This work was supported by the National Natural Science Foundation of China (81601608) and NUPTSF (NY216024).

All animal experiments were performed in accordance with the protocols approved by the OG Pharmaceutical guidelines for Animal Care and Use of Laboratory Animals. The protocols followed the guidelines of Ethics Committee for laboratory animals of OG Pharmaceutical.
1. Nanoparticle shelled microbubbles (NSMs) synthesis
<ol>
	<li>Disperse magnetic nanoparticles (Fe3O4, iron oxide) in deionized water to form a 10 mg/mL stock solution.</li>
	<li>Place the tube containing th...

<ol>
	<li>Kievit, F. M., Zhang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+nanotheranostics:+improving+imaging+and+therapy+by+targeted+delivery+across+biological+barriers.">Cancer nanotheranostics: improving imaging and therapy by targeted delivery across biological barriers.</a> Advanced Materials. 23 (36), 217-247 (2011).</li><li>Wu, H., 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=Fe3O4-Based+Multifunctional+Nanospheres+for+Amplified+Magnetic+Targeting+Photothermal+Therapy+and+Fenton+Reaction.">Fe3O4-Based Multifunctional Nanospheres for Amplified Magnetic Targeting Photothermal Therapy and Fenton Reaction.</a> ACS Biomaterials Science &amp; Engineering. 5 (2), 1045-1056 (2018).</li><li>Thorat, N. 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Progressive Comparison of Iron Oxide and Gold Nanoparticles Heating in Water, in Cells, and In Vivo.</a> Advanced Functional Materials. 28 (37), 1803660 (2018).</li><li>Xu, C., 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=Magnetic+Hyperthermia+Ablation+of+Tumors+Using+Injectable+Fe(3)O(4)/Calcium+Phosphate+Cement.">Magnetic Hyperthermia Ablation of Tumors Using Injectable Fe(3)O(4)/Calcium Phosphate Cement.</a> ACS Applied Materials &amp; Interfaces. 7 (3), 13866-13875 (2015).</li><li>Wilhelm, S., 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=Analysis+of+nanoparticle+delivery+to+tumours.">Analysis of nanoparticle delivery to tumours.</a> Nature Reviews Materials. 1, 16014 (2016).</li><li>Chen, H., Zhang, W., Zhu, G., Xie, J., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rethinking+cancer+nanotheranostics.">Rethinking cancer nanotheranostics.</a> Nature Reviews Materials. 2, (2017).</li><li>Rapoport, N. Y., Kennedy, A. M., Shea, J. E., Scaife, C. L., Nam, K. 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Here, we presented a protocol of fabricating iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. The IONPs were densely packed around the air core to form a magnetic shell, which can be controlled by the external magnetic field for targeting. Once delivered, the release of IONPs can be achieved by ultrasound trigger. The released IONPs can be activated by both NIR light and AFM for PTT and MHT, or the combin...

Cancer is one of the deadliest diseases, causing millions of deaths every year worldwide and huge economic losses1. In clinics, conventional anticancer therapies, such as surgical resection, radiotherapy, and chemotherapy still cannot provide a satisfactory therapeutic efficacy2. Limitations of these therapies are high toxic side effects, high recurrence rate and high metastasis rate3. For example, chemotherapy is suffered from the low delivery efficiency of chemo drugs precisely to the tumor site4. The inability of drugs to penetrate deep into the tumor tissue across the biological barriers, including extracellular matrix and high tumor interstitial fluid pressure, is also responsible for the low therapeutic efficacy5. Besides, the tumor resistance usually happens in the patients who received treatment by single chemotherapy6. Therefore, techniques where thermal ablation of tumor occurs, such as photothermal therapy (PTT) and magnetic hyperthermia therapy (MHT), have shown promising results to reduce tumor resistance and have been emerging in clinical trials7,8,9.
PTT triggers thermal ablation of cancer cells by the action of photothermal conversion agents under the irradiation of the laser energy. The generated high temperature (above 50 &#730;C) induces complete cell necrosis10. Very recently, iron oxide nanoparticles (IONPs) were demonstrated to be a photothermal conversion agent that can be activated by near-infrared (NIR) light11. &#160;Despite the low molar absorption coefficient in the near infrared region, IONPs are candidates for low-temperature (43&#160; &#730;C) photothermal therapy, a modified therapy to reduce the damage caused by heat exposure to normal tissues and to initiate antitumor immunity against tumor metastasis12. One of the limitations of PTT is the low penetration depth of the laser. For deep seated tumors, alternating magnetic field (AFM) induced heating of iron oxide nanoparticles, also called magnetic hyperthermia, is an alternative therapy for PTT13,14. The main advantage of MHT is the high penetration of magnetic field15. However, the required relatively high concentration of IONPs remains a major disadvantage for its clinical application. The delivery efficiency of nanomedicine (or nanoparticles) to solid tumors in animals has been 1-10% due to a series of obstacles including circulation, accumulation, and penetration16,17. Therefore, a controlled and targeted IONPs delivery strategy with the ability to achieve high tissue penetration is of great interest in cancer treatment.
Ultrasound mediated nanoparticle delivery has shown its ability to facilitate the penetration of nanoparticles deep into the tumor tissue, due to the phenomenon called microbubble cavitation18,19. In the present study, we fabricate IONPs shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. The NSM contains an air core and a shell of iron oxide nanoparticles, with a diameter of approximately 5.4 &#181;m. The NSMs can be magnetically guided to the tumor site. Then the release of IONPs is triggered by ultrasound, accompanied by microbubble cavitation and microstreaming. The momentum received from the microstreaming facilitates the penetration of IONPs into the tumor tissue. The PTT and MHT can be achieved by NIR laser irradiation or AFM application, or with the combination of both.

<table border="0" cellpadding="0" cellspacing="0" style="width:1331px;" width="1330">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:337px;">808 nm laser power</td>
			<td style="width:315px;">Changchun New Industries Optoelectronics Tech</td>
			<td style="width:263px;">MDL-F-808-5W-18017023</td>
			<td style="width:416px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Calcein-AM</td>
			<td>Thermo Fisher SCIENTIFIC</td>
			<td>C3099</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fetal bovine serum</td>
			<td>Invitrogen</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fluorescence Microscope</td>
			<td>Olympus</td>
			<td>IX71</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Function generator</td>
			<td>Keysight</td>
			<td>33500B series</td>
			<td style="width:416px;">20 MHz, 2 channels with arbitrary waveform generation capability</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;">Gelatin gel</td>
			<td>Sigma</td>
			<td>9000-70-8</td>
			<td style="width:416px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Heating machine</td>
			<td>Shuangping</td>
			<td>SPG-06- II</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Homemade focused transducer</td>
			<td></td>
			<td></td>
			<td>Frequency=855, R-X=36.2W+5.8W, |Z|-&#952;=37W+8&#176;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Homogenizer</td>
			<td>SCILOGEX</td>
			<td>D-160</td>
			<td>8000-30000 rpm</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Hydrophone</td>
			<td>T&#38;C</td>
			<td>NH1000</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ICR male mice</td>
			<td>OG Pharmaceutical. Co. Ltd</td>
			<td></td>
			<td>8-week-old</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Inductively coupled plasma optical emission spectrometry</td>
			<td>PerkinElmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Infrared thermal imaging camera.</td>
			<td>FLIR</td>
			<td>E50</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Iron(II,III) oxide</td>
			<td>Alfa Aesar</td>
			<td>1317-61-9</td>
			<td>50-100nm APS Powder</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Laser power meter</td>
			<td colspan="2">Changchun New Industries Optoelectronics Tech</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Oscilloscope</td>
			<td>Keysight</td>
			<td>DSOX3054T</td>
			<td>Bandwidth 500 MHz, Sampling Rate 5 GS/S, 4 channels</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">RF Power Amplifier</td>
			<td>T&#38;C</td>
			<td>AG1020</td>
			<td style="width:416px;">The signal source can also be connected to an external signal source. The gain can be adjusted from 0 to 100%. It has multiple functions such as frequency sweep, pulse, and triangle.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Roswell Park Memorial Institute-1640</td>
			<td>KeyGEN BioTECH</td>
			<td>KGM31800</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium dodecyl sulfate</td>
			<td>Sigma</td>
			<td>151-21-3</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The triple-responsive nanoparticle-shelled microbubbles (NSMs) used in this study were prepared by agitating the mixture of the surfactant and IONPs. The IONPs (50 nm) self-assembled at the interface of liquid and gas core, to form a densely packed magnetic shell. The morphology of NSMs is shown in Figure. 1A. The resulted NSMs presented a spherical shape and with an average diameter of 5.41 &#177; 1.78 &#956;m (Figure 1B). The results indicated the NSMs were prepared successfully. When ...

Watch this Scientific Journal Video about Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy at JoVE.com

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

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 ...

This protocol holds great promise for improving post-nanomedicine delivery and the anti-cancer efficacies of nanoparticles in cancer treatment. This technique synergizes magnetic, acoustic, and optical responsivenesses into one nanotherapeutic platform for the control and targeted delivery of nanomedicine, and facilitates the combination of photothermal and magnetic hyperthermia therapy. Demonstrating the procedure will be Siyu Wang, a magnetic, acoustic, and optical-triple-responsive microbubbles for magnetic hyperthermia and photothermal combination cancer therapy fellow from my laboratory.For nanoparticle shelled microbubble formation, uniformly disperse magnetic iron oxide nanoparticles in deionized water to generate a 10 milligram per milliliter stock solution and load the nanoparticle solution into an ultrasonic cleaning machine for 20 minutes. At the end of the sonication, add 150 microliters of deionized water, 150 microliters of 10 millimolar sodium dodecyl sulfate, and 400 microliters of the sonicated iron oxide nanoparticle solution in a 1.5 milliliter centrifuge tube. Next, fix a homogenizer with a scaffold in an ice bath and place the nanoparticle solution into the ice bath.Immerse the homogenizer probe in the nanoparticle solution and homogenize the suspension for three minutes at 20, 000 revolutions per minute. At the end of the homogenization, allow the solution to stabilize for 12 hours at room temperature before placing the tube into a magnetic holder to adsorb the nanoparticle shelled microbubbles to the tube wall. Replace the supernatant with one milliliter of fresh deionized water three times to wash the nanoparticle shelled microbubbles.After the last wash, slightly shake the tube and transfer 10 microliters of the nanoparticle shelled microbubbles onto a clean glass slide. Use a fluorescence microscope and a 20X magnification to image the nanoparticle shelled microbubbles. After imaging, open the image in the microscope software and use the ruler to set a red line with the same length as the ruler.Click set and scale to enter the length of the ruler and draw lines of the same lengths at the diameters of at least 200 individual microbubbles. Then click report and view report. To measure the acoustic response of the microbubbles, dilute 200 microliters of the nanoparticle shelled microbubbles in 800 microliters of deionized water in a 1.5 milliliter tube and connect the function generator, amplifier, impedance matching, and homemade focus transducer.Place the transducer in the center of the bottom of the artificial cuboid sink and connect the hydrophone with an oscilloscope to monitor the output ultrasound intensity. Add enough deionized water to submerge the transducer and adjust the function generator to the sweep mode. Tune in the frequency range from 10 to 900 kilohertz and set the amplitude to 20 voltage peak to peak.Use the amplifier to adjust the power of the ultrasound to 0.1%and the cycle duration to four seconds with a one-second time interval. Place the tube of nanoparticles into the scaffold on the top of the homemade focus transducer and attach the magnet to the bottom of the tube. Turn on the function generator and the amplifier power.After five 25-second ultrasound cycles, switch off the function generator and remove the magnet. Then replace the nanoparticle solution with one milliliter of deionized water and repeat the ultrasound and treatment. To set up the laser for optical treatment of the microbubbles, first turn on the laser power supply.After several minutes, fix a fiber-coupled 808 nanometer laser diode onto a retort stand and use an optical fiber to direct the laser beam to the sample stage. Use a convex lens to focus on the sample stage to achieve a six millimeter diameter light spot and measure the power output with the laser power meter. Then adjust the power to one watt per square centimeter.To perform a photothermal measurement, prepare one milliliter volumes of different concentrations of the iron oxide nanoparticles in individual 1.5 milliliter centrifuge tubes and place the first tube at the focused region of the laser beam. Record the baseline temperature of the sample and turn on the laser and infrared thermal imaging camera. Irradiate the sample continuously for 10 minutes while recording the temperature in real time.Then turn off the laser and the camera and wait for the temperature of the region to return to the baseline before measuring the other sample concentrations in the same manner. For a magnetic hyperthermia measurement in an aqueous solution, prepare different iron oxide nanoparticle dilutions as demonstrated and place one dilution in the center of a water cold magnetic induction copper coil. Turn on the alternating magnetic field and the infrared thermal imaging camera and continuously induce the sample for 10 minutes while recording the temperature in real time.At the end of the treatment, turn off the alternating magnetic field and the camera. When the temperature of the copper coil has returned to baseline, measure the next sample. Nanoparticle shelled microbubbles typically demonstrate a spherical shape with an average diameter of about 5.41 micrometers.Although the microbubbles remain intact for up to a year, a stepwise release of iron can be achieved by increasing the number of ultrasound cycles. Iron oxide nanoparticle-mediated photothermal measurement in aqueous solution reveals a rapid increase in iron oxide nanoparticle temperature over time with a 30 degree Celsius temperature increase achieved upon 10 minutes of exposure to near infrared laser light at a five milligram per milliliter iron concentration. Compared to the control group, no differences in morphology or live cell number are observed when breast cancer cell lines are incubated with a high concentration of iron, suggesting a good bioavailability of the iron oxide nanoparticles.Upon irradiation, the nanoparticle-treated cancer cells became rounded in shape and demonstrated decreased viability indicating apoptosis. Five minutes after irradiation, the temperature of the gelatin injection areas rapidly increases by about 20 degrees Celsius. When exposed to alternating magnetic field therapy, thermal imaging of different concentrations of iron oxide nanoparticle reveals an alternating magnetic field response characteristic of nanoparticle shelled microbubbles.Further, wholesale imaging of mice exposed to alternating magnetic field therapy reveals significant rapid temperature changes within the area of interest. During the nanoparticle solution agitation, make sure that the homogenizer probe remains completely immersed within the solution. This protocol can also achieve and improving the penetration into tumor tissues to address the challenges of nanomedicine delivery in cancer treatment.

magnetic-acoustic-optical-triple-responsive-microbubbles-for-magnetic

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Bioengineering

2.9K 조회수.  Nanjing University of Posts & Telecommunications. This protocol holds great promise for improving post-nanomedicine delivery and the anti-cancer efficacies of nanoparticles in cancer treatment. This technique synergizes magnetic, acoustic, and optical responsivenesses into one nanotherapeutic platform for the control and targeted delivery of nanomedicine, and facilitates the combination of photothermal and magnetic hyperthermia therapy. Demonstrating the procedure will be Siyu Wang, a magnetic, acoustic, and optical-triple-responsive microbu...