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.
