This research is funded by an operating grant from the Canadian Institutes of Health Research (CIHR) to C.A. and D.A.J. The authors acknowledge the Canadian Foundation for Innovation and Princess Margaret Cancer Foundation for funding the STTARR research facility that enables the imaging and therapy research components of this work.

1. Liposome Preparation
<ol>
	<li>Dissolve the lipids 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-stearoyl-2-hydroxy-sn-glycero-3-phosphatidylcholine (MSPC or S-lyso-PC) and N-(carbonyl-methoxypolyethyleneglycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (mPEG2000-DSPE) in chloroform. For example, for preparation of 10 ml of HTLC, weigh out 314.4 mg DPPC, 39.4 mg MSPC, and 83.9 mg mPEG2000-DSPE into an amber glass vial. Then, dissolve the lipids in 2 ml of chloroform and heat up the vial for 30 sec in a 60 &#176;C water bath.</li>
	<li>Remove the chloroform using a rotary evaporator. Place the resulting lipid film under high-pressure vacuum O/N.</li>
	<li>For 10 ml of HTLC, hydrate the lipid film with 5 ml 0.1N Tris buffer (pH 7.4) for 1 hr. At the same time, weigh out 162.4 mg 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) lipid and 100 mg CDDP powder and hydrate with 5 ml 0.1N Tris buffer containing 30% ethanol (pH 7.4) for 1 hr.</li>
	<li>For the Gd-HTLC formulation, weigh out the same amount of DPPG lipid and 10 mg CDDP powder, then hydrate with 2.5 ml Gd-HP-DO3A solution (279.3 mg/ml ) plus 2.5 ml 0.1N Tris buffer containing 30% ethanol (pH 7.4) for 1 hr. During hydration, the mixtures must be kept in amber vials and placed on a hotplate at 70 &#176;C with constant stirring and vortexing every 10 min.</li>
	<li>Combine the lipid mixture and the lipid drug mixture, and again hydrate for 1 hr. Vortex every 15-20 min.</li>
	<li>Assemble the 10-ml extruder with two stacks of 200 nm polycarbonate filters. Connect the thermobarrel of the extruder to a circulating water bath at 70 &#176;C, and connect the extruder to a compressed nitrogen tank.</li>
	<li>Immediately after hydration, transfer the mixture into the extruder chamber. Open the nitrogen flow (set pressure to 200 psi) to extrude the liposomes through the membranes. Collect the liposomes in a 50 ml tube. Keep the tube in a hot water bath (70 &#176;C) at all times during the extrusion process. Repeat this process 5 times.</li>
	<li>Disassemble the extruder and change to two stacks of 100 nm polycarbonate filters. Reassemble the extruder and set the pressure to 400 psi. Repeat Step 5, except extrude the liposomes 10 times. Collect the sample from the final extrusion into a 15 ml tube.</li>
	<li>Cool down the liposomes to RT, and centrifuge at 1,000 x g for 3 min to precipitate insoluble CDDP.</li>
	<li>Dialyze the liposomes O/N against 0.9% saline in dialysis tubing with a 15,000 molecular weight cutoff (MWCO) under sterile conditions.</li>
	<li>Dilute 10 &#181;l of the liposomes in 990 &#181;l distilled water. Use dynamic light scattering to measure the size distribution of HTLC and Gd-HTLC. Perform 3 measurements with 10 runs each.</li>
	<li>Dilute 40 &#181;l of the liposomes in 3,960 &#181;l distilled water. Make 5-6 standard solutions of platinum and gadolinium in the concentration range of 0.1 to 20 &#181;g/ml . Measure the platinum and gadolinium concentrations using inductively coupled plasma-atomic emission spectrometer (ICP-AES) at 3 different wavelengths. Perform 3 measurements at each wavelength for average result.</li>
	<li>Determine the gel to liquid crystalline phase transition temperature (Tm) of the HTLC liposome formulation using a differential scanning calorimeter (DSC). Load 5-10 mg of HTLC into a DSC pan and use an empty pan as a reference. Use a scan rate of 5 &#176;C/min to ramp up the temperature from 0 &#176;C to 60 &#176;C.</li>
	<li>Wrap the liposomes in aluminum foil to prevent light exposure and store at 4 &#176;C.</li>
</ol>
2. In vitro Release from Liposomes
<ol>
	<li>Prepare spin columns.
	<ol>
		<li>Incubate 50 g gel filtration beads in 400 ml 0.9% saline at RT for 3-4 hr. Roll up a piece of glass wool, wet with 0.9% saline and place it into the tip of a 1 ml syringe. Press the glass wool to fill approximately 0.05-0.1 ml of the syringe. Use a glass pipette to gradually add about 1 ml gel filtration media into the syringe.</li>
		<li>Place the syringe into a 15 ml tube and centrifuge at 1,000 x g for 3 min. Remove the spin column and place it into a 15 ml tube.</li>
	</ol>
	</li>
	<li>Sample 400 &#181;l of HTLC or Gd-HTLC into 8 1-dram glass vials and incubate in a temperature controlled water bath at either 37 &#176;C or 42 &#176;C. Take out one vial at each time point (i.e., 5, 15, 30 and 60 min) and immediately place it on ice.</li>
	<li>Add 100 &#181;l of 0.9% saline into the prepared spin column, then add 100 &#181;l of HTLC or Gd-HTLC before incubation (control) or after incubation in the water bath into the spin column. Centrifuge at 1,000 x g for 3 min. Remove the syringe from the tube and dilute the solution in the tube for ICP-AES analysis.</li>
</ol>
3. Implantation of Subcutaneous Xenograft of Cervical Tumor
<ol>
	<li>All animal studies were conducted according to animal use protocols approved by the Animal Care Committee of the University Health Network (UHN).</li>
	<li>Perform all animal studies in a Biosafety Cabinet (BSC). Autoclave all surgical tools before any surgery. After each surgery, wipe the surgical tools with 70% ethanol and sterilize again using a hot bead sterilizer.</li>
	<li>For euthanasia, place the animal in an insulated CO2 chamber, then perform cervical dislocation.</li>
	<li>For general anesthesia using 100% oxygen, use 5% isofluorane for induction and 2% for maintenance. To ensure the depth of anesthesia, apply pressure with forceps to palmar surface of the footpad to observe animal response. Apply eye lubricant to prevent dryness while under anesthesia.</li>
	<li>For an animal that has undergone surgery, place the animal in a clean cage without the company of other animals. Monitor until sufficient consciousness is regained.</li>
	<li>Culture the ME-180 cells in alpha-minimal essential medium (&#945;-MEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics.</li>
	<li>Harvest the cells and keep the cells in complete media before inoculation. Count the cells using a hemocytometer.</li>
	<li>Inoculate each donor mouse to bear an intramuscular (i.m.) ME-180 (human cervical cancer cells) tumor. 
	Note: Growth at the i.m. site has been selected in order to ensure the development of well vascularized tumors. Purchase female SCID mice (aged 6-8 weeks, approximately 20 g) from an in-house breeding facility.
	<ol>
		<li>Anesthetize a female SCID mouse and inject 1 x 106 ME-180 cells into the gastrocnemius muscle of the hind limb using a 27 G needle.</li>
		<li>Measure tumor size using a caliper until the tumors have reached 9-12 mm in their longest dimension. Terminate the study if the tumor has exceeded 12 mm, or if the tumor mass compromises normal behavior, ambulation, food and water intake.</li>
	</ol>
	</li>
	<li>Implant tumor pieces from donor mice into recipient mice.
	<ol>
		<li>Anesthetize a donor mouse using 5% isofluorane. Euthanize the mouse using cervical dislocation under anesthesia. Remove the donor tumor from the donor mouse. Cut the tumor into cubic fragments of 2-3 mm3.</li>
		<li>Anesthetize a recipient mouse using 5% isofluorane. Inject 100 &#956;l of 0.5 mg/ml meloxicam subcutaneously before surgery. Apply iodine surgical scrub solution, then 70% ethanol, and lastly iodine solution to the shaved skin. Shave the left hind limb of the recipient mouse. Make an incision at the level of skin. Insert one donor tumor piece subcutaneously through the incision. Close the incision using 1-2 wound clip(s).</li>
		<li>Remove the clips 3-5 days after implantation. Subcutaneous tumor is required for using the laser-based heating setup.</li>
		<li>Allow tumors to grow for 2-3 weeks before heating treatment.</li>
	</ol>
	</li>
</ol>
4. Design, Assembly and Calibration of a Conformal Laser Delivery Illuminator for In Vivo Heating
<ol>
	<li>Achieve tissue heating using a 763 nm diode laser coupled to a conformal illuminator using an optical fiber.
	<ol>
		<li>The illuminator provides uniform superficial laser illumination of the xenograft tumor. It is composed of a 30 x 20 x 17 mm block of highly reflective material containing three adjoined light integrating chamber spheres with one chamber in the middle (16 mm in diameter) and 2 small chambers on either side (16 mm in length and 5 mm in diameter) (Figure 1).</li>
		<li>In order for light to pass from one chamber into the other, two small 5 mm diameter holes were cut between the small outer chambers and the larger middle chamber. Light is delivered from the laser into the outer chambers using a 400 &#181;m cut-end fiber connected to the laser that is passed into the chamber through a 600 &#181;m diameter hole.</li>
		<li>Due to the nature of light interaction with the chamber walls, light delivered into one of the small chambers is spatially homogenized then passes through the interior ports into the larger chamber where it is further spatially homogenized. Light then exits the 10 mm diameter port on the middle chamber.</li>
	</ol>
	</li>
	<li>Calibrate the illuminator light delivery with respect to the laser power setting using a NIST calibrated 50 mm integrating sphere to measure the delivered power.
	<ol>
		<li>Calculate the light distribution in the tumor using Monte Carlo simulations based on a simplified geometry of the tumor. Generate the Monte Carlo code in a custom algorithm written with a commercial computational software package and base on the standard code of Monte Carlo modeling of light transport in multi-layered tissues of Jacques et al.14.</li>
		<li>Model the tumor as a hemisphere lying on a flat skin line with a diameter matching the average tumor dimension of 7 mm at the skin surface and a height of 5 mm. Model homogeneous illumination of the surface by randomly launching photons throughout the exposed hemisphere and direct at the center of the sphere.</li>
		<li>Use optical properties including absorption, &#181;a = 0.025 mm-1, scattering, &#181;s = 10 mm-1, anisotropy factor, g = 0.9 and refractive index, n=1.4. Use one million photons in the calculation.</li>
		<li>Perform these measurements before any in vivo heating experiments with no further modifications after initial calibration.</li>
	</ol>
	</li>
</ol>
5. Conformal Heating of Tumor using Custom-designed Laser Chamber Setup
<ol>
	<li>Connect one end of a laser fiber to the laser device and the other end to the illuminator.</li>
	<li>Anesthetize the animal using 5% isofluorane. Insert a 27 G injection catheter into the lateral tail vein of the mouse. Insert a 22 G catheter into the center of the tumor. Place a fiber optic temperature probe into the hollow catheter to monitor temperature change.</li>
	<li>Cover the entire tumor with the illuminator (Figure 2). First set the power to 0.8-1 W. Turn on the laser and wait for the temperature to rise. Maintain the temperature of the tumor at 42 &#176;C by manually adjusting the laser power between 0.1-0.8 W.</li>
</ol>
6. Temperature Distribution Evaluated Through MR Thermometry (MRT)
<ol>
	<li>Measure the temperature distribution of the heated tumor using the laser-based heating setup through proton resonance frequency shift (PRF-shift) MRT on a 7 Tesla preclinical MR imaging system15 in conjunction with the fiber optic temperature probe measurements.</li>
	<li>Acquire thermometry images at 10 sec intervals using a 2D-FLASH pulse sequence (echo time 4.5 msec; repetition time 156.25 msec) with 312 x 312 &#181;m in-plane resolution and 2 mm slice thickness in a single slice at the level of the fiber optic temperature probe.</li>
</ol>
7. MR Monitoring of Agent Release
<ol>
	<li>Perform T1-weighted imaging prior to and 20 min after administration of Gd-HTLC.</li>
	<li>Implant two tumors, one on each of the hind limbs, of a female SCID mouse using the method described above in Section 3 for the implantation. Preheat the tumor on the left hind limb for 5 min at 42 &#176;C prior to injection of Gd-HTLC at a dose of 66.3 mg/kg Gd-HP-DO3A and 1.4 mg/kg CDDP, then heat the tumor for an additional 20 min post-injection. Perform injection during heating treatment. Use the tumor on the right hind limb as an unheated control.</li>
	<li>Acquire dynamic MR images (36 x 20 mm field-of-view, 200 x 200 &#181;m in-plane resolution, 2 mm slice thickness; MR sequence protocol) to monitor Gd-HP-DO3A release at 12 sec intervals for the entire 20 min post administration of Gd-HTLC, beginning 30 sec prior to injection.</li>
	<li>Contour the tumor (heated and unheated) and muscle volumes. Calculate the mean MR signal of all voxels within each contoured volume.</li>
</ol>

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Hyperthermia in cancer treatment: a primer. , (2006).</li><li>Landon, C. D., Park, J. Y., Needham, D., Dewhirst, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+Drug+Delivery+and+Hyperthermia:+The+Materials+Design+and+Preclinical+and+Clinical+Testing+of+Low+Temperature-Sensitive+Liposomes+Used+in+Combination+with+Mild+Hyperthermia+in+the+Treatment+of+Local+Cancer.">Nanoscale Drug Delivery and Hyperthermia: The Materials Design and Preclinical and Clinical Testing of Low Temperature-Sensitive Liposomes Used in Combination with Mild Hyperthermia in the Treatment of Local Cancer.</a> Open Nanomed. 3, 38-64 (2011).</li><li>Koning, G. A., Eggermont, A. M., Lindner, L. H., ten Hagen, T. 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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-activated+thermosensitive+liposomal+cisplatin+(HTLC)+results+in+effective+growth+delay+of+cervical+carcinoma+in+mice.">Heat-activated thermosensitive liposomal cisplatin (HTLC) results in effective growth delay of cervical carcinoma in mice.</a> J Control Release. 178, 69-78 (2014).</li><li>Lammers, T., Kiessling, F., Hennink, W. 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Liposomes were first developed in the 1960s as drug delivery vehicles that carry hydrophilic drugs in their internal aqueous volume and hydrophobic drugs within their lipid bilayer2. In addition to use in therapeutic applications, liposomes have been explored for diagnostic applications when labeled with radionuclides or loaded with imaging contrast agents17. In recent years, theranostics and therapeutic-diagnostic pairs have been pursued to provide opportunities for image-guided patient stratificat...

The pathophysiology of solid tumors results in the enhanced permeability and retention (EPR) of nanoscale systems. This has led to the development of many drug delivery systems that take advantage of this effect to target the tumor tissue while minimizing systemic side effects1. Liposomal delivery technologies have been widely investigated for drug or imaging probes2. Although liposomes have significantly reduced the systemic toxicity compared to conventional chemotherapy, there have been few improvements in clinical efficacy3,4. Studies have shown that the limited efficacy is due to a lack of drug release from the carrier4,5. As a result, development of liposomes that are activated to release the encapsulated drug in response to external stimuli has attracted considerable attention. Hyperthermia has been employed for decades as a relatively safe treatment modality for cancer patients6. Therefore the development of thermosensitive liposomes with heat as an external trigger has been a logical combination with significant potential for clinical translation. Indeed, the lysolipid-containing thermosensitive liposome formulation of doxorubicin, known as LTSL-DOX, has now reached clinical evaluation7.
Recent clinical data with LTSL-DOX has shown that the protocol for heat delivery is a critical factor that can heavily influence patient outcomes8. In humans, radiofrequency, microwave, laser and ultrasound transducers are used to apply hyperthermia locally at tumor sites9. In preclinical studies requiring heating of subcutaneous tumors, heating catheters10,11 and water baths12,13 are most often employed. In this manuscript, we introduce a new method for heating subcutaneous tumors using a custom designed laser-based heating setup, which enables more conformal heating of the tumor volume.&#160;Using MR compatible materials, the setup is small enough to fit within the bore of a small animal MR imager, allowing real time monitoring of changes in tissue temperature during the laser heating.
The MR contrast agent, gadoteridol (Gd-HP-DO3A), was co-encapsulated with CDDP into a thermosensitive liposome formulation of CDDP (HTLC), known as Gd-HTLC, for real-time MR image-guided monitoring and assessment of heat-activated drug release and validation of the heating protocol. Our results demonstrate that the laser-based heating apparatus efficiently activated the release of encapsulated agents from the Gd-HTLC formulation while being monitored through MR imaging.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Rotary evaporator</td>
			<td>Heidolph Instruments GmbH &#38; Co.KG</td>
			<td>Laborota 4000</td>
			<td></td>
		</tr>
		<tr>
			<td>High pressure extruder</td>
			<td>Northern Lipids Inc.</td>
			<td>T.001</td>
			<td>10 mL thermobarrel</td>
		</tr>
		<tr>
			<td>Heating circulator</td>
			<td>VWR International LLC.</td>
			<td>11305</td>
			<td>Connected to extruder</td>
		</tr>
		<tr>
			<td>Polycarbonate membrane filter</td>
			<td>Whatman</td>
			<td>110605;110606</td>
			<td></td>
		</tr>
		<tr>
			<td>Differential scanning calorimeter (DSC)</td>
			<td>TA Instruments</td>
			<td>Q100</td>
			<td></td>
		</tr>
		<tr>
			<td>Inductively coupled plasma-atomic emission spectrometer (ICP-AES)</td>
			<td>PerkinElmer</td>
			<td>Optima 7300DV</td>
			<td></td>
		</tr>
		<tr>
			<td>Zetasizer</td>
			<td>Malvern Instruments Ltd.</td>
			<td>Nano-ZS</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell incubator</td>
			<td>NuAire Inc.</td>
			<td>NU-5800</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclip wound clip applier</td>
			<td>Becton Dickinson</td>
			<td>427630</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclip wound clip remover</td>
			<td>Becton Dickinson</td>
			<td>427637</td>
			<td></td>
		</tr>
		<tr>
			<td>Wound clips</td>
			<td>Becton Dickinson</td>
			<td>427631</td>
			<td>9 mm</td>
		</tr>
		<tr>
			<td>763 nm Laser device</td>
			<td>Biolitec</td>
			<td>Ceralas CD 403 laser</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser probe</td>
			<td>Thorlabs Inc.</td>
			<td>FT400EMT</td>
			<td>With SMA and flat cleave connectors</td>
		</tr>
		<tr>
			<td>Spectralon (illuminator)</td>
			<td>Labsphere Inc.</td>
			<td>FAST-SL-5CMX5CM</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>CSTM-SL-5CMX5CM</td>
			<td></td>
		</tr>
		<tr>
			<td>7 Tesla prelinical magnetic resonance (MR) imaging system</td>
			<td>Bruker Corporation</td>
			<td>Biospec 70/30</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber optic temperature sensor</td>
			<td>LumaSense Technologies Inc.</td>
			<td>Luxtron FOT Lab Kit</td>
			<td></td>
		</tr>
		<tr>
			<td>Integrating sphere</td>
			<td>Newport Corporation</td>
			<td>819C</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical power meter</td>
			<td>Newport Corporation</td>
			<td>1830-R</td>
			<td></td>
		</tr>
	</tbody>
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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.

The HTLC liposomes are manufactured using common methods, including lipid film formation, hydration, extrusion and dialysis. During steps involving CDDP, caution should be taken not to expose CDDP to any aluminum material, as CDDP will be deactivated through the formation of a black deposit. An illustration of HTLC is shown in Figure 3. The physico-chemical properties of HTLC were summarized in a manuscript recently published in the Journal of Controlled Release16. The gadolinium and ...

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

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 ...

custom-designed-laser-based-heating-apparatus-for-triggered-release

Research

JoVE Journal

Bioengineering

9.0K Views.  University of Toronto. AN MRI-compatible 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.

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

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Magnetically-Assisted Remote Controlled Microcatheter Tip Deflection under Magnetic Resonance Imaging

X-ray fluoroscopy-guided endovascular procedures have several significant limitations, including difficult catheter navigation and use of ionizing radiation, which can potentially be overcome using a magnetically steerable catheter under MR guidance.
	The main goal of this work is to develop a microcatheter whose tip can be remotely controlled using the magnetic field of the MR scanner. This protocol aims to describe the procedures for applying current to the microcoil-tipped microcatheter to produce consistent and controllable deflections.
	A microcoil was fabricated using laser lathe lithography onto a polyimide-tipped endovascular catheter. In vitro testing was performed in a waterbath and vessel phantom under the guidance of a 1.5-T MR system using steady-state free precession (SSFP) sequencing. Various amounts of current were applied to the coils of the microcatheter to produce measureable tip deflections and navigate in vascular phantoms.
	The development of this device provides a platform for future testing and opportunity to revolutionize the endovascular interventional MRI environment.

Current applied to an endovascular microcatheter with microcoil tip made by laser lathe lithography can achieve controllable deflections under magnetic resonance (MR) guidance, which may improve speed and efficacy of navigation of vasculature during various endovascular procedures.

X-ray fluoroscopy-guided  endovascular procedures have several significant limitations, including  difficult catheter navigation and use of ionizing ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH&#8226;, NO&#8226;). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 &#181;T (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

Magnetic and Thermal-sensitive Poly(N-isopropylacrylamide)-based Microgels for Magnetically Triggered Controlled Release

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were designed and fabricated for magnetically triggered release. PNIPAAm-based magnetic microgels with a spherical structure were produced via a temperature-induced emulsion followed with physical-crosslinking by mixing PNIPAAm, polyethylenimine (PEI), and Fe3O4-NH2 magnetic nanoparticles. Because of their dispersity, the Fe3O4-NH2 nanoparticles were embedded inside the polymer matrix. The amine groups exposed on the Fe3O4-NH2 and PEI surface supported the spherical structure by physically crosslinking with the amide groups of the PNIPAAm. The hydrophobic anti-cancer drug curcumin can be dispersed in water after encapsulation into the microgels. The microgels were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), and UV-Vis spectral analysis. Furthermore, magnetically triggered release was studied under an external high frequency magnetic field (HFMF). A significant &#34;burst release&#34; of curcumin was observed after applying the HFMF to the microgels due to the magnetic inductive heating (hyperthermia) effect. This manuscript describes the magnetically triggered controlled release of Cur-PNIPAAm/Fe3O4-NH2 encapsulated curcumin, which can be potentially applied for tumor therapy.

Magnetic and Thermal-sensitive PolyN-isopropylacrylamide-based Microgels for Magnetically Triggered Controlled Release

This manuscript describes the preparation of magnetic and thermal-sensitive microgels via a temperature-induced emulsion without chemical reaction. These sensitive microgels were synthesized by mixing poly(N-isopropylacrylamide) (PNIPAAm), polyethylenimine (PEI) and Fe3O4-NH2 nanoparticles for the potential use in magnetically and thermally triggered drug-release.

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were ...

Metabolic Support of Excised, Living Brain Tissues During Magnetic Resonance Microscopy Acquisition

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic resonance (MR) microscopy data. While it is possible to perform MR collections on living, excised mammalian tissue, such experiments have traditionally been constrained by resolution limits and are thus incapable of visualizing tissue microstructure. Conversely, MR protocols that did achieve microscopic image resolution required the use of fixed samples to accommodate the need for static, unchanging conditions over lengthy scan times. The current protocol describes the first available MR technique that enables imaging of living, mammalian tissue samples at microscopic resolutions. Such data is of great importance to the understanding of how pathology-based contrast changes occurring at the microscopic level influence the content of macroscopic MR scans such as those used in the clinic. Once such an understanding is realized, diagnostic methods with greater sensitivity and accuracy can be developed, which will translate directly to earlier disease treatment, more accurate therapy monitoring and improved patient outcomes.
While the described methodology focuses on brain slice preparations, the protocol is adaptable to any excised tissue slice given that changes are made to the gas and perfusate preparations to accommodate the tissue&#39;s specific metabolic needs. Successful execution of the protocol should result in living, acute slice preparations that exhibit MR diffusion signal stability for periods up to 15.5 h. The primary advantages of the current system over other MR compatible perfusion apparatuses are its compatibility with the MR microscopy hardware required to attain higher resolution images and ability to provide constant, uninterrupted flow with carefully regulated perfusate conditions. Reduced sample throughput is a consideration with this design as only one tissue slice may be imaged at a time.

The current protocol describes a method by which users can maintain viability of acute hippocampal and cortical slice preparations during the collection of magnetic resonance microscopy data.

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Apparatus for Harvesting Tissue Microcolumns

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns of full-thickness skin tissue. The small size of the microcolumns allows donor sites to heal quickly without causing donor site scarring, while harvesting full-thickness tissue enables the incorporation of all cellular and extracellular components of skin tissue, including those associated with deeper dermal regions and the adnexal skin structures, which have yet to be successfully reproduced using conventional tissue engineering techniques. The microcolumns can be applied directly into skin wounds to augment healing, or they can be used as the autologous cell/tissue source for other tissue engineering approaches. The harvesting needles are made by modifying standard hypodermic needles, and they can be used alone for harvesting small amounts of tissue or coupled with a simple suction-based collection system (also made from commonly available laboratory supplies) for high-volume harvesting to facilitate studies in large animal models.

Here we describe a protocol for producing harvesting needles that can be used to collect full-thickness skin tissue without causing donor site scarring. The needles can be combined with a simple collection system to achieve high-volume harvesting.

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns ...

Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic toxicity and increases circulation time of the drugs. In a novel approach to microbubble-assisted drug delivery, nanoparticles are incorporated in or on microbubble shells, enabling local and triggered release of the nanoparticle payload with ultrasound. A thorough understanding of the release mechanisms within the vast ultrasound parameter space is crucial for efficient and controlled release. This set of presented protocols is applicable to microbubbles with a shell containing a fluorescent label. Here, the focus is on microbubbles loaded with poly(2-ethyl-butyl cyanoacrylate) polymeric nanoparticles, doped with a modified Nile Red dye. The particles are fixed within a denatured casein shell. The microbubbles are produced by vigorous stirring, forming a dispersion of perfluoropropane gas in the liquid phase containing casein and nanoparticles, after which the microbubble shell self-assembles. A variety of microscopy techniques are needed to characterize the nanoparticle-stabilized microbubbles at all relevant timescales of the nanoparticle release process. Fluorescence of the nanoparticles enables confocal imaging of single microbubbles, revealing the particle distribution within the shell. In vitro ultra-high-speed imaging using bright-field microscopy at 10 million frames per second provides insight into the bubble dynamics in response to ultrasound insonation. Finally, nanoparticle release from the bubble shell is best visualized by means of fluorescence microscopy, performed at 500,000 frames per second. To characterize drug delivery in vivo, the triggered release of nanoparticles within the vasculature and their extravasation beyond the endothelial layer is studied using intravital microscopy in tumors implanted in dorsal skinfold window chambers, over a timescale of several minutes. The combination of these complementary characterization techniques provides unique insight into the behavior of microbubbles and their payload release at a range of time and length scales, both in vitro and in vivo.

The presented protocols can be used to characterize the response of fluorescently-labeled microbubbles designed for ultrasound-triggered drug delivery applications, including their activation mechanisms as well as their bioeffects. This paper covers a range of in vitro and in vivo microscopy techniques performed to capture the relevant length and timescales.

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic ...