Preparation of placenta-targeted nanoparticles and confirmation of the targets of nanoparticles
Placenta-targeted nanoparticles were prepared as described previously (Zhang et al., 2018). Briefly, the first step was preparation of CBX-loaded nanoparticles (CBX-NPs). CBX-NPs were synthesized from PLGA, soybean lecithin, CBX and DSPE-PEG (2000)-COOH using a previously reported single-step sonication method (Zhang et al., 2018). The second step is conjugation of chondroitin sulfate A (CSA)-binding peptide to CBX-NPs (CBX-CSA) and conjugation of the scramble peptide (SCR) to CBX-NPs (CBX-SCR). The conjugation efficiency of the peptides was confirmed via the bicinchoninic acid (BCA) assay. The third step is to define characterization of the nanoparticles. The size, surface charge, polydispersity, and size distribution of the NPs were determined using a Beckman Coulter Delsa TM Nano C (Beckman, USA) at room temperature. The morphology and particle size of these NPs were further analyzed using a transmission electronic microscope (TEM, JEM-100XII, JEOL, Japan) with the negative stain method (Zhang et al., 2018). Finally, the encapsulation efficiency and loading efficiency of CBX in the NPs was determined. A dialysis experiment was performed to analyze CBX release from the different CBX formulations. The CSA-binding peptide-conjugated nanoparticles loaded with indocyanine green (ICG-CSA-NPs) and ICG without peptide conjugation (ICG-NPs) were prepared according to the same procedures.
Pregnant rats at GD13.5 were intravenously administered ICG-CSA-NPs and ICG-NPs (5mg/kg). Forty-eight hours after injection, the animals were anaesthetized, and ICG signal in uteri was imaged using a Bruker In-Vivo Xtreme system (Perkin Elmer) as described previously (Zhang et al., 2018). Then, the animals were sacrificed, the fetus, placenta, lung, kidney, heart, spleen and liver were collected and fixed by PAF(4%). The tissues were embedded into OCT and freezing sliced (15μm). ICG fluorescence images were captured using a Zeiss LSM 780 confocal microscope. This experiment was blinded to the operators.