Intratumor injected gold molecular clusters for NIR-II imaging and cancer therapy

Abstract

Surgical resections of solid tumors guided by visual inspection of tumor margins have been performed for over a century to treat cancer. Near-infrared (NIR) fluorescence labeling/imaging of tumor in the NIR-I (800 to 900 nm) range with systemically administrated fluorophore/tumor-targeting antibody conjugates have been introduced to improve tumor margin delineation, tumor removal accuracy, and patient survival. Here, we show Au₂₅ molecular clusters functionalized with phosphorylcholine ligands (AuPC, ~2 nm in size) as a preclinical intratumorally injectable agent for NIR-II/SWIR (1,000 to 3,000 nm) fluorescence imaging-guided tumor resection. The AuPC clusters were found to be uniformly distributed in the 4T1 murine breast cancer tumor upon intratumor (i.t.) injection. The phosphocholine coating afforded highly stealth clusters, allowing a high percentage of AuPC to fill the tumor interstitial fluid space homogeneously. Intra-operative surgical navigation guided by imaging of the NIR-II fluorescence of AuPC allowed for complete and non-excessive tumor resection. The AuPC in tumors were also employed as a photothermal therapy (PTT) agent to uniformly heat up and eradicate tumors. Further, we performed in vivo NIR-IIb (1,500 to 1,700 nm) molecular imaging of the treated tumor using a quantum dot-Annexin V (QD-P³-Anx V) conjugate, revealing cancer cell apoptosis following PTT. The therapeutic functionalities of AuPC clusters combined with rapid renal excretion, high biocompatibility, and safety make them promising for clinical translation.

Keywords: NIR-II imaging; gold nanoclusters; imaging-guided surgery; molecular imaging of apoptosis; photothermal therapy.

Fig. 1.

(A) Crystallographic representation of Au₂₅ cluster structure using UCSF Chimera program (version 1.16) based on published crystal structure data (61). Color codes of the elements: Au: yellow, S: green. Schematic illustration of AuGSH cluster surface modification with PC ligand to result in AuPC conjugate. For simplicity, only the conjugation of γ-glutamate carboxylic functional group to PC ligand is shown while the conjugation to glycine carboxylic group in GSH is omitted. The structures of GSH and PC ligands are shown inside the frame. (B) UV−Vis absorption spectra of AuGSH and AuPC clusters in PBS buffer pH7.4. The spectra of cluster solutions (500 µg/mL) were measured using a 2-mm quartz cuvette. The inset shows the spectra in UV region and highlights the absorption of PC ligand. (C) PLE spectra of AuGSH cluster at various excitation wavelengths. (D) Visible-NIR-I and NIR-II fluorescence spectra of AuGSH cluster in PBS buffer pH7.4 (120 µg/mL) excited at 550 nm.

Fig. 2.

(A) Schematic representation of in vivo NIR-II imaging-guided tumor resection of intratumorally administered AuPC clusters in PBS buffer pH7.4 (1×, 300 µg) into a mouse bearing 4T1 tumor on a right hindlimb (3 to 6 wk old female Balb/c, n = 8). (B) Pre-surgical, intra-operative and post-surgical NIR-II imaging were perform using wide-field NIR-II fluorescence imaging system (excited by an 808-nm laser at a power density of 70 mW/cm², exposure times 3 and 7 ms for 1× and 5× magnification, respectively, using an 1,100-nm long-pass filter). The tumor area marked by a rectangle was imaged using 5× magnification (exposure time 7 ms). For surgical resection, the skin covering the tumor was carefully removed allowing NIR-II imaging of the exposed tumor. The tumor was then excised in one step. Scale bars are 1 cm and 2 mm for 1× and 5× objectives, respectively. (C) TBR ratio (Left graph) was calculated using the fluorescence intensity of tumor and nearby background after resection. The Right graph is the cross-sectional profile marked in white dotted lines represented in mean fluorescent intensity (MFI) values. Data are presented as box plots (median line, 25th and 75th percentiles; whiskers, outlier). (D) H&E staining and NIR-I fluorescence imaging of resected tumor tissue in Cy5 channel (AuPC emission). 20× objective, scale bar is 0.5 mm. The tissue sections were ~20 µm.

Fig. 3.

(A) Schematic representation of TIF collection by centrifugation from resected tumors i.t. injected with AuGSH (1×, 300 µg in PBS buffer pH 7.4), AuPC (1×, 300 µg in PBS buffer pH 7.4), and ICG (5 mM in DI-water). (B) Ex vivo NIR-II imaging of resected tumors before and after centrifugation (n = 3). The imaging was done under an 808-nm laser excitation at a power density of 70 mW/cm² using an 1,100-nm long-pass filter, 5× objective, scale bar is 2 mm. The exposure times were 5 and 3 ms for AuPC/AuGSH and ICG, respectively. (C) Color photographs (Top) of collected TIF extracts inside Eppendorf vials. NIR-II images of collected extracts (Bottom, 10× diluted solutions, 3 ms-exposure time). (D) Tumor MFI difference before and after the centrifugation. (E) The calculated percentages of NIR-II brightness in collected extracts (10× diluted solutions, 3 ms-exposure time). Error bars represent SD of three repeated experiments.

Fig. 4.

(A) The temperature change of AuPC cluster solutions (60 µL, 25 mg/mL in PBS buffer pH7.4) during continuous 30 min irradiation with an 808-nm laser at a power density of 300 mW/cm² (n = 3). The inset shows thermal pictures of AuPC solution inside Eppendorf vial before (0 min) and 10 min after laser irradiation recorded with a thermal camera. (B) NIR-II PL spectra of AuPC cluster solution (50× dilution) before and after irradiation using an 808-nm laser at a power density of 35 mW/cm². The Inset shows bar graphs of NIR-II peak intensity change post-irradiation and are presented as mean values ± SD. The data were analyzed by Tukey’s test.

Fig. 5.

(A) Schematic representation of PTT treatment of intratumorally administered AuPC cluster into a mouse bearing 4T1 tumor on a right hindlimb (6 wk old female Balb/c, n = 18). Saline-injected mice irradiated with laser were used as a control group (n = 16). PTT treatment was done by an 808-nm laser irradiation at a power density of 300 mW/cm² for 30 min. (B) Wide-field NIR-II imaging before and after PTT and on later timepoints was done under an 808-nm laser excitation at a power density of 70 mW/cm² using an 1,100-nm long-pass filter. The exposure time was 40 ms. The Insets show low exposure (1 ms) NIR-II images. Arrows point to the tumor and inguinal lymph node (iLN). 1× objective, scale bar is 1 cm. (C) Thermal camera pictures of mice before and during PTT treatment, scale bar is 1 cm. (D) Color pictures of mice before and after PTT. Morphological changes on the tumor surface become evident 1-d post-treatment with obvious scab formation. Temperature increase (E) and tumor volume changes in AuPC (F) and control (G) groups during and post-PTT treatment, respectively. (H) Kaplan–Meier survival analyses of AuPC and control groups.

Fig. 6.

(A) In vivo apoptosis imaging post PTT treatment of intratumorally administered AuPC cluster into a mouse bearing 4T1 tumor on a right hindlimb (6 wk old female Balb/c, n = 3). QD-P³-Anx V and QD-P³ were intravenously administered to the AuPC-mediated PTT group (n = 3) 1.5 h after the treatment. Systemic administration of QD-P³-Anx V to saline-injected mice with (+laser) or without (−laser) laser irradiation was used as control groups (n = 3 each). PTT treatment was done by an 808-nm laser irradiation at a power density of 300 mW/cm² for 30 min. Wide-field NIR-II imaging 1.5 and 24 h post-i.v. administration of probes was done under an 808-nm laser excitation at a power density of 70 mW/cm² using a 1,500-nm long-pass filter. The exposure times were 40 ms and 100 ms for 1.5 h and 24 h post-i.v., respectively. 1× objective, scale bar is 1 cm. (B) The calculated T/NT values for probes at 24 h post-i.v. injection. Data are presented as box plots (median line, 25th and 75th percentiles, whiskers, outlier). Absolute P values were derived using One-Way ANOVA. (C) Flow cytometry analyses of Annexin V-FITC/PI double-stained tumor cells 24 h post-PTT. (D) Apoptosis cell death in AuPC and control groups. (E) Ex vivo confocal imaging of apoptosis 24 h post-PTT treatment of intratumorally administered AuPC cluster and saline. The cryosections of tumor tissues (~30-µm thickness) were stained with TUNEL assay kit and DAPI and imaged in DAPI (ex: 405 nm, em: 430 to 470 nm), FITC (TUNEL, ex: 488 nm, em: 500 to 540 nm), and Cy5 (AuPC cluster, ex: 640 nm, em: 650 to 750 nm) channels. 20× objective, scale bar is 50 μm.