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>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#181;Henries (&#181;H), allowing for frequencies ranging between 30-300 kHz. Cool the generator using recycled water through a centrifugal boost pump, pressure regulated to 50 psi.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#176;C.</li>
	<li>Use fiber optic probes to monitor temperatures within the tumor, the animal&#8217;s core, and the animal environment or for in vitro studies, monitoring the temperature of the cell pellet, and the water surrounding the tubes.</li>
	<li>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.</li>
</ol>
2. Hyperthermia in vitro
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Add 0.5 mL of trypsin to each well being treated, and gently swirl. Use a microscope to check that the cells are detached.</li>
	<li>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.</li>
	<li>Spin tubes at 60 x g for 2-3 min to allow cells to pellet. Retain the pellet in the media.</li>
	<li>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 &#176;C. Monitor the temperature within the tube and the water bath using separate fiber optic temperature probes.</li>
	<li>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).</li>
	<li>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 &#176;C (CEM43 of 30).</li>
	<li>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.</li>
	<li>If necessary, for the next experimental procedure, lyse the cells for RNA or protein expression analysis.</li>
</ol>
3. Hyperthermia in vivo
<ol>
	<li>Cell culture and inoculation
	<ol>
		<li>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.</li>
		<li>Trypsinize cells and collect using pure RPMI media (no FBS or pen/strep).</li>
		<li>Count cells and create a solution for the desired concentration of cells, based on the inoculation volume, and mouse numbers.</li>
		<li>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.</li>
		<li>Check for the lack of response to the righting reflexes.</li>
		<li>Shave the right flank using an electric shaver.</li>
		<li>Clean the injection area with an alcohol wipe. Inject 1-2 x 106 cells, using a 100 &#181;L glass syringe with a 28 G needle, dispersed in 50 &#181;L of media intradermally on the shaved right flank of anesthetized.</li>
	</ol>
	</li>
	<li>Tumor growth/Nanoparticle injection
	<ol>
		<li>Measure tumors in 3 dimensions using calipers (length, width, and depth), and calculate volumes by (length x width x depth x &#960;)/6.</li>
		<li>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).</li>
		<li>Anesthetize mice that will be receiving mNPs as described in 3.1.4.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>AMF treatment
	<ol>
		<li>Anesthetize the mouse and place on a heating pad to maintain core temperature.</li>
		<li>Check for the lack of response to the righting reflexes. Remove the ear tag or any other metal objects on the mouse.</li>
		<li>Gently place a lubed fiber optic temperature probe into the rectum of the mouse.</li>
		<li>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.</li>
		<li>Insert a 3-sensor fiber optic temperature probe into the catheter. The catheter protects the fiber optic temperature probe sensors.</li>
		<li>Tape the rectal and intratumoral probe to the tail of the animal to ensure they remain in place.</li>
		<li>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.</li>
		<li>Place the tube within the coil set up and reconnect the anesthesia.</li>
		<li>Place a fiber optic temperature probe loosely into the tube to measure the environment temperature.</li>
		<li>Turn on the chiller, and ensure coolant is being circulated.</li>
		<li>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.</li>
		<li>Turn on the magnet at a low power percentage.</li>
		<li>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 &#176;C. Regulate the core temperature using the conditioned air jacket.</li>
		<li>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.</li>
		<li>Shut off the AMF once desired dose, as previously determined by the user (for example CEM43 40), is achieved within the tumor.</li>
		<li>Once the AMF is shut down, remove the tube from the coil.</li>
		<li>Remove the mouse from the tube, extracting the various probes &#38; catheter. If necessary, tag the animal with a new metal ear tag.</li>
		<li>Once treatments are completed, shut down the chiller.</li>
		<li>Recover the animals from anesthesia ensuring no complications. Monitor their behavior to ensure return to normal.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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...

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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 ...

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Research

JoVE Journal

Medicine

4.2K Views.  Dartmouth College. Dit protocol presenteert technieken en methodologie die nodig zijn voor de nauwkeurige afgifte van magnetische nanodeeltjeshyperthermie met behulp van een geavanceerd leverings- en monitoringsysteem.

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

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 & 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 ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect resulting in significant reductions in systemic toxicity. Nonetheless, insufficient release of encapsulated drug from liposomes has limited their clinical efficacy. Temperature-sensitive liposomes have been engineered to provide site-specific release of drug in order to overcome the problem of limited tumor drug bioavailability. Our lab has designed and developed a heat-activated thermosensitive liposome formulation of cisplatin (CDDP), known as HTLC, to provide triggered release of CDDP at solid tumors. Heat-activated delivery in vivo was achieved in murine models using a custom-built laser-based heating apparatus that provides a conformal heating pattern at the tumor site as confirmed by MR thermometry (MRT). A fiber optic temperature monitoring device was used to measure the temperature in real-time during the entire heating period with online adjustment of heat delivery by alternating the laser power. Drug delivery was optimized under magnetic resonance (MR) image guidance by co-encapsulation of an MR contrast agent (i.e.,&#160;gadoteridol) along with CDDP into the thermosensitive liposomes as a means to validate the heating protocol and to assess tumor accumulation. The heating protocol consisted of a preheating period of 5 min prior to administration of HTLC and 20 min heating post-injection. This heating protocol resulted in effective release of the encapsulated agents with the highest MR signal change observed in the heated tumor in comparison to the unheated tumor and muscle. This study demonstrated the successful application of the laser-based heating apparatus for preclinical thermosensitive liposome development and the importance of MR-guided validation of the heating protocol for optimization of drug delivery.

AN MRI-compatible&#160;custom-designed laser-based heating apparatus has been developed to provide local heating of subcutaneous tumors in order to activate release of agents from thermosensitive liposomes specifically at the tumor region.

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect ...

Bioengineering

Magnetic Resonance-Guided High Intensity Focused Ultrasound Generated Hyperthermia: A Feasible Treatment Method in a Murine Rhabdomyosarcoma Model

Magnetic resonance-guided high intensity focused ultrasound (MRgHIFU) is an established method for producing localized hyperthermia. Given the real-time imaging and acoustic energy modulation, this modality enables precise temperature control within a defined area. Many thermal applications are being explored with this noninvasive, nonionizing technology, such as hyperthermia generation, to release drugs from thermosensitive liposomal carriers. These drugs can include chemotherapies such as doxorubicin, for which targeted release is desired due to the dose-limiting systemic side effects, namely cardiotoxicity. Doxorubicin is a mainstay for treating a variety of malignant tumors and is commonly used in relapsed or recurrent rhabdomyosarcoma (RMS). RMS is the most common solid soft tissue extracranial tumor in children and young adults. Despite aggressive, multimodal therapy, RMS survival rates have remained the same for the past 30 years. To explore a solution for addressing this unmet need, an experimental protocol was developed to evaluate the release of thermosensitive liposomal doxorubicin (TLD) in an immunocompetent, syngeneic RMS mouse model using MRgHIFU as the source of hyperthermia for drug release.

Presented here is a protocol to use controlled hyperthermia, generated by magnetic resonance-guided high intensity focused ultrasound, to trigger drug release from temperature-sensitive liposomes in a rhabdomyosarcoma mouse model.

Magnetic resonance-guided high intensity focused ultrasound (MRgHIFU) is an established method for producing localized hyperthermia. Given the real-time ...

Cancer Research

Nanodrug Targeting Oncogenic miRNA-10b for Treating Metastatic Breast Cancer

Monitoring of Nanodrug Accumulation in Murine Breast Cancer Metastases

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have been shown to be associated with the metastatic potential of breast cancer and are implicated in tumor cell migration, invasion, and viability. However, it can be difficult to deliver an inhibitory RNA molecule to the tissue of interest. To overcome this challenge and deliver active antisense oligonucleotides to tumors, we utilized magnetic iron oxide nanoparticles as a delivery platform. These nanoparticles target tissues with increased vascular permeability, such as sites of inflammation or cancer. Delivery of these nanoparticles can be monitored in vivo by magnetic resonance imaging (MRI) due to their magnetic properties. Translation of this therapeutic approach into the clinic will be more accessible because of its compatibility with this relevant imaging modality. They can also be labeled with other imaging reporters such as a Cy5.5 near-infrared optical dye for correlative optical imaging and fluorescence microscopy. Here, we demonstrate that nanoparticles labeled with Cy5.5 and conjugated to therapeutic oligomers targeting oncogenic miRNA-10b (termed MN-anti-miR10b, or &#34;nanodrug&#34;) administered intravenously accumulate in metastatic sites, opening a possibility for therapeutic intervention of metastatic breast cancer.

Here, we describe the protocol for in vivo delivery of magnetic iron oxide nanoparticles carrying RNA oligomers to metastatic breast cancer in animal models, providing a clinically viable approach for the therapeutic silencing of oncogenic nucleic acids.

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have ...

Iron Oxide Nanoparticles for Image-Guided Therapy Delivery in Murine Glioblastoma

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a significant hurdle in the delivery of therapies to GBM. Reported here is an image-guided, iron oxide-based therapeutic delivery nano platform capable of bypassing this physiological barrier by virtue of size and accumulating in the tumor region, delivering its payload. This 25 nm nano platform consists of crosslinked dextran-coated iron oxide nanoparticles labeled with Cy5.5 fluorescent dye and containing antisense oligonucleotide as a payload. The magnetic iron oxide core enables tracking of the nanoparticles through in vivo magnetic resonance imaging, while Cy5.5 dye allows tracking by optical imaging. This report details the monitoring of the accumulation of this nanoparticle platform (termed MN-anti-miR10b) in orthotopically implanted glioblastoma tumors following intravenous injection. In addition, it provides insight into the in vivo delivery of RNA oligonucleotides, a problem that has hampered the translation of RNA therapeutics into the clinic.

Author Spotlight: Innovative Cancer Therapies with Iron Oxide Nanoparticles for Glioblastoma Treatment

The blood-brain barrier is a significant hurdle in the delivery of therapies for glioblastoma, a disease for which there is no cure. Here, we report an in vivo image-guided iron oxide therapeutic nano platform that can bypass this physiological barrier by virtue of size and accumulate in the tumor.

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a ...

Using Magnetometry to Monitor Cellular Incorporation and Subsequent Biodegradation of Chemically Synthetized Iron Oxide Nanoparticles

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized in cells and then left within. One challenge is to assess their fate in the intracellular environment with reliable and precise methodologies. Herein, we introduce the use of the vibrating sample magnetometer (VSM) to precisely quantify the integrity of magnetic nanoparticles within cells by measuring their magnetic moment. Stem cells are first labeled with two types of magnetic nanoparticles; the nanoparticles have the same core produced via a fast and efficient microwave-based nonaqueous sol gel synthesis and differ in their coating: the commonly used citric acid molecule is compared to polyacrylic acid. The formation of 3D cell-spheroids is then achieved via centrifugation and the magnetic moment of these spheroids is measured at different times with the VSM. The obtained moment is a direct fingerprint of the nanoparticles&#8217; integrity, with decreasing values indicative of a nanoparticle degradation. For both nanoparticles, the magnetic moment decreases over culture time revealing their biodegradation. A protective effect of the polyacrylic acid coating is also shown, when compared to citric acid.

Iron oxide nanoparticles are synthesized via a nonaqueous sol gel procedure and coated with anionic short molecules or polymer. The use of magnetometry for monitoring the incorporation and biotransformations of magnetic nanoparticles inside human stem cells is demonstrated using a vibrating sample magnetometer (VSM).

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized ...

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

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

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