Dosing and Testing Regimens in vivo

For dose choice, we firstly concentrated the RBC-NP-50 as high as possible, and then we found the highest injectable concentration was nearly of 50 mg/mL (cell membrane protein) otherwise would go to gel status. On the other hand, the acceptable injection volume regarding mouse ethics is less than 500 μL; thus we selected 400 μL per mouse. The average weight of pregnant mouse at later stage of gestation is about 50 g, and we thus figured out a single high dose (denoted 1HD) of RBC-NP-50 at 400 mg/kg. Parallelly, a thrice successive low dose (denoted 3LD) of RBC-NP-50 at 200 mg/kg was chosen as another experimental group. Each injection of 3LD was half compared to 1HD, but 1.5-fold higher than 1HD in total. For dosing frequency, we first demonstrated that RBC-NP-50 could accumulate in placenta and fetus and reach plateau around GD17 (Figure 2E and F). Refering to an average parturition time point of mouse is about GD21, we thus picked the GD17 as the final injection time both for 1HD and 3LD. Taken together, pregnant mice at GD17 were administrated with RBC-NP-50 at 400 mg/kg by i.v.; or pregnant mice were repeatedly successive dosed with 200 mg/kg of RBC-NP-50 by i.v. at GD15, 16, and 17 for three times. At each dosing regimen, mice treated with an identical volume of 8% (w/v) sucrose were used as controls.

Biodistribution of RBC-NP at different sizes in pregnant mice. (A) Hydrodynamic size (diameter) and zeta potential (mV) of different nanoparticles before and after RBC membrane coating. n = 3, mean ± SD. (B) Colloidal stability of different RBC-NP particles in PBS over 7 days. n = 3, mean ± SD. Fluorescent images and statistical analysis showing different accumulations in placentas (C) and fetuses (D) of pregnant mice after being injected with various sizes of DiD-labelled RBC-NP particles by i.v., including RBC-NP-50, RBC-NP-180, RBC-NP-400, and RBC-NP-1000. n = 5, mean ± SD. Fluorescent images and statistical analysis showing different accumulations in placentas (E) and fetuses (F) of pregnant mice after being injected with DiD-labelled RBC-NP-50 at different gestation day (GD), including GD14, 15, 16, and 17. n = 5, mean ± SD.

For testing regimens, at 48 h (GD19) following the final injection of either 1HD or 3LD of RBC-NP-50, pregnant mice in all groups were euthanized and the related parameters both from maternal mice and their fetuses were tested; or the related measurements upon maternal and juvenile mice were monitored/tested throughout the duration until 21 d post-partum/birth.