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Bioengineering

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

Published: September 10th, 2018

DOI:

10.3791/58219

1Laboratory for Reproductive Health, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, 2College of Veterinary Medicine, Hunan Agricultural University, 3Key Laboratory of Chemical Engineering Process and Technology for High-efficiency Conversion, College of Chemistry and Material Sciences, Heilongjiang University, 4Department of Obstetrics and Gynecology, Wayne State University School of Medicine

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 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.

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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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 elsewhere

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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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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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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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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

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