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

Summary

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.

Abstract

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.

Introduction

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 mortality^1,2,3, and very few drugs have been proven to be effective for treating pregnancy disorders^4,5. 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 systems^13,14,15. 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 tool^16,17 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.

Protocol

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

1. 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 elsewhere¹⁸.

2. In vivo Fluorescence Imaging

1. Preparation of pregnant mice
   1. Place female CD-1 mice (8-12 weeks) with a fertile male of the same strain in one cage (male: female=1:2) in the afternoon and check the vaginal plugs the following morning. If a vaginal plug is observed, define the mouse as embryonic day 0.5 (E0.5).
   2. House pregnant mice alone in a pathogen-free animal room with a 14 h light/10 h dark cycle and provide free access to food and water until E14.5.
2. Intravenous injection of nanoparticles
   1. Before the procedure, sterilize the nanoparticles by filtration through a 0.22 μm syringe filter. Weigh the pregnant mouse at E11.5 to determine the quantity and volume of nanoparticle injection.
      Note: The nanoparticle injection volume should be less than 1% (volume/weight) of the body weight of the pregnant mouse. For example, the nanoparticle injection volume should be less than 0.25 mL in a 25 g mouse.
   2. To dilate the tail vein, warm the tail for 5-10 min with a heating pad.
   3. Before injection, aspirate the INPs or plCSA-INPs into a 28 g insulin syringe.
   4. Transfer the pregnant mouse to a holding device that restrains the mouse while allowing access to the tail vein. Clean the tail with an alcohol swab. Then insert the syringe into the tail vein. Slowly inject the INPs or plCSA-INPs (5 mg/kg ICG equivalent) with even pressure over 5-10 s.
      Note: Stop injecting if a blister appears on the tail because this outcome indicates that the needle is not in the vein. Syringes should not be shared between mice to minimize disease transmission and cross-contamination.
   5. Record the injection time. Meanwhile, apply gentle pressure to the injection site until the bleeding stops, which normally takes 30-60 s.
3. In vivo imaging
   1. 30 min after the injection, image the pregnant mice using the in vivo fluorescence imaging system.
   2. Anesthetize the pregnant mice with an oxygen flow rate of 1.0 L/min and isoflurane at 2-4% in an associated chamber of the anesthesia unit and verify full anesthesia by slow and regular breathing. Then, move them into the imaging chamber. Place the anesthetized pregnant mice into the imaging chamber, keeping the animals in a supine position.
   3. Place a nose cone over the mouth and nose to allow the inhalation of 1-2% isoflurane with an oxygen flow rate of 1.0 L/min to maintain anesthesia.
   4. Select 2D-fluorescence and photographic parameters to image the ICG fluorescence signals. Set the exposure to auto and the excitation/emission wavelengths to 710/820 nm.
   5. At the end of the imaging procedure, turn off the isoflurane influx to stop the anesthesia, and carefully return the pregnant mice to their cages.
   6. 48 h after nanoparticle injection, anesthetize the pregnant mice with isofluorane, and then sacrifice the dam by cervical dislocation. Collect the fetuses and placentas using Graefe forceps, Graefe tissue forceps, and dissecting scissors.
   7. Place the placentas and fetuses into the imaging chamber, and image using the method described in step 2.3.4.

3. HFUS Evaluation of Embryonic Development

1. Animal models
   1. Obtain and prepare the pregnant mice as described in step 2.1.
   2. Use HFUS to image pregnant mice at E 6.5 (Protocols 3.2 and 3.3.3). First, confirm pregnancy by visualizing embryos on day E6.5, and then randomly allocate the pregnant mice into three groups: the MNP group, plCSA-MNP group, and phosphate-buffered saline (PBS) group.
   3. Inject PBS, MNPs or plCSA-MNPs (1 mg/kg MTX equivalent) into the tail veins of the pregnant mice every other day starting at E6.5 as described in step 2.2.
2. Preparation for imaging
   1. 24 h after the injection of nanoparticles, image the pregnant mice using the HFUS imaging system.
   2. Anesthetize the pregnant mice as described in step 2.3.2. Turn on the integrated temperature controls of the imaging platform and preheat the platform to 37-42 °C. Secure the pregnant mice in a supine position on the platform using tape.
   3. Place the nose cone connected to the anesthesia unit over the snout. Apply 2% isoflurane with an oxygen flow rate of 1.0 L/min to maintain steady anesthesia.
   4. Chemically remove hair from the abdomen using a depilatory cream. Wipe out the residual cream thoroughly with water-soaked gauze, and then coat the abdomen with acoustic coupling gel.
3. Imaging procedure
   1. Place the 40 MHz transducer in the mechanical arm.
   2. Adjust the transducer position to obtain longitudinal images of the fetus and placenta with the region of interest located in the focal zone.
   3. B-Mode imaging and analysis
      Note: See Movie 1.
      1. Click the B-Mode button and lower the transducer over the abdomen until the fetus and placenta come into view. Press Scan/Freeze to initiate/stop imaging, press Cine store to store the cine loop, and press Frame store to store frame images.
      2. Click the Measure button to analyze the gestational sac length (GS), fetal crown rump length (CRL), biparietal diameter (BPD), abdominal circumference (AC), placental diameter (PD) and placental thickness (PT).
   4. PW-Doppler imaging and analysis
      Note: See Movie 1.
      1. Using the same scan projection, click the PW button, place the sampling volume box in the center of the umbilical artery, and press Scan/Freeze to initiate imaging. Click Cine store to collect umbilical artery images.
      2. Click the Measure button to calculate the umbilical artery peak velocity (UA).
   5. Color Doppler mode imaging and analysis
      1. Using the same scan projection, click the Color button and adjust the transducer position to obtain images of the fetal heart. Press Scan/freeze to initiate imaging and Cine store to collect images.
      2. Click the Measure button to calculate the fetal heart rate (HR).

4. HPLC Analysis

1. Tissue preparation
   1. Inject the pregnant mice with a single dose of MNPs or plCSA-MNPs (1 mg/kg MTX equivalent) at late pregnancy (e.g., E14.5) as described in step 3.1.3.
   2. After 24 h, anesthetize the mice by an intraperitoneal injection of avertin at 240 μg/body weight (g). Ensure no response to a foot pinch to verify that the mice are fully anesthetized.
   3. Spray the chest area with 75% ethanol. Perform cardiac perfusion (cut the right atria and perfuse through the left ventricle) as previously described in detail^19,20 with 50 mL of ice-cold 0.9% saline for 10 min to remove unbound nanoparticles.
   4. Euthanize the dam. Perform a cesarean section to collect the fetuses and placentas using Graefe forceps, dissecting scissors, and Graefe tissue forceps, and store the tissues at -80 °C prior to analysis.
   5. Prepare the homogenization solution (10% perchloric acid) and keep on ice. Collect approximately 200 mg of tissue, and add 500 μL of homogenization solution to each sample. Homogenize the samples using a homogenizer at full speed for 30 s, and repeat this procedure twice.
   6. Centrifuge the samples at 14,000 ×g for 20 min at 4 °C. Filter the supernatant (approximately 300 μL) through a 0.45 μm syringe filter, and transfer the resulting liquid to an HPLC vial. Place sample vials into an autosampler tray for injection.
2. Preparation of standards
   1. Prepare the following solution for the mobile phase: 40 mM potassium phosphate dibasic (pH 4.5) and acetonitrile (88:12, v/v). Filter the solution through a 0.45 μm pore size syringe filter and transfer the resulting liquid to a clean HPLC reservoir bottle.
      Note: Adjust the pH with 0.1 M phosphoric acid. Use ultrasonic vibration for 15 min to degas the mobile phase each time prior to use.
   2. Weigh 10 mg of MTX into a 1.5 mL centrifuge tube. Add 1 mL of 1 M sodium hydroxide.
   3. Vortex at high speed until the MTX dissolves completely.
      Note: This is the primary stock and can be stored at -20 °C for several months.
   4. To create the secondary MTX stock (500 μg/mL), dilute 50 μL of the primary stock in 950 μL of the mobile phase.
      Note: Store on ice until use, and prepare fresh daily. It is important to use the mobile phase for the preparation of standards to avoid peaks resulting from mixing dissimilar solutions after sample injection.
   5. Make further dilutions to create the standards (Table 1). Store the standards on ice and prepare fresh daily. Run the standards in series with the experimental samples.

Number	Final concentration (μg/mL)	500 μg/mL standard, μL	Mobile phase(μL)
1	0.5	1	999
2	1	2	998
3	2.5	5	995
4	10	20	980
5	25	50	950
6	50	100	900
7	100	200	800

Table 1. Prepare of standard curve for MTX. The final concentration of MTX standard solution is from 0.5-100 μg/mL.

3. HPLC instrumentation and operation parameters
   Note: Samples were analyzed on an HPLC system equipped with a solvent pump, a UV spectrophotometric detector (313 nm), and a C18 column (250×4.6 mm, 5 μm particle size).
   1. Turn on the HPLC degasser to remove air from the system. Turn on the flow, equilibrating the column with the mobile phase for 30 min to reduce baseline noise.
   2. Set the temperature of the column to 25 °C, inject 20 μL sample volumes at a flow rate of 1 mL/min, and click the Run Method to start the analysis.
   3. When the runs are complete, manually change the mobile phase to HPLC-grade acetonitrile. Run for approximately 15 min to protect the system.
      Note: Failure to perform this step following the recommended running time could result in damage to the column.
   4. For quantitative analysis, calculate the areas under the standard MTX peaks of interest using the HPLC system software.

Results

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...

Discussion

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 ...

Materials

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