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

Summary

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

Abstract

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 ˚C (fever-level) to 1,000 ˚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  (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 ˚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.

Introduction

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 nanoparticles^7,8. 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 effect^10,11,12. 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 method^12,14,15. 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 °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 parameters^5,16. The CEM calculation works best for temperatures between 39-57 °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 °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’s core body temperature maintained at a normal level throughout the treatment. Allowing for the animal’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.

Protocol

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

1. Design custom AMF antenna (coil) to be a closed loop, choosing shapes to create the desired magnetic field. Use inductance formulas and characteristics from power generator choice to design compatible coils to generate the desired field. Use different designs for in vitro and in vivo experiments.
2. Ensure the AMF antenna inductance falls within the acceptable range of the power generator. Add or subtract capacitors to match (tune) the antenna to the power generator.
3. For in vitro experiments, design a 14-turn helical coil, inner diameter 2 cm and length 14 cm, that can contain 1.5 mL tubes, allowing for the treatment of multiple samples simultaneously. Insulate the coil with a vinyl polymer and use a polystyrene spacer to separate the coil from the tubes. Details of design specification and considerations are present in the Supplementary File 1.
4. For in vivo experiments, acquire a custom built whole-body helical coil from a manufacturer with proprietary design information. Use 8 mm square tubing (as it creates a more uniform field within the bore of the coil), and a concentrator at the targeted treatment area. Make the concentrator 5.0 cm long, with a total of 5 turns resulting in a 3.6 cm inner diameter, 5.2 cm outer diameter, and have its location at the targeted treatment area. Surround the coil with a polycarbonate shell.
5. Use an AMF generator with adjustable power and frequency, rated at 10 kW or greater as the power source. Inductance match the power source and antennae/coils to a range of 0.62 to 1.18 µHenries (µH), allowing for frequencies ranging between 30-300 kHz. Cool the generator using recycled water through a centrifugal boost pump, pressure regulated to 50 psi.
6. Cool the coils with a 5.6 ton cooling capacity chiller that pumps 25% ethylene glycol-based heat transfer fluid diluted with water through the AMF antenna. Set the temperature of the chiller to such that the antenna does not heat or cool the sample.
7. For the animal containment, construct a tubular holder that can be suspended in the center of the coil with a 0.5 cm air gap between the holder and the coil surface. Connect an adjustable conditioned air pump that circulates air through the shell around the coil and set it to maintain a normal animal core temperature. Connect the anesthesia machine to the tubular animal holder near the head of the animal to ensure proper delivery of anesthesia.
8. For cell containment, create an apparatus that circulates water from a water bath through the spacer where tubes are placed. Set the temperature of this water bath such that the tubes are surrounded by water at 37 °C.
9. Use fiber optic probes to monitor temperatures within the tumor, the animal’s core, and the animal environment or for in vitro studies, monitoring the temperature of the cell pellet, and the water surrounding the tubes.
10. Use magnetic iron oxide nanoparticles that are 100 nm in size for all experiments.
    NOTE: Concentration and specific absorption rate (SAR) are two characteristics that must be considered when choosing nanoparticles, as they directly affect the possible heating and thermal dose¹⁸.

2. Hyperthermia in vitro

1. Culture B16F10 murine melanoma cells in RPMI media with 10% FBS and 1% Pen/strep. Plate 150, 000 cells/well in 6-well plates, with 2 mL of complete medium.
2. Determine the appropriate treatment for each well, i.e., cells without mNPs and no AMF, cells with mNPs and no AMF, cells without mNPs and AMF, cells with mNPs and AMF.
   NOTE: Additionally, ensure there are appropriate controls if combining hyperthermia with another therapy. AMF is performed in a standard research bench laboratory retrofitted with the needed power and cooling capabilities.
3. 24 h following plating, add mNPs to the appropriate wells as determined in the previous step. Add mNPs to a concentration of 3 mg iron/mL. Ensure the mNPs are distributed throughout the well, either by creating a stock media/mNP solution (removing old media, adding this solution) or by adding the mNPs directly and gently swirling plates for homogenous distribution.
4. Begin the treatment, 48 h after the addition of mNPs, when wells are ~80% confluent, by removing the media and washing wells with fresh media. Remove the media.
5. Add 0.5 mL of trypsin to each well being treated, and gently swirl. Use a microscope to check that the cells are detached.
6. Add 1 mL of media to each well to collect the cells into 1.5 mL tubes. Collect all the cells from the well (~1 x 10⁶ cells). Use a clearly labeled separate tube for each well.
7. Spin tubes at 60 x g for 2-3 min to allow cells to pellet. Retain the pellet in the media.
8. Place tubes in the spacer full of water within the coil. Set the temperature of the water bath such that the media and cell pellet are maintained at 37 °C. Monitor the temperature within the tube and the water bath using separate fiber optic temperature probes.
9. Turn on the chiller, check that the coolant is flowing through the coil. Turn on the power source and adjust the percent of maximum to field desired. Operate the 14-turn solenoid coil, powered by 10 kW generator, at 165 kHz and 23.87 kA/m (300 Oe).
10. Place a separate fiber optic temperature probe into one of the tubes. Treat the cells until the previously determined protocol thermal dose. An example is 30 min at 43 °C (CEM43 of 30).
11. Resuspend cells in the media that is in their tubes and re-plate into new 6 well plates. Clearly label the new plates. The goal is to re-plate all the cells collected (~1 x 10⁶ cells).
    NOTE: New 6 well plates should be used to ensure the cells being cultured received treatment. If the old plates are used there could still be cells left on the plates that were not successfully trypsinized.
12. If necessary, for the next experimental procedure, lyse the cells for RNA or protein expression analysis.

3. Hyperthermia in vivo

1. Cell culture and inoculation
   1. Culture B16F10 murine melanoma cells in RPMI media with 10% FBS and 1% Pen/strep. Use plates/dishes that will provide enough cells for inoculation of the desired number of animals. For example, 10, 100 mm dishes, plated at 100, 000 cells will be confluent with enough cells for 20 mice injections within 48 h.
   2. Trypsinize cells and collect using pure RPMI media (no FBS or pen/strep).
   3. Count cells and create a solution for the desired concentration of cells, based on the inoculation volume, and mouse numbers.
   4. Anesthetize 6-week old female C57Bl/6 mice using vaporized isoflurane and oxygen. Place animals into a plexiglass box with 5% isoflurane and 95% oxygen until induced. Once induced, remove animal and use a face cone at 2% isoflurane to complete steps 3.1.5-3.1.7 and 3.3.3-3.3.6.
      NOTE: For anesthesia during the treatment use a built-in anesthesia containment. Follow standard institutional protocols for mouse anesthesia. Prior to animal experimentation ensure appropriate IACUC approval. After anesthesia, return the animal to cage and monitor recovery to ensure no complications.
   5. Check for the lack of response to the righting reflexes.
   6. Shave the right flank using an electric shaver.
   7. Clean the injection area with an alcohol wipe. Inject 1-2 x 10⁶ cells, using a 100 µL glass syringe with a 28 G needle, dispersed in 50 µL of media intradermally on the shaved right flank of anesthetized.
2. Tumor growth/Nanoparticle injection
   1. Measure tumors in 3 dimensions using calipers (length, width, and depth), and calculate volumes by (length x width x depth x π)/6.
   2. When tumor volumes reach 120 mm³ (+/- 20 mm³), place the animals on study. Design the study, ensuring there are appropriate control and treatment groups including combination therapy cohorts (i.e., control, mNPH, radiation, and the combination).
   3. Anesthetize mice that will be receiving mNPs as described in 3.1.4.
   4. Clean the area with an alcohol wipe. Inject mNPs into the tumor 3 h before AMF treatment. Inject a volume such that the dose is 7.5 mg of iron/cm³ of tumor.
      NOTE: Unpublished data from the lab suggests maximal mNP uptake occurs at 3-6 h.
3. AMF treatment
   1. Anesthetize the mouse and place on a heating pad to maintain core temperature.
   2. Check for the lack of response to the righting reflexes. Remove the ear tag or any other metal objects on the mouse.
   3. Gently place a lubed fiber optic temperature probe into the rectum of the mouse.
   4. Place a catheter into the tumor, removing the needle. Cut the catheter such that it does not stick out of the tumor too much.
      NOTE: The fiberoptic temperature catheters are placed and removed while the mice are under general anesthesia i.e., the catheters are only in place during the tumor heating procedure. Mice are given a single subcutaneous dose of an NSAID analgesia medication, ketoprofen (5 mg/kg), at the time of the procedure. We have not observed short or long-term discomfort or morbidity associated with the placement of the catheters.
   5. Insert a 3-sensor fiber optic temperature probe into the catheter. The catheter protects the fiber optic temperature probe sensors.
   6. Tape the rectal and intratumoral probe to the tail of the animal to ensure they remain in place.
   7. Place the mouse into a 50 mL tube, head to the bottom. The tube should have a hole near the head where the anesthesia will be connected and delivered.
   8. Place the tube within the coil set up and reconnect the anesthesia.
   9. Place a fiber optic temperature probe loosely into the tube to measure the environment temperature.
   10. Turn on the chiller, and ensure coolant is being circulated.
   11. Check and ensure the computer software is displaying the various temperatures and begin recording to allow for a CEM43 calculation to be displayed in real time. The required CEM43 is the dose previously determined.
       NOTE: Before the magnet is turned on, ensure no metal items are attached to the animal, as these will heat rapidly. Additionally, ensure that everyone in the room does not have a pacemaker and it is safe for them to be there.
   12. Turn on the magnet at a low power percentage.
   13. Ensure that the fiber optic temperature probes are recording temperature changes. Temperatures will increase once the AMF is activated as the field increases. Ensure the core temperature of the animal remains at 38 °C. Regulate the core temperature using the conditioned air jacket.
   14. Adjust the strength of the magnetic field by changing the power on the generator, using the built-in control dial, which in turn controls the temperature level in the tumor.
   15. Shut off the AMF once desired dose, as previously determined by the user (for example CEM43 40), is achieved within the tumor.
   16. Once the AMF is shut down, remove the tube from the coil.
   17. Remove the mouse from the tube, extracting the various probes & catheter. If necessary, tag the animal with a new metal ear tag.
   18. Once treatments are completed, shut down the chiller.
   19. Recover the animals from anesthesia ensuring no complications. Monitor their behavior to ensure return to normal.

Results

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...

Discussion

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...

Materials

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