NIR-II nanoprobes in-vivo assembly to improve image-guided surgery for metastatic ovarian cancer

Abstract

Local recurrence is a common cause of treatment failure for patients with solid tumors. Tumor-specific intraoperative fluorescence imaging may improve staging and debulking efforts in cytoreductive surgery and, thereby improve prognosis. Here, we report in vivo assembly of the second near-infrared window (NIR-II) emitting downconversion nanoparticles (DCNPs) modified with DNA and targeting peptides to improve the image-guided surgery for metastatic ovarian cancer. The NIR-II imaging quality with DCNPs is superior to that of clinically approved ICG with good photostability and deep tissue penetration (8 mm). Stable tumor retention period experienced 6 h by in vivo assembly of nanoprobes can be used for precise tumor resection. Superior tumor-to-normal tissue ratio is successfully achieved to facilitate the abdominal ovarian metastases surgical delineation. Metastases with ≤1 mm can be completely excised under NIR-II bioimaging guidance. This novel technology provides a general new basis for the future design of nanomaterials for medical applications.

Fig. 1

Schematic illustration of NIR-II nanoprobes fabrication for ovarian metastasis surgery under NIR-II bioimaging guidance. Schematic of stepwise fabrication of DNA and FSH_β modified DCNPs (DCNPs-L₁-FSH_β) and in vivo assembly of DCNPs-L₁-FSH_β (first injection) and DCNPs-L₂-FSH_β (second injection) with improved tumor targeting and rapid hepatic and renal clearance after two-staged in sequence injections. Metastatic ovarian tumors can be clearly observed and precisely removed with NIR-II image-guided surgery

Fig. 2

In vitro assembly of NIR-II nanoprobes. Transmission electron microscope (TEM) images of as-made NaGdF₄: 5% Nd@NaGdF₄ nanocrystals (a), DNA (L₁) modified DCNPs (b) and DNA complementary induced assembly between DCNPs-L₁-FSH_β and DCNPs-L₂-FSH_β in PBS (c). d The NIR-II fluorescence spectrum of DCNPs, DCNPs-L₁-FSH_β, and self-assembled DCNPs. Inset, NIR-II fluorescence images of the corresponding samples. e Size distribution of DCNPs, DCNPs-L₁-FSH_β, and self-assembled DCNPs determined by dynamic light scattering. f, g Photostability of DCNPs (f) and ICG (g) in a variety of biological media at 37 °C under continuous 808 nm laser exposure at a power density of 0.2 W cm⁻². h NIR-II fluorescence images show complete attenuation of NIR-I light (ICG) by 3 mm, while NIR-II signals (DCNPs) are able to be detected through 8 mm of phantom tissues. Representative images are for n = 5 per group. i Signal to background ratios of DCNPs and ICG as a function of tissue phantom depth. Scale bars represent 10 nm in a, b and 50 nm in (c). Mean ± s.d. for n = 5 (*P < 0.05 vs. ICG, two-sided Student’s t test)

Fig. 3

In vivo assembly of NIR-II nanoprobes. a NIR-II fluorescence bioimaging (1000 nm long-pass filter) of the nude mice with murine epidermal tumor by single caudal vein first injection and two-staged in sequence injection (first + second) (interval between two injection is 8 h) under 808 nm excitation (fluence rate = 40 mW cm⁻²). The concentration of DCNPs in single injection is same to the sum of that for two-staged injection. Liver distribution of nanoprobes (b), tumor targeting efficiency (c), and T/N ratio (d) with single first injection and two-staged in sequence injection, respectively. The red dotted line in d indicates the Rose criterion. (^*P < 0.05 vs. first, two-sided Student’s t test). e Fluorescence images of DCNPs-L₁(Cy5)-FSH_β + DCNPs-L₂(Cy7)-FSH_β (i) and DCNPs-L₁(Cy5)-FSH_β + DCNPs-L₁(Cy7)-FSH_β (ii) in epidermal tumor of the nude mice and the corresponding harvest organs, blood and tumors (left to right and to bottom: heart, liver, spleen, lung, kidney, tumor, and blood). f T/N ratio of the in vivo assembly (first + second), active targeting (DCNPs-L₁-RGD + DCNPs-L₂-RGD), passive targeting (DCNPs-L₁ + DCNPs-L₂), and preassembly (first + second) groups. (^*P < 0.05 vs. DCNPs-L₁-RGD + DCNP-L₂-RGD, two-sided Student’s t test). g Blood circulation and urine excretion of nanoprobes after second injection. The T/N ratio is tumor-to-liver ratio in this epidermal tumor model experiments. All scale bars, 1 cm. Representative images are for n = 5 per group. Mean ± s.d. for n = 5

Fig. 4

Optimal surgery time window of in vivo assembly. a Comparison of MRI and NIR-II fluorescence imaging (1000 nm long-pass filter) of subcutaneous human ovarian adenocarcinoma. b Correlation for the tumor size ratio between MRI and NIR-II fluorescence bioimaging method. c Tumor retention of assembled DCNPs from 20 to 30 h PI. d H&E staining results of the tumors resected in 20–28 h PI under NIR-II fluorescence bioimaging guidance. e NIR-II fluorescence image-guided tumor surgery with the guided dose of clinic approved ICG probe (1.5 mg kg⁻¹). Tumor margin was confirmed by the H&E staining result. Scale bars, 5 mm in NIR-II bioimaging and MRI results, 0.2 mm in H&E staining images. Representative images are for n = 5 per group. Mean ± s.d. for n = 5

Fig. 5

NIR-II image-guided surgery. a Optical photo of human ovarian adenocarcinoma peritoneal metastases model (i), the corresponding NIR-II fluorescence bioimaging (1000 nm long-pass filter) results obtained at 22 h PI (ii) and the enlargement of the NIR-II nanoprobes labeled large peritoneal metastatic tumors (Nos. 1–8) and ultrasmall lesions (Nos. 9–13) (iii). Scale bar, 1 cm. b T/N ratios plotted as a function of different labeled peritoneal metastatic tumors, red dotted line is according to the Rose criterion. c H&E staining results of tumor margin in Nos. 1–8 (scale bars, 0.2 mm) and metastatic lesions in Nos. 9–13 (scale bars, 0.5 mm). Tumors were resected under NIR-II fluorescence bioimaging guidance in a. d NIR-II fluorescence bioimaging results of the popliteal lymph node metastasis at 22 h PI. Scale bar, 1 cm. e T/N ratios plotted as a function of different PI of the first injection, red dotted line is according to the Rose criterion. f H&E staining results of popliteal lymph node metastasis (Scale bar, 0.5 mm). Tumors were resected under NIR-II fluorescence bioimaging guidance in d. The T/N ratio is tumor-to-normal peripheral tissue ratio in this peritoneal metastases model and popliteal lymph node metastasis model experiments. Representative images are for n = 5 per model. Mean ± s.d. for n = 5