Name: in vitro and in vivo delivery of magnetic nanoparticle hyperthermia using a custom-built delivery system
Uploaded: 2020-07-02T14:30:00
Description: Watch this Scientific Journal Video about In Vitro and In Vivo Delivery of Magnetic Nanoparticle Hyperthermia Using a Custom-Built Delivery System at JoVE.com

The study was funded by grant numbers: NCI P30 CA023108 and NCI U54 CA151662.

The Dartmouth College Animal Care and Use Program is accredited by the American Association for the Accreditation of Laboratory Animal Care (iAAALAC) and adheres to all UDSA and NIH (Office of Laboratory Animal Welfare) guidelines and regulations. All in vivo studies were approved by the Dartmouth College Institutional Animal Care and Use Committee (IACUC). Euthanasia procedure adhere to the 2020 AVMA Guidelines for the Euthanasia of Animals.
1. Instrumentation/design of the system
...

<ol>
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V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitigation+of+eddy+current+heating+during+magnetic+nanoparticle+hyperthermia+therapy.">Mitigation of eddy current heating during magnetic nanoparticle hyperthermia therapy.</a> International Journal of Hyperthermia. 32 (7), 735-748 (2016).</li><li>Johannsen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+hyperthermia+of+prostate+cancer+using+magnetic+nanoparticles:+Presentation+of+a+new+interstitial+technique.">Clinical hyperthermia of prostate cancer using magnetic nanoparticles: Presentation of a new interstitial technique.</a> International Journal of Hyperthermia. 21 (7), 637-647 (2005).</li><li>Horsman, M. R., Overgaard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperthermia:+a+Potent+Enhancer+of+Radiotherapy.">Hyperthermia: a Potent Enhancer of Radiotherapy.</a> Clinical Oncology. 19 (6), 418-426 (2007).</li><li>Petryk, A. A., Giustini, A. J., Gottesman, R. E., Kaufman, P. A., Hoopes, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+nanoparticle+hyperthermia+enhancement+of+cisplatin+chemotherapy+cancer+treatment.">Magnetic nanoparticle hyperthermia enhancement of cisplatin chemotherapy cancer treatment.</a> International Journal of Hyperthermia. 29 (8), 845-851 (2013).</li><li>Peeken, J. C., Vaupel, P., Combs, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+hyperthermia+into+modern+radiation+oncology:+What+evidence+is+necessary.">Integrating hyperthermia into modern radiation oncology: What evidence is necessary.</a> Frontiers in Oncology. 7, 132 (2017).</li><li>Petryk, A. A., Giustini, A. J., Gottesman, R. E., Trembly, B. S., Hoopes, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+magnetic+nanoparticle+and+microwave+hyperthermia+cancer+treatment+methodology+and+treatment+effect+in+a+rodent+breast+cancer+model.">Comparison of magnetic nanoparticle and microwave hyperthermia cancer treatment methodology and treatment effect in a rodent breast cancer model.</a> International Journal of Hyperthermia. 29 (8), 819-827 (2013).</li><li>Stigliano, R. V., Shubitidze, F., Petryk, A. A., Tate, J. A., Hoopes, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+nanoparticle+hyperthermia:+predictive+model+for+temperature+distribution.">Magnetic nanoparticle hyperthermia: predictive model for temperature distribution.</a> Energy-based Treatment of Tissue and Assessment VII. 8584, 858410 (2013).</li><li>Dewhirst, M., Viglianti, B. L., Lora-Michiels, M., Hoopes, P. J., Hanson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+dose+requirement+for+tissue+effect:+experimental+and+clinical+findings.">Thermal dose requirement for tissue effect: experimental and clinical findings.</a> Thermal Treatment of Tissue: Energy Delivery and Assessment II. 4954, 37 (2003).</li><li>Duval, K. E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunogenetic+effects+of+low+dose+(CEM43+30)+magnetic+nanoparticle+hyperthermia+and+radiation+in+melanoma+cells.">Immunogenetic effects of low dose (CEM43 30) magnetic nanoparticle hyperthermia and radiation in melanoma cells.</a> International Journal of Hyperthermia. 36, 37-46 (2019).</li><li>Giustini, A. J., Petryk, A. A., Cassim, S. M., Tate, J. A., Baker, I., Hoopes, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+Nanoparticle+Hyperthermia+in+Cancer+Treatment.">Magnetic Nanoparticle Hyperthermia in Cancer Treatment.</a> Nano LIFE. 01, 17-32 (2010).</li><li>Hoopes, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumoral+iron+oxide+nanoparticle+hyperthermia+and+radiation+cancer+treatment.">Intratumoral iron oxide nanoparticle hyperthermia and radiation cancer treatment.</a> Thermal Treatment of Tissue: Energy Delivery and Assessment IV. 6440, (2007).</li><li>Semiatin, S. L., Zinn, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coil+design+and+fabrication+basic+design+and+modifications.">Coil design and fabrication basic design and modifications.</a> Heat Treating. , 32-41 (1988).</li><li>Maxwell, J. 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The design and implementation of this system provides the ability to conduct accurate and reproducible in vitro and in vivo magnetic nanoparticle hyperthermia experiments. It is critical that the system is designed such that the AMF frequency and field strength are adequately matched to the magnetic nanoparticle type, concentration, and the tissue location and temperature desired. Additionally, the accurate monitoring of the temperature in real time is crucial for safety and the calculation of an accurate thermal dose (c...

Hyperthermia has historically been used in cancer therapy, either alone or in combination with other treatments. Although it has a long history of use, the most advantageous method for delivering this treatment is still being debated and is dependent on the disease site and location. Methods for hyperthermia delivery include microwave, radiofrequency, focused ultrasound, laser, and metallic nanoparticles (such as gold or iron oxide)1,2,3,4. These methods of delivery can lead to a range of treatment temperatures from fever-level through to hundreds of degrees C. The biological effect of hyperthermia depends primarily on the temperatures used and the duration of the treatment5. For this manuscript and purpose, we are focusing on magnetic nanoparticle hyperthermia (mNPH). This method allows for focused, localized, well monitored, and controlled temperature changes, using non-toxic, FDA approved, iron oxide nanoparticles.
One pitfall of other hyperthermia modalities is a lack of precise cellular targeting; hyperthermia does not a have an inherently high therapeutic ratio, therefore, careful thermometry and targeting is necessary6. mNPH allows for systemic or intratumoral injection of mNPs, with heat only being generated where the mNPs are located, thus targeting the treatment to the tumor directly. mNPH can be effective when the magnetic nanoparticles are located inside or outside of the cell. For cancer therapy, the general overview of mNPH is that the magnetic nanoparticles are injected (intratumorally or intravenously), then an alternating magnetic field is applied, causing the nanoparticle magnetic poles to constantly realign, leading to a localized heating of the cells and tissue associated with the nanoparticles7,8. By adjusting the volume of nanoparticles and the frequency/strength of the alternating magnetic field (AMF), it is possible to carefully control the temperature generated within the tissue.
This treatment works well in tumors that are near the body surface, as deeper tumors require stronger AMF so the risk of eddy current heating increases9. There is evidence of hyperthermia being used clinically as a monotherapy, however, oftentimes hyperthermia is combined with radiation therapy or chemotherapy, leading to a more targeted anti-cancer effect10,11,12. Clinical evidence of hyperthermia working in combination with radiation therapy is reviewed in a previous publication13. Our lab has successfully treated a variety of animals, from mice to pigs and spontaneous canine cancers, using the mNPH method12,14,15. This protocol is designed for those interested in investigating the effects of localized hyperthermia treatment, either alone or in combination with other therapies.
One of the most important factors in hyperthermia is being able to measure and understand, in real time, the thermal dose being delivered to the target/tumor tissue. A standard way of calculating and comparing dose is through demonstration of the cumulative equivalent minutes of heating at 43 &#176;C; this algorithm allows for the comparison of doses independent of the delivery system, maximum and minimum temperatures (within a specific range) and heat up/cool down parameters5,16. The CEM calculation works best for temperatures between 39-57 &#176;C5. For example, in some of the studies we have performed, we have chosen a thermal dose of CEM43 30 (i.e., 30 min at 43 &#176;C). Choosing this dose allowed us to look at a safe, effective, immunogenetic effects in vitro, both alone, and in combination with a single dose of radiation17.
With magnetic nanoparticle hyperthermia, there are several factors that need to be considered in building an appropriate delivery system. The instrumentation design includes important safety factors, such as the use of a chiller to ensure the magnetic field delivery equipment remains cool even when operated at high power, and fail-safe procedures that prevent the system from being turned on if all temperature, power assessment, and control systems have not been activated. Additionally, there are important biological factors that need to be considered for both in vivo and in vitro situations. When using cultured cells, it is necessary to treat in growth media and maintain at a consistent viable temperature to avoid physiological changes that could affect results. For individual nanoparticle types, it is important to know the specific absorption rate (SAR) when calculating AMF based heating parameters. Similarly, it is important to know the mNP/Fe concentration, in cells and tissues, that is necessary to achieve the desired heating. In vivo methods require even more attention to detail since the animal must be maintained under anesthesia during treatment and the animal&#8217;s core body temperature maintained at a normal level throughout the treatment. Allowing for the animal&#8217;s body temperature to drop, as happens under anesthesia, can affect the overall results, with respect to the thermal dose of the tissue being treated.
In this manuscript, we discuss the methods used to design and construct a versatile magnetic nanoparticle hyperthermia system, as well as important use factors that need to be considered. The system described allows for the robust, consistent, biologically appropriate, safe, and well-controlled delivery of magnetic nanoparticle hyperthermia. Finally, it should be noted that the mNPH studies we conduct often involve other therapies such as radiation, chemotherapy, and immunotherapy. For these results to be meaningful, it is important to determine how the delivered heat can affect the efficacy and/or safety-toxicity of other modalities (or vice versa) and the well-being of the animal. For this reason and the dosimetry and therapeutic situations previously mentioned, it is essential to pay strict attention to the magnetic nanoparticle hyperthermia dosing accuracy and the continuous core and target temperature measurements. The goal of this protocol is to provide a straightforward, consistent method and description for the delivery of safe and effective magnetic nanoparticle hyperthermia.

<table>
	<tbody>
		<tr>
			<td>.25% Trypsin</td>
			<td>Corning</td>
			<td>45000-664</td>
			<td>available from many companies</td>
		</tr>
		<tr>
			<td>1.5 mL tubes</td>
			<td>Eppendorf</td>
			<td>Eppendorf 22363204</td>
			<td>available from many companies</td>
		</tr>
		<tr>
			<td>B16F10 murine melanoma cells</td>
			<td>American Type Culture Collection</td>
			<td>CRL-6475</td>
			<td></td>
		</tr>
		<tr>
			<td>C57/Bl6 mice</td>
			<td>Charles river</td>
			<td>027C57BL/6</td>
			<td>6-week-old female mice</td>
		</tr>
		<tr>
			<td>Chiller</td>
			<td>Thermal Care</td>
			<td>NQ 5 series</td>
			<td>chiller that cools the coil</td>
		</tr>
		<tr>
			<td>Coolant fluid</td>
			<td>Dow Chemical Company</td>
			<td>Dowtherm SR-1</td>
			<td>antenna cooling fluid</td>
		</tr>
		<tr>
			<td>Fetal Bovine serum</td>
			<td>Hyclone</td>
			<td>SH30071</td>
			<td>available from many companies</td>
		</tr>
		<tr>
			<td>fiber optic probes, software and chassis</td>
			<td>FISO</td>
			<td></td>
			<td>FISO evolution software used to read the temperatures</td>
		</tr>
		<tr>
			<td>IR camera</td>
			<td>Flir</td>
			<td></td>
			<td>infrared camera to monitor unintentional heating</td>
		</tr>
		<tr>
			<td>iron oxide nanoparticles</td>
			<td>micromod Partikeltechnologie GmbH</td>
			<td>Bionized NanoFerrite</td>
			<td>dextran coated iron oxide nanoparticles</td>
		</tr>
		<tr>
			<td>mouse coil, solenoid</td>
			<td>Fluxtrol</td>
			<td>custom built</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin/streptomycin</td>
			<td>Corning</td>
			<td>45000-652</td>
			<td>available from many companies</td>
		</tr>
		<tr>
			<td>RF generator</td>
			<td>Huttinger</td>
			<td>TIG 10/300</td>
			<td>power source</td>
		</tr>
		<tr>
			<td>RPMI media</td>
			<td>Corning</td>
			<td>45000-396</td>
			<td>available from many companies</td>
		</tr>
	</tbody>
</table>

in vitro and in vivo delivery of magnetic nanoparticle hyperthermia using a custom-built delivery system

Hyperthermia has long been used in the treatment of cancer. Techniques have varied from the intra-tumoral insertion of hot iron rods, to systemically delivered tumor antibody-targeted magnetic nanoparticles, at temperatures from 39 &#730;C (fever-level) to 1,000 &#730;C (electrocautery) and treatment times from seconds to hours. The temperature-time relationship (thermal dose) dictates the effect with high thermal doses resulting in the tissue ablation and lower thermal doses resulting in sublethal effects such as increased blood flow, accumulation of drugs and immune stimulation. One of the most promising current medical therapies is magnetic nanoparticle hyperthermia&#160; (mNPH). This technique involves activating magnetic nanoparticles, that can be delivered systemically or intratumorally, with a non-invasive, non-toxic alternating magnetic field. The size, construct and association of the magnetic nanoparticles and the frequency and field strength of the magnetic field are major heating determinants. We have developed sophisticated instrumentation and techniques for delivering reproducible magnetic nanoparticle hyperthermia in large and small animal models and cultured cells. This approach, using continuous, real time temperature monitoring in multiple locations, allows for the delivery of well-defined thermal doses to the target tissue (tumor) or cells while limiting non-target tissue heating. Precise control and monitoring of temperature, in multiple sites, and use of the industry standard algorithm (cumulative equivalent minutes at 43 &#730;C /CEM43), allows for an accurate determination and quantification of thermal dose. Our system, which allows for a wide variety of temperatures, thermal doses, and biological effects, was developed through a combination of commercial acquisitions and inhouse engineering and biology developments. This system has been optimized in a manner that allows for the rapid conversion between ex vivo, in vitro, and in vivo techniques. The goal of this protocol is to demonstrate how to design, develop and implement an effective technique and system for delivering reproducible and accurate magnetic nanoparticle therapy (mNP) hyperthermia.

In vitro studies 
Cells will only achieve and maintain the desired temperature and thermal dose if the amount and concentration of the magnetic nanoparticles/iron and the AMF are appropriately matched. When using magnetic nanoparticles to heat cells in vitro (and in vivo), it should be noted that to achieve hyperthermia in cells with internalized magnetic nanoparticles, a specific level of intracellular mNP/Fe will be necessary, and number and proximity of mNP loaded cells, to each other, will be ne...

Watch this Scientific Journal Video about In Vitro and In Vivo Delivery of Magnetic Nanoparticle Hyperthermia Using a Custom-Built Delivery System at JoVE.com

In Vitro and In Vivo Delivery of Magnetic Nanoparticle Hyperthermia Using a Custom-Built Delivery System

This protocol presents techniques and methodology necessary for the accurate delivery of magnetic nanoparticle hyperthermia using a sophisticated delivery and monitoring system.

Hyperthermia has long been used in the treatment of cancer. Techniques have varied from the intra-tumoral insertion of hot iron rods, to systemically ...

This method is significant because it provides the most robust and reproducible way to deliver magnetic nanoparticle hyperthermia treatment in preclinical models. This technique allows for the precise and consistent delivery of nanoparticle hyperthermia by accurately monitoring and controlling the local environment, animal physiology and thermal dose. When attempting this protocol, keep in mind that nanoparticle heating must remain confined to the target tissue.It is essential to use abundant and appropriate thermometry to ensure reproducibility and safety. 24 hours after plating B16 F10 murine melanoma cells add magnetic nanoparticles to a final concentration of three milligrams ion per milliliter. Ensure that the nanoparticles are distributed evenly throughout the well by creating a stock solution of media and magnetic nanoparticles in advance.48 hours after adding the nanoparticles, add 0.5 milliliters of trypsin to each well being treated and gently swirl the plate. Use a microscope to check that the cells are detached. Then, add one milliliter of media to each well and collect all the cells into 1.5 milliliter tubes using a clearly labeled separate tube for each well.Spin the tubes at 60G for two to three minutes to pellet the cells. Then place tubes in the spacer full of water within the coil. Set the temperature of the water bath to maintain the media and cell pallet at 37 degrees Celsius.Set up separate fiber optic temperature probes to monitor the temperature within the tube and the water bath. Turn on the chiller and check that the coolant is flowing through the coil. Then turn on the power source and adjust the percent of maximum to the desired field.Operate the 14 turns solenoid coil powered by a 10 kilowatt generator at 165 kilohertz and 23.87 kiloamperes per meter. Treat the cells until a previously determined protocol thermal dose is reached. After treatment, re-suspend the pelleted cells in the media within the tubes and replate them into new, clearly labeled six-well plates.After anesthetizing the mouse, clean the tumor with an alcohol wipe. Inject the magnetic nanoparticles into the tumor three hours before AMF treatment. After three hours, anesthetize the mouse again and check for the lack of response to the righting reflexes.Remove the ear tag or any other metal objects on the mouse and gently place a lubed fiber optic temperature probe into its rectum. Place the catheter into the tumor. Remove the needle.Then cut the catheter so that it does not stick out of the tumor too much. Insert a three-sensor fiber optic temperature probe into the catheter, which will protect the sensors. Tape the rectal and intratumoral probe to the tail of the animal.Place the mouse into a 50 milliliter tube which should have a hole near the head where the anesthesia can be connected and delivered. Place the tube within the coil setup and reconnect the anesthesia. Place a fiber optic temperature probe loosely into the tube to measure the temperature of the environment.Then turn on the chiller and ensure that the coolant is being circulated. Verify that the computer software is displaying the various temperatures and begin recording to allow for a CEM43 calculation to be displayed in real time where the required CEM43 dose should have been previously determined. Turn on the magnet at a low power percentage and ensure that the fiber optic temperature probes are recording temperature changes.In short, that the core temperature of the animal remains at 38 degrees Celsius, regulating it with the conditioned air jacket. Adjust the strength of the magnetic field by changing the power which controls the temperature level in the tumor. Shut off the AMF once the desired dose is achieved.Remove the tube from the coil and remove the mouse from the tube. Then, shut down the chiller. Extract the probes and catheters and if necessary, tag the animal with a new metal ear tag.Monitor the mouse during recovery from anesthesia, ensuring that it resumes normal behavior and that there are no complications. For in vivo hypothermia, it is essential to place as many fiber optical temperature probes as possible in strategic sites for real-time efficacy and safety assessment. These probes make it possible to record temperature throughout the experiment, allowing for accurate dosimetry and thermal history.Curves generated during an in vivo experiment are shown here, highlighting the capability to closely monitor temperature and adjust the system to maintain tumor temperatures within the desired range. The volcano plots show differential expressed genes following in vitro and in vivo magnetic nanoparticle hyperthermia treatment, demonstrating how molecular techniques can be used to monitor the hyperthermia effects. Accurate monitoring of tumor temperatures, animals core temperature, and thermal dose are necessary for consistent delivery of treatment.Following hyperthermia, different analyses can be performed to understand the effect and mechanism of the treatment. And other therapies can be implemented for a greater therapeutic effect.

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