumor temperature and cycling of laser administration to maintain hyperthermic range throughout the procedure. Figure B shows the average difference in internal tumor temperature between gold nanarod injected and control, or PBS injected mice throughout the five minute exposure to near-infrared light. Without the injection of gold nanorods, tumors exposed to near-infrared light will not heat into mild hyperthermic range.

PBS injected tumors plateau around 39 to 40 degrees Celsius in contrast to gold nanorod injected tumors which reach hyperthermic temperatures of 42 to 48 degrees. Our gold nanorod mediated targeted hyperthermic therapy induces tumor volume shrinkage across multiple mirroring cancer models within 48 to 72 hours post-treatment. Figure A shows tumor volume measurements in 4T1, a breast cancer model following gold nanorod mediated targeted hyperthermic therapy.

Figure B shows B16 F10, a melanoma model, and figure C shows CT26, a colorectal cancer model. A reduction in, or slowing of tumor volume growth within 48 to 72 hours following treatment, is indicative of a positive result. When performing this protocol, accurate real-time monitoring of internal tumor temperature is crucial to ensure that mild hyperthermic range is achieved, and that treatment is being delivered consistently.

Following this procedure, daily tumor volume measurements should be performed to record changes in tumor volume. Tumor regression within two to three days is a positive indicator of treatment success.