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.