This work was supported by grants from the National Natural Sciences Foundation (81771617) and the Natural Science Foundation of Guangdong Province (2016A030313178) awarded to X.F.; a grant from the Shenzhen Basic Research Fund (JCYJ20170413165233512) awarded to X.F; and the Eunice Kennedy Shriver National Institute of Child Health &#38; Human Development of the National Institutes of Health under Award Number R01HD088549 (the content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health) to N.N.

All mouse experiments strictly followed protocols (SIAT-IRB-160520-YYS-FXJ-A0232) approved by the Animal Care and Use Committee of Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences.
1. Synthesis of Placental Chondroitin Sulfate A-Targeted Lipid-Polymer Nanoparticles
<ol>
	<li>Synthesize MTX- and ICG-loaded lipid-polymer nanoparticles (MNPs and INPs respectively) and plCSA-BP-conjugated nanoparticles (plCSA-MNPs and plCSA-INPs) as described in detail elsewhere<sup class=...

<ol>
	<li>Rodger, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Association+of+Factor+V+Leiden+and+Prothrombin+Gene+Mutation+and+Placenta-Mediated+Pregnancy+Complications:+A+Systematic+Review+and+Meta-analysis+of+Prospective+Cohort+Studies.">The Association of Factor V Leiden and Prothrombin Gene Mutation and Placenta-Mediated Pregnancy Complications: A Systematic Review and Meta-analysis of Prospective Cohort Studies.</a> PLOS Medicine. 7 (6), e1000292 (2010).</li><li>Rodger, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inherited+thrombophilia+and+pregnancy+complications+revisited.">Inherited thrombophilia and pregnancy complications revisited.</a> Obstetrics &amp; Gynecology. 112 (2 Pt 1), 320-324 (2008).</li><li>Brenner, B., Aharon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombophilia+and+adverse+pregnancy+outcome.">Thrombophilia and adverse pregnancy outcome.</a> Clinics in Perinatology. 34 (4), 527-541 (2007).</li><li>Fisk, N. M., McKee, M., Atun, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relative+and+absolute+addressability+of+global+disease+burden+in+maternal+and+perinatal+health+by+investment+in+R&amp;D.">Relative and absolute addressability of global disease burden in maternal and perinatal health by investment in R&amp;D.</a> Tropical Medicine &amp; International Health. 16 (6), 662-668 (2011).</li><li>Fisk, N. M., Atun, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Market+failure+and+the+poverty+of+new+drugs+in+maternal+health.">Market failure and the poverty of new drugs in maternal health.</a> PLOS Medicine. 5 (1), e22 (2008).</li><li>Kaitu'u-Lino, T. u. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+nanoparticle+delivery+of+doxorubicin+into+placental+tissues+to+treat+ectopic+pregnancies.">Targeted nanoparticle delivery of doxorubicin into placental tissues to treat ectopic pregnancies.</a> Endocrinology. 154 (2), 911-919 (2013).</li><li>King, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-homing+peptides+as+tools+for+targeted+delivery+of+payloads+to+the+placenta.">Tumor-homing peptides as tools for targeted delivery of payloads to the placenta.</a> Science Advances. 2 (5), e1600349 (2016).</li><li>Beards, F., Jones, L. E., Charnock, J., Forbes, K., Harris, L. 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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+delivery+to+the+human+and+mouse+uterus+using+immunoliposomes+targeted+to+the+oxytocin+receptor.">Drug delivery to the human and mouse uterus using immunoliposomes targeted to the oxytocin receptor.</a> American Journal of Obstetrics and Gynecology. 216 (3), e281-e283 (2017).</li><li>Zhang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placenta-specific+drug+delivery+by+trophoblast-targeted+nanoparticles+in+mice.">Placenta-specific drug delivery by trophoblast-targeted nanoparticles in mice.</a> Theranostics. 8 (10), 2765-2781 (2018).</li><li>Fan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+monitoring+of+placenta-specific+transgene+expression+by+bioluminescence+imaging.">Noninvasive monitoring of placenta-specific transgene expression by bioluminescence imaging.</a> PloS One. 6 (1), e16348 (2011).</li><li>Murata, M., Tahara, K., Takeuchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+in+vivo+imaging+of+surface-modified+liposomes+to+evaluate+their+behavior+after+pulmonary+administration.">Real-time in vivo imaging of surface-modified liposomes to evaluate their behavior after pulmonary administration.</a> European Journal of Pharmaceutics and Biopharmaceutics. 86 (1), 115-119 (2014).</li><li>Ito, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+whole-body+multimodality+imaging+of+gastric+cancer+peritoneal+metastasis+combining+fluorescence+imaging+with+ICG-labeled+antibody+and+MRI+in+mice.">New whole-body multimodality imaging of gastric cancer peritoneal metastasis combining fluorescence imaging with ICG-labeled antibody and MRI in mice.</a> Gastric Cancer. 17 (3), 497-507 (2014).</li><li>Mazza, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposome-Indocyanine+Green+Nanoprobes+for+Optical+Labeling+and+Tracking+of+Human+Mesenchymal+Stem+Cells+Post-Transplantation+In+Vivo.">Liposome-Indocyanine Green Nanoprobes for Optical Labeling and Tracking of Human Mesenchymal Stem Cells Post-Transplantation In Vivo.</a> Advanced Healthcare Materials. 6 (21), (2017).</li><li>Greco, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+frequency+ultrasound+for+in+vivo+pregnancy+diagnosis+and+staging+of+placental+and+fetal+development+in+mice.">High frequency ultrasound for in vivo pregnancy diagnosis and staging of placental and fetal development in mice.</a> PloS One. 8 (10), e77205 (2013).</li><li>Spurney, C. F., Leatherbury, L., Lo, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+ultrasound+database+profiling+growth,+development,+and+cardiovascular+function+in+C57BL/6J+mouse+fetuses.">High-frequency ultrasound database profiling growth, development, and cardiovascular function in C57BL/6J mouse fetuses.</a> Journal of the American Society of Echocardiography. 17 (8), 893-900 (2004).</li><li>Zhang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+characterization+of+placental+chondroitin+sulfate+A+(plCSA)+-targeting+lipid-polymer+nanoparticles.">Synthesis and characterization of placental chondroitin sulfate A (plCSA) -targeting lipid-polymer nanoparticles.</a> Journal of Visualized Experiments. , (2018).</li><li>Devraj, K., Guerit, S., Macas, J., Reiss, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+In+Vivo+Blood-brain+Barrier+Permeability+Assay+in+Mice+Using+Fluorescently+Labeled+Tracers.">An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers.</a> Journal of Visualized Experiments. (132), (2018).</li><li>Beeton, C., Chandy, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mononuclear+cells+from+the+central+nervous+system+of+rats+with+EAE.">Isolation of mononuclear cells from the central nervous system of rats with EAE.</a> Journal of Visualized Experiments. (10), 527 (2007).</li><li>Watson, E. D., Cross, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+structures+and+transport+functions+in+the+mouse+placenta.">Development of structures and transport functions in the mouse placenta.</a> Physiology. 20 (3), 180-193 (2005).</li><li>Frangioni, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+near-infrared+fluorescence+imaging.">In vivo near-infrared fluorescence imaging.</a> Current Opinion in Chemical Biology. 7 (5), 626-634 (2003).</li><li>Flores, L. E., Hildebrandt, T. B., Kuhl, A. A., Drews, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+detection+and+staging+of+spontaneous+embryo+resorption+by+ultrasound+biomicroscopy+in+murine+pregnancy.">Early detection and staging of spontaneous embryo resorption by ultrasound biomicroscopy in murine pregnancy.</a> Reproductive Biology and Endocrinology. 12, 38 (2014).</li><li>Khankin, E. V., Hacker, M. R., Zelop, C. M., Karumanchi, S. 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In this manuscript, we outline a three-method system for determining whether plCSA-BP-guided nanoparticles are an efficient tool for targeting the delivery of drugs to the placenta. The use of in vivo imaging to monitor the infrared fluorescent ICG signal confirmed the placental targeting specificity of plCSA-BP. Using HFUS and HPLC, we demonstrated that plCSA-BP-conjugated nanoparticles can efficiently deliver MTX only to the placenta cells, not to the fetus.
In the in vivo ...

Placenta-mediated pregnancy complications, including pre-eclampsia, pregnancy loss, placental abruption and small gestational age (SGA), are common and lead to substantial fetal and maternal morbidity and mortality1,2,3, and very few drugs have been proven to be effective for treating pregnancy disorders4,5. The development of strategies for more selective and safer placenta-targeted drug delivery during pregnancy remains challenging in modern drug therapy.
In recent years, several reports have focused on the targeted delivery of drugs to uteroplacental tissues by coating nanoparticles with peptides or antibodies as placenta-targeted tools. These include an anti-epidermal growth factor receptor (EGFR)6 antibody, tumor-homing peptides (CGKRK and iRGD)7, placenta-targeted peptides8, placental vasculature-targeted peptides9 and antibodies against the oxytocin receptor10.
Here, we demonstrate that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) can be used for the targeted delivery of nanoparticles and their drug payloads to the placenta11. The plCSA-BP-guided nanoparticles are complementary to the reported uteroplacental targeting methods because they target the placental trophoblast.
As a non-invasive method, in vivo imaging has been used to monitor placenta-specific gene expression in mice12, and indocyanine green (ICG) has been widely used to track nanoparticles using fluorescence imaging systems13,14,15. Thus, we intravenously injected plCSA-BP-conjugated nanoparticles loaded with ICG (plCSA-INPs) to visualize the plCSA-INP distribution in pregnant mice with a fluorescence imager. We then intravenously injected methotrexate (MTX)-loaded plCSA-NPs into pregnant mice. High-frequency ultrasound (HFUS), another non-invasive, real-time imaging tool16,17 was used to monitor fetal and placental development in the mice. Finally, we used high-performance liquid chromatography (HPLC) to quantify MTX distribution in the placentas and fetuses.
In this protocol, we describe in detail the three-method system used to assess the efficiency of placenta-targeted drug delivery by plCSA-BP-guided nanocarriers.

<table border="0" cellpadding="0" cellspacing="0" style="width:785px;" width="786">
	<tbody>
		<tr>
			<td>CD-1 mice</td>
			<td>Beijing Vital River</td>
			<td>201</td>
			<td>Female (8-12 week)</td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>BD</td>
			<td>328421</td>
			<td>for IV injection</td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Sinopharm Chemical</td>
			<td>10009218</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>Soybean lecithin</td>
			<td>Avanti Polar Lipids</td>
			<td>441601</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>DSPE-PEG-COOH</td>
			<td>Avanti Polar Lipids</td>
			<td>880125</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>PLGA</td>
			<td>Sigma-Aldrich</td>
			<td>719897</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>Ultrasonic processor</td>
			<td>Sonics</td>
			<td>VCX130</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>Methotrexate (MTX)</td>
			<td>Sigma-Aldrich</td>
			<td>V900324</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>Indocyanine green (ICG)</td>
			<td>Sigma-Aldrich</td>
			<td>1340009</td>
			<td>for in vivo imaging</td>
		</tr>
		<tr>
			<td>phosphate-buffered saline (PBS)</td>
			<td>Hyclone</td>
			<td>SH30028.01</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS spectrum instrument</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>for in vivo imaging</td>
		</tr>
		<tr>
			<td>Ultrasound transmission gel</td>
			<td>Guanggong</td>
			<td>ZC4252418</td>
			<td>for ultrasound imaging</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Lunan Pharmaceutical</td>
			<td>I0040</td>
			<td>for maintain the anesthesia</td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Nair</td>
			<td>TMG001</td>
			<td>for removing fur</td>
		</tr>
		<tr>
			<td>40 MHz transducer</td>
			<td>VisualSonics</td>
			<td>MS550S</td>
			<td>for ultrasound imaging</td>
		</tr>
		<tr>
			<td>High-frequency ultrasound imaging system</td>
			<td>VisualSonics</td>
			<td>Vevo2100</td>
			<td>for ultrasound imaging</td>
		</tr>
		<tr>
			<td>Avertin</td>
			<td>Sigma-Aldrich</td>
			<td>T48402</td>
			<td>for anesthesia</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Mindray</td>
			<td>SK-500III</td>
			<td>forcardiac perfusion</td>
		</tr>
		<tr>
			<td>0.9% saline solution</td>
			<td>Meilunbio</td>
			<td>MA0083</td>
			<td>forcardiac perfusion</td>
		</tr>
		<tr>
			<td>1.5 mL Polypropylene tubes</td>
			<td>AXYGEN</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>-80 &#176;C freezer</td>
			<td>Thermo Fisher Scientific</td>
			<td>88600V</td>
			<td></td>
		</tr>
		<tr>
			<td>Centriguge</td>
			<td>Cence</td>
			<td>H1650R</td>
			<td></td>
		</tr>
		<tr>
			<td>Perchloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>311421</td>
			<td>for precipitating protein</td>
		</tr>
		<tr>
			<td>Homogenizer</td>
			<td>SCIENTZ</td>
			<td>SCIENTZ-48</td>
			<td>for homogenizing tissue</td>
		</tr>
		<tr>
			<td>Syringe filter (0.45 &#956;m)</td>
			<td>Millipore</td>
			<td>SLHV033RS01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sinopharm Chemical</td>
			<td>10019763</td>
			<td>for solving MTX</td>
		</tr>
		<tr>
			<td>HPLC vials</td>
			<td>Waters</td>
			<td>670650620</td>
			<td>for HPLC</td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Sinopharm Chemical</td>
			<td>20032117</td>
			<td>for HPLC</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>JKchemical</td>
			<td>932537</td>
			<td>for HPLC</td>
		</tr>
		<tr>
			<td>C18 column</td>
			<td>Waters</td>
			<td>186003966</td>
			<td>for HPLC</td>
		</tr>
		<tr>
			<td>HPLC system</td>
			<td>Shimadzu</td>
			<td></td>
			<td>for HPLC</td>
		</tr>
	</tbody>
</table>

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.

In this manuscript, plCSA-BP-conjugated nanoparticles loaded with MTX (plCSA-MNPs) or ICG (plCSA-INPs) were intravenously injected into pregnant mice. In vivo imaging revealed strong ICG signals in the region of the uterus 30 min after plCSA-INP injection. The INPs were mainly localized to the liver and spleen region (Figure 1A). At 48 h after plCSA-INP injection, pregnant mice were sacrificed, revealing ICG signals only in the placenta, while with n...

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

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 ...

This method can help answer key questions in the nanomedicine field about the effectiveness and the safety of drug loaded on nanoparticle delivered to the mother and the fetus. The main advantage of this technique is that it facilitates an accurate biochemical measurement of the amount of drug delivered to the placenta. The implications of this technique extend toward the therapy of placenta-mediated pregnancy complications because it's important to ensure precise targeting of the drug delivery to the placenta.Visual demonstration of this method is critical, as high-frequency ultrasound evaluation of embryonic development requires patience and experience to master. For intravenous nanoparticle injection, transfer an Embryonic Day 14.5 pregnant mouse with heat-dilated tail veins into a restrainer, and clean the tail with an alcohol swab. Then insert the needle tip of a 28-gauge insulin syringe into a dilated tail vein, and slowly depress the plunger over five to 10 seconds with even pressure to deliver the nanoparticles into the tail vein.30 minutes after the injection, place the anesthetized pregnant mouse into the imaging chamber of an in vivo fluorescence imaging system in the supine position. Select 2D fluorescence and the appropriate photographic parameters for imaging the indocyanine green fluorescence signals, and set the exposure to auto and the excitation and emission wavelengths to 710 and 820 nanometers, respectively. Then image the animal, and return the pregnant mouse to its cage.48 hours after the injection, use Graefe forceps, Graefe tissue forceps, and dissecting scissors to collect the fetuses and placentas, and image the harvested tissues as just demonstrated for the whole animal. For high-frequency ultrasound evaluation of embryonic development, 24 hours after nanoparticle injection, secure the anesthetized pregnant dam onto a 37 to 42-degree Celsius preheated ultrasound imaging platform in the supine position, and place the 40-megahertz transducer into the mechanical arm. Then adjust the transducer position to obtain longitudinal images of the fetus and placenta with the region of interest within the focal zone.For B-mode imaging and analysis, select the B-mode and lower the transducer over the abdomen until the fetus and placenta come into view. Click Scan/Freeze to initiate and stop the imaging, Cine Store to store the cine loop, and Frame Store to store the frame images. Then click Measure to analyze the gestational sac length, fetal crown rump length, biparietal diameter, abdominal circumference, placental diameter, and placental thickness.For pulse wave Doppler imaging and analysis, using the same scan projection, first click Color, then select Pulse Wave, and place the sampling volume box in the center of the umbilical artery. Click Scan/Freeze to initiate imaging and Cine Store to collect the umbilical artery images. Then click Measure to calculate the umbilical artery peak velocity.For color Doppler mode imaging and analysis, using the same scan projection, click Color, and adjust the transducer position to obtain images of the fetal heart as demonstrated. Then click Measure to calculate the fetal heart rate of the stored cine image loop. For high-pressure liquid chromatography, or HPLC, analysis, first inject an Embryonic Day 14.5 pregnant mouse with a single dose of nanoparticles as demonstrated.After 24 hours, perfuse the heart of the pregnant animal with 50 milliliters of ice-cold 0.9%saline for 10 minutes to remove any unbound nanoparticles, and collect the fetuses and placentas for minus 80-degree Celsius storage. On the day of the analysis, add 500 microliters of freshly prepared homogenization solution to each 200-milligram tissue sample, and homogenize the samples two times at full speed for 30 seconds per dissociation. After the second homogenization, centrifuge the samples and filter the supernatants through a 0.45-micrometer syringe filter into an HPLC vial.Place the sample vials into an autosampler tray for injection, and turn on the HPLC degasser to remove air from the system. Next, turn on the flow to equilibrate the column with the mobile phase for 30 minutes to reduce baseline noise, and set the temperature of the column to 25 degrees Celsius. Then inject 20-microliter sample volumes at a one milliliter per minute flow rate within 30 minutes, and click Run Method to begin the analysis.When the runs are complete, manually change the mobile phase to HPLC-grade acetonitrile, and run the acetonitrile for approximately 15 minutes to protect the system. In vivo imaging reveals strong indocyanine green signals in the uterine tissue 30 minutes after placental chondroitin sulfate-bound fluorescence-conjugated nanoparticle injection, while the unbound nanoparticles mainly localize to the liver and splenic regions. 48 hours after injection, fluorescent signal is observed in the placenta of placental chondroitin sulfate-bound nanoparticle injected animals, with no signals detected within the fetus.High-frequency ultrasound monitoring of placental chondroitin sulfate-bound fluorescence-conjugated nanoparticle injected animals reveals a significant decrease in key fetal and placental developmental landmarks. Interestingly, treatment with methotrexate-bound nanoparticles also slightly impairs fetal and placental development, indicating that the nanoparticles may improve the delivery of methotrexate to the placenta via the enhanced permeability and retention effect. The measurement of methotrexate concentrations by HPLC indicates a retention time of seven minutes within the placentas of methotrexate-bound nanoparticle-injected mice.24 hours after injection, the placental methotrexate levels in the methotrexate-bound nanoparticle-injected group are significantly lower than that measured in the placental chondroitin sulfate and methotrexate-bound nanoparticle group, with no methotrexate detected in the fetuses of the latter. Methotrexate can still be detected in the placenta 48 hours after placental chondroitin sulfate and methotrexate-bound nanoparticle injection, demonstrating that placental chondroitin sulfate and methotrexate-bound nanoparticles cannot cross the placenta, thus minimizing potential adverse effects on the fetus. While attempting this procedure, it's important to remember that the placenta begin to form around Embryonic Day 9 1/2, and therefore the in vivo imaging experiment is better performed on Embryonic Day 10 1/2 or later.Following this procedure, other methods like immunofluorescent staining and analysis can be performed to answer additional questions about localization of the nanoparticles within the placenta. After its development, this technique paved the way for researcher in the field of reproductive medicine to explore the possibility of placenta-targeting treatment in pregnancy complications.

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Research

JoVE Journal

Bioengineering

9.6K visualizações.  Chinese Academy of Sciences. This method can help answer key questions in the nanomedicine field about the effectiveness and the safety of drug loaded on nanoparticle delivered to the mother and the fetus. The main advantage of this technique is that it facilitates an accurate biochemical measurement of the amount of drug delivered to the placenta. The implications of this technique extend toward the therapy of placenta-mediated pregnancy complications because it's important to ensure precise targeting of the drug delive...