【Adv.Funct.Mater.】肿瘤信噪比显著提升至8倍，一种低蛋白结合、肾脏可清除的泛癌近红外探针#

Molecular shielding strategy to prepare low protein binding, highly soluble, tumor-targeting, and renal-clearable MS-ICGs. The schematic also shows the overall molecular design of MS-ICGs by presenting the molecular structure of 6D-ICG, with 6 negatively charged aspartic acid moieties, as an example in the middle panel. Created with Biorender.com and ChemDraw.#

Solubility and optical properties of MS-ICGs and ICG. (a) Photograph of ICG and MS-ICGs dissolved in a mixture of n-octanol and PBS after phase separation. (b) pH-dependent n-octanol/water partition coefficients (LogD) of ICG and MS-ICGs. (c) Absorbance and (d) fluorescence spectra of MS-ICGs and ICG at concentrations ranging from 1 to 50 µm. (e) Monomer peak absorbance (at 782 nm) of MS-ICGs and ICG. (f) Fluorescence intensity at 808 nm of MS-ICGs and ICG. Data are presented as mean ± standard error of the mean (SEM). Studies were run in triplicates. Statistical analyses in (e, f) were performed using one-way analysis of variance (ANOVA) with Tukey post hoc test. ****p < 0.0001.#

Cellular uptake mechanism of MS-ICGs and ICG. Confocal images (left) of 4T1 and A375 cells incubated with 10 µm of ICG or 6D-ICG for 2 h, with or without pretreatment with 250 µm OATP inhibitor BSP for 4 h. Bar graphs (right) are quantified cellular uptake of ICG or 6D-ICG at different conditions. Data are presented as mean ± standard error of the mean (SEM). For each condition in right panels, ∼50–100 cells were counted. Statistical analysis was performed using Student’s t-test. ****p < 0.0001.#

在理化性质表征中，研究人员首先通过正辛醇-缓冲液分配实验和光谱分析验证了屏蔽效应。如图2所示，未经修饰的游离ICG由于两亲性结构在水溶液中极易发生严重聚集，导致荧光发生猝灭，且其主要分布在有机相中。相比之下，经过亲水性氨基酸修饰后的衍生物呈现出极佳的水溶性，其吸收光谱展现出清晰的单体峰。随着修饰天冬氨酸数量的增加，分子的聚集程度显著降低，其中修饰了六个天冬氨酸的6D-ICG表现最为优异，在高达50微摩尔的浓度下也几乎不发生聚集，其荧光强度相比游离ICG呈现出数倍的爆发式提升。

Reduced protein binding, prolonged blood circulation, and renal clearance of MS-ICGs. (a) Absorbance and (b) Fluorescence maxima of ICG and MS-ICGs (25 µm) in 10% human plasma. (c) Native gel electrophoresis analysis of MS-ICGs or ICG (5 µm) after incubation in PBS with or without 10% human plasma for 1 h. Coomassie blue staining (left panel) and fluorescence of ICG (right panel) were used to detect proteins and MS-ICGs or free ICG, respectively. (d) Percent of unbound molecule in PBS or 10% human plasma. (e) Remaining injected dose of MS-ICGs or ICG in mouse blood at different time points post intravenous injection (50 nmol). ND is not detected. The box highlights an enlarged view of the data at 24 h. (f) Concentration of MS-ICGs or ICG in mouse urine samples collected at different time points after intravenous injection (50 nmol). Data are presented as mean ± standard error of the mean (SEM). Studies were run in triplicates in (a, b, d). Four mice for each group in blood circulation and urine clearance studies were used in (e, f). In urine clearance studies, enough urine samples could not be collected from all mice at all time points. Therefore, 6 h data for 6D-ICG in (f) contains 2 data points. All other conditions in (f) have at least 3 data points. Statistical analysis was performed using one-way ANOVA in (d, e, f) with Tukey post hoc test. In (e, f) Student’s t-test was applied for the time points with 2 detectable conditions (2, 6, and 24 h in (e), and 24 h in (f)). n.s., not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.#

在细胞层面，研究团队深入探讨了这些新型分子的细胞摄取机制。如图3所示，在小鼠乳腺癌和人黑色素瘤细胞中，天冬氨酸修饰的探针展现出了良好的细胞内化能力。当研究人员引入广谱OATP抑制剂溴磺酞进行前处理后，细胞对6D-ICG的摄取量发生了显著断崖式下跌。这一确凿的竞争性抑制结果有力地证明了，屏蔽修饰并未破坏ICG分子固有的OATP结合活性，新探针依然完美保留了通过OATP通道介导的肿瘤细胞特异性内化机制。

随后，体内药代动力学和生物分布实验进一步彰显了分子盾牌的威力。如图4所示，在10%的人类血浆环境中，游离ICG由于迅速与白蛋白结合而诱发了显著的光谱红移和荧光变化，而在原生凝胶电泳中，高亲水性的6D-ICG约有80%依然维持在完全游离、未结合蛋白的自由状态。这种不与血浆蛋白“粘连”的特性彻底重塑了其体内行为，游离ICG在小鼠静脉注射后5分钟内即被清出血液，而6D-ICG的血液循环半衰期大幅延长至约2小时。更关键的是，得益于极低的蛋白结合率，6D-ICG成功突破了肾小球的尺寸滤过屏障，在小鼠尿液中检测到了极高的荧光信号，实现了高效的肾脏快速清扫，从而极大地缓解了传统染料在肝、脾等单核巨噬细胞系统的非特异性淤积。

(a) Representative IVIS images of mice at different time points post injection of MS-ICGs or ICG (50 nmol). Blue circles indicate the location of implanted 4T1 tumors. (b) Quantified mean fluorescence intensities of ICG signals at tumor sites at different time points, calculated using LivingImage software. (c) Tumor-to-background ratios at different time points. (d) Ex vivo IVIS images of excised tumors and major organs 2 days after injection of MS-ICGs or ICG. (e) Mean fluorescence intensity of ICG in various tissues collected from mice injected with MS-ICGs or ICG. (f) Confocal microscope images of 4T1 tumor sections from mice injected with 6D-ICG. Data are presented as mean ± standard error of the mean (SEM). In (a–c) 5 mice per group were used for 4D-ICG and 6D-ICG, and 3 mice per group were used for other groups. In (e) 8 mice were used for 6D-ICG, 2 mice were used for free ICG, and 3 mice per group were used for other groups. Statistical analysis was performed using one-way ANOVA in (e) with Tukey post hoc test. n.s., not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.#

这种药代动力学性质的根本性改善，最终转化为极其震撼的体内肿瘤成像效果。如图5所示，研究人员将探针静脉注射到接种了乳腺癌肿瘤的小鼠体内，并在长达2天的时间内进行连续荧光追踪。注射了游离ICG的小鼠在任何时间段都未能显现出清晰的肿瘤轮廓。与之形成鲜明对比的是，6D-ICG在注射后4小时便开始在肿瘤部位高效富集，随着血液及健康组织中的探针通过肾脏不断清扫，背景噪声迅速消退。在注射后24至48小时，肿瘤部位的荧光信号达到了巅峰，肿瘤与背景信号比（TBR）惊人地达到了8。离体器官组织切片的高分辨荧光共聚焦成像进一步证实，6D-ICG不仅在肿瘤微环境中实现了极其均匀的穿透分布，而且被特异性地内化到了表达红色荧光蛋白的癌细胞内部。

Proposed tumor accumulation mechanism of molecularly shielded 6D-ICG. Upon systemic administration, free ICG quickly binds to plasma proteins or forms aggregates, whereas 6D-ICG, remains in unbound state, improving its blood circulation and enabling renal clearance. Prolonged blood circulation of 6D-ICG compared to free ICG enables its strong accumulation in solid tumors through OATPs-mediated uptake by cancer cells. Created with Biorender.com.#

为了验证该探针是否具有普遍的临床实用价值，研究团队将其拓展到了包含人类结直肠癌、子宫颈癌、黑色素瘤以及小鼠肺癌、结肠癌和前列腺癌在内的六种截然不同的实体瘤模型中。如图7所示，在历经2天的体内循环与清扫后，6D-ICG无一例外地在所有测试的恶性肿瘤区域实现了高度特异性的富集，离体荧光强度显著超越了周围的健康组织，甚至大大高于传统的肝脏代谢背景，雄辩地证明了其作为一种泛癌种近红外手术影像引导探针的广谱适用性。

6D-ICG specifically accumulates in a broad range of tumor models. (a) Representative IVIS images of mice bearing HCT-116, HeLa, A375, LLC1, MC38, and TRAMP-C2 tumors 2 days after injection of 6D-ICG (50 nmol). Blue circles indicate tumor locations. (b) Ex vivo IVIS images of tumors and major organs collected 2 days after injection of 6D-ICG. (c) Mean fluorescence intensity of ICG in different tissues. Data are presented as mean ± standard error of the mean (SEM). In (c) 3 mice per group were used for each tumor type. Statistical analysis was performed using one-way ANOVA with Tukey post hoc test. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.#

最后，研究团队将最具挑战性的大鼠颅内原位胶质瘤模型引入了验证阶段。如图8所示，通过对比增强核磁共振成像确诊成瘤后，静脉注射6D-ICG成功实现了对颅内脑肿瘤的精准高对比度显像，其荧光信号与肿瘤细胞自带的红色荧光实现了完美的空间重叠，肿瘤与健康脑组织的信号比达到了7.9。大面积全脑切片扫描结果表明，在健康且血脑屏障完好的脑组织中完全没有该探针的非特异性残留，而在血脑屏障遭到破坏的胶质瘤核心及边界区域，荧光边界极为陡峭和清晰，成功为手术切除标定了极其精准的“绿色盲区”边缘。

6D-ICG specifically accumulates in an intracranial glioblastoma model. (a) Contrast enhanced T1-MRI image of a rat brain and the corresponding IVIS images showing mCherry and ICG signals within the tumor. (b) Mean fluorescence intensity of 6D-ICG in the tumor, healthy brain and other tissues. (c) Representative microscopic images of whole brain sections with dotted lines outlining the brain border and tumor margin, along with enlarged views of tumor tissue (site 1), tumor border (site 2), and normal brain tissue (site 3). Data are presented as mean ± standard error of the mean (SEM). In (b) three rats were used. Statistical analysis for (b) was performed using one-way ANOVA with Tukey post hoc test. *p < 0.05; **p < 0.01.#