【ACS Nano】山西大学阴彩霞团队|相较传统方法提前42小时预警，基于口服纳米聚集体探针的急性肾损伤超早期无创尿检新策略#

Scheme 1. (a) Design of the Oral Nanoaggregate Probe MB-ES; (b) Schematic Diagram of the Oral Metabolic Pathway of MB-ES; and (c) Application of the MB-ES-Based Oral-to-Urinalysis Analysis Strategy in the Early Noninvasive Diagnosis of AKI#

Figure 1. Design and characterization of MB-ES. (a) Schematic diagram of the in vivo metabolism of orally administered MB. (b) Fluorescence intensity of MB (10 μM) at 696 nm in buffers of different pH. λex: 620 nm. (c) Schematic diagram of the protonation process of MB at pH 1. (d) Chemical structure of MB-ES. (e) Fluorescence intensity of MB-ES (10 μM) at 696 nm in buffers of different pH. λex: 620 nm. (f) Hydrodynamic size distribution of MB-ES. (g) Representative SEM images of MB-ES. (h) Hydrodynamic size distribution of MB-ES under different pH conditions. (i) Schematic diagram of the dispersion state of MB-ES in PBS. (j) Confocal microscopy images of NCM-460 and HCT-116 cells after incubation with MB-ES (10 μM) or HC-Cl-BC (10 μM) for 30 min or 2 h. Error bars represent standard deviation (SD) (n = 3).#

这一酯酶特异性激活的响应机制在体外实验中得到了完美证实。如图2所示，在加入酯酶后，探针在665纳米处的吸收峰明显增强，位于696纳米处的近红外荧光强度大幅跃升达137倍。通过高效液相色谱与质谱联用分析，研究人员清晰地观察到了前体探针的消耗和游离亚甲基蓝的生成。在动力学和滴定实验中，荧光强度随着酯酶浓度的增加呈极佳的线性关系，且该水解反应能被特异性酯酶抑制剂显著阻断。这表明探针具有极高的化学稳定性和优异的酶催化水解特异性。

Figure 2. Responsiveness of MB-ES to esterase. (a) Schematic diagram of MB-ES responding to esterase and releasing MB. (b) UV–vis absorption spectra of MB-ES (10 μM) upon reaction with esterase in PBS buffer (10 mM, pH 7.4) at 37 °C. Insets: Color change of MB-ES after reaction with esterase. (c) Fluorescence emission spectra of MB-ES (10 μM) upon reaction with esterase in PBS buffer (10 mM, pH 7.4) at 37 °C. Insets: NIR fluorescence images of MB-ES before and after reaction with esterase. (d) HPLC spectra of MB-ES incubated with esterase in PBS (10 mM, pH 7.4) at 37 °C. HPLC traces of the pure compounds (MB-ES and MB) are also shown for comparison. (e) MS analysis (m/z: 270–300) of the products from the reaction of MB-ES with esterase. (f) Time-dependent fluorescence changes of MB-ES (10 μM) at 696 nm after the addition of esterase (5 U/mL). (g) Plot of the fluorescence intensity of MB-ES (10 μM) at 696 nm versus varying concentrations of esterase (0.5–5 U/mL). (h) Fluorescence responses of MB-ES (10 μM) at 696 nm to different analytes. (i) Fluorescence intensity changes of MB-ES (10 μM) at 696 nm after the addition of a mixture of 5 U/mL esterase and AEBSF (2 and 4 mM) for 30 min at 37 °C. (j) Fluorescence stability of MB-ES (10 μM) at 696 nm in PBS buffer at 37 °C. λex: 620 nm. Error bars represent standard deviation (SD) (n = 3).#

在确保探针具备良好体内生物安全性的基础上，研究团队进一步阐明了其在小鼠体内的动态代谢机制。如图3所示，健康小鼠在口服灌胃探针后，小肠区域迅速出现明亮的近红外荧光，并随着时间的推移向后段移动。在给药90分钟后，小鼠膀胱区域的荧光信号逐渐增强。尿液质谱和荧光分析确认，尿液中排出的物质正是探针在肠道被特异性激活后释放、并经由血液循环最终通过肾小球滤过排出的亚甲基蓝。这套“肠道激活-肾脏排泄”的独特代谢路径，为肾功能检测奠定了扎实的生理学基础。

Figure 3. Biocompatibility and in vivo metabolic process of MB-ES. The levels of (a) ALT, (b) AST, (c) BUN, and (d) sCr in the serum of mice from the healthy control group and the oral MB-ES group. (e) Body weight changes in mice from the healthy control and oral MB-ES groups. (f) Representative H&E-stained images of major organs (heart, liver, spleen, lung, kidney, and colon) of mice from the oral MB-ES group. (g) In vivo fluorescence images of healthy mice were acquired at 0, 20, 40, 60, 90, 120, 150, and 180 min following oral gavage of MB-ES (200 μM, 300 μL). (h) In vivo fluorescence images of healthy mice were acquired at 0, 20, 40, 60, 90, 120, 150, and 180 min following oral gavage of PBS (10 mM, pH 7.4, 300 μL). (i) Mean fluorescence intensity in the bladder region corresponding to panels (g) and (h). (j) MS analysis (m/z: 250–300) of gastric contents from mice after oral gavage of MB-ES. (k) Urine collection diagram and urine fluorescence images. (l) MS analysis (m/z: 270–300) of urine collected from healthy mice after oral gavage of MB-ES. (m) Schematic diagram of the in vivo metabolism of orally administered MB-ES. Error bars represent standard deviation (SD) (n = 3). Statistical analyses were performed with Student’s t test. n.s.: nonsignificant.#

基于该代谢通路，研究团队在顺铂诱导的肾小管损伤小鼠模型上开展了诊断效能评估。如图4所示，在顺铂注射后的不同时间点，对小鼠口服探针并进行活体成像。实验发现，早在急性肾损伤诱导后6小时，小鼠膀胱区域的荧光信号即出现显著降低。随着病程进展，膀胱内甚至完全无法检测到荧光信号，而肾脏离体成像则显示出由于滤过受阻导致的荧光物质微量滞留。与之形成鲜明对比的是，传统的血清肌酐和尿素氮指标以及肾脏组织病理切片在损伤发生48小时内均未表现出明显的异常，这表明该“口服-尿检”方法成功将诊断窗口期提前了42小时。接受者操作特征曲线（ROC）分析显示，该无创荧光尿检方法的曲线下面积高达0.99，远优于传统血液生化指标。

Figure 4. Oral-to-urinalysis diagnosis of cisplatin-induced AKI. (a) Schematic diagram of cisplatin-induced AKI mouse model. (b) Timeline of cisplatin-induced AKI diagnosis using an oral-to-urinalysis approach. (c) Representative ventral and (d) dorsal fluorescence images of living mice at 6, 12, 24, 48, and 72 h after cisplatin treatment, following oral gavage of MB-ES (200 μM, 300 μL). (e) Mean fluorescence intensity in the bladder region corresponding to panel (c). (f) Comparison of the mean fluorescence intensity in the bladder at 120 min post administration. (g) Schematic diagram of urine collection and urine fluorescence images from different groups of mice 2 h after oral gavage of MB-ES. (h) Mean fluorescence intensity of urine samples from different groups of mice. (i) Body weight changes of mice in the control group and cisplatin treatment group. (j) The concentration of MB in the plasma of different groups of mice 1 h after oral gavage of MB-ES (100 mg/kg). (k) Ex vivo fluorescence images of kidneys from different groups of mice 2 h after oral gavage of MB-ES. Blood urea nitrogen (BUN) (I) and serum creatinine (sCr) (m) levels of mice at different time points post cisplatin treatment. (n) Receiver operating characteristic (ROC) analysis for the diagnostic specificity and sensitivity of BUN, sCr, fluorescence imaging, and urinalysis in discriminating between healthy mice (n = 6) and cisplatin-treated mice (n = 15). (o) Representative photomicrographs of H&E staining of kidney sections from mice in different groups. Error bars represent standard deviation (SD) (n = 3). Statistical analyses were performed with Student’s t test. n.s.: nonsignificant, *P < 0.05, **P < 0.01, ***P < 0.001.#

为了验证该方法的普适性，研究团队引入了更为复杂的单侧输尿管梗阻机械性损伤模型。如图5所示，梗阻小鼠在手术后6小时便表现出膀胱荧光强度的急剧减弱，而此时小鼠的血清肌酐依旧保持在完全正常的生理范围内，这进一步证明了该方法对不同病因引起的急性肾脏滤过功能受损均具有极高的超早期诊断敏感性。

Figure 5. Oral-to-urinalysis diagnosis of UUO-induced AKI. (a) Schematic diagram of UUO-induced AKI mouse model. (b) Timeline of UUO-induced AKI diagnosis using an oral-to-urinalysis approach. (c) Representative ventral and (d) dorsal fluorescence images of living mice at 6 h, 2 days, and 5 days after surgery, following oral gavage of MB-ES (200 μM, 300 μL). (e) Mean fluorescence intensity in the bladder region corresponding to panel (c). (f) Comparison of the mean fluorescence intensity in the bladder at 120 min post administration. (g) Schematic diagram of urine collection and urine fluorescence images from different groups of mice 2 h after oral gavage of MB-ES. (h) Mean fluorescence intensity of urine samples from different groups of mice. (i) Ex vivo images of kidneys from different groups of mice 2 h after oral gavage of MB-ES, along with their corresponding fluorescence images. Blood urea nitrogen (BUN) (j) and serum creatinine (sCr) (k) levels of mice at different time points post-surgery. (l) Representative photomicrographs of H&E staining of left kidney sections from mice in different groups. Error bars represent standard deviation (SD) (n = 3). Statistical analyses were performed with Student’s t test. n.s.: nonsignificant, *P < 0.05, **P < 0.01, ***P < 0.001.#

为了让这一科学发现能够造福普通公众，研究团队开发了便携式肾功能评估平台。如图6所示，研究人员引入了亲水性的两亲性共聚物Pluronic F127来提高探针的溶解度，并设计了专用的尿液比色试纸条。健康小鼠在口服共溶制剂后，由于亚甲基蓝的顺利排出，尿液试纸条呈现出明亮的深蓝色；而肾损伤小鼠的尿液试纸条颜色则显著变浅甚至不显色。该视觉比色方法实现了对肾小球滤过功能的快速直观评估。

Figure 6. Portable renal function assessment based on oral-to-urinalysis. (a) Schematic diagram of implementation steps for portable renal function assessment based on oral-to-urinalysis. (b) Schematic diagram of solubilization of MB-ES using amphiphilic polymer Pluronic F127. (c) Schematic diagram of test strip analysis for cisplatin-induced AKI and images of stained test strips. (d) Schematic diagram of test strip analysis for UUO-induced AKI and images of stained test strips.#