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>

<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...

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 ...

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Research

JoVE Journal

Medicine

4.2K Views.  Dartmouth College. This protocol presents techniques and methodology necessary for the accurate delivery of magnetic nanoparticle hyperthermia using a sophisticated delivery and monitoring system.

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 &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 ...

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) ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...