【JACS】湖南大学张晓兵团队|15分钟精准锁定光疗后中性粒细胞#

(a) Multisignal imaging methods in previous work. I: Single-signal probe. II: Dual-channel probes. III: Reported multimodal imaging probes. (b) Schematic diagram of the “spatiotemporal asynchronous” strategy in this study. The fluorescence is restored by the first response. The fluorescence is quenched and the photoacoustic signal is enhanced in response to the second responder. The fluorescence and photoacoustic are turned on sequentially. (c) Chemical structure of HD-LN. (d) The correlation between tumor prognostic metastasis and neutrophil elastase release during phototherapy is monitored in real time by HD-LN.#

Figure 1. (a) Basic optical properties of HD-HD5. Φs is the relative fluorescence quantum yield estimated by using indocyanine green (ICG, Φs = 0.13 in DMSO), λem = 680 nm, Slit width: _d_ex = _d_em = 5 nm. (b) Normalized absorption, (c) emission, and (d) photoacoustic spectra of different compounds in buffer solution (5 μM, PBS/EtOH = 1/1, v/v, pH = 9.0). (e) Fluorescence and photoacoustic imaging of different compounds (5 μM). (f) Construction of gradient multimode signal dyes. Linear relationship between cooling time and negative natural logarithm of temperature drive for (g) HD-NE (20 μM), (h) HD-NH****2 (20 μM) in PBS buffer (pH = 7.4). (i) PA signals of HD-NE and HD-NH****2 at different wavelengths (10 μM, PBS/EtOH = 8/2, v/v). (j) PA intensities of HD-NE (10 μM) and HD-NH****2 (10 μM) at 720 nm. (k) Fluorescence intensity of HD-NE (10 μM) and HD-NH****2 (10 μM), λex = 640 nm. (l) Linearity of the PA signals of HD-NE and HD-NH****2 at 720 nm versus concentration. Error bars, ± SEM n = 3. ****p < 0.0001.#

为了验证该探针的级联响应可行性，研究人员合成了集成有肿瘤靶向链段及双特异性酶响应位点的智能探针HD-LN并进行了体外酶解实验。如图2所示，探针首先与肿瘤高表达的亮氨酸氨基肽酶（LAP）发生反应，切除其羟基保护基团，不仅使近红外荧光发射强度显著恢复，还同步激活了其产生单线态氧的光动力治疗活性。随后，在模拟中性粒细胞招募环境并加入中性粒细胞弹性蛋白酶（NE）后，探针的荧光迅速淬灭，而其在七百二十纳米处的光声信号则大幅度增强。高相色谱分析与特异性阻断实验进一步证实，这种独特的荧光淬灭伴随光声增强的现象完全源于两步特异性酶切反应，展现出极高的靶向选择性。

Figure 2. In vitro characterization of HD-LN. (a) Schematic illustration of the activation mechanism for the sequential activation probe HD-LN. (b) UV/vis absorption spectra, (c) fluorescence spectra, (d) Normalized PA intensities and (e) HPLC traces of HD-LN (10 μM) after incubation with/without LAP (200 U/L), NE (5 μg/mL), or LAP&NE at 37 °C in the PBS buffer (pH = 7.4). λex = 660 nm. (f) Fluorescence intensity (at 720 nm) of HD-LN (10 μM) and (g) PA intensity (at 720 nm) of HD-NE (10 μM) in the presence of various species: Cas3 (0.2 U/mL); Cat B (0.3 U/mL); Cat K (0.3 U/mL); Cat S (0.3 U/mL); DPP4 (0.5 μg/mL); DPP9 (0.5 μg/mL); FAP α (0.5 μg/mL); GGT (0.5 U/L); NE (10 μg/mL) and LAP (200 U/L). For HD-LN, the tested species were incubated with the probe at 37 °C for 1 h before measurement. For HD-NE, the tested species were incubated with the probe at 37 °C for 4 h before measurement. Slit width: _d_ex = _d_em = 10 nm. Error bars, ± SEM n = 3.#

随后，研究团队在细胞和活体层面对探针的监测能力进行了深度验证。在共培养细胞模型中，如图3所示，经历光疗应激的乳腺癌细胞能够显著诱导共培养的中性粒细胞过度表达中性粒细胞弹性蛋白酶并触发细胞外陷阱的释放，而利用HD-NE探针能够通过流式细胞术与多模态成像清晰地定量表征这一活化过程。在荷瘤小鼠体内，静脉注射HD-LN三小时后，探针在肿瘤部位实现了高效富集并激发出强烈的荧光信号，如图4所示。此时施加光动力治疗，在光疗结束仅一小时内，便能观察到肿瘤区域荧光迅速衰减、而光声信号显著上升的异步转化现象。而在预先注射弹性蛋白酶抑制剂的对照组中，这种信号转换被完全阻断。这一关键在体实验直接证明，光动力治疗在极短时间内便会诱发肿瘤微环境内中性粒细胞的快速聚集与弹性蛋白酶的集中释放。

为了阐明中性粒细胞募集与光疗预后不良的因果关系，研究团队设计了多组联合治疗方案。如图5所示，单纯接受光动力治疗的小鼠，虽然原位肿瘤生长受到一定抑制，但其肺部和肝部均出现了大面积的远端转移灶，且肿瘤组织内杀伤性CD8阳性T细胞的浸润极低。免疫荧光染色表明，这与光疗激发的密集细胞外陷阱屏障密切相关。相反，当将光动力治疗与中性粒细胞弹性蛋白酶抑制剂联合使用时，不仅肿瘤生长受到了极其显著的协同抑制，原位及远端的肺、肝转移也彻底消失。这表明阻断弹性蛋白酶能够打破细胞外陷阱构筑的物理屏障，促使大量T细胞顺利浸润至肿瘤内部发挥免疫杀伤作用。

Figure 4. (a) Real-time fluorescence intensity in the tumor region of mice following intravenous injection of HD-LN (200 μM, 50 μL). (b) Fluorescence intensity of various organs in mice 3 h after intravenous injection of HD-LN (200 μM, 50 μL). (c) Schematic illustration of HD-LN application for in vivo imaging. (d) Real-time PA imaging (top) and fluorescence imaging (bottom) in tumor-bearing mice after HD-LN (200 μM, 50 μL) injection. (e) Real-time PA imaging (top) and fluorescence imaging (bottom) of tumor-bearing mice in which the tumor region was irradiated by a 660 nm laser (0.7 W/cm2) for 15 min at the third hour after HD-LN (200 μM, 50 μL) injection. (f) Normalized PA intensity at 720 nm and (g) normalized FL intensity in the tumor region of mice in d, e. (h) Mice were injected intratumorally with SV (6 μg) and 4 h later, HD-LN (200 μM, 50 μL) was injected into the tail vein. At 3 h after HD-LN injection, the tumor region of mice was irradiated using a 660 nm laser (0.7 W/cm2) for 15 min. Imaging of (i) PA and (j) FL signals in the tumor region of mice. The 3 h + L group was imaged 2 h after light irradiation. Error bars, ± SEM n = 3. ***p < 0.001, ****p < 0.0001.#

Figure 5. (a) Schematic illustration of HD-LN and SV treatment. (b) Immunofluorescence staining of MPO and Ly6G in tumors of 4T1 tumor-bearing mice from different treatment groups. Green, red and blue fluorescence indicate Ly6G, MPO and nucleus staining, respectively, scale bar = 200 μm. (c, d) Normalized fluorescence intensity in b. (e) The tumor volume within 15 days. (f) Tumor volume and (g) tumor weight of mice in each group on day 15. The CD206 immunohistochemistry staining in the (h) lungs and (j) liver of mice in each group on day 15, scale bar: 200 μm. (i, k) Quantification of the CD206-positive area in panels h and j. (l) Immunofluorescence staining detection of CD8+ T cells (red) infiltration in different groups of tumors, blue channel: DAPI, scale bar = 200 μm. Error bars, ± SEM n = 3. “ns” for not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with the HD-LN+SV.#

得益于HD-LN在体评估弹性蛋白酶活性的独特优势，该探针还被成功应用于抗炎及靶向药物的在体高效筛选。如图6所示，通过实时追踪不同药物处理后小鼠肿瘤部位的荧光与光声信号变化趋势，研究人员在活体水平上快速鉴定出胡椒碱和红花素等具有优异体内抗炎活性的药物能够显著抑制弹性蛋白酶的表达。这一发现不仅验证了探针作为在体药物筛选平台的实用价值，也为临床开发新型微环境调节剂提供了新方向。

Figure 6. (a) Molecular mechanisms of HD-LN for real-time in vivo drug screening of NE inhibitors. (b) Schematic timeline for real-time monitoring of drug therapy and phototherapy combination therapy. (c) After intratumoral treatment with carthamidin (10 mg/kg, 50 μL), piperine (10 mg/kg, 50 μL) and indomethacin (10 mg/kg, 50 μL) , HD-LN was i.v. injected. PA images (top) and fluorescence images (bottom) were obtained at 0 h, 3 and 5 h. At the third hour after HD-LN injection, the tumor area was irradiated for 15 min with a 660 nm laser (0.7 W/cm2). (d) Normalized PA intensities at 720 nm in c. (e) Normalized fluorescence intensities in c. (f) Western blotting analysis of NE in tumors after combined treatment with different drugs and phototherapy. (g) ImageJ normalized quantitative analysis of NE/GAPDH ratios in f. (h) Photograph of the tumor after treatment, scale bar = 10 mm. (i) Tumor volume of mice in each group at the end of the experiment. Error bars, ± SEM n = 3. **p < 0.01, ***p < 0.001, ****p < 0.0001.#