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 the IONPs solution in an ultrasonic cleaning machine for 20 min. Obtain a uniformly dispersed IONPs solution before use.</li>
	<li>Add 150 &#956;L of deionized water, 150 &#956;L of 10 mM sodium dodecyl sulfate (SDS), and 400 &#956;L of a stock solution of IONPs from step 1.1. in 1.5 mL centrifuge tube.&#160;</li>
	<li>Fix the homogenizer with a scaffold in an ice bath.</li>
	<li>Place the tube of the mixture in an ice bath and position the homogenizer probe precisely to be immersed in the mixture solution.</li>
	<li>Adjust the homogenizer speed to 20,000 rpm and turn on the homogenizer for 3 min.</li>
	<li>Turn off the homogenizer and remove the tube from the ice bath.</li>
	<li>Place the tube to the tube rack for 12 h stabilization at room temperature.</li>
	<li>Adsorb the resulted NSMs to the tube wall by a magnet and remove the supernatant. Then replenish with 1 mL of fresh deionized water.</li>
	<li>Repeat the wash process for three times and re-suspend NSMs in 1 mL of fresh distilled water.</li>
	<li>Transfer 10 &#956;L of NSMs suspension to a clean glass slide after slightly shaking.</li>
	<li>Use a fluorescence microscope at 20x magnification to visualize NSMs morphology. Ensure taking photos at random area.</li>
	<li>Measure the diameter of the NSMs from the photos using an open access Nano Measurer 1.2 software. Count at least 200 microbubbles.
	<ol>
		<li>Click &#8220;File&#8221; and &#8220;Open&#8221; to select the image file to be processed. After the image is imported, press the &#8220;ruler&#8221;. Draw a red line with the same length as the ruler.</li>
		<li>Then press &#8220;Settings&#8221; | &#8220;Ruler&#8221; and enter the length of the ruler. Draw lines of the same lengths as the diameters of the individual microbubbles in the image. Complete all the measurements, then click on &#8220;Report&#8221; | &#8220;View Report&#8221;.</li>
	</ol>
	</li>
</ol>
2. Acoustic response of NSMs
<ol>
	<li>Dilute 200 &#956;L of NSMs with 800 &#956;L deionized water, then transfer into a 1.5 mL centrifuge tube to form a stock solution.</li>
	<li>Connect the function generator, amplifier, impedance matching, and homemade focused transducer. Place the transducer in the center at the bottom of the artificial cuboid sink and connect the hydrophone with an oscilloscope to monitor the output ultrasound intensity (Figure 1). Add deionized water into the sink to immerse the transducer.</li>
	<li>Adjust the function generator to the sweep mode, tune the frequency range from 10 kHz to 900 kHz and set the amplitude to 20 Vpp (Voltage peak-peak). Adjust the power of ultrasound to 0.1% by the amplifier. Each cycle&#8217;s duration is 4 s with a time interval of 1 s.</li>
	<li>Prepare 1 mL NSMs stock solution samples in the tube and fix the tube with a scaffold on the top of the homemade focused transducer. Attach the magnet to the bottom of the tube and attract the NSMs.</li>
	<li>Turn on the power of the function generator and the amplifier. Switch off the function generator after application of 5 cycles (25 s) of ultrasound. Remove the magnet and collect 1 mL of the solution containing the released IONPs. Add 1 mL of deionized water to the centrifuge tube.</li>
	<li>Repeat step 2.5. until NSMs in the tube collapse completely.</li>
	<li>Quantify all the released IONPs by the inductively coupled plasma optical emission spectrometry (ICP-OES) as described previously13.</li>
</ol>
3. Optical response of NSMs
NOTE: In this work, a laser system containing 808 nm laser power and an infrared thermal imaging camera previously described by Xu et al. is utilized20.
<ol>
	<li>Laser system preparation
	<ol>
		<li>Turn on the power supply of the laser and allow it to warm for several minutes. Fix a fiber coupled 808 nm laser diode on a retort stand.</li>
		<li>Direct the laser beam to the sample stage through an optical fiber and focus on the sample stage to achieve a 6 mm light spot (in diameter) by a convex lens.</li>
		<li>Measure the power output with a laser power meter and adjust the power to 1 W/cm2.</li>
		<li>Fix the infrared thermal imaging camera on the tripod. Turn on the camera and check the if working (e.g., monitor the focused region of interest (ROI) temperature). Turn off the power supply and the infrared thermal imaging camera.</li>
	</ol>
	</li>
	<li>Photothermal measurement in aqueous solution
	<ol>
		<li>Prepare the 1 mL sample at different IONPs concentration (1.05 mg/mL, 1.35 mg/mL, 3.65 mg/mL, 5 mg/mL) in a 1.5 mL centrifuge tube.</li>
		<li>Place the tube of interest at the focused region of the laser beam and record the baseline temperature of the sample.</li>
		<li>Turn on the laser power and the infrared thermal imaging camera and irradiate the sample for 10 min continuously. At the same time, record the temperature in real time.</li>
		<li>Turn off the laser power and infrared thermal imaging camera after 10 min of irradiation. Wait for the temperature of the region to return to the baseline.</li>
		<li>Repeat 3.2.2 to 3.2.4 for the measurement of other samples. 
		NOTE: Use deionized water at 20 &#176;C as a control for the photothermal measurement.</li>
	</ol>
	</li>
	<li>Photothermal measurement in cultured cells 
	NOTE: Murine breast cancer cells (4T1) were selected as a model to investigate the inhibition effect by photothermal treatment.
	<ol>
		<li>Feed the cells with Roswell Park Memorial Institute-1640 (RPMI-1640) medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin. Set the culturing environment as 37 &#176;C and 5% CO2.</li>
		<li>Culture 4T1 cells in T25 flasks and passage the cells in a 1:2 ratio when 90% confluence is reached. 
		NOTE: The subculture ratio can be adjusted according to the specific cell conditions in different labs.</li>
		<li>Remove and discard the culture medium. Rinse the cell layer with 1x PBS solution to remove the residual serum containing trypsin inhibitor.</li>
		<li>Add 2 mL of Trypsin-EDTA solution (0.25%) to the flask for detachment. Then add 3 mL of 1640 medium and aspirate cells by gently pipetting.</li>
		<li>Collect 5 mL of the cell suspension and centrifuge at 500 x g for 3 min.</li>
		<li>Remove the supernatant and add 1 mL of fresh 1640 medium to form a cell suspension.</li>
		<li>Add 100 &#956;L of the cell suspension to a confocal dish containing 1 mL of the culture medium. Ensure that the concentration of cell suspension is 9 x 105/mL, and the final cell concentration in cell culture confocal dish is 8.1 x 104/mL.</li>
		<li>Place the inoculated cell culture confocal dish in an incubator for 24 h.</li>
		<li>Dilute different concentration (1.05 mg/mL, 1.35 mg/mL, 3.65 mg/mL, 5 mg/mL) of the IONPs sample to make 1 mL solution with serum-free 1640 medium.</li>
		<li>Aspirate the culture medium from the confocal dish and add the prepared sample solution.</li>
		<li>Turn on the laser power. Focus the laser beam at the center of the dish and adjust the output power to 1 W/cm2. Turn on the infrared thermal imaging camera, irradiate the cells in the confocal dish for 10 min continuously. Record the temperature in real time for the focused region.</li>
		<li>Turn off the laser and infrared thermal imaging camera. Transfer the irradiated dish to the incubator for another 24 h.</li>
		<li>Remove and discard the culture medium, add 1 mL of the fresh culture medium in the confocal dish. Add 5 &#956;L of Calcein-AM solution (1 mg/mL) into the dish.</li>
		<li>Incubate the confocal dish at 37 &#176;C and 5% CO2 for 15 min. Rinse the cell layer with 1x PBS solution twice.</li>
		<li>Observe and image the cells by a confocal fluorescence microscope with an excitation wavelength of 488 nm and an emission wavelength of 500-540 nm.</li>
		<li>Choose 5 areas in the confocal images randomly and count the number of live 4T1 cells in each area manually. Quantify the viability of 4T1 cells by comparing the number of live cells in all experimental groups with the control group. 
		NOTE: Use the serum-free 1640 medium at 20 &#176;C as a control for the photothermal measure. Use the sample without laser irradiation as cell viability control group.</li>
	</ol>
	</li>
	<li>Photothermal measurement in vivo
	<ol>
		<li>Prepare 3 of 8-week-old ICR male mice with a mean weight of 25 &#177; 2 g.</li>
		<li>Add 2 g of gelatin powder to 20 mL of deionized water. Heat the solution to 40 - 50 &#176;C, dissolve the gelatin gel completely to form a transparent and clear solution.</li>
		<li>Add 100 mg IONPs to the solution from 3.4.2.</li>
		<li>Heat the gel to 40 &#8211; 50 &#176;C, immediately inject 500 &#956;L of the gelatin solution into the right breast pad of the mouse.</li>
		<li>Turn on the laser power and focus the laser beam at the interest region (right breast pad of the mice). Adjust the output power to 1 W/cm2. Turn on the infrared thermal imaging camera, irradiate the interest region for 10 min continuously. Record the temperature of the region of interest in real time.</li>
		<li>Turn off the laser and infrared thermal imaging camera.</li>
		<li>Euthanize mice by CO2 asphyxiation and cervical vertebra dislocation or any method approved by the Institute&#8217;s animal research committee.</li>
	</ol>
	</li>
</ol>
4. Magnetic hyperthermia measurement
NOTE: Here, a magnetic hyperthermia system previously described by Wu et al. is utilized (21).
<ol>
	<li>Prepare a magnetic hyperthermia system include an alternating magnetic field (AFM) generator and an infrared thermal imaging camera.
	<ol>
		<li>Turn on the chiller for 10 min and then power on the moderate radio frequency heating machine (i.e., AFM generator).</li>
		<li>Set the parameters of the machine as follows: frequency (f) = 415 kHz, magnetic field intensity = 1.8 kA/m.</li>
	</ol>
	</li>
	<li>Magnetic hyperthermia measurement in aqueous solution
	<ol>
		<li>Prepare 1 mL of the sample at different IONPs concentration (1.05 mg/mL, 1.35 mg/mL, 3.65 mg/mL, 5 mg/mL) in a 1.5 mL centrifuge tube.</li>
		<li>Place the tube in the center of a water-cooled magnetic induction copper coil.</li>
		<li>Turn on the alternating magnetic field (AFM) and the infrared thermal imaging camera. Induce the sample for 10 min continuously and record the temperature in real time. 
		NOTE: The camera is located at the top of the sample, supplying a cross-sectional view of the sample.</li>
		<li>Turn off the AFM and the infrared thermal imaging camera. Wait for the temperature of the copper coil to come back to the baseline for 10 min. 
		NOTE: Be careful of high temperature, avoid direct contact with hands and wait for cooling before removing samples.</li>
		<li>Repeat 4.2.2 to 4.2.4 for the measurement of the other samples.</li>
		<li>Power off the moderate radio frequency heating machine (AFM) and chiller. 
		NOTE: Use the deionized water at 20 &#176;C as a control for magnetic hyperthermia measurement.</li>
	</ol>
	</li>
	<li>Magnetic hyperthermia measurement in vivo
	<ol>
		<li>Prepare 3 of 8-week-old ICR male mice with a mean weight of 25 &#177; 2 g.</li>
		<li>Prepare 20 mL of 10 % gelatin gel containing 5 mg/mL IONPs solution.</li>
		<li>Heat the gelatin gel to 40 - 50 &#176;C, immediately inject 500 &#956;L of the gelatin solution into the right breast pad of the animal. 
		NOTE: Magnetic-induced hyperthermia experiments were conducted with the heating machine using the same parameters as the in vitro test.</li>
		<li>Turn on the alternating magnetic field (AFM) and the infrared thermal imaging camera. Place the right breast pad of the mice in the center of a water-cooled magnetic induction copper coil.</li>
		<li>Turn on the infrared thermal imaging camera, image the interest region (right breast pad of the mice) for 10 min continuously and record the temperature of the interest region in real time.</li>
		<li>Turn off the power switch of the machine and infrared thermal imaging camera after 10 min of induction. Wait for the temperature of the copper coil to return to the baseline for 10 min.</li>
		<li>Repeat 4.3.4 to 4.3.6 for the measurement of other samples.</li>
		<li>Power off the moderate radio frequency heating machine (AFM) and chiller. 
		NOTE: Be careful of high temperature, avoid direct contact with hands and wait for cooling before removing samples.</li>
		<li>Euthanize mice by CO2 asphyxiation and cervical vertebra dislocation or any method approved by the Institute&#8217;s animal research committee.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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 ...

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

Research

JoVE Journal

Bioengineering

3.0K Views.  Nanjing University of Posts & Telecommunications. 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.

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

Magnetic Tweezers for the Measurement of Twist and Torque

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques include so-called force spectroscopy approaches such as atomic force microscopy, optical tweezers, flow stretching, and magnetic tweezers. Amongst these approaches, magnetic tweezers have distinguished themselves by their ability to apply torque while maintaining a constant stretching force. Here, it is illustrated how such a &#8220;conventional&#8221; magnetic tweezers experimental configuration can, through a straightforward modification of its field configuration to minimize the magnitude of the transverse field, be adapted to measure the degree of twist in a biological molecule. The resulting configuration is termed the freely-orbiting magnetic tweezers. Additionally, it is shown how further modification of the field configuration can yield a transverse field with a magnitude intermediate between that of the &#8220;conventional&#8221; magnetic tweezers and the freely-orbiting magnetic tweezers, which makes it possible to directly measure the torque stored in a biological molecule. This configuration is termed the magnetic torque tweezers. The accompanying video explains in detail how the conversion of conventional magnetic tweezers into freely-orbiting magnetic tweezers and magnetic torque tweezers can be accomplished, and demonstrates the use of these techniques. These adaptations maintain all the strengths of conventional magnetic tweezers while greatly expanding the versatility of this powerful instrument.

Magnetic tweezers, a powerful single-molecule manipulation technique, can be adapted for the direct measurements of the twist (using a configuration called freely-orbiting magnetic tweezers) and torque (using a configuration termed magnetic torque tweezers) in biological macromolecules. Guidelines for performing such measurements are given, including applications to the study of DNA and associated nucleo-protein filaments.

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

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

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

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

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

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 ...

Magnetic Levitation Coupled with Portable Imaging and Analysis for Disease Diagnostics

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for specialized equipment and trained personnel require that many diagnostic tests be performed at remote, centralized clinical laboratories. Magnetic levitation is a simple yet powerful technique and can be applied to levitate cells, which are suspended in a paramagnetic solution and placed in a magnetic field, at a position determined by equilibrium between a magnetic force and a buoyancy force. Here, we present a versatile platform technology designed for point-of-care diagnostics which uses magnetic levitation coupled to microscopic imaging and automated analysis to determine the density distribution of a patient's cells as a useful diagnostic indicator. We present two platforms operating on this principle: (i) a smartphone-compatible version of the technology, where the built-in smartphone camera is used to image cells in the magnetic field and a smartphone application processes the images and to measures the density distribution of the cells and (ii) a self-contained version where a camera board is used to capture images and an embedded processing unit with attached thin-film-transistor (TFT) screen measures and displays the results. Demonstrated applications include: (i) measuring the altered distribution of a cell population with a disease phenotype compared to a healthy phenotype, which is applied to sickle cell disease diagnosis, and (ii) separation of different cell types based on their characteristic densities, which is applied to separate white blood cells from red blood cells for white blood cell cytometry. These applications, as well as future extensions of the essential density-based measurements enabled by this portable, user-friendly platform technology, will significantly enhance disease diagnostic capabilities at the point of care.

We present a magnetic levitation technique coupled with automated imaging and analysis in both a smartphone-compatible device and a device with embedded imaging and processing. This is applied to measure the density distribution of cells with two demonstrated biomedical applications: sickle cell disease diagnosis and separating white and red blood cells.

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

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 ...

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps compared to standard metallographic sample preparation procedures. The paper proposes a novel bespoke rig for dynamic domain imaging with in situ BH (magnetic hysteresis) measurements and elaborates the protocols for the sensor preparation and the use of the rig to ensure accurate BH measurement. The protocols for static and ordinary dynamic domain imaging (without in situ BH measurements) are also presented. The reported method takes advantage of the convenience and high sensitivity of the traditional Bitter method and enables in situ BH measurement without interrupting or interfering with the domain wall movement processes. This facilitates establishing a direct and quantitative link between the domain wall movement processes&#8211;microstructural feature interactions in ferritic steels with their BH loops. This method is anticipated to become a useful tool for the fundamental study of microstructure&#8211;magnetic property relationships in steels and to help interpret the electromagnetic sensor signals for non-destructive evaluation of steel microstructures.

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements.

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...