Near-infrared fluorescent nanoprobe enables noninvasive, longitudinal monitoring of graft outcome in RPE transplantation

Abstract

Objectives: Retinal pigment epithelium (RPE) cell transplantation holds therapeutic promise for retinal degenerative diseases, but longitudinal monitoring of graft survival and efficacy remains clinically challenging. The aim of this study is to develop a simple and effective method for the therapeutic quantification of RPE cell transplantation and immune rejection in vivo.

Methods: A nanoprobe was developed and modified to label donor RPE cells, and used to monitor the position and intensity of the fluorescence signal in vivo. Immunofluorescence staining and single-cell RNA sequencing (scRNA-seq) were used to characterize the cell types showing the fluorescence signal of the nanoprobe and to determine the composition of the immune microenvironment associated with subretinal transplantation.

Results: The spatial distribution of the fluorescence signal of the nanoprobe corresponded with the site of transplantation, but the signal intensity decreased over time, while the signal distribution extended to the choroid. Additionally, the nanoprobe fluorescence signal was detected in the liver and spleen during long-term monitoring. Conversely, in mice administered the immunosuppressive drug cyclosporine A, the decrease in signal intensity was slower and the expansion of the signal distribution was less pronounced. Immunofluorescence analysis revealed a significant temporal increase in the proportion of macrophages with nanoprobe-labeled cells following transplantation. The stability and cell-penetrating ability of the nanoprobe enables the labeling of immune cell niches in RPE transplantation. Additionally, scRNA-seq analysis of nanoprobe-labeled cells identified MDK and ANXA1 signaling pathway in donor RPE cells as initiators of the immune rejection cascade, which were further amplified by macrophage-mediated pro-inflammatory signaling.

Conclusion: Near-infrared fluorescent nanoprobes represent a reliable method for in vivo tracing of donor RPE cells and long-term observation of nanoprobe distribution can be used to evaluate the degree of immune rejection. Molecular analysis of nanoprobe-labeled cells facilitates the characterization of the dynamic immune cell rejection niche and the landscape of donor-host interactions in RPE transplantation.

Keywords: RPE transplantation; immune rejection; in vivo tracking; macrophage; near-infrared fluorescent nanoprobe.

Figure 1

Properties and toxicity of the nanoprobe. (A) Schematic illustration of the nanoprobe preparation process. (B) Emission spectrum of the nanoprobe. (C) Particle size distribution of nanoprobes. (D) Comparison of cell viability between w/ probe group (nanoprobe) and w/o probe group (blank). (E) Immunofluorescence images of fRPE cells in vitro incubated with the nanoprobe and the proportion of probe-positive fRPE cells at D1 and D14 assessed by flow cytometry analysis. (F) Bulk RNA sequencing analysis of probe-labeled fRPE cells revealed gene expression profiles comparable to those of unlabeled cells.

Figure 2

NIR ratiometric imaging and fundus multimodal imaging of nanoprobe-labeled cells. (A) Experimental scheme for nanoprobe imaging in vivo using the IVIS Series Lumina III imaging system (n = 3). (B) Representative images of mice in the blank, w/o cyclosporin A (CsA) and w/CsA groups at D0, D1, D3, D6, and D12 after cell transplantation captured by the IVIS Series Lumina III imaging system. (C) Line chart showing a slower decline of signal intensity in the w/CsA group compared to the w/o CsA group. (D) Experimental scheme for nanoprobe imaging by the small animal ophthalmic multimodal imaging system (n = 3). (E) Fundus fluorescence images and optical coherence tomography (OCT) of mice injected subretinally with nanoprobe-labeled cells at D3 and D7 after cell transplantation. (F) Fundus fluorescence images of both GFP and near-infrared (NIR) channels at different time points. The symbols **denote p = 0.01 and ****denote p = 0.0001.

Figure 3

Distribution dynamics of probe signal in vivo and in vitro. (A) Immunofluorescence staining of RPE65 in retinal cryosections of mice injected subretinally with nanoprobe-labeled cells. The distribution of the nanoprobe signal (white arrow) transferred from the subretinal space to the choroid layer. Scale bar, 50 μm. (B) Experimental scheme for the non-contact co-culture assay. Flow cytometry analysis showing the proportion of probe-labeled BV2 microglial cells. (C) Experimental scheme for the contact co-culture assay. Flow cytometry analysis showing the proportion of CFSE/probe-labeled cells. (D) Immunofluorescence staining of IBA1 in retinal cryosections of mice injected subretinally with nanoprobe-labeled cells with or without intraperitoneal injection of cyclosporin A (CsA). Scale bar, 50 μm. (E) Nanoprobe signals (white arrow) in liver and spleen captured in vivo (n = 3).

Figure 4

Single-cell intercellular communication between donor fRPE cells and transplantation niche cells. (A) Flow cytometry analysis of CD45⁺ and Probe⁺ cells in retina/RPE/choroid at D1 and D5 after transplantation. (B) Probe⁺ live cells were subjected to scRNA-seq analysis. UMAP plot of Probe⁺ cells from mice after transplantation showing 8 clusters. Stacked bar plot showing the composition of different cell types in Probe⁺ cells at D1 and D5. (C) Dot plot illustrating donor cell communicating with each cluster. (D) Dot plot illustrating macrophage activated by each cluster at D1 and D5. The communication probability of each group was represented by dot size and color intensity.

Figure 5

Graphical illustration of probe kinetics that predicts fate of donor RPE cells in vivo. In fundus fluorescence imaging, the attenuation and dispersion of the probe signal in the prolonged monitoring indicate that donor cells are engulfed by macrophages, which subsequently reside in situ or migrate to the spleen and liver through the bloodstream. The fate of donor cells can be categorized into three distinct groups according to the pathways followed: (1) cells functionally integrating into the host RPE layer without being rejected; (2) cell debris being phagocytosed by tissue-resident macrophages and residing within the retina or choroid; and (3) those activating the peripheral immune system by the circulating probe-labeled macrophage.