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.
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Method Article
Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods
In This Article
Summary
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 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 ....
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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
- 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
- Preparation of pregnant mice....
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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.......
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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 .......
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Disclosures
X.F. and B.Z. are inventors on patent application PCT/CN2017/108646 submitted by SIAT that covers a placenta-specific drug delivery method and its application. All other authors declare that they have no competing interests.
Acknowledgements
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 & 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.
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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 |
References
- Rodger, M. A., et al. 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. PLOS Medicine. 7 (6), e1000292(2010).
- Rodger, M. A., et al.
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