Name: magnetic-, acoustic-, and optical-triple-responsive microbubbles for magnetic hyperthermia and pothotothermal combination cancer therapy
Uploaded: 2020-05-22T14:30:00
Description: Watch this Scientific Journal Video about Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy at JoVE.com

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>
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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">
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		<col />
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		<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.

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Bioengineering

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 ...

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 ...

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various factors affect the relaxation rates, such as buffer composition, solution pH, temperature, and magnetic field. In this last regard, the spin-lattice relaxation time can be measured at clinical field strengths, but at lower fields, where these compounds are dispensed from the polarizer and transported to the MRI, the relaxation is even faster and difficult to measure. To have a better understanding of the amount of magnetization lost during transport, we used fast field-cycling relaxometry, with magnetic resonance detection of 13C nuclei at ~0.75 T, to measure the nuclear magnetic resonance dispersion of the spin-lattice relaxation time of hyperpolarized [1-13C]pyruvate. Dissolution dynamic nuclear polarization was used to produce hyperpolarized samples of pyruvate at a concentration of 80 mmol/L and physiological pH (~7.8). These solutions were rapidly transferred to a fast field-cycling relaxometer so that relaxation of the sample magnetization could be measured as a function of time using a calibrated small flip angle (3&#176;-5&#176;). To map the T1 dispersion of the C-1 of pyruvate, we recorded data for different relaxation fields ranging between 0.237 mT and 0.705 T. With this information, we determined an empirical equation to estimate the spin-lattice relaxation of the hyperpolarized substrate within the mentioned range of magnetic fields. These results can be used to predict the amount of magnetization lost during transport and to improve experimental designs to minimize signal loss.

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

We present a protocol to measure the magnetic field dependence of the spin-lattice relaxation time of 13C-enriched compounds, hyperpolarized by means of dynamic nuclear polarization, using fast field-cycled relaxometry. Specifically, we have demonstrated this with [1-13C]pyruvate, but the protocol could be extended to other hyperpolarized substrates.

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various ...