【JACS】南京大学叶德举、北海道大学Takanori Suzuki等|100倍荧光增强！双锁定制纳米探针助力结直肠癌术中精准切除

文章标题： A Cascade-Responsive AND-Logic-Activatable Nanoprobe for Intraoperative Fluorescence Imaging of Colorectal Cancer
通讯作者： Luyan Wu, Min Feng, Takanori Suzuki, Deju Ye
文章链接： https://doi.org/10.1021/jacs.6c04349

文章概要

引言

手术彻底切除肿瘤病灶是提高癌症患者生存率的关键，但传统的白光视觉检查难以识别微小肿瘤边缘或隐匿性病灶。虽然荧光成像技术为外科医生提供了“导航”辅助，但目前临床常用的荧光探针如吲哚菁绿（ICG）往往处于 “常开”状态且缺乏特异性，导致肿瘤与背景比（TBR）较低。为了解决这一痛点，南京大学叶德举教授团队联手合作者开发了一种名为duNP-DA的级联响应“与门”逻辑激活近红外荧光纳米探针。该探针能够针对结直肠癌微环境中的酸性pH和高水平硫化氢（H2S） 进行特异性激活，从而实现肿瘤病灶的精准边界划定。

Figure 1. Schematic overview of the cascade-responsive, AND-logic-activatable NIR FL nanoprobe duNP-DA for enhanced intraoperative cancer imaging. (a) Conventional dual-locked probes that require two biomarkers to activate fluorescence yet suffer from slow reaction kinetics and nonspecific uptake. (b) Our proposed cascade-responsive AND-logic-activatable nanoprobe, which requires cascade extracellular (TME pH ∼6.5) and intracellular dual triggers (lysosomal pH ∼5.0 + H2S) for activation, ensuring minimal background and high specificity. (c) Chemical structure of the dication EM 12+ (λabs = 500–1000 nm, a broad quencher) and its proposed rapid two-electron reduction into diene EM 1 (λabs < 500 nm, nonquencher for NIR fluorophores) mediated by H2S, featuring secondary reaction kinetics of 9780 ± 140 M–1 s–1 under physiological conditions. (d) Chemical structure of the pH-activatable NIR fluorophore IR-N, illustrating its activation to emit FL at 800 nm upon protonation. (e) Overview of duNP-DA preparation and the resultant cascaded response mechanism to the acidic extracellular TME (pH 6.5) and intracellular conditions of H2S and lysosomal pH (5.0). The surface decoration of duNP-DA with negative DA groups creates an electrostatic repulsion toward [HS–] anions. Under acidic TME conditions, these DA groups are cleaved, yielding duNP-NH2 with positively charged –NH3+ groups, which enhances the electrostatic attraction toward [HS–]. This interaction increases the local concentration of [HS–] and accelerates the reactivity. duNP-NH2 remains FL800 off when interacting with either H2S or lysosomal pH (5.0) individually but emits strong FL800 emission only when both conditions are met simultaneously. (f) Illustration of the application of duNP-DA for enhanced intraoperative imaging of CRC. Following intravenous injection, negatively charged duNP-DA migrates to tumor tissues via the enhanced permeability and retention (EPR) effect. Here, acidic TME conditions prompt DA hydrolysis, converting it to positively charged duNP-NH2, which facilitates tumor cell endocytosis and lysosomal accumulation. Inside tumor cells, the simultaneous presence of H2S and acidic lysosomal environment enable rapid activation of the probe, resulting in a strong FL800 signal. In contrast, duNP-DA remains quenched and shows minimum uptake in normal tissue cells due to neutral pH and low H2S levels, thus ensuring a high TBR for accurate intraoperative FL imaging.

主要实验及结论

研究人员首先巧妙设计了探针的化学结构，将其封装在具有负电荷表面修饰的纳米颗粒中。这种设计让探针在血液循环中保持稳定且不发光，有效地降低了全身性的噪声干扰。当探针通过血液循环到达结直肠癌区域时，肿瘤胞外微环境的酸性首先诱发探针表面的电荷翻转，使其从负电荷变为正电荷，这一过程极大地增强了肿瘤细胞对探针的摄取效率。

Figure 2. Characterization of duNP-NH2. (a) Schematic diagram of the conversion of EM 12+ to EM 1 after reduction by H2S. (b) HPLC traces of EM 1, EM 12+, and EM 12+ (9 μg/mL) after incubation with NaHS (100 μM) for 30 s. (c) UV/vis-NIR absorption spectra of EM 1, 12+, and 12+ (9 μg/mL) after incubation with NaHS (100 μM) in H2O for 30 s. (d) Normalized time-dependent decline of UV/vis-NIR absorption (584 nm) of EM 12+ (4 μg/mL) following incubation with varying concentrations of NaHS in PBS (pH 7.4). (e) Schematic diagram of the FL activation of IR-N by protonation. (f) FL spectra of pH-moNP-NH2 (3.2 μg/mL) with different pH. (g) Measurement of the p_K_a values of pH-moNP-NH2 (p_K_a = 5.5). (h) General procedure for the preparation of duNP-NH2. (i,j) DLS analysis (i) and TEM image (j) of duNP-NH2. Scale bar: 200 nm. (k,l) UV/vis-NIR absorption spectra, photographs (inset) (k) and FL spectra (l) of duNP-NH2 (54/3.2 μg/mL 12+/IR-N) before (−) and after (+) reaction with NaHS (100 μM) at different pH (I: −NaHS, pH 7.4; II: −NaHS, pH 5.0; III: +NaHS, pH 7.4; IV: +NaHS, pH 5.0). (m) Time-dependent changes of the UV–vis absorption (584 nm) of duNP-NH2 (4/0.22 μg/mL 12+/IR-N) upon incubation with different concentrations of NaHS (0, 30, 40, 50, and 60 μM). (n) Normalized FL intensities of duNP-NH2 (I) and H2S-moNP-NH2 (II) at PBS or mouse serum. (o) FL imaging of duNP-NH2 (red) and lysosome (green) in HCT116 cells. Scale bar: 10 μm. (p,q) FL imaging (p) and average FL intensity (q) of HCT116 cells incubated with duNP-NH2 together with PBS (I), 300 μM ZnCl2 (II), 1 mM NaHS (III), 200 μM L-Cys (IV), or 20 mM NH4Cl (V). Scale bar: 20 μm. (r,s) No-washing FL imaging (r) and normalized FL intensity (s) of HCT116 cells incubated with H2S-moNP-NH2 (I) and duNP-NH2 (II) for 3 h. Scale bar: 20 μm. The FL regions inside and outside the cell were chosen for the region-of-interest (ROI) measurement to quantify intracellular and extracellular FL. (t) Differentiation of H2S-rich tumor cells (HCT116 cells) and H2S-deficient normal cells (HUVEC cells) using duNP-NH2 or pH-moNP-NH2. Scale bar: 20 μm. Data are mean ± standard deviation (n = 3). Statistical differences in (m,p,r) were analyzed by a Student’s two-sided t-test.

Figure 3. Characterization of duNP-DA. (a) Proposed mechanism of duNP-DA for response to an acidic TME inversing the surface charge. (b) Schematic illustration of the control probe duNP-SA. (c,d) DLS analysis (c) and TEM image (d) of duNP-DA. (e,f) UV/vis-NIR absorption spectra (e) and zeta potentials (f) of duNP-NH2 and duNP-DA. (g) Changes of zeta potential of duNP-SA (I) and duNP-DA (II) after incubation at pH 7.4, 6.5, and 5.5 for 0–180 min. (h) Comparison of the of duNP-SA (I) and duNP-DA (II) toward H2S after preincubation of each nanoprobe at indicated pH for 30 min. duNP-SA and duNP-DA were first incubated at pH 7.4, 6.5, or 5.5 for 30 min, then washed via ultrafiltration and resuspended in PBS buffer (pH 7.4) prior to the addition of NaHS to measure k__2. (i–k) FL images (i), flow cytometry analysis (j), and corresponding mean fluorescence intensity (MFI) (k) of HCT116 cancer cells after incubation with duNP-DA or duNP-SA at different conditions for 3 h. I: duNP-SA, pH 7.4; II, duNP-SA, pH 6.5; III, duNP-DA, pH 7.4; IV, duNP-DA, pH 6.5. (l) FL imaging of HUVEC cells untreated or pretreated with NaHS (1 mM, 1 h), followed by incubation with duNP-NH2 or duNP-DA (54/3.2 μg/mL 12+/IR-N) for 3 h. Scale bar: 20 μm. Data are mean ± s.d. (n = 3). Statistical differences in (f), (h), and (k) were analyzed by a Student’s two-sided t-test.

在细胞实验中，探针展现出了惊人的激活能力。只有当溶酶体酸性pH和内源性H2S同时存在时，探针内部的荧光淬灭机制才会被彻底打破，产生超过100倍的荧光增强。在小鼠模型中，duNP-DA不仅成功描绘了直径小至2毫米的微小肿瘤，更在手术导航中立下奇功：它成功引导切除了肉眼完全无法发现的深层腹膜转移结节，术后病理分析也证实了切除的精准度。

Figure 4. FL imaging and FL-guided tumor resection of duNP-DA for s.c. HCT116 colon tumor. (a) FL images of s.c. HCT116 colon tumors in vivo following administration (i.v.) of pH-moNP-DA (I), H2S-moNP-DA (II), duNP-SA (III), duNP-DA (IV), and duNP-DA + L-Cys (V). The FL images were acquired with λex/λem = 780/845 nm. (b,c) Average FL intensity of tumors (b) and tumor-to-background ratio (TBR) (c) in living mice at 0, 4, 8, 12, 24, 48, and 96 h after different treatments. Red arrows identify the tumor locations, and black circles on the leg muscles indicate the background locations. (d) FL imaging of HCT116 tumor tissue slices resected from mice 48 h postinjection (iv) of different NPs. Scale bar: 200 μm. (e–g) Representative FL images (e), average FL intensity of tumors (f), and TBR (g) of HCT116 tumors at a mean size of 5.4 ± 1.1, 14.4 ± 1.7, 30.1 ± 3.7, and 54.9 ± 2.8 mm3 24 h postinjection (i.v.) of duNP-DA. Three mice were imaged for each group. Red arrows identify the tumor locations, and black circles on the back muscles indicate the background locations. (h) Schematic illustration of FL-guided tiny HCT116 colon tumor resection. (i) Representative photograph (BF) and FL images of the tumor and surrounding tissues 24 h postinjection (i.v.) of duNP-DA. Screen-capture images of the intraoperative mouse at indicated stage (After anesthesia, Expose tumor, Excise tumor, and After resection, respectively). (j) Representative photograph (bright field) and H&E staining of tiny tumor resected by FL-guided surgery. The red dashed lines indicate normal tissue at the margins. Scale bar: 50 μm. Data are mean ± s.d. (n = 3). Statistical differences in (c) were analyzed by a Student’s two-sided t-test.

Figure 5. FL imaging of orthotopic and peritoneal metastasis colon tumors using duNP-DA in mice. (a) Schematic illustration of the duNP-DA for FL imaging of orthotopic CT-26-Luc tumor-bearing mice. (b,c) Representative FL images (b) and FL intensity (c) of orthotopic colon tumors 24 h postinjection (i.v.) of duNP-DA. The yellow arrow indicates the tumor locations, and the blue dash box on the adjacent normal tissue indicates the background locations. (d) Average FL intensity of main organs and tumors (T: tumor, He: heart, Li: liver, Sp: spleen, Lu: lung, Ki: kidneys, St: stomach, S. I.: small intestine, Col: colon) resected from mice 24 h postinjection (i.v.) of duNP-DA. (e) Representative FL image and H&E staining of the orthotopic colon tumor. Scale bar: 200 μm. (f) Schematic illustration of the duNP-DA for FL imaging of peritoneal metastasis CT-26-Luc tumor-bearing mice. (g,h) Representative FL images (g) and FL intensity (h) of peritoneal metastasis CT-26 tumors 24 h postinjection (i.v.) of duNP-DA. Yellow arrows indicate the tumor locations, and the blue dash box on the adjacent normal tissue indicates the background locations. (i–k) Representative FL images (i), FL intensity (j), photograph (bright field), and H&E staining (k) of tiny peritoneal metastasis CT-26 tumors 24 h postinjection (i.v.) of duNP-DA. The yellow arrow indicates the tumor locations, and the blue dash box on the adjacent normal tissue indicates the background locations. (l) Schematic illustration of the duNP-DA for FL-guided resection of peritoneal metastatic colon tumors in mice. (m) Representative photograph (BF) and FL images of the tumor and surrounding tissues 24 h postinjection (i.v.) of duNP-DA. Screen-capture images of an intraoperative mouse at indicated stage: after anesthesia (I), exposed abdominal cavity (II), excised tumor 1 (III), excised tumor 2 (IV), and after resection of tumors (V). (n) H&E staining of peritoneal metastatic tumor 1 and tumor 2 resected by fluorescence-guided surgery. Data are mean ± s.d. (n = 3). Statistical differences in (c,d) were analyzed by a Student’s two-sided t-test.

为了验证临床转化的潜力，研究团队对来自临床患者的22份结直肠癌标本进行了盲法测试。实验结果令人振奋，该探针在区分肿瘤与正常组织方面表现出了100%的诊断灵敏度和特异性。更重要的是，探针的荧光强度与人工智能（AI）量化的肿瘤细胞密度呈现出极强的相关性（Pearson’s r = 0.9103），这意味着探针不仅能告诉医生哪里是癌，还能反映出癌症的严重程度。

Figure 6. FL imaging of clinical colon cancer tissues in CRC specimens. (a) Schematic diagram showing the detection of colon cancer tissues in human specimens using duNP-DA. (b,c) FL images (b) and FL intensity (c) of colon tumor tissues and adjacent normal tissues specimen incubated with duNP-DA (54/3.2 μg/mL 12+/IR-N) for different times. (d–f) Representative FL images (d), normalized FL intensity (e), and TBR (f) of colon tumor center and adjacent tissues specimen incubated with following NPs of duNP-DA (I) and H2S-moNP-DA (II) for 3 h and then rinsed with PBS buffer three times. The FL image was collected with λex/em = 780/845 nm. Scale bar: 4 mm. Normal tissue is selected as the background. (g) Representative photograph (BF) and FL images of colon tissue specimens resected from a CRC patient. Scale bar: 2 mm. The red dash box indicates the tumor locations, and the blue dash box on the adjacent normal tissue indicates the background locations. (h) FL images of colon cancer tissue slices dissected from the CRC specimen after incubation with duNP-DA (red) for 3 h and stained with DAPI (blue). The yellow dotted line indicates the junction of tumor cells and normal cells. Scale bar: 50 μm. (i) H&E staining of the colon tissue slice dissected from the CRC specimen. The dash box indicates the enlarged areas, in which the red box labeled ROI 1 shows the tumor tissue and the blue box labeled ROI 2 indicates the normal colon tissue in the resected colon tissue slice, respectively. Scale bar: 100 μm. (j) Normalized FL intensity and TBR of tumor and normal colon tissue specimens resected from CRC patients. The red dash box in (g) indicates the tumor location, and the blue dash box indicates the location of the normal colon tissues. (k,l) Representative photograph (BF), FL images, and H&E staining (k) and FL intensity and TBR (l) of tumor-invasive and normal lymph nodes specimen resected from a CRC patient. The red dash box in (k) indicates the location of tumor, and the blue dash box indicates the location of normal tissue selected as the background. Scale bar: 1 mm. (m,n) Representative photograph (BF), FL images, and H&E staining (m) and FL intensity and TBR (n) of tumor-invasive peritoneum specimen resected from a CRC patient. The red arrows indicate the tumor site. The red dash box in (m) indicates the location of tumor, and the blue dash box indicates the location of normal tissue selected as the background. Scale bar: 2 mm. Data are mean ± s.d. (n = 3). Statistical differences in (e,i) were analyzed by a Student’s two-sided t-test.

总结及展望

该研究成功构建了一种级联响应的纳米探针平台，为结直肠癌的术中精准导航提供了新的利器。duNP-DA探针通过胞外电荷翻转与胞内双靶标逻辑激活的有机结合，克服了传统探针假阳性高、背景干扰强的局限。尽管目前该探针在给药时间窗口和临床实时性上仍有优化空间，但其展示出的卓越肿瘤识别能力和AI定量潜力，预示着它在未来个性化精准外科手术中具有广阔的应用前景，有望显著降低癌症复发风险并改善患者预后。

【JACS】南京大学叶德举、北海道大学Takanori Suzuki等|100倍荧光增强！双锁定制纳米探针助力结直肠癌术中精准切除 ​

文章概要 ​

引言 ​

主要实验及结论 ​

总结及展望 ​

【JACS】南京大学叶德举、北海道大学Takanori Suzuki等|100倍荧光增强！双锁定制纳米探针助力结直肠癌术中精准切除

文章概要

引言

主要实验及结论

总结及展望