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>

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<li>Baronzio, G. F., Hager, E. D. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBaronzio%2C+GF+%22Hyperthermia+in+cancer+treatment%3A+a+primer%22">Hyperthermia in cancer treatment: a primer</a>. , Springer Science. (2006).</li>
<li>Landon, C. D., Park, J. Y., Needham, D., Dewhirst, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanoscale+Drug+Delivery+and+Hyperthermia%3A+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%5BTitle%5D)+AND+%22Open+Nanomed%22%5BJournal%5D)">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>Celsion Corporation. , Celsion Announces Updated Overall Survival Data from HEAT Study of ThermoDox
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</ol>

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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><tbody><tr><td>Rotary evaporator</td><td>Heidolph Instruments GmbH &amp;amp; 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></table>

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 ...

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JoVE Journal

Bioengineering

9.0K Views.  University of Toronto.  AN MRI-compatibele maat ontworpen laser gebaseerde verwarmingsapparaten ontwikkeld plaatselijke verhitting van subcutane tumoren verschaffen om afgifte van stoffen uit warmte gevoelige liposomen activeert specifiek op het tumorgebied. 

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

One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles

We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide &alpha;, was conjugated to a phospholipid and then self-assembled to liposome-like spherical vesicles. Due to the extremely high density of porphyrin in the porphyrin-lipid bilayer, porphysomes generated large extinction coefficients, structure-dependent fluorescence self-quenching, and excellent photothermal efficacy. In our formulation, porphysomes were synthesized using high pressure extrusion, and displayed a mean particle size around 120 nm. Twenty-four hr post-intravenous injection of porphysomes, the local temperature of the tumor increased from 30 &deg;C to 62 &deg;C rapidly upon one minute exposure of 750 mW (1.18 W/cm2), 671 nm laser irradiation. Following the complete thermal ablation of the tumor, eschars formed and healed within 2 weeks, while in the control groups the tumors continued to grow and all reached the defined end point within 3 weeks. These data show how porphysomes can be used as potent photothermal therapy (PTT) agents.

We developed novel intrinsic multifunctional nanovesicles called porphysomes, which have structure-dependent fluorescence self-quenching and unique photothermal properties, thus functioning as potent photothermal therapy agents. We formulated porphysomes using high pressure extrusion and investigated their photothermal therapy efficacy in a xenograft tumor model.

We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide &alpha;, was conjugated to a ...

Controllable Nucleation of Cavitation from Plasmonic Gold Nanoparticles for Enhancing High Intensity Focused Ultrasound Applications

In this study, plasmonic gold nanoparticles were simultaneously exposed to pulsed near-infrared laser light and high intensity focused ultrasound (HIFU) for the controllable nucleation of cavitation in tissue-mimicking gel phantoms. This in vitro protocol was developed to demonstrate the feasibility of this approach, for both enhancement of imaging and therapeutic applications for cancer. The same apparatus can be used for both imaging and therapeutic applications by varying the exposure duration of the HIFU system. For short duration exposures (10 &#181;s), broadband acoustic emissions were generated through the controlled nucleation of inertial cavitation around the gold nanoparticles. These emissions provide direct localization of nanoparticles. For future applications, these particles may be functionalized with molecular-targeting antibodies (e.g. anti-HER2 for breast cancer) and can provide precise localization of cancerous regions, complementing routine diagnostic ultrasound imaging. For continuous wave (CW) exposures, the cavitation activity was used to increase the localized heating from the HIFU exposures resulting in larger thermal damage in the gel phantoms. The acoustic emissions generated from inertial cavitation activity during these CW exposures was monitored using a passive cavitation detection (PCD) system to provide feedback of cavitation activity. Increased localized heating was only achieved through the unique combination of nanoparticles, laser light and HIFU. Further validation of this technique in pre-clinical models of cancer is necessary.

This protocol demonstrates the controllable nucleation of cavitation in gel phantoms, through simultaneous exposure to both near-infrared pulsed laser light and high intensity focused ultrasound (HIFU). The cavitation activity can then be used for enhancing imaging and/or therapeutic uses of HIFU.

In this study, plasmonic gold nanoparticles were simultaneously exposed to pulsed near-infrared laser light and high intensity focused ultrasound (HIFU) ...

Engineering

Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control over the drug release. However, most of the relevant technologies are still in the development process and are unprocurable by clinics. Here, we describe a facile fabrication of these photo-responsive NPs with commercially available gold NPs and thermo-responsive liposomes. Calcein is used as a model drug to evaluate the encapsulation efficiency and the release kinetic profile upon heat/light stimulation. Finally, we show that this photo-triggered release is due to the membrane disruption caused by microbubble cavitation, which can be measured with hydrophone.

This protocol describes a simple preparation method for gold nanoparticle integrated photo-responsive liposomes with the commercially available materials. It also shows how to measure the microbubble cavitation process of the synthesized liposomes upon the treatment of pulsed laser.

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control ...

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

Plasmonic Photothermal Cancer Therapy: Nanoparticle-embedded Tumor-tissue-mimicking Phantoms for Visualizing Photothermal Temperature Distribution

Plasmonic photothermal therapy (PPTT), an emerging cancer treatment, involves delivering nanoparticles (NPs) to a tumor, followed by near-infrared (NIR) irradiation to generate localized heat that destroys cancer cells. Before administering PPTT, the therapeutic parameters -- NP concentration, irradiation intensity, and duration -- need to be estimated. For this, numerical simulations are performed. However, to ensure robust computation, these simulations must be validated through photothermal experiments on tumor-tissue-mimicking phantoms replicating the optical properties of tumor tissue. For PPTT, therapeutic parameters are governed by the scattering and absorption of incident radiation by the tissue and NPs. Therefore, validation experiments can be conducted on phantoms mimicking the reduced scattering coefficient (&#181;s&#39;) and absorption coefficient (&#181;a) of the target tumor/tissue.
Specifically, this protocol provides instructions for preparing phantoms mimicking &#181;s&#39; and &#181;a of breast tumor injected with gold nanorods, surrounded by normal breast tissue. The protocol also details NIR irradiation, temperature monitoring, and validation of numerical results by comparing spatiotemporal temperatures with those measured using thermocouples. The protocols presented in this study facilitated the preparation of hydrogel-based cylindrical breast tumor-tissue phantoms with dimensions (&#981;40 x 12 mm) and a central tumor region (&#981;20 x 6 mm), comprising 1% agarose as the base matrix and intralipid as the scattering constituent and tumor region embedded with gold nanorods at 25 &#181;g/mL concentration. Representative results from a case study illustrate the application of fabricated phantoms for validating numerical simulations for PPTT. The study concludes that the demonstrated protocols are valuable for conducting photothermal experiments aimed at optimizing and planning therapeutic parameters prior to in vivo experiments and validating numerical simulations for PPTT.

This article presents the protocol for preparing tumor-tissue phantoms that replicate optical properties for plasmonic photothermal therapy. It details phantom preparation, photothermal evaluations, and validation of the developed numerical model based on photothermal temperature measurements for assessing therapeutic parameters, offering an ethical, cost-effective alternative to in vivo studies for preliminary testing.

Plasmonic photothermal therapy (PPTT), an emerging cancer treatment, involves delivering nanoparticles (NPs) to a tumor, followed by near-infrared (NIR) ...

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading chemotherapeutic drugs, such as doxorubicin (DOX). To achieve this, an ethanolic lipid mixture and ammonium sulfate solution are injected into a staggered herringbone micromixer (SHM) microfluidic device. The solutions are rapidly mixed by the SHM, providing a homogeneous solvent environment for liposomes self-assembly. Collected liposomes are first annealed, then dialyzed to remove residual ethanol. An ammonium sulfate pH-gradient is established through buffer exchange of the external solution by using size exclusion chromatography. DOX is then remotely loaded into the liposomes with high encapsulation efficiency (&#62; 80%). The liposomes obtained are homogenous in size with Z-average diameter of 100 nm. They are capable of temperature-triggered burst release of encapsulated DOX in the presence of mild hyperthermia (42 &#176;C). Indocyanine green (ICG) can also be co-loaded into the liposomes for near-infrared laser-triggered DOX release. The microfluidic approach ensures high-throughput, reproducible and scalable preparation of LTSLs.

The protocol presents the optimized parameters for preparing thermosensitive liposomes using the staggered herringbone micromixer microfluidics device. This also allows co-encapsulation of doxorubicin and indocyanine green into the liposomes and the photothermal-triggered release of doxorubicin for controlled/triggered drug release.

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading ...

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 ...

Real-Time Magnetic Resonance Guided Focused Ultrasound for Painful Bone Metastases

Bones are one of the most common sites of cancer metastasis, which usually causes pain and impairs quality of life. Radiation therapy combined with opioids is the standard treatment for painful bone metastases. This treatment achieves effective pain control in 60&#8722;74% of patients, but limited treatment choices with limited benefits are available for recurrent or residual painful bone metastases after radiotherapy. More than 40% of patients still experience moderate to severe bone pain after reirradiation. Magnetic resonance-guided focused ultrasound (MRgFUS) combines high-intensity focused ultrasound, which achieves thermal ablation of bone metastases and subsequent pain reduction, with real-time magnetic resonance (MR) thermometry to monitor the temperature of anatomic MR images, with an accuracy of 1 &#176;C, spatial resolution of 1 mm, and temporal resolution within 3 s. As well as being increasingly used clinically for controlling metastatic bone pain, the use of MRgFUS for other diseases has also been tested. However, the use of MR software as a thermometer is the only technique available to verify the accuracy of the software and assure energy delivery. Here, we describe an efficient method of quality assurance we developed for thermal detection and energy delivery before each MRgFUS treatment and also propose a modified workflow to expedite the treatment course as well as to reduce patients&#39; pain during the procedure.

Magnetic resonance could offer real-time monitoring of the position and temperature of focused ultrasound in thermal ablation for painful bone metastases, regardless of cancer type or previous local treatments. Our innovative method of quality assurance could facilitate the application of this effective and safe treatment.

Bones are one of the most common sites of cancer metastasis, which usually causes pain and impairs quality of life. Radiation therapy combined with ...

Medicine

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.

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 ...

Continuous-wave Thulium Laser for Heating Cultured Cells to Investigate Cellular Thermal Effects

An original method to heat cultured cells using a 1.94 &#181;m continuous-wave thulium laser for biological assessment is introduced here. Thulium laser radiation is strongly absorbed by water, and the cells at the bottom of the culture dish are heated through thermal diffusion. A laser fiber with a diameter of 365 &#181;m is set about 12 cm above the culture dish, without any optics, such that the laser beam diameter is almost equivalent to the inner diameter of the culture dish (30 mm). By keeping a consistent amount of culture medium in each experiment, it is possible to irradiate the cells with a highly reproducible temperature increase.
To calibrate the temperature increase and its distribution in one cell culture dish for each power setting, the temperature was measured during 10 s of irradiation at different positions and at the cellular level. The temperature distribution was represented using a mathematical graphics software program, and its pattern across the culture dish was in Gaussian form. After laser irradiation, different biological experiments could be performed to assess temperature-dependent cell responses. In this manuscript, viability staining (i.e., distinguishing live, apoptotic, and dead cells) is introduced to help determine the threshold temperatures for cell apoptosis and death after different points in time.
The advantages of this method are the preciseness of the temperature and the time of heating, as well as its high efficiency in heating cells in a whole cell culture dish. Furthermore, it allows for study with a wide variety of temperatures and time durations, which can be well-controlled by a computerized operating system.

An original experimental setup for heating cells in a culture dish using 1.94 &#181;m continuous-wave laser radiation is introduced here. Using this method, the biological responses of retinal pigment epithelial (RPE) cells after different thermal exposures can be investigated.

An original method to heat cultured cells using a 1.94 &#181;m continuous-wave thulium laser for biological assessment is introduced here. Thulium laser ...

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

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