【Adv.Mater.】哈工大陈冠英|颠覆癌症代谢！光控纳米“排酸阀”：实现肿瘤体积暴跌6倍与实时Barometry Bar#

引言#

质子在调节癌细胞行为、代谢和信号传导通路中起着至关重要的作用，这使得调控细胞内质子浓度（pH值）成为一种极具前景的癌症治疗策略。然而，如何在体内实现空间和时间上的精准质子释放，并进行实时动态监测，一直是临床转化面临的巨大挑战。传统的化学调控手段往往依赖被动扩散，容易破坏正常组织稳态，且极易因长期给药引发肿瘤的适应性耐药。为了突破这一瓶颈，该研究创新性地开发了一种近红外光控的纳米质子输送系统（UFPL）。该系统巧妙地利用上转换纳米颗粒作为能量供体，通过光致互变异构反应在病灶处触发强烈的质子风暴。这种急性酸应激能瞬间斩断癌细胞高达50%的葡萄糖摄取，并通过抑制关键的mTOR信号通路激活过度自噬，进而引发线粒体功能障碍并导向细胞凋亡。研究人员将这一全新的细胞死亡通路定义为质子介导的自噬诱导凋亡（PAA）。更令人兴奋的是，该平台首次实现了治疗过程的实时可视化定量反馈，在小鼠实验中成功实现了肿瘤体积惊人的6倍缩减，为恶性肿瘤的精准诊疗开辟了全新的生化维度。

Synthesis and characterizations of the photoacid (PA) and UFPL nanoagent. (a) Schematic illustration of the synthetic route for PA (top) and the final UCNPs@Fc–PA–LF (UFPL) nanoagent (bottom). (b) Transmission electron microscopy (TEM) images of NaYbF4: Tm, NaYbF4: Tm@NaYF4 (UCNPs), UCNPs@Fc (UF), UCNPs@Fc-PA (UFP), and UCNPs@Fc@PA@LF (UFPL) nanoparticles (from left to right). (c), (d) Molecular dynamics (MD) simulations depicting the interfacial assembly of PA on the NaYF4 surface, revealing dynamic adsorption, migration, and equilibration. Time-resolved snapshots (0–40 ns) show PA molecules progressively adsorbing and organizing into a densely packed monolayer, reaching saturation at 40 ns. (e) Structural model illustrating PA molecules adsorbed onto the NaYF4 crystal surface. (f) Slab models of the (001) surface of hexagonal-phase NaYF4 showing the adsorption of PA onto NaF and YF surfaces, with corresponding surface binding energies calculated for PA adsorption. Yellow, cyan, and grey spheres represent Na, Y, and F atoms, respectively.#

随后，团队深入探究了近红外光驱动的质子释放动力学与伴随的发光动态恢复Barometry Bar机制。围绕上转换发光光谱与产酸剂吸收光谱的重叠积分分析，光谱数据证实两者存在极高的共振能量转移效率（450纳米处高达43.9%）。第一性原理时变密度泛函理论（TD-DFT）与自旋轨道耦合计算从理论上揭示了产酸剂在吸收上转换光子后，经历电子云重排与异构化，触发活性氢解离释放质子的热力学可行性。在溶液实验中，通过酸敏感探针监测发现，在980纳米近红外光照射下，溶液pH值在30分钟内从7.41骤降至5.12，质子释放速率达每分钟0.43微摩尔每升。与此同时，原本作为淬灭剂的二茂铁在局部高浓度质子的攻击下发生生物降解，使纳米颗粒在800纳米处的近红外发光强度显著飙升达8.3倍。发光强度的倒数与环境pH值展现出完美的线性反比关系，从而在国际上首次建立起基于发光恢复的体内原位pH光测条形码Barometry Bar框架。

Principles of proton production and in vitro acidification experiment of UFPL nanoagent. (a) UCNPs–PA Förster Resonance Energy Transfer Mechanism. (b) Absorption spectra of PA (blue), Fc (violet) and normalized emission of UCNPs (red), highlighting the overlap integral of 6.27 × 1013·nm4·M−1·cm−1, which indicates the potential for energy transfer between the two species. (c) Corresponding lifetime decays at 361, 450, and 475 nm, and FRET efficiency, respectively. (d) First-principle and time-dependent density functional theory (TD-DFT) calculated energy level diagram, spin-orbit matrix element (<_T_n|_H_SO|_S_1>), and the corresponding spin-orbital coupling (SOC) constants of PA molecules. (e) The energy levels of the highest occupied molecular orbitals (HOMOs)/lowest unoccupied molecular orbitals (LUMOs) of PA (red) and product (SP) (blue) (bottom). (f) Molecular electrostatic potential surface (MEPs) of PA and SP. (g) The change in reaction energy from PA to SP upon exposure to emissions from UCNPs under NIR irradiation.#

紧接着，实验深入解析了UPL纳米颗粒诱导的质子介导自噬诱导凋亡（PAA）分子生物学层级机制。在细胞层面，利用细胞内酸敏感荧光探针清晰观测到了近红外光照介导的细胞内强酸性红绿荧光转变。共聚焦显微镜图像显示，强酸环境促使纳米颗粒迅速逃逸内吞体，皮尔森相关系数显著降低。细胞内ATP检测显示能量工厂产出骤降40%，印证了代谢受到严重破坏。蛋白质印迹（Western Blot）分析从分子水平确证，强酸应激导致葡萄糖转运蛋白Glut1显著下调，细胞对葡萄糖的摄取呈现出显著的pH依赖性抑制。这种剧烈的“饥饿应激”迫使mTOR通路发生显著下调，上调了自噬关键蛋白ATG5并促进了LC3-I向LC3-II的强烈转化。Cyto-ID荧光染色与生物透射电镜图像互为印证，在光照治疗组中清晰观测到了大量成簇出现的自噬体与空泡化结构。通过引入自噬抑制剂3-MA的对照实验进一步证实，阻断自噬后细胞存活率大幅回升51%，自噬通路被阐明是导致线粒体膜电位崩溃并最终执行Caspase-3剪切凋亡的专一性绝对上游开关。

Proton-induced UFPL nanoagent degradation and UCL recovery. (a) Schematic diagram of laser path applied in real-time detection of H+ concentration using RB Base (RB) as an acid indicator. (b) pH variation and RB Base fluorescence peak area under NIR alone (left) and NIR + UFPL (2 mg/mL, right) during irradiation and subsequent dark incubation. (c) Schematic representation of upconversion luminescence (UCL) recovery in UFP due to Fc biodegradation induced by H+. (d) UCL at 800 nm for the UFP, UFP + H2O2, UF + H2O2 + NIR, UFP + H2O2 + NIR, and Fc + H2O2 + NIR groups. (e) Relative change in UCL intensity at 800 nm and pH change for the UFP nanoagent in simulated tumor microenvironment (0.05 mm H2O2) under NIR irradiation (0.5 W/cm2) within 14 min. (f) Linear correlation of 1/pH with the relative UCL intensity of the UFP nanoagent.#

为了确保机制分析的系统性与无遗漏性，研究人员利用转录组测序（RNA-Seq）深入挖掘了治疗后细胞内的生物信息学全局变化。测序数据经过差异表达基因分析揭示出多达1075个显著变化的基因，其中包含366个表达下调基因和627个表达上游基因，在火山图与热图上呈现出黑白分明的两极分化。随后的KEGG通路富集气泡图和GO功能圈图高度一致地指向了代谢通路、自噬通路与凋亡通路的三重富集。蛋白质-蛋白质相互作用网络进一步揪出了Cdkn1b等关键调节核心。最终，基因集富集分析（GSEA）完美印证了“糖解信号通路”的显著抑制和“自噬信号通路”的强烈激活，从高通量转录组维度再次严丝合缝地固化了质子流触发PAA通路的分子级证据链。

In vitro investigation of the therapeutic mechanism of UPL nanoagents. (a) A schematic illustrates the NIR-triggered proton-mediated autophagy-induced apoptosis (PAA) mechanism, wherein acute acidic stress suppresses glucose uptake and inhibits mTOR signaling, thereby triggering excessive autophagy characterized by autophagosome formation. This process functionally leads to mitochondrial dysfunction, ultimately activating the autophagy-induced apoptosis. (b) pH-responsive fluorescence of SNARF-4F, showing an emission shift from yellow (neutral pH) to green (acidic pH). (c) Confocal laser scanning microscopy (CLSM) images of GL261 cells incubated with UPL nanoagents following prolonged NIR irradiation (8 min). (d) CLSM images of GL261 cells co-stained with Lysotracker Red (lysosomes, red), FITC-labeled nanoagents (green), and DAPI (nuclei, blue), with corresponding Pearson correlation coefficient analysis. (e) Intracellular ATP levels and (f) Glut1 expression in GL261 cells treated with PBS, NIR, and UPL + NIR. (g) Schematic representation of colorimetric glucose quantification using a Glucose Assay Kit, where absorbance of the blue-green Schiff compound reflects glucose concentration, as shown in (h) for different treatment groups. (i) Quantitative analysis of glucose uptake in glucose-starved cells under varying pH conditions. (j) Western blot analysis of mTOR, ATG5, LC3-I/II, P62, and LAMP-1/LAMP-2 expression in PBS-, NIR-, and UPL + NIR-treated cells. (k) CLSM images showing CYTO-ID green fluorescence for autophagy detection in GL261 cells under PBS, NIR, and UFPL + NIR treatments, with the corresponding detection mechanism illustrated. (l) Bio-TEM images of GL261 cells after PBS, UFL, or UPL + NIR treatment, highlighting autophagosomes (yellow arrows) and vacuoles (orange arrows). (m) Flow cytometry analysis of Lysotracker fluorescence intensity across PBS, NIR, and UPL + NIR groups.#

RNA-seq analysis of UPL nanoagent. (a) Schematic representation of the RNA-seq analysis used to investigate cell bioinformatics following UFPL + NIR treatment. The analysis includes: (b) Heat map showing changes in mRNA levels of differentially expressed genes post-treatment; (c) Volcano plot visualizing gene expression differences; (d) Comparison of the number of upregulated and downregulated genes in the UFPL + NIR group vs. control groups; (e) KEGG bubble plot displaying the top 9 enriched pathways following UFPL + NIR treatment (blue: metabolic pathways; red: apoptotic pathways; black: pathways associated with both metabolism and apoptosis); (f) Protein-protein interaction network of genes related to autophagy, apoptosis, and glucose uptake; (g, h) GO circle diagrams illustrating the biological functions of differentially expressed genes after UFPL + NIR treatment. Gene Set Enrichment Analysis (GSEA) identified differentially expressed genes between the control and UFPL + NIR groups, with red and blue lines indicating upregulated and downregulated genes, respectively (i, j).#

在体内验证阶段，团队成功实施了肿瘤积聚的近红外上转换发光成像跟踪与光控产酸治疗评估。在活体动物层面，通过低功率近红外光扫描，上转换发光成像纵向追踪了纳米颗粒在荷瘤小鼠体内的时空分布，发现在静脉注射11小时后脑部胶质瘤部位的积聚量达到峰值，确立了黄金治疗窗口。在连续光照10分钟的过程中，小鼠原位肿瘤发光强度增强了5.6倍，微电极测得肿瘤内部pH从6.61剧烈下滑至5.86，展现出极为显著的体内酸靶向操作能力。体内生物发光成像与脑部切片病理分析共同见证了震撼的治疗效果：相较于持续恶化扩张的对照组，光治疗组的肿瘤体积与重量实现极其罕见的6倍暴跌，小鼠的30天生存率从0%逆天提升至60%。最后，体内代谢动力学研究证实，超过82%的纳米颗粒在5天内通过尿液和粪便安全排出体外，主要脏器切片无任何器质性损伤，完美兼顾了超凡的抗癌疗效与极高的临床安全性。

In vivo UCL imaging of UFPL nanoagent. (a) Schematic illustration of NIR-induced Fc collapse and UCL recovery for H+ visual quantification in vivo. (b) In vivo UCL images of tumor-bearing mice after intravenous injection of UFPL nanoagent (15 mg/mL, 50 µL) at various time points. (c) Corresponding UCL intensity in the tumor and liver. (d) In vivo UCL images of tumor-bearing mice at various time points (0–14 min) post-NIR irradiation, taken 11 h after UFPL nanoagent injection. (e) Relative UCL fluorescence (RFI) intensities and pH value at tumor sites within 14 min of irradiation time, along with their Pearson correlation coefficient. (f) Linear correlation between the reciprocal pH (1/pH) and relative in vivo UCL intensity of the UFP nanoagent during the 0–10 min irradiation period. (g) Linear relationship between tumor inhibition rate (Y%) and both UCL RFI and ΔpH.#

In vivo therapy performance. (a) Schematic illustration showing the establishment of GL261 tumor-bearing mice. (b) In vivo bioluminescence images of orthotopic tumor-bearing C57 mice treated with PBS, NIR, and UPL + NIR, respectively, over 14 days (days 0, 7, 9, 11, and 14). (c) Tumor volume and (d) tumor weight ratio of GL261 tumor-bearing mice after treatment with PBS, NIR, UFL, and UPL + NIR, respectively. (e) Schematic of UFPL-mediated tumour acidification (left) and intratumoral pH measurement using a microelectrode (right). (f) pH changes inside the tumor after UFPL nanoagent injection and 980 nm NIR irradiation over 60 min. (g) Multispectral fluorescence imaging (580 nm and 640 nm) of intratumoral pH after UFPL nanoagent injection, followed by 10 min of NIR irradiation. (h) Immunohistochemical analysis of LC3B and p62 in tumor sections treated with PBS, NIR, and UPL + NIR, with corresponding quantitative analysis shown in (i). Data in (d) are presented as mean ± SD (n = 5). The p values were calculated using Tukey’s post-test (***p < 0.001, **p < 0.01, or *p < 0.05). Adopted in vivo UPL, UFL, and UFPL nanoagent dose: 15 mg/mL, 50 µL.#