Effects of O+ and a Non-O+ Blood Type, Number Concentration, and Membrane Phosphatidylserine Flipping on the Circulation Dynamics and Biodistribution of Microsized Erythrocyte-Derived Optical Particles in Mice

Abstract

Erythrocyte-derived microparticles containing near-infrared (NIR) dyes such as indocyanine green present a promising cell-based platform for optical imaging and phototherapeutics. Using real-time intravital NIR fluorescence imaging of mice vasculature, we investigated the effects of blood type, specifically O⁺ and B⁺, used in fabricating these particles, the number concentration (N_v) of the particles, and the relocalization of phosphatidylserine (PS) to the outer leaflet of the particles' membrane on the resulting circulation dynamics following a single retro-orbital injection. Additionally, we quantified the biodistribution of particles in various organs. We found that the fluorescence emission half-life for particles engineered from O⁺ blood type extended from 11.4 ± 3.0 to 43.1 ± 9.6 min with increased N_v from a low range of 0.4-0.6 to high range of 1.4-1.6 million particles/per μL, when only 30-55% of the particles demonstrated externalized PS. For these particles, the liver and gallbladder, lungs, and spleen showed similar levels of accumulation at 60 min post administration. When >90% of O⁺-particles showed PS externalization, or when the particles were fabricated from B⁺ blood type despite PS externalization in 30-55% of the particles, the emission half-life was reduced to 15.8 ± 5.9 and 18.1 ± 4.6 min, respectively. There was lower accumulation of these particles in the spleen as compared to the liver and gallbladder and the lungs. In vitro experiments demonstrated increased PS externalization correlated to a more efficient uptake of the particles by macrophages. These findings emphasize the importance of blood type, N_v, and PS in engineering erythrocyte-derived particles for future clinical applications.

Keywords: cell-based therapeutics; drug delivery; erythrocyte engineering; fluorescence imaging; microparticles; near-infrared.

Figure 1.

Optical characteristics of $\mu \text{NETs}$. (A) Illustrative extinction, and (B) Fluorescence emission spectra (dots) and Gaussian fits (solid curves) of 300x diluted Low-Medium, and High PS⁺, High $N_{\mathrm{v}}$, O⁺-$\mu \text{NETs}$ in response to photoexcitation at 755±2.5 nm. (C) ∆MFI associated with Low-Medium PS⁺, O⁺- (n=3), and High PS⁺, O^+--$\mu \text{NETs}$ (n=3). Error bars represent standard deviation, p<0.05 (*). (D, E) Respective confocal fluorescence images of dilute Low-Medium PS⁺, O⁺-, and High PS⁺, O⁺-$\mu \text{NETs}$. (F, G) Respective brightfield images of ~1000x diluted Low-Medium PS⁺, High $N_{\mathrm{v}}$, O⁺- and High PS⁺, High $N_{\mathrm{v}}$, O⁺-$\mu \text{NETs}$.

Figure 2.

Representative fluorescence images of 300x diluted (A) Low-Medium PS⁺ and (B) High PS⁺, High $N_{\mathrm{v}}$, O⁺-$\mu \text{NETs}$. Panels are falsely colored for ICG (red) and annexin V–AF488 (green) fluorescence, and merged emissions. Quantification of externalized PS represented with dot plots of SSC-A vs fluorescence detected PS from diluted (C) RBCs; (D) Low-Medium PS⁺, O⁺-, and (E) High PS⁺, O⁺-$\mu \text{NETs}$ stained with AV-AF488. (F) ΔMFI associated with RBCs (n=3); Low-Medium PS⁺, O⁺- (n=3); and High PS⁺ O⁺-$\mu \text{NETs}$ (n=3). Error bars represent standard deviation. Single and triple asterisks correspond to statistically significant differences at p<0.05 and p<0.001, respectively.

Figure 3.

Representative fluorescence images of mice vasculature at 1, 10, 30, and 60 minutes post retro-orbital injection of (A) Low-Medium PS⁺, High $N_{\mathrm{v}}$, O⁺-, (B) Low-Medium PS⁺, Medium $N_{\mathrm{v}}$, O⁺-, and (C) Low-Medium PS⁺, Low $N_{\mathrm{v}}$, O⁺-$\mu \text{NETs}$. (D) Normalized intensity over time for Low-Medium PS⁺, O⁺-$\mu \text{NETs}$ injected at high (n=4), medium (n=3), and low (n=5) $N_{\mathrm{v}}$. Each data point is an average value of the intensity. Error clouds represent standard deviations.

Figure 4.

Representative fluorescence images of mice vasculature at 1, 10, 30, and 60 minutes post retro-orbital injection of (A) 400 μM free ICG, (B) Low-Medium PS⁺, High $N_{\mathrm{v}}$, B⁺-, and (C) High PS⁺, High $N_{\mathrm{v}}$, O⁺-$\mu \text{NETs}$. (D) Normalized intensity over time for 400 μM free ICG (n=3); Low-Medium PS⁺, High $N_{\mathrm{v}}$, B⁺- (n=5), and High PS⁺, High $N_{\mathrm{v}}$, O⁺- $\mu \text{NETs}$ (n=4). Each data point is an average value of the intensity. Error clouds represent standard deviations.

Figure 5.

(A) Biodistribution of ICG in the liver and gallbladder, lungs, and spleen at 30 minutes post retro-orbital injection (n=3) as quantified by the spectrally-integrated fluorescence emission. (B) Biodistribution of Low-Medium PS⁺, Low $N_{\mathrm{V}}$, O⁺- (n=3), Low-Medium PS⁺, High $N_{\mathrm{V}}$, O⁺- (n=4), Low-Medium PS⁺, High $N_{\mathrm{V}}$, B⁺- (n=4), and High PS⁺, High $N_{\mathrm{V}}$, O⁺-$\mu \text{NETs}$ (n=4) as quantified by the metric s (Equation 1). Error bars represent standard deviations. Statistically significant differences between the indicated pairs at p<0.05 (*), p<0.01 (**), p<0.001 (***) are shown.

Figure 6.

Representative fluorescence images of MH-S macrophages at 120 minutes of incubation with (A) Low-Medium PS⁺, high $N_{\mathrm{V}}$, O⁺, and (B) High PS⁺, O⁺ $\mu \text{NETs}$. Images associated with DAPI, ICG, and merged fluorescence are presented. (C) Average fluorescence measured in ≥ 200 cells for each type of $\mu \text{NETs}$, normalized to μNET ICG fluorescence (Equation 3). Error bars represent quartile regions. Statistically significant differences between the indicated pairs at p<0.05 (*) are shown.