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>
	<li>Chen, X., Tan, L., Liu, T., Meng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-Nanomaterials+for+Tumor+Microwave+Hyperthermia:+Design,+Preparation,+and+Application.">Micro-Nanomaterials for Tumor Microwave Hyperthermia: Design, Preparation, and Application.</a> Current Drug Delivery. 14 (3), 307-322 (2016).</li><li>Luo, W., 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=Effects+of+radiofrequency+ablation+versus+other+ablating+techniques+on+hepatocellular+carcinomas:+A+systematic+review+and+meta-analysis.">Effects of radiofrequency ablation versus other ablating techniques on hepatocellular carcinomas: A systematic review and meta-analysis.</a> World Journal of Surgical Oncology. 15 (1), 126 (2017).</li><li>Ter Haar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+and+sound:+Focused+ultrasound+in+the+clinic.">Heat and sound: Focused ultrasound in the clinic.</a> International Journal of Hyperthermia. 31 (3), 223-224 (2015).</li><li>Salunkhe, A. B., Khot, V. M., Pawar, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+Hyperthermia+with+Magnetic+Nanoparticles:+A+Status+Review.">Magnetic Hyperthermia with Magnetic Nanoparticles: A Status Review.</a> Current Topics in Medicinal Chemistry. 14 (5), 572-594 (2014).</li><li>Dewhirst, M. W., Viglianti, B. L., Lora-Michiels, M., Hanson, M., 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=Basic+principles+of+thermal+dosimetry+and+thermal+thresholds+for+tissue+damage+from+hyperthermia.">Basic principles of thermal dosimetry and thermal thresholds for tissue damage from hyperthermia.</a> International Journal of Hyperthermia. 19 (3), 267-294 (2003).</li><li>Roizin-Towle, L., Pirro, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+response+of+human+and+rodent+cells+to+hyperthermia.">The response of human and rodent cells to hyperthermia.</a> International Journal of Radiation Oncology, Biology, Physics. 20 (4), 751-756 (1991).</li><li>Hergt, R., Dutz, S., M&#252;ller, R., Zeisberger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+particle+hyperthermia:+Nanoparticle+magnetism+and+materials+development+for+cancer+therapy.">Magnetic particle hyperthermia: Nanoparticle magnetism and materials development for cancer therapy.</a> Journal of Physics Condensed Matter. 18 (38), (2006).</li><li>Kumar, C. S. S. R., Mohammad, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+nanomaterials+for+hyperthermia-based+therapy+and+controlled+drug+delivery.">Magnetic nanomaterials for hyperthermia-based therapy and controlled drug delivery.</a> Advanced Drug Delivery Reviews. 63 (9), 789-808 (2011).</li><li>Stigliano, R. 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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+physical+lines+of+force.">On physical lines of force.</a> The London, Edinburgh, and Dublin Philosophical Magazine and Journal of Science. 21 (139), 161-175 (1861).</li></ol>

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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Research

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Medicine

Ex Vivo Culture of Patient Tissue &amp; Examination of Gene Delivery

This video describes the use of patient tissue as an ex vivo model for the study of gene delivery. Fresh patient tissue obtained at the time of surgery is sliced and maintained in culture. The ex vivo model system allows for the physical delivery of genes into intact patient tissue and gene expression is analysed by bioluminescence imaging using the IVIS detection system. The bioluminescent detection system demonstrates rapid and accurate quantification of gene expression within individual slices without the need for tissue sacrifice. This slice tissue culture system may be used in a variety of tissue types including normal and malignant tissue and allows us to study the effects of the heterogeneous nature of intact tissue and the high degree of variability between individual patients. This model system could be used in certain situations as an alternative to animal models and as a complementary preclinical mode prior to entering clinical trial.

Ex Vivo Culture of Patient Tissue &amp; Examination of Gene Delivery

This article describes the culture of patient tissue slices for gene delivery studies and subsequent analysis of gene expression using IVIS bioluminescence imaging.

This video describes the use of patient tissue as an ex vivo model for the study of gene delivery.  Fresh patient tissue obtained at the time of surgery ...

Surgical Technique for Spinal Cord Delivery of Therapies: Demonstration of Procedure in Gottingen Minipigs

This is a compact visual description of a combination of surgical technique and device for the delivery of (gene and cell) therapies into the spinal cord. While the technique is demonstrated in the animal, the procedure is FDA-approved and currently being used for stem cell transplantation into the spinal cords of patients with ALS. While the FDA has recognized proof-of-principle data on therapeutic efficacy in highly characterized rodent models, the use of large animals is considered critical for validating the combination of a surgical procedure, a device, and the safety of a final therapy for human use. The size, anatomy, and general vulnerability of the spine and spinal cord of the swine are recognized to better model the human. Moreover, the surgical process of exposing and manipulating the spinal cord as well as closing the wound in the pig is virtually indistinguishable from the human. We believe that the healthy pig model represents a critical first step in the study of procedural safety.

Short visual description of the surgical technique and device used for the delivery of (gene and cell) therapies into the spinal cord. The technique is demonstrated in the animal but is entirely translatable and currently being used for human application.

This is a  compact visual description of a combination of surgical technique and device  for the  delivery of (gene and cell) therapies into the spinal ...

Intramyocardial Cell Delivery: Observations in Murine Hearts

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue revascularization and cell survival. In addition, further observations revealed that specific stem cells, such as cardiac stem cells, mesenchymal stem cells and cardiospheres have the ability to integrate within the surrounding myocardium by differentiating into cardiomyocytes, smooth muscle cells and endothelial cells.
Here, we present the materials and methods to reliably deliver noncontractile cells into the left ventricular wall of immunodepleted mice. The salient steps of this microsurgical procedure involve anesthesia and analgesia injection, intratracheal intubation, incision to open the chest and expose the heart and delivery of cells by a sterile 30-gauge needle and a precision microliter syringe.
Tissue processing consisting of heart harvesting, embedding, sectioning and histological staining showed that intramyocardial cell injection produced a small damage in the epicardial area, as well as in the ventricular wall. Noncontractile cells were retained into the myocardial wall of immunocompromised mice and were surrounded by a layer of fibrotic tissue, likely to protect from cardiac pressure and mechanical load.

Intramyocardial cell delivery in murine models of cardiovascular diseases, such as hypertension or myocardial infarction, is widely used to test the therapeutic potential of different cell types in regenerative studies. Therefore, a detailed description and a clear visualization of this surgical procedure will help to define the limits and advantages of cardiovascular cell therapeutic analyses in small rodents.

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue ...

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences between the murine and human respiratory systems, especially in their upper respiratory tracts (URT). In some models, these differences in the murine nasal cavity can have a significant impact on disease progression and presentation in the lower respiratory tract (LRT) when using intranasal instillation techniques, potentially limiting the usefulness of the mouse model to study these diseases. For these reasons, it would be advantageous to develop a technique to instill bacteria directly into the mouse lungs in order to study LRT disease in the absence of involvement of the URT. We have termed this lung specific delivery technique intubation-mediated intratracheal (IMIT) instillation. This noninvasive technique minimizes the potential for instillation into the bloodstream, which can occur during more invasive traditional surgical intratracheal infection approaches, and limits the possibility of incidental digestive tract delivery. IMIT is a two-step process in which mice are first intubated, with an intermediate step to ensure correct catheter placement into the trachea, followed by insertion of a blunt needle into the catheter to mediate direct delivery of bacteria into the lung. This approach facilitates a &#62;98% efficacy of delivery into the lungs with excellent distribution of reagent throughout the lung. Thus, IMIT represents a novel approach to study LRT disease and therapeutic delivery directly into the lung, improving upon the ability to use mice as surrogates to study human respiratory disease. Furthermore, the accuracy and reproducibility of this delivery system also makes it amenable to Good Laboratory Practice Standards (GLPS), as well as delivery of a wide range of reagents which require high efficiency delivery to the lung.

Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System

Intubation-mediated intratracheal (IMIT) instillation of reagents is an excellent, noninvasive method for studying respiratory disease, as well as a method for instilling therapeutic reagents directly into the lung. It is a rapid and highly reproducible method which is suitable for preclinical testing.

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Intravascular Delivery of Biologics to the Rat Kidney

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage Renal Disease with high risk of death for cardiovascular events. Moreover, recent clinical studies have shown that renovascular structure and function may have a great impact on functional renal recovery after surgery. Here, we describe a protocol for the delivery of drugs into the renal artery of rats. This procedure offers significant advantages over the frequently used systemic administration as it may allow a more localized therapeutic effect. In addition, the use of rodents in pharmacodynamic analysis of preclinical studies may be cost effective, paving the way for the design of translational experiments in larger animal models. Using this technique, infusion of rat recombinant Vascular Endothelial Growth Factor (VEGF) protein in rats has induced activation of VEGF signaling as shown by increased expression of FLK1, pAKT/AKT, pERK/ERK. In summary, we established a protocol for the intrarenal delivery of drugs in rats, which is simple and highly reproducible.

The administration of drugs for recovery of kidney function requires control of the localization and distribution of the therapeutic compound. Here, we describe in detail a simple technique for intrarenal delivery of drugs in rats. This procedure may be easily performed with no mortality and high reproducibility.

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage ...

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling molecules that affect multiple organs; they modulate gut motility and hormone production, and alter vascular tone, glucose metabolism, lipid metabolism, and energy utilization. Changes in fecal bile acids may alter the gut microbiome and promote colon pathology including cholerrheic diarrhea and colon cancer. Key regulators of fecal bile acid composition are the small intestinal Apical Sodium-dependent Bile Acid Transporter (ASBT) and fibroblast growth factor-19 (FGF19). Reduced expression and function of ASBT decreases intestinal bile acid up-take. Moreover, in vitro data suggest that some FDA-approved drugs inhibit ASBT function. Deficient FGF19 release increases hepatic bile acid synthesis and release into the intestines to levels that overwhelm ASBT. Either ASBT dysfunction or FGF19 deficiency increases fecal bile acids and may cause chronic diarrhea and promote colon neoplasia. Regrettably, tools to measure bile acid malabsorption and the actions of drugs on bile acid transport in vivo are limited. To understand the complex actions of bile acids, techniques are required that permit simultaneous monitoring of bile acids in the gut and metabolic tissues. This led us to conceive an innovative method to measure bile acid transport in live animals using a combination of proton (1H) and fluorine (19F) magnetic resonance imaging (MRI). Novel tracers for fluorine (19F)-based live animal MRI were created and tested, both in vitro and in vivo. Strengths of this approach include the lack of exposure to ionizing radiation and translational potential for clinical research and practice.

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Tools to diagnose bile acid malabsorption and measure bile acid transport in vivo are limited. An innovative approach in live animals is described that utilizes combined proton (1H) plus fluorine (19F) magnetic resonance imaging; this novel methodology has translational potential to screen for bile acid malabsorption in clinical practice.

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...