Supported by NIH R01 HL74082, the Hartwell Foundation, and the Focused Ultrasound Surgery Foundation.

1. Microbubble Production
<ol>
	<li>To prepare albumin microbubbles ( MBs), place a 1% solution of serum albumin in normal saline in a flask with a blanket of gas (octafluoropropane) above the aqueous phase. Briefly sonicate the solution (30 sec) with an ultrasound disintegrator equipped with an extended &frac12;" titanium probe. This formulation is similar to Optison (GE Heathcare), which is provided in a concentration range of 0.5- 1.2 x 109 MBs/ml. Determine mean MB diameter with a Multisizer Coulter Counter. The albumin mean MB diameter used in this study was 1.93um &plusmn;1.63um.</li>
	<li>To fabricate lipid MBs, prepare an aqueous dispersion of 1 mg/ml polyethyleneglycol-40 stearate (Sigma Chemical Co., St. Louis, MO) and 2 mg/ml distearoyl phosphatidylcholine (Avanti Polar Lipids, Alabaster, AL) and sonciate as previously described (1.1) with decafluorobutane gas. Determine mean MB diameter with a Multisizer Coulter Counter. The mean lipid MB diameter used in this study was 2.01um &plusmn;1.29um.</li>
</ol>
2. Nanoparticle Fabrication
<ol>
	<li>These methods were adapted from the water-in-oil-in-water emulsion solvent evaporation technique described by Davda (2002) and Chappell (2008).</li>
	<li>Prepare a 2% Poly(vinyl alcohol) PVA solution by dissolving 20g of PVA in 1000 ml of deionized (DI) water. Allow solution to fully dissolve on a stir plate overnight. Centrifuge solution at 1000 rpm for 10 minutes and filter with a 0.22&mu;m sterile filter to remove any residual undissolved PVA.</li>
	<li>To fabricate bovine serum albumin (BSA) loaded poly(lactic-co-glycolic acid) (PLAGA) nanoparticles (NPs), dissolve 180mg of uncapped 85:15 PLAGA in 6ml methylene chloride (MC) in a glass scintillation vial. Vortex the MC/PLAGA solution for 2 minutes.</li>
	<li>Dissolve desired payload (15mg of BSA) in 1.5 ml of PBS. Add the PBS/BSA solution in two parts to the PLAGA/MC solution with intermittent vortexing. Place solution on ice for 5minutes and sonicate at 45W for 120 seconds. </li>
	<li>Add the MC/PLAGA/payload/PBS solution to 24ml of 2% PVA, in two portions with intermediate vortexing. </li>
	<li>Placing the solution on ice for 5minutes and sonicate at 45W for 120.</li>
	<li>Stir the NP emulsion for 12 hours on a stir plate in a fume hood to allow evaporation of MC and NP stabilization. </li>
	<li>Centrifuge the suspension for 25minutes at 20,000 rpm at 4&deg;C twice to isolate NPs and remove residual PVA. Resuspend the pellet in 8ml of DI water and centrifuge for 10 minutes at 1000 rmp at 4&deg;C to isolate the 100nm population of NPs.</li>
	<li>Isolate the supernatant and flash freeze it at -80&deg; C. Lyophilize the frozen sample for 48 hrs. Store the lyophilized particles in a dessicator at -80&deg; C until time of use.</li>
</ol>
3. Composite Delivery Vehicle Fabrication (Protocol Adapted from VisEn Chemistry Notes)
<ol>
	<li>Conversion of VivoTag680 to carboxy surface functionality
	<ol>
		<li>Combine one vial of VivoTag680 with 50 &mu;L of 1.0 M HEPES, pH 7 and Succinic anhydride (2.5 mg, 25 &igrave;mol) in 25 &mu;L DMSO. Add 50 &mu;L of 1.0 M NaOH to this solution, mixed thoroughly; allow solution to react for 2 hours at room temperature protected from light. Purify particles using Bio-Rad BioGel (P100, medium) eluting with 0.1 M MES buffer, pH 6.0. Collect the green band.</li>
	</ol>
	</li>
	<li>Activation of carboxy-modified VivoTag680 
	<ol>
		<li>Combine carboxy-modified VivoTag680 obtained in 0.1 M MES buffer, pH 6, with 1 mg of 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 2.2 mg of N-hydroxysulfosuccinimide (Sulfo-NHS). Allow to react for 2 h at room temperature.</li>
		<li>Purify activated particles from excess activating agent (EDC) by gel filtration using Bio-Rad BioGel (P100, medium) eluting with 0.1 M MES buffer, pH 6.0 and collect the green band. Immediately conjugate the activated particles to amine-containing molecules (e.g. BSA-loaded Nanoparticle). Allow the solution to react for 2 hours at room temperature. Amine conjugated NPs were purified from excess EDC by centrifuging at 20,000 rpm for 60min at 4 &deg;C, pouring off the supernatant after centrifugation and resuspending in 0.1 MES buffer.</li>
	</ol>
	</li>
	<li>Stop here to fabricate VivoTag680 tagged NPs for drug delivery studies.</li>
	<li>Conjugation of carboxy-modified PLGA-BSA-VivoTag680 Nanoparticles (NP680) to Albumin Microbubbles 
	<ol>
		<li>Combine NP680 obtained in 0.1 M MES buffer, pH 6 with 1 mg of 1-Ethyl-3-(3 dimethylaminopropyl)-carbodiimide (EDC) and 2.2 mg of N-hydroxy-sulfosuccinimide (Sulfo-NHS). Allow the solution to react for 1 hour at room temperature.</li>
		<li>Purify activated nanoparticles from excess EDC by centrifuging at 20,000 rpm for 60min at 4 &deg;C, pouring off the supernatant after centrifugation. Resuspend nanoparticles in 0.1 MES buffer.</li>
		<li>Wash albumin MBs three times in degassed PBS to remove excess BSA from solution.</li>
		<li>Immediately conjugate the activated particles to amine-containing molecules (e.g. Albumin Microbubbles). Allow NP680 solution to react for 2 hour at room temperature. Purify NP680 conjugated microbubbles (MNCA) from unbound NP680s by washing three times with degassed PBS.</li>
		<li>Determine the concentration of MNCAs using a Coulter Counter.</li>
	</ol>
	</li>
</ol>
4 .Tumor Model
All animal experiments were in compliance with an animal protocol approved by the University of Virginia Animal Care and Use Committee.
<ol>
	<li>C6 Giloma rat tumor cell line was provided by Dr. Jason Sheehan of UVA (Charlottesville, VA). 
 <ol>
 <li>Cells line was tested to ensure cells were mycoplasma free.</li>
 </ol>
</li>
	<li>Maintain the cell line in F-12K Nutrient Mixture supplemented with 16% horse serum, 3% fetal bovine serum (FBS) and 1% Pen-Strep, (Gibco, USA) at 37&deg;C and 5% CO2.</li>
	<li>Inoculate C57BLJ6/Rag1 mice (Jackson) with 3 x 106 C6 Giloma tumor cells suspended in 300 &mu;l of PBS subcutaneously in the left hindlimb. Allow tumor to grow for 12 days to reach a maximum diameter of 8-10mm.</li>
</ol>
5. In Vivo Ultrasound Application
<ol>
	<li>Anesthetize mice with an intraperitoneal (IP) combination injection of ketamine hydrochloride (60 mg/kg body weight) and xylazine (0.1 mg/kg body weight) prior to treatment.</li>
<li>Prepare a maintenance anesthetic of ketamine hydrochloride (20 mg/kg body weight) and xylazine (0.3 mg/kg body weight). Administer as needed.</li>
<li>Cannulate the tail vein of each animal for the intravenous (I.V.) administration of a MB, MB/NP or MNCA solution.</li>
	<li>Tumor perfusion measurements
	<ol>
		<li>I.V. infuse a lipid MB solution (1x108 MBs/g body weight in 0.3 ml of 0.9% saline) at a rate of 15&mu;l/min with a continuous infusion pump (Harvard Apparatus PHD 2000; Harvard Apparatus, Holliston, MA).</li>
		<li>An Acuson Sequoia 512 ultrasonography system (Siemens Medical Solutions, Mountain View, CA) equipped with an 8 13 MHz linear 15L8 probe was used in this study to quantify contrast enhanced tumor perfusion. Couple the 15L8 probe to the tumor with a water-based ultrasound gel (Parker Laboratories Aquasonic 100; Parker Laboratories, Inc., Fairfield, NJ). Scan the tumor in B-Mode to obtain the best imaging plane.</li>
		<li>In contrast pulse sequence (CPS) mode, acquire a continuous video capturing microbubbles signal intensity 5 seconds prior, and 20 following, the high-amplitude "burst" pulse at a frame rate of 13Hz. Repeat measurements in four imaging planes.</li>
		<li>Wait ten minutes to infuse albumin MBs and initiate therapeutic low frequency treatment to allow lipid MBs to clear from the circulation.</li>
	</ol>
	</li>
	<li>Therapeutic Ultrasound Treatment
	<ol>
		<li>Ten minutes following tumor perfusion measurements (5.3) couple a 0.75'' diameter 1 MHz unfocused transducer (A314S; Panametrics, Waltham, MA) to the skin above the flank tumor. I.V. infuse an albumin MB (1 x105 MBs/g body weight in 0.3 ml of 0.9% saline), MB/NP (1 x105 MBs/g and 0.2 ug NPs/g body weight in 0.3 ml of 0.9% saline) or MNCA solution (1 x105 MNCAs/g body weight in 0.3 ml of 0.9% saline).</li>
		<li>Drug Delivery Treatment
		<ol>
			<li>Insonate for sixty minutes with the "1-Burst" pulse sequence (5.4.2.2) during a continuous infusion of NPs, a co-injection of MBs and NPs, or MNCAs (5.4.1).</li>
			<li>The "1-Burst" pulsing sequence consists of 100 consecutive 1 MHz sinusoids (duty cycle= 0.00002) each of 1V peak to- peak amplitude from a waveform generator (AFG-310; Tektronix, Inc., Beaverton, OR). Amplify the waveform signal by a 55 dB RF with a power amplifier (ENI 3100LA; Electronic Navigation Industries, Richardson, TX).</li>
		</ol>
		</li>
		<li>Ablative Treatment
		<ol>
			<li>Identical to 5.4.2.1, however insonate with the "5-Burst-Medium" or "5-Burst - Extended" pulsing sequence</li>
			<li>The "5-Burst-Medium" pulsing sequence consists of 5000 1 MHz sinusoids (duty cycle= 0.005) each of 1V peak to- peak amplitude . The "5-Burst-Exteneded" pulsing sequence consists of 10000 1 MHz sinusoids (duty cycle= 0.01) each of 1V peak to- peak amplitude.</li>
			<li>During the time course of the experiment monitor tumor temperature by inserting a needle thermocouple probe (Omega T-type, HYP1-30-1/2-T-G-60-SMPW-M) 2cm into the tumor. Record temperature measurements every five minutes.</li>
		</ol>
	</li>
	</ol>
	</li>
	<li>Repeat tumor perfusion measurements as described in 5.3 ten minutes following therapeutic treatment.</li>
</ol>
6. Tumor Perfusion Quantification
<ol>
	<li>Using the CPS program on the Sequoia, enter ACQ mode. Select the region of interest encompassing the complete tumor volume. Perfusion recovery curves will be generated of the form y = A (1 - e -&beta;t) + C (Sadlowski 2002; Chromas 2001; Yeh 2004) that are fit to the CPS data. Evaluate &beta;, a relative measure of red blood cell velocity, pre-treatment and post-treatment.</li>
	<li>Export CPS files for off-line analysis. Convert data into avi files. Break avi files into frame-by-frame image sequences. Using Compaq Visual Fortan, or a similar software package, generate a time averaged image of all frames from the destructive pulse to 8 seconds following the destructive pulse. </li>
	<li>To determine perfused tumor area, using ImageJ software, apply a threshold of 247 to the 8-bite time averaged CPS image and quantify the percent enhanced pixels within the tumor volume. Overlay the corresponding B-mode image with the time averaged thresholded image to accurately define tumor area.</li>
	<li>Average the percent perfused area for a minimum of four CPS clips to obtain a final perfused area at a given time point for a particular tumor. </li>
	<li>Take the product of percent perfused area (6.4) and &beta; (.1) to determine relative tumor blood flow.</li>
</ol>
7. Nanoparticle Biodistribution in Tumor
<ol>
	<li>Twelve days prior to treatment with fluorescently labeled NPs place mice on a low alfasprot diet (Harlan, Indianapolis IN) to reduce autofluorescence caused by normal mouse chow when imaging.</li>
	<li>Shave the imaging site (flank to liver) to remove all hair.</li>
<li>Remove residual hair with a chemical hair removal agent, such as Nair.
<ol>
<li>Rinse of all residual chemical hair remover or a chemical burn may occur.</li>
</ol>
</li>
<li>Image mice on the FMT system (VisEn Medical) before treatment and 0, 1, 4, and 24 hours after treatment as described in 5.4.2.</li>

	<li>Prior to imaging anesthetize mice with an IP combination injection of ketamine hydrochloride (60 mg/kg body weight) and xylazine (0.1 mg/kg body weight). Place mouse on the imaging cartridge. Acquire reflectance images in white light and fluorescent modes. Carry out fluorescent tomographic imaging in the VT680 channel.</li>
	<li>Employ the FMT software to generate 3D reconstructions of the imaging data utilizing a normalized Born equation. Following the reconstruction, select volumes of interest (VOI's) by drawing a region of interest (ROI's) in all 3 imaging planes (X, Y, Z). A mean fluorescent value and total VOI volume and fluorochrome concentration, due to fluorescently tagged BSA NPs, will be generated for VOI's encompassing the feet and liver.</li>
</ol>
8. Tumor Growth Rate
<ol>
	<li>Evaluate tumor volume by taking daily measurements using digital calibers. Calculate tumor volumes using an ellipsoid approximation; V= 1/6 &#960; abc. Where a, b, and c are the maximum diameters of the tumor measured in three orthogonal planes.</li>
</ol>
9. Tumor Processing and Analysis
<ol>
	<li>Seven days following treatment euthanize animals. Cannulate the left ventricle and exsanguinate blood with 10ml of 2% Heparinized Tris CaCl2 Buffer (.68mM) perfusion, followed by 10ml Tris CaCl2 Buffer (.68mM), each for 10 minutes at 100 mmHg.</li>
	<li>Perfusion-fix tissue with an infusion of 4% paraformaldehyde in PBS (4&deg;C) for 10minutes at 100 mmHg. Allow sample to fix for 60 minutes. </li>
	<li>Excise specimens, embed in paraffin, and cut into 5-micron sections. </li>
	<li>Perform standard hematoxylin-eosin staining to evaluate histological changes that may have occurred as a result of the ultrasound exposures. </li>
	<li>To detect apoptotic cells, use a terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay (ApoptTag kit, Intergen Co., Norcross, GA, USA).</li>
</ol>
10. Representative Results
1. Nanoparticle Fabrication (2.0)
<ol>
	<li>If this protocol is preformed properly NPs will be spherical in shape, as determined by scanning electron microcopy (SEM), and have a Gaussian distribution around 100nm, as determined by light scattering techniques. Representative results are shown in Figure 1.</li>
</ol>
2. Targeted Drug Delivery (5.4.1)
<ol>
	<li>Based on published studies performed in our laboratory (Song 2002, Chappell 2008) and unpublished results present in Figure 2, ultrasonic microbubble destruction results in enhanced delivery of intravascular nanoparticles to tumor tissue. We have also shown that our MNCA delivery technique results in heightened nanoparticle delivery immediately following treatment.</li>
</ol>
3. Tumor Ablation (5.4.3)
<ol>
	<li>We have shown that prolonged ultrasonic microbubble destruction, at relatively low duty cycles (0.005-0.01), results in mechanical ablation of the tumor microvasculature and regression of tumor growth. If this protocol is performed properly one should expect changes in (i) rate of tumor growth (Figure 3); (ii) tumor hemodynamics (Figure 4) ; (iii) necrotic area ; and (iv) apoptosis .</li>
</ol>
<img src="/files/ftp_upload/2145/2145fig1.jpg" alt="Figure 1" /> Figure 1. Characterization of PLAGA nanoparticles bearing BSA. (A) SEM image of properly fabricated nanoparticles. (B) SEM image of improperly fabricated nanoparticles.
<img src="/files/ftp_upload/2145/2145fig2.jpg" alt="Figure 2" /> Figure 2. (A) Fluorescence-mediated tomography (FMT) images of ultrasound treated (top) and control treated (bottom) subcutaneous gliomas immediately following treatment with a MNCAs, where nanoparticles (NPs) are bearing 680 fluorescence signals are superimposed on grayscale planar excitation light image.
<img src="/files/ftp_upload/2145/2145fig3.jpg" alt="Figure 3" /> Figure 3. Representative fold change in tumor growth following microbubble insonation with the five-burst extended pulsing protocol.
<img src="/files/ftp_upload/2145/2145fig4.jpg" alt="Figure 4" /> Figure 4. B-Mode (A,C) and contrast-enhanced ultrasonography (B,D) images of a subcutaneous C6 glioma tumor in a mouse. In the initial pretreatment image (A) the boundary of mostly hypoechoic tumor has been traced in blue. Time average enhancement after intravenous injection of a contrast agent is shown in (B) pretreatment. Posttreatment, before contrast injection, tumor is again mostly hypoechoic (D). After contrast injection, there is significantly less enhancement in the tumor.

<ol>
	<li>Chappell, J., Song, J., Burke, C., Klibanov, A., Price, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+delivery+of+nanoparticles+bearing+fibroblast+growth+factor-2+by+ultrasonic+microbubble+destruction+for+therapeutic+arteriogenesis.">Targeted delivery of nanoparticles bearing fibroblast growth factor-2 by ultrasonic microbubble destruction for therapeutic arteriogenesis.</a> Small. 10, 1769-1777 (2008).</li><li>Chomas, J. E., Pollard, R., Wisner, E., Ferrara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subharmonic+Phase-Inversion+for+Tumor+Perfusion+Estimation.">Subharmonic Phase-Inversion for Tumor Perfusion Estimation.</a> IEEE. 2, 1713-1716 (2001).</li><li>Davda, J., Labhasetwar, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+nanoparticle+uptake+by+endothelial+cells.">Characterization of nanoparticle uptake by endothelial cells.</a> Int J Pharm. 233, 51-51 (2002).</li><li>Hynynen, K., McDannolod, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+MR+imaging-guided+focal+opening+of+the+blood-brain+barrier+in+rabbits.">Noninvasive MR imaging-guided focal opening of the blood-brain barrier in rabbits.</a> Radiology. 220, 640-646 (2001).</li><li>McDannold, N., Vykhodtseva, N., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbubble+contrast+agent+with+focused+ultrasound+to+create+brain+lesions+at+low+power+levesl:+MR+imaging+and+histological+study+in+rabbits.">Microbubble contrast agent with focused ultrasound to create brain lesions at low power levesl: MR imaging and histological study in rabbits.</a> Radiology. 241, 95-106 .</li><li>McDannold, N., Vykhodtseva, N., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+the+blood-brain+barrier+with+focused+ultrasound:+Association+with+cavitation+activity.">Targeted disruption of the blood-brain barrier with focused ultrasound: Association with cavitation activity.</a> Phys Med Biol. 51, 793-807 .</li><li>McDannold, N., Clement, G. T., Black, P., Jolesz, F., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcranial+magnetic+resonance+imaging-+guided+focused+ultrasound+surgery+of+brain+tumors:+initial+findings+in+3+patients.">Transcranial magnetic resonance imaging- guided focused ultrasound surgery of brain tumors: initial findings in 3 patients.</a> Neurosurgery. 66, 323-332 (2009).</li><li>Meairs, S., Alonso, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+microbubblesand+the+blood+brain+barrier.">Ultrasound microbubblesand the blood brain barrier.</a> Progr Biophys Mol Biol. 93, 354-362 (2007).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Optison, products insert. , (2009).</li><li>Sadlowskie, A., Chromas, J., Pollard, R., Bloch, S., Griffey, S., Wisner, W., Ferrara, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mean+Flow+Rate+and+Intergrated+Perfusion+Estimates+Obtained+with+Contrast-Assisted+Ultrasound.">Mean Flow Rate and Intergrated Perfusion Estimates Obtained with Contrast-Assisted Ultrasound.</a> IEEE Ultrasonics Symposium. , 1977-1980 (2002).</li><li>Song, J., Chappell, J. C., Qi, M., VanGieson, E. J., Kaul, S., Price, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+injection+site,+microvascular+pressure+and+ultrasound+variables+on+microbubble-mediated+delivery+of+microspheres+to+muscle.">Influence of injection site, microvascular pressure and ultrasound variables on microbubble-mediated delivery of microspheres to muscle.</a> J Am Coll. Cardiol. 39, 726-731 (2002).</li><li>Sheikov, N., McDannold, N., Vykhodtseva, N., Jolesz, F., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanisms+of+blood-brain+barrier+opeing+induced+by+ultrasound+in+the+presences+of+microbubbles.">Cellular mechanisms of blood-brain barrier opeing induced by ultrasound in the presences of microbubbles.</a> Ultrasound Med. Biol. 30, 979-989 (2004).</li><li>Tanter, J., Fink, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focusing+and+steering+through+absorbing+and+aberrating+layers:+Application+to+ultrasonic+propagation+through+the+skull.">Focusing and steering through absorbing and aberrating layers: Application to ultrasonic propagation through the skull.</a> J Acoust Soc Am. 103, 2403-2410 (1998).</li><li>Yeh, C. K., Kruse, D. E., Lim, M. 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No conflicts of interest declared.

Critical Steps
Cannulation of mouse tail vein:
Intravenous injection into the mouse tail vein can be a challenging procedure. However, a tail vein catheter can greatly improve the likelihood of a successful injection. To make the catheter, repeatedly bend back and forth a 25 gauge needle until it breaks from the hub. Insert the blunt end into the end of PE 20 tubing and seal the connection with silicon glue. To prepare the catheter for cannulation, attach a syringe ...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>ApoptTag kit</td>
<td>&nbsp;</td>
<td>Intergen Co.</td>
<td>S7110</td>
<td>&nbsp;</td>
<tr>
<td>un-capped 85:15 poly(lactic-co-glycolic acid) (PLAGA)</td>
<td>&nbsp;</td>
<td>Lakeshore Biomaterials</td>
<td>Custom</td>
<td>&nbsp;</td>
<tr>
<td>Vivo Tag 680</td>
<td>&nbsp;</td>
<td>VisEn Medical</td>
<td>10120</td>
<td>Used to Tag BSA</td>
</tr>
<tr>
<td>Poly(vinyl alcohol)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>363136</td>
<td>&nbsp;</td>
<tr>
<td>MicroTip Sonicator</td>
<td>&nbsp;</td>
<td>Misonix</td>
<td>S-4000</td>
<td>&nbsp;</td>
<tr>
<td>Sequoia</td>
<td>&nbsp;</td>
<td>Simons Medical</td>
<td>P.O.A</td>
<td>Equipped with CPS </td>
</tr>
<tr>
<td>FreeZone 2.5</td>
<td>&nbsp;</td>
<td>Labconco</td>
<td>7670020</td>
<td>Equipped with Nitrogen Trap</td>
</tr>
<tr>
<td>Methylene chloride (CH2Cl2)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>D37-500</td>
<td>&nbsp;</td>
<tr>
<td>FMT 250</td>
<td>&nbsp;</td>
<td>VisEn Medical</td>
<td>P.O.A</td>
<td>&nbsp;</td>
<tr>
<td>F-12K Nutrient Mixture</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>21127-022</td>
<td>&nbsp;</td>
<tr>
<td>polyethyleneglycol-40 stearate </td>
<td>&nbsp;</td>
<td>Sigma Chemical </td>
<td>9004-99-3</td>
<td>&nbsp;</td>
<tr>
<td>distearoyl phosphatidylcholine</td>
<td>&nbsp;</td>
<td>Avanti Polar Lipids</td>
<td>770365</td>
<td>&nbsp;</td>
<tr>
<td>Multisizer Coulter Counter</td>
<td>&nbsp;</td>
<td>Beckman Coulter </td>
<td>P.O.A</td>
<td>&nbsp;</td>
<tr>
<td>Waveform Generator </td>
<td>&nbsp;</td>
<td>Tektronix, Inc.</td>
<td>AFG-310</td>
<td>&nbsp;</td>
<tr>
<td>water-based ultrasound gel </td>
<td>&nbsp;</td>
<td>Parker Laboratories</td>
<td>Aquasonic 100</td>
<td>&nbsp;</td>
<tr>
<td>Infusion pump </td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>Harvard Apparatus PHD 2000</td>
<td>&nbsp;</td>
<tr>
<td>1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) </td>
<td>&nbsp;</td>
<td>Pierce Biotechnology</td>
<td>25952-53-8</td>
<td>&nbsp;</td>
<tr>
<td>N-hydroxysulfosuccinimide (Sulfo-NHS)</td>
<td>&nbsp;</td>
<td>Pierce Biotechnology</td>
<td>106627-54-7</td>
<td>&nbsp;</td>
<tr>
<td>Succinic anhydride</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>603902</td>
<td>&nbsp;</td>
<tr>
<td>Power Amplifier </td>
<td>&nbsp;</td>
<td>Electronic Navigation Industries</td>
<td>ENI 3100LA</td>
<td>&nbsp;</td>
<tr>
<td>Needle Thermocouple Probe </td>
<td>&nbsp;</td>
<td>Omega</td>
<td>HYP1-30-1/2-T-G-60-SMPW-M</td>
<td>&nbsp;</td>
<tr>
<td>BioGel (P100, medium)</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>150-4170</td>
<td>&nbsp;</td>
<tr>
<td>.75&#x2019;&#x2019; diameter 1 MHz unfocused transducer</td>
<td>&nbsp;</td>
<td>Panametrics</td>
<td>A314S </td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

contrast ultrasound targeted treatment of gliomas in mice via drug-bearing nanoparticle delivery and microvascular ablation

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the microvasculature are controlled by varying ultrasound pulsing parameters.  Specifically, we are testing whether such approaches may be used to treat malignant brain tumors through drug delivery and microvascular ablation.  Preliminary studies have been performed to determine whether targeted drug-bearing nanoparticle delivery can be facilitated by the ultrasound mediated destruction of "composite" delivery agents comprised of 100nm poly(lactide-co-glycolide) (PLAGA) nanoparticles that are adhered to albumin shelled microbubbles. We denote these agents as microbubble-nanoparticle composite agents (MNCAs). When targeted to subcutaneous C6 gliomas with ultrasound, we observed an immediate 4.6-fold increase in nanoparticle delivery in MNCA treated tumors over tumors treated with microbubbles co-administered with nanoparticles and a 8.5 fold increase over non-treated tumors. Furthermore, in many cancer applications, we believe it may be desirable to perform targeted drug delivery in conjunction with ablation of the tumor microcirculation, which will lead to tumor hypoxia and apoptosis. To this end, we have tested the efficacy of non-theramal cavitation-induced microvascular ablation, showing that this approach elicits tumor perfusion reduction, apoptosis, significant growth inhibition, and necrosis. Taken together, these results indicate that our ultrasound-targeted approach has the potential to increase therapeutic efficiency by creating tumor necrosis through microvascular ablation and/or simultaneously enhancing the drug payload in gliomas.

Watch this Scientific Journal Video about Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation at JoVE.com

Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation

Insonation of microbubbles is a promising strategy for tumor ablation at reduced time-averaged acoustic powers, as well as for the targeted delivery of therapeutics. The purpose of the present study is to develop low duty cycle ultrasound pulsing strategies and nanocarriers to maximize non-thermal microvascular ablation and payload delivery to subcutaneous C6 gliomas.

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the ...

contrast-ultrasound-targeted-treatment-gliomas-mice-via-drug-bearing

Research

JoVE Journal

Bioengineering

15.9K vistas.  University of Virginia. Insonación de microburbujas es una estrategia prometedora para la ablación del tumor en un promedio de reducción de tiempo de potencias acústicas, así como para la distribución selectiva de la terapéutica. El propósito del presente estudio es desarrollar estrategias de ultrasonido de baja deber ciclo de pulsación y nanotransportadores para maximizar no de ablación térmica microvascular y la entrega a la carga por vía subcutánea C6 gliomas.

Video: Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation

Introduction to the Ultrasound Targeted Microbubble Destruction Technique

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid microbubble contrast agents7-9. In mice these vector-carrying microbubbles can be administered intravenously or directly to the left ventricle of the heart. In larger animals they can also be infused through an intracoronary catheter. The subsequent delivery from the circulation to a target organ occurs by acoustic cavitation at a resonant frequency of the microbubbles. It seems likely that the mechanical energy generated by the microbubble destruction results in transient pore formation in or between the endothelial cells of the microvasculature of the targeted region10. As a result of this sonoporation effect, the transfection efficiency into and across the endothelial cells is enhanced, and transgene-encoding vectors are deposited into the surrounding tissue. Plasmid DNA remaining in the circulation is rapidly degraded by nucleases in the blood, which further reduces the likelihood of delivery to non-sonicated tissues and leads to highly specific target-organ transfection.

Ultrasound Targeted Microbubble Destruction (UTMD) can be used to direct site-specific delivery of bioactive molecules, including therapeutic genes, to target organs accessible to ultrasound, such as the heart and liver1-6.

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Real-time Monitoring of High Intensity Focused Ultrasound (HIFU) Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An oscillatory motion is generated at the focus of a 93-element and 4.5 MHz center frequency HIFU transducer by applying a 25 Hz amplitude-modulated signal using a function generator. A 64-element and 2.5 MHz imaging transducer with 68kPa peak pressure is confocally placed at the center of the HIFU transducer to acquire the radio-frequency (RF) channel data. In this protocol, real-time monitoring of thermal ablation using HIFU with an acoustic power of 7 W on canine livers in vitro is described. HIFU treatment is applied on the tissue during 2 min and the ablated region is imaged in real-time using diverging or plane wave imaging up to 1,000 frames/second. The matrix of RF channel data is multiplied by a sparse matrix for image reconstruction. The reconstructed field of view is of 90&#176; for diverging wave and 20 mm for plane wave imaging and the data are sampled at 80 MHz. The reconstruction is performed on a Graphical Processing Unit (GPU) in order to image in real-time at a 4.5 display frame rate. 1-D normalized cross-correlation of the reconstructed RF data is used to estimate axial displacements in the focal region. The magnitude of the peak-to-peak displacement at the focal depth decreases during the thermal ablation which denotes stiffening of the tissue due to the formation of a lesion. The displacement signal-to-noise ratio (SNRd) at the focal area for plane wave was 1.4 times higher than for diverging wave showing that plane wave imaging appears to produce better displacement maps quality for HMIFU than diverging wave imaging.

Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

Patient-Specific Polyvinyl Alcohol Phantom Fabrication with Ultrasound and X-Ray Contrast for Brain Tumor Surgery Planning

Phantoms are essential tools for clinical training, surgical planning and the development of novel medical devices. However, it is challenging to create anatomically accurate head phantoms with realistic brain imaging properties because standard fabrication methods are not optimized to replicate any patient-specific anatomical detail and 3D printing materials are not optimized for imaging properties. In order to test and validate a novel navigation system for use during brain tumor surgery, an anatomically accurate phantom with realistic imaging and mechanical properties was required. Therefore, a phantom was developed using real patient data as input and 3D printing of molds to fabricate a patient-specific head phantom comprising the skull, brain and tumor with both ultrasound and X-ray contrast. The phantom also had mechanical properties that allowed the phantom tissue to be manipulated in a similar manner to how human brain tissue is handled during surgery. The phantom was successfully tested during a surgical simulation in a virtual operating room.
The phantom fabrication method uses commercially available materials and is easy to reproduce. The 3D printing files can be readily shared, and the technique can be adapted to encompass many different types of tumor.

This protocol describes the fabrication of a patient specific skull, brain and tumor phantom. It uses 3D printing to create molds, and polyvinyl alcohol (PVA-c) is used as the tissue mimicking material.

Phantoms are essential tools for clinical training, surgical planning and the development of novel medical devices. However, it is challenging to create ...