Preparation of Red Blood Cell Membrane-Coated Upconversion Nanoparticles and The Targeting Multimodality Imaging on 4T1 Breast Cancer

Objectives: To date, upconversion nanoparticles (UCNPs) have been widely applied in tumor imaging, drug targeting studies and many other biomedical areas. However, the shorter blood clearance time and immunogenicity of nanoparticles limit their further application in vivo. As a safer and more biocompatible vector, the natural cell membrane has become the research hotspot. In this study, folic acid (FA) inserted red blood cell membrane-coated upconversion nanoparticles (FA-RBC-UCNPs) were designed to enhance nanoparticles’ biocompatibility and tumor targeting ability, so to achieve the targeting multimodality imaging of 4T1 breast cancer.

Methods: RBC-vesicles were prepared by hypotonic treatment and subsequential extrusion. To encapsulate UCNPs into RBC-vesicles, the mixture was repeatedly extruded through a 400-nm porous polycarbonate membrane on a mini-extruder. The physical characteristics, optical property and the protein content on RBC-UCNPs were confirmed. 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-2000] (DSPE-PEG-FA) was incubated with empty RBCm to form DSPE-PEG-modified RBC membranes. FA-PEG-DSPE-RBCm was reconstructed into vesicles and then coated onto UCNPs (named FA-RBC-UCNPs). CCK-8 assay was used to evaluate the cytotoxicity. MRI and upconversion luminescence (UCL) imaging were conducted at different time points after and UCNPs, RBC-UCNPs and FA-RBC-UCNPs injected into 4T1 tumor bearing mice. DSPE-PEG-N₃ was inserted into RBCm. N₃(FA)-RBC-UCNPs and N₃-RBC-UCNPs were intravenously injected into the 4T1 tumor-bearing mice for pre-positioning 36 h earlier. After preparation of Al¹⁸F-NETA-L-DBCO, the chelator was injected for click chemistry in vivo. Micro PET imaging and bio-distribution study were performed at 0.5, 1 and 2h after injection of Al¹⁸F-NETA-L-DBCO. Systematic toxicity evaluation was performed over a span of 30 d.

Results: After coating with the RBC membranes, the hydrodynamic diameter of UCNPs increased from 73.5 nm to 187.8 nm. SDS-PAGE protein analysis showed that the main protein bands of RBC-UCNP were basically same as that of natural RBC lysate. UV absorption spectra demonstrated RBC-UCNPs showed an additional characteristic absorption near 400 nm compared to UCNPs. UCL emission spectra showed the fluorescence property of UCNP still maintained after RBC-vesicles coating. CCK-8 assay exhibited UCNPs, RBC-UCNPs or FA-RBC-UCNPs had no obvious cytotoxicity to cancer cells. MRI and UCL imaging demonstrated that FA-RBC-UCNPs group has the highest tumor uptake in 4T1 tumor bearing mice. Relative lower fluorescence signal of liver and spleen ware observed in FA-RBC-UCNPs compared to RBC-UCNPs and UCNPs. PET imaging indicated that N₃(FA)-RBC-UCNPs group equipped higher tumor uptake of the tracer at various times compared with N₃-RBC-UCNPs group. However, the control group, only injected Al¹⁸F-NETA-L-DBCO without pre-positioning, demonstrated no obvious uptake at the tumor site in the whole imaging process. In all three groups, the liver and spleen were not visible, but part of the intestinal were with tracer concentration. Neither death nor significant difference in body weight was noted. Blood biochemistry, hematology tests, and histology analysis all suggested a good in vivo biocompatibility of RBC-UCNPs.

Conclusions: The RBC-UCNPs were successfully constructed with no significant cytotoxicity and biological toxicity. In vivo tumor targeted multimodality imaging was successfully enhanced by inserted DSPE-PEG-FA. Pre-targeting methods successfully detected the 4T1 tumor by combining short half-life nuclide with biomimetic nanoparticles. Our method provides a new potential biomedical application depending on the biomimetic nanoparticles. Funding: This work was supported by the National Natural Science Foundation of China (No. 81630049, 81801738 and 81501532).