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

Guanzhou Di¹Chen Lu¹Mengting Xue¹Weiqi Li¹Runmin Xie¹Xinpei Yuan¹Xu Zhen²Min Wu²Xiying Mao^1*Songtao Yuan^1*

• ¹Department of Ophthalmology, First Affiliated Hospital, Nanjing Medical University, Nanjing, China
• ²State Key Laboratory of Analytical Chemistry for Life Science, Nanjing University, Nanjing, Jiangsu Province, China

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.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.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.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.