Fabrication of Red Blood Cell-Based Multimodal Theranostic Probes for Second Near-Infrared Window Fluorescence Imaging-Guided Tumor Surgery and Photodynamic Therapy

Abstract

The therapeutic efficacy of fluorescence image-guided tumor surgery and photodynamic therapy (PDT) is impaired by the penetration depth limitation, low signal-to-noise ratio of traditional first near-infrared window (NIR I) fluorescence and the hypoxic tumor microenvironment. Here, a "red blood cell-based multimodal probe" was proposed to achieve enhanced tumor targeting and retention of fluorescent probes after an intravenous injection, so that second near-infrared window (NIR II) fluorescence bioimaging-guided complete tumor resection and high-efficiency photodynamic therapy could then be realized. Methods: The hexanoic acid ester-modified rose bengal (RB-HA), RGD (Arginine-Glycine-Aspartic) peptide and avidin were covalently coupled onto amine-modified upconversion nanoparticles (UCNPs) via EDC/NHS reaction (UCNPs@RB@RGD@avidin). Afterwards, the complex of ICG with bovine serum albumin (BSA) was loaded into RBCs through hypotonic dialysis (RBC@ICG). Then, the membrane proteins of RBC@ICG were biotinylated by biotin-modified phospholipids (RBC@ICG@biotin). Finally, the RBCp (Red Blood Cell based probe) was obtained by crosslinking UCNPs@RB@RGD@avidin to RBC@ICG@biotin through the interaction of avidin and biotin. The obtained multimodal RBCp was extensively characterized, both in vitro and in vivo, including analysis of chemical, physical and fluorescent features, O₂ delivery ability, tumor accumulation, NIR II fluorescence bioimaging ability, photodynamic therapeutic efficiency, and biosafety. Results: The RBCp experienced efficient tumor targeting and long tumor retention for almost 4 h after intravenous injection, and the superior signal-to-noise ratio at the optimal time window can be used for guiding precise tumor resection under an 808-nm laser irradiation to facilitate lymph popliteal metastasis surgical delineation. Meanwhile, the RBCp can provide laser-responsive O₂ release to enhance the PDT efficiency of popliteal lymph node metastasis under NIR II fluorescence bioimaging guidance. These excellent performances obviously lead to remarkably enhanced synergistic therapeutic effects of tumor surgery and metastatic inhibition. Conclusion: The proposed strategy will develop a new platform to increase surgical resection completeness and improve PDT efficiency, resulting in the successful and complete inhibition of tumor and metastasis, which could offer a promising approach for the clinical translation of malignant tumor treatment.

Keywords: indocyanine green; oxygen delivery; photodynamic therapy; second near-infrared window fluorescence; tumor surgery.

Figure 1

A schematic illustration of RBCp fabrication. With an 808-nm laser irradiation, RBCp can continuously release ICG and O₂, which can be applied for NIR II fluorescence bioimaging-guided tumor surgery as well as to enhance NIR II fluorescent bioimaging-guided photodynamic therapy.

Figure 2

Characterization of RBCp. TEM images of core nanocrystals of NaGdF₄:Yb,Er (A) and NaGdF₄:Yb,Er@NaGdF₄ with the core-shell nanostructure (B). (C) SEM images of RBC-based multimodal probes and (D) the enlarged image with higher magnification of two RBC-based multimodal probes attached with UCNPs; insert: RBCs without UCNPs modification. (E) The emission spectrum of UCNPs under a 980-nm laser irradiation and the absorption spectrum of RB. (F) The emission spectrum of aqueous ICG solution under an 808-nm laser irradiation; insert: NIR II fluorescence image of ICG observed by InGaAs camera (100 ms exposure time, 1000 nm long-pass filter).

Figure 3

NIR II fluorescence imaging and laser activated O₂ release of RBCp. (A) NIR I and NIR II fluorescent images of ICG show complete attenuation of NIR I light within 4 mm, while the NIR II light is able to be detected through 8 mm of phantom tissues. The corresponding fluorescence intensities (B) and signal-to-background ratios (C) of both NIR I and NIR II fluorescence as a function of tissue phantom depth. (D) In vitro O₂ generation of RBCp with and without an 808-nm laser irradiation; pure RBCs with an 808-nm laser irradiation were set as a control group. (E) O₂ release of RBCp in hypoxic HepG2 cells with and without exposure to an 808-nm laser for 10 min. All of the images were obtained by a confocal laser scanning microscope. (F) 2D photoacoustic images of epidermal liver tumors with RBCp and RBC injection to detect the blood O₂ saturation in the vasculature, by an 808-nm laser irradiation for 0, 5 and 10 min. Representative images are from n = 4. Error bars indicate SD (n = 4).

Figure 4

Optimal NIR II fluorescence imaging time window for surgery. (A) NIR II fluorescence bioimaging (100 ms exposure time, 1000 nm long-pass filter) of a subcutaneous tumor-bearing nude mouse with a single tail vein RBCp injection under an 808-nm excitation (0.2 W/cm²). (B) Liver distribution and tumor targeting efficiency of RBCp. (C) Tumor-to-liver ratio after an intravenous injection of RBCp. (D) Tumor-to-liver ratio of RBCp, RBCp without UCNPs modification and RBCp without RGD motif targeting. (E) H & E staining results of the tumors removed from 10 - 14 h post injection (PI) under the guidance of NIR II fluorescence bioimaging. Representative images are from n = 4. Error bars indicate SD (n = 4).

Figure 5

NIR II fluorescence imaging guided tumor surgery. NIR II fluorescence bioimaging results (12 h PI) of epidermal tumors with sizes of 7 mm³ (A) and 3 mm³ (C) and NIR II fluorescence bioimaging results after the surgical resection of tumors. (B, D) H&E staining results of the tumor and normal tissue boundary in A and C, respectively. (E) The NIR II bioimaging results (12 h PI) of popliteal lymph node metastasis. (F) H & E staining results of popliteal lymph node metastasis. The metastasis was resected under the navigation of NIR II fluorescence bioimaging.

Figure 6

NIR II fluorescence imaging guided PDT of RBCp. (A) NIR II fluorescence bioimaging-guided PDT of popliteal lymph node metastasis-bearing mice by RBCp injection and alternate irradiation with 808-nm and 980-nm laser. (B) Popliteal lymph node metastasis growth profiles after a quantitative NIR II fluorescence analysis of all images in A. (C) Weight change curves of popliteal lymph node metastasis-bearing mice after different treatments for 18 days. (D) H&E and TUNEL staining images of the tumors after 3 days of indicated treatments. Scale bars represent 100 μm. Representative images are from n = 4. Error bars indicate SD (n = 4).