分子工程增强AIEgens的光敏性，在缺氧环境下提高光动力治

Wan Q, Zhang R, Zhuang Z, et al. Molecular Engineering to Boost AIE‐Active Free Radical Photogenerators and Enable High‐Performance Photodynamic Therapy under Hypoxia[J]. Advanced Functional Materials, 2020, 30(39): 2002057.文章大致概况：这篇文章主要目的是通过分子工程设计出能够提高光动力治疗的分子，并且在缺氧的肿瘤环境中也能够起到很好的作用。要想设计出这样的分子，需要对相关原理有一定的了解，并且对之前类似分子的弊端有所改进。根据Jablonski图，研究者确定增加分子内电荷转移(ICT)，可以最小化单重态和三重态之间的能级差(ΔEst)，这样就能够产生更多的三重态能量分子，从而产生更多的ROS，达到治疗的目的。而在缺氧环境下用于PDT比较好的材料都是无机的，副作用和降解问题难以解决，少数的有机材料却因为在生理环境下会出现ACQ效应，导致其效果较差。而AIE材料具有相反的效果，因此可以拿来做分子的合成。经过推导，研究者设计出阴离子-π+AIEgens这样的分子来满足以上的需求。接着研究者对这种分子的合成，理化性质做了详细的描述，并对其产生ROS的能力、三重态表征、细胞成像和活体动物成像一一做了实验和叙述。最终证明了这种分子有一定的应用价值。

文章结构图

研究背景及相关知识：

荧光成像引导光动力疗法（PDT）能够实现精确的药物追踪和肿瘤可视化的无创治疗，是目前研究热点。光动力治疗简单的理解就是往人体肿瘤内注入相关光敏分子，分子受到光的刺激后能够产生一定量的自由基，氧自由基能够对肿瘤细胞进行杀伤。治疗的效果则主要取决于这种分子能否高效的进入肿瘤细胞内，并且能够产生足够多的自由基。PDT使用的这些光敏剂（PSs）主要通过I型（电子转移）或II型（能量转移）光化学机理产生细胞毒性的活性氧自由基（ROS）。微波和X射线进行PDT活化也可以产生ROS，并且可以更好地克服光穿透问题。而大多数有机PSs主要通过II型途径产生单线态氧（1O2）。

两种产生ROS的途径 Ⅱ型途径的问题在于由于其高氧依赖性和1O2的高耗氧量，因为实体肿瘤中的往往处于严重缺氧状态（氧压<5mmHg），这样就会降低有机PSs的抗癌效果。 为了克服肿瘤内缺氧，现在有高压氧疗法以增加肿瘤内的氧含量、MnO 2催化产生的氧以及低氧反应性药物，并且已经取得了一定的效果，但是这种方法具有一定的生物毒性和高氧性癫痫发作的不良副作用。因此开发出较少的O2依赖性PSs用于PDT治疗癌症是非常有必要的。 I型机制涉及受激PS与相邻底物之间的电子转移以产生自由基（例如O2-和HO·），并且PS与蛋白质、DNA或脂质的相互作用增强了PDT的效率，从而导致细胞凋亡，这种机制可以在低氧的情况下进行。I型产生的ROS，超氧自由基（O2-·）通过细胞内超氧化物歧化酶（SOD）介导的歧化转化为过氧化氢（H2O2）和O2，然后生成的H2O2可与细胞内亚铁离子反应生成高细胞毒性羟基自由基（HO·），进一步加剧细胞损伤并增强PDT的抗癌效果。这种级联反应提高了总体PDT效率，因此这种自由基产生剂已成为缺氧肿瘤治疗的的候选材料。然而，这类物质大多是含金属的配合物（如TiO 2和ZnO），缺点是可以被UV光激活、有潜在的副作用以及较差的生物降解性。而有同样效果的机分子（例如，酞菁，二氢卟酚和苯并吩噻嗪）是非常少见的，而且大多数有机物是疏水性的，在生理环境中会出现聚集引起的荧光猝（ACQ）。为了解决聚集荧光淬灭的问题，我们自然能够想到与之完全相反的AIEgens。在溶液中，由于从最低激发单重态（S1）到基态（S0）的非辐射内部转化（IC）消耗了AIEgens的激发态能量，因此在溶液中的荧光较弱。分子聚集时，这种非辐射性IC会受到分子内运动的限制，从而导致S1能量通过辐射跃迁或系统间穿越（ISC）通道衰减和释放。根据Jablonski图，该过程提供了足够的空间来增强相对荧光强度和三重态能量的产生，以实现理想的ROS产生，这是由辐射衰减（Kr）、非辐射IC（KIC）和ISC（KISC）之间的三个竞争关系所产生的。在这种紧密的竞争关系中，II型过程比I型过程更快，所以尚没有使用AIE进行自由基ROS生成器的分子工程设计。

Jablonski图高效的ISC可以确保产生足够的三重态能量，并创建一个富电子环境以向激发的PS提供电子，这是设计I型 ROS的关键。最小化单重态和三重态之间的能级差(ΔEst)是通过制造强烈的分子内电荷转移(ICT)态来加速ISC提高三重态能量产生的有效途径。电荷相互作用在生物体系中普遍存在，最近报道的阴离子-π+AIEgens具有良好的生物相容性、亲水性和亚细胞靶向性，这种性质的材料比较满足PDT的要求。除了这项优势以外，阴离子-π+ AIEgen在从用于激发光敏剂的阴离子还原剂产生富电子条件方面具有先天优势。因此，构建具有较强ICT特性和抑制KIC和促进KISC能力的富电子阴离子-π+AIEgens，将增强激发三重态PSs对电子的俘获作用，实现自由基ROS发生器。

如何设计所需的分子？

为了体现这一设计思想，设计者从不同的构筑单元制备了阴离子-π+AIEgen：

• 转子型还原剂三苯胺及其甲氧基取代衍生物(MTPA)连接到苯并-2，1，3-噻二唑(BZ)/萘并[2，3-c][1，2，5]噻二唑(NZ)基团上，作为协同的AIE活性供体，抑制非辐射IC，激活聚合态的辐射通道和ISC通道；

• 苯乙烯吡啶作为受体，确保激发三重态能量转换的强度；

• 具有很强还原性的碘离子和协同供体，在聚集的微环境中创造富电子的条件，并为激发的PSS提供电子。

• 最后的实验和理论结果都表明富电子的阴离子-π+AIEgens能够产生更多的ICT，更多的自由基活性氧(ROS)以及更明亮的荧光。在常氧/低氧环境下，新型阴离子-π+PSS(AIE-PSS)能够产生更多的氧自由基ROS，使其具有优异的体内外荧光成像和光动力学效率。本研究的目的是能够指导 typeⅠROS的设计，以克服传统PDT存在的问题。



• 四种Anion-πAIEgens的分子式及其电子贡献能力的排行

实验具体过程

四种化合物制作过程及光物理性质4，7-二溴苯并[c][1，2，5]噻二唑(BrBZ)购自商业来源，受体4，9-二溴萘并[2，3-c][1，2，5]噻二唑(BrNZ)是根据以前的文献制备的。基于不对称设计，通过铃木碳-碳偶联将电子给体TPA和MTPA单元与受体偶联，以获得64-82%的理想产率的中间体(TBZBr、MTBZBr、TNZBr和MTNZBr)。(4-甲酰基苯基)硼酸进一步偶联到这些中间体的另一侧，以87-92%的产率获得含醛中间体，其进一步与先前制备的1，4-二甲基吡啶-1-碘化鎓盐反应，以超过90%的产率制备最终产物(TBZPy、MTBZPy、TNZPy和MTNZPy)。所有产品均经核磁共振氢谱确认，最终产品经高分辨率质谱进一步表征。 首先用紫外可见光谱和荧光光谱研究了四种化合物的光学性质。MTBZPy的最大吸收峰位于476nm，这比TBZPy的最大吸收峰(447纳米)更长，这是因为用更强的供体MTPA取代了TPA。这表明在增强给电子能力之后改善了ICT效果。当NZ取代BZ单元时，由于TNZPy的吸收波长(511nm)比BZ基化合物的吸收波长长，给电子能力进一步提高。MTNZPy显示出最强的ICT效果，因为其最长吸收波长更长(528nm)。此外，在THF溶剂中，TNZPy (662nm)和MTNZPy (686nm)的发射峰的波长比TBZPy (625nm)和MTBZPy (659nm)的发射峰的波长更长，这证实了NZ基荧光团比BZ基荧光团的ICT更强。因此这几种化合物产生ICT的能力遵循了同样的模式，正如预期的那样:TBZPy < MTBZPy < TNZPy < MTNZPy密度泛函理论(DFT)计算表明，最高占有分子轨道(HOMOs)的电子云主要分布在TPA和MTPA单元上，略微离域到BZ和NZ嵌段，而最低未占据分子轨道(LUMOs)由苯乙烯基吡啶阳离子主导。因此，HOMO和LUMO的空间分离证实了TBZ/MTBZ和TNZ/MTNZ区块可以作为合作的电子供体，因为富含电子的TPA/MTPA和BZ/ NZ部分结合在一起形成一个整体。为了清楚地比较化合物的ICT性质，对它们在不同极性的各种溶剂中的基态和激发态性质进行了表征。当溶剂极性逐渐增加时，最大吸收带没有明显变化，表明化合物在基态对极性环境不敏感。而激发态对极性环境的高度敏感性，可以观察到显著的发射红移。值得注意的是，MTNZPy在激发态对溶剂极性表现出非典型的敏感性，这与其基态下的HOMO和LUMO之间几乎垂直的二面角有关。TBZPy和MTBZPy的二面角分别为54.6°和52.0°，而TNZPy和MTNZPy的二面角分别增加到86.9°和89.5°。因此，基于BZ的荧光团的发射红移(65和66nm)比基于NZ的荧光团的发射红移(39和10nm)相对更宽，并且伴随着溶剂极性的增加。这些明显的红移显示了典型的ICT特征。此外，Lippert-Mataga模型通过比较Stoke shift (va − vf)与环境极性(δf)的斜率来比较它们的ICT强度。TBZPy的斜率为11，060cm-1，小于MTBZPy (14，633cm-1)和TNZPy (22，321cm-1)的斜率，表明增强的给电子能力应对应于增强的ICT强度。此外，在M06-2X/6-31G(d，p)水平上计算了它们的偶极矩(μ)。TNZPy比TBZPy (26.7 D)和MTBZPy (29.5 D)显示更大的μ (30.4 D)，这与它们的实验斜率一致，而MTNZPy具有最大的33.5 D。这一结果表明，当受体相同时，协作性供体的供电子能力大小顺序：TBZPy< MTBZPy< TNZPy< MTNZPy 研究这四种化合物的光学性质，荧光在二甲基亚砜（DMSO）中显示弱荧光（ΦF为0.03~1.1％），但聚集后荧光增强（ΦF为2.2–9.5％），表明它们具有潜在的AIE。以TNZPy为例，它在DMSO中显示出非常弱的荧光，这可能归因于非分子通道的频繁分子内旋转，非辐射通道具有很强的ICT和高能量消耗。当加入水作为劣溶剂时，由于分子旋转有限和阻止了激发态能量通过非辐射途径消耗，其荧光强度显著增加，表现出典型的TNZPy的AIE效果。其他三种化合物表现出类似的AIE行为。所有AIEgen在溶液和聚集体中都表现出较短的荧光寿命(1.70-13.78 ns)。它们在聚集态的Kr值远高于单分子态，表明加速辐射衰变通道主要响应于它们的AIE，这是由于非辐射衰变通道的限制。然而，集合体形式的KIC+ISC也较高(，并且NZ基团的KIC+ISC明显高于BZ基材料，这可能有助于NZ基料的KISC过程更高效、更快，因为它们的产生ICT的能力更强。

光触发ROS产生

为了确定“ICT越多是否会导致更多的自由基ROS生成”，使用荧光指示剂2‘，7’-二氯二氢荧光素二乙酸酯(H2DCF-DA)来评估总ROS生成。由于化合物在可见光区域有很强的吸收，因此使用容易获得无害的白光作为辐射光源来触发ROS生成。当四种AIEgen在DMSO中溶解时，ROS的产生效率很低。然而，含有AIE-PS纳米颗粒的H2DCF-DA溶液的荧光被触发，并且在很短的照射时间内(20s)，其强度迅速增加，反映了ROS的高效和快速产生。这证实了聚集促进了ISC，且促进了ROS的产生，因为聚集抑制了非辐射通道。AIEgens聚集后总ROS生产效率的趋势与其ICT强度的趋势相关，表明我们提出的“增加ICT强度以促进ROS产生”的观点是可信的。研究继续详细研究活性氧的产生，一种常见的专有1O2探针ABDA，被用来选择性地检测II型1O2。在光照射下，ABDA(空白组)在378 nm处的吸收峰略有下降，两个NZ基的AIE-PSS的峰也略有下降，而TBZPy和MTBZPy的峰值迅速下降，表明基于BZ的AIE-PSS产生1O2的效率很高。接下来，比较了(RB)PS和四种化合物的1O2产量。两种BZ基AIE-PSS比RB染料具有更高的1O2效率，而TBZPy 比MTBZPy具有更高的1O2效率。 然而和以上结论有矛盾的是，总ROS产量的排序是TBZPy<MTBZPy<TNZPy<MTNZPy，这与ABDA和SOSG指标的结果相矛盾，因此NZ-PSS也可能产生I型(自由基为主)ROS。根据之前的研究，我们通过荧光探针DHR123和还原型维生素C(VC)作为指示剂来验证自由基ROS的产生。结果表明纯DHR123溶液的荧光强度略有变化，在加入TBZPy和MTBZPy后，荧光强度急剧增加，然后趋于平稳。然而，NZ基团的AIE-PSS的强度稳步增加，最终超过了BZ基团的AIE-PSS，这意味着在基于NZ基团的AIE-PSS中存在自由基ROS。用自由基清除剂VC验证了DHR123荧光强度的增强确实是由自由基ROS引起的。MTNZPy在添加VC后显示出降低的发射强度，另外两种AIE-PSS(MTBZPy和TNZPy)显示出类似的强度趋势。然而，在含有TBZPy的DHR123溶液中加入Vc并没有影响DHR123的荧光强度，这表明TBZPy主要产生1O2类自由基。TBZPy和RB在添加VC清除剂后具有不变的发射强度，但其他三个(MTBZPy、TNZPy和MTNZPy)的发射强度显著降低，表明MTBZPy能够同时产生1O2和自由基ROS，但以NZ-基团的AIE-PSS主要产生自由基ROS。研究可以确认DHR123探针对1O2和自由基ROS有反应，因为商用的1O2(RB)在与DHR123接触时也有反应。另一种荧光指示剂羟基苯基荧光素的分析证实了NZ-AIE-PSS中显著的自由基ROS生成，并显示与基于BZ的ROS生成相比，NZ基团的AIE-PSS中的自由基ROS生成效率更高。为了进一步为基于NZ-基团的AIE-PSS产生自由基ROS提供坚实的证据，使用了电子顺磁共振(EPR)光谱分析。BMPO和BMPO+AIEgen(TNZPy和MTNZPy)在黑暗中均不产生任何EPR信号，而当BMPO+TNZPy/MTNZPy溶液照射时，可以观察到明显的EPR信号，这与顺磁自由基物种的产生有关。因此，我们可以确认低ICT的TBZPy只产生了1o2。根据MTBZPy和NZ基团的AIE-PSS的结果，一旦ICT的强度被增强的供电子能力所增强，1O2物种就会转化为自由基ROS，这与研究提出的“更多的ICT导致更多的自由基ROS的产生”是一致的。

三重态激发态表征

为了全面研究能量分布对四种AIE-PSS产生ROS效率的影响，在CAM-B3LYP/6-31G+(d，p)水平上，采用含时密度泛函理论(TD-DFT)研究了四种AIE-PSS通过ISC的三重态能量转换。低激发态单重态和三重态之间的ΔEst(S1/T1-2)表明，在该系列中，随着ICT强度从TBZPy到MTNZPy，AIE-PSs的ΔEst降低，这意味着大部分激发能量可以通过ISC通道消耗，这与基于光谱的实验中NZ基团的微弱荧光是一致的。MTBZPy表现出比TBZPy更小的ΔEst，导致ROS的产生大于TBZPy的ROS产生，但仍远低于两种自由基ROS产生效率更高的NZ-基酶产生的ROS。四种AIE-PSS均表现出显著的三重态，实验ΔEst值的变化趋势与理论预测相符。MTNZPy具有最小的ΔEst(0.08eV)，并且由于其高效的ISC，应该能够响应最高的ROS生成。因此，理论和实验结果支持“富电子阴离子-π+PSS促进自由基活性氧生成的ICT”的观点。

细胞成像

由于两种基于NZ-的AIE-PSS比基于BZ-的AIE-PSS产生自由基活性氧的效率更高，所以我们在后续的生物学实验中采用了这两种基于NZ-的AIE-PSS。针对其独特的AIE特性，进行了体外细荧光成像的研究。动态光散射(DLS)显示，TNZPy和MTNZPy纳米聚集体的平均流体动力学直径分别为43 nm和90 nm，较小的尺寸分布确保了这些纳米粒子很容易进入细胞，并具有增强的渗透性和保留性。然后研究了AIE-PSS进入细胞的过程。对两种AIE-PSS与HeLa细胞孵育不同时间(2、4和6h)的详细观察表明，两种AIE-PSS均逐渐穿透细胞，且具有明显的动态过程。在最初的2h内，可在膜周围观察到一些红色颗粒荧光点，随着时间的推移，红色荧光面积逐渐增大，初级颗粒荧光点转变为红色网状荧光，弥漫在整个细胞质中，与细胞背景形成良好的图像对比度，证实AIE-PSS已成功进入细胞。此外，这些AIE-PSS同时对线粒体和溶酶体显示出更好的细胞器靶向性。

常氧和缺氧条件下体外光动力疗法的实验研究

对癌细胞的PDT效率应通过标准的MTT法和活/死细胞共染色方法进行研究。用CCK-8法评价了两种AIE-PSS对HeLa和T24细胞的暗毒性，显示出良好的生物相容性和足够的安全性。然后，进一步评价肿瘤细胞在常氧和缺氧条件下的光动力效率(PDT)。在常氧下，当MTNZPy的孵育强度为6μm时，癌细胞几乎被完全消灭，但是在更高强度（8μm）的TNZPy上实现了相似的治疗结果，这表明MTNZPy的PDT性能优于TNZPy。在低氧条件下，通过在8%氧气环境中培养肿瘤细胞可以模拟肿瘤的体外低氧环境，据报道肿瘤细胞的含氧量约为0-4%。研究表明在低氧条件下，两种AIE-PSS都保持了良好的PDT结果。MTNZPy的PDT效率仍然高于TNZPy，因为它产生了更高的自由基ROS。活/死细胞共染色实验进一步证明了这两种AIE-PSS在常氧和低氧环境下的PDT结果。两种AIE-PSS在低氧环境下保持了比常氧环境下更好的PDT效率，不仅对HeLa细胞，而且在常氧和低氧环境下的T24细胞都保持了优异的致死性。

治疗机制

根据以往的研究，PSS在光触发下产生的ROS可以迅速破坏细胞器的生物学功能，导致细胞凋亡、坏死和自噬。本研究发现这两个AIE-PSS对溶酶体和线粒体具有动态、特异的靶向性。这两种AIE-PSS在PDT过程中是否会同时引起溶酶体和线粒体细胞器的破坏尚不清楚。由于吖啶橙(AO)与不同细胞器接触时会产生多色荧光变化，因此可采用吖啶橙(AO)作为细胞器完整性指示剂。PSS单独或在光处理的细胞中显示非常强烈的颗粒状AO荧光，表明溶酶体隔间仍然完好无损。当细胞与AIE-PSS在光照射下孵育时，AO荧光消失，溶酶体和线粒体的完整性严重破坏，形成自噬小泡。此外，细胞核还出现了形态变小、皱缩，胞浆成分渗漏，细胞塌陷等这些现象。这些结果证实了光动力触发的线粒体和溶酶体破坏，通过细胞器的协同破坏提高了PDT的结果。

活体肿瘤荧光成像与PDT性能

由于AIE-PSS在缺氧环境中具有近红外(NIR)发射和理想的PDT性能，因此研究对T24皮肤瘤裸鼠进行了小动物荧光成像和实体瘤抑制实验。瘤内注射AIE-PSS后，可以通过荧光信号清楚地观察到肿瘤部位，并与周围组织区分开来。在注射后24小时，肿瘤部位仍然清晰可见，表明AIE-PSS有效地保留了肿瘤。注射后24h处死小鼠，获取小鼠各器官和肿瘤组织的体外荧光图像。肿瘤部位仍有较强的肿瘤荧光信号，而肝、肾、心、脾、肺、肠等其他器官未检测到荧光信号，表明AIE-PSS在肿瘤部位有较好的保留。接下来，当肿瘤体积达到约50mm3时，在瘤内注射24小时后进行体内PDT治疗。对照组肿瘤体积在整个治疗过程中明显增大，PBS治疗组肿瘤生长不受光照影响，但相对对照组增长缓慢，说明光照对肿瘤的抑制作用较小。此外，单独使用TNZPy和MTNZPy治疗的小鼠显示出不可抑制的肿瘤生长，证明了这些化合物的生物安全性。但照射15min(0.15W cm−2)后，各处理组肿瘤生长受到明显抑制，肿瘤缩小、结痂。相应的肿瘤照片证实了AIE-PSS的抗肿瘤作用。重要的是，在整个PDT治疗过程中，没有一只小鼠出现体重异常变化，H&E染色切片图像显示，PDT治疗后主要器官的形态学没有明显变化，但明显抑制了肿瘤的分化。这些数据表明，这两种AIE-PSS是生物相容性的，可用于体内系统，具有临床治疗的潜力。 英文版：The main purpose of this article is to design molecules that can improve photodynamic therapy through molecular engineering, and can also play a good role in hypoxic tumor environments.To design such a molecule, it is necessary to have a certain understanding of the relevant principles and to improve the drawbacks of the previous similar molecules.According to the Jablonski diagram, the researchers determined that increasing the intramolecular charge transfer (ICT) can minimize the energy level difference (ΔEst) between the singlet state and the triplet state, so that more triplet energy molecules can be generated, thereby generating more ROS, to achieve the purpose of treatment.However, the better materials for PDT in an oxygen-deficient environment are inorganic, and the side effects and degradation problems are difficult to solve. However, a small number of organic materials have an ACQ effect in a physiological environment, resulting in poor results. The AIE material has the opposite effect, so it can be used for molecular synthesis.After deduction, the researchers designed molecules such as anions -π+AIEgens to meet the above requirements.Then the researchers described the synthesis and physicochemical properties of this molecule in detail, and performed experiments and descriptions of its ability to generate ROS, triplet characterization, cell imaging, and live animal imaging. Finally proved that this kind of molecule has certain application value.

Research background and related knowledge:

Fluorescence imaging guided photodynamic therapy (PDT) can achieve precise drug tracking and non-invasive treatment of tumor visualization, and is currently a research hotspot. The simple understanding of photodynamic therapy is to inject relevant photosensitive molecules into human tumors. The molecules can generate a certain amount of oxygen free radicals after being stimulated by light, and oxygen free radicals can kill tumor cells. The effect of treatment mainly depends on whether this molecule can enter tumor cells efficiently and can generate enough free radicals.These photosensitizers (PSs) used in PDT mainly generate cytotoxic reactive oxygen radicals (ROS) through type I (electron transfer) or type II (energy transfer) photochemical mechanisms. PDT activation by microwave and X-rays can also generate ROS, and can better overcome the problem of light penetration. Most organic PSs mainly produce singlet oxygen (1O2) through type II pathways.The problem with the type II approach is that due to its high oxygen dependence and high oxygen consumption of 1O2, solid tumors are often in a severe hypoxic state (oxygen pressure <5mmHg), which will reduce the anti-cancer effect of organic PSs.In order to overcome hypoxia in tumors, there are methods such as hyperbaric oxygen therapy to increase the oxygen content in tumors, MnO 2 catalyzed oxygen and hypoxic reactive drugs, etc., and certain effects have been achieved, but this method has certain effects Biological toxicity and adverse side effects of hyperoxic seizures. Therefore, it is very necessary to develop fewer O2-dependent PSs for PDT to treat cancer. Type I mechanism involves the transfer of electrons between stimulated PS and adjacent substrates to generate free radicals (such as O2- and HO·), and the interaction of PS with proteins, DNA or lipids enhances the efficiency of PDT, resulting in apoptosis, this mechanism can be carried out under hypoxia.Type I ROS, superoxide radicals (O2-·) are converted into hydrogen peroxide (H2O2) and O2 through intracellular superoxide dismutase (SOD)-mediated disproportionation, and then the generated H2O2 can interact with intracellular sub-substances. Iron ions react to produce highly cytotoxic hydroxyl radicals (HO·), which further aggravates cell damage and enhances the anti-cancer effect of PDT. This cascade reaction increases the overall PDT efficiency, so this free radical generator has become a candidate material for the treatment of hypoxic tumors.However, most of these substances are metal-containing complexes (such as TiO 2 and ZnO). The disadvantages are that they can be activated by UV light, have potential side effects, and have poor biodegradability. Organic molecules with the same effect (for example, phthalocyanine, chlorin and benzophenothiazine) are very rare, and most organic substances are hydrophobic, and fluorescence quenching caused by aggregation will occur in a physiological environment ( ACQ).In order to solve the problem of aggregation fluorescence quenching, we can naturally think of AIEgens, which is the opposite. In solution, since the non-radiative internal conversion (IC) from the lowest excited singlet state (S1) to the ground state (S0) consumes the excited state energy of AIEgens, the fluorescence in the solution is weak. When molecules gather, this non-radiative IC will be limited by the movement of the molecules, which will cause the S1 energy to attenuate and release through radiation transitions or inter-system crossing (ISC) channels. According to the Jablonski diagram, the process provides enough space to enhance the relative fluorescence intensity and triplet energy generation to achieve the ideal ROS generation, which is caused by radiation attenuation (Kr), non-radiation IC (KIC) and ISC (KISC) Between the three competing relationships.In this close competition, the type II process is faster than the type I process, so AIE has not been used for molecular engineering design of free radical ROS generators.An efficient ISC can ensure that sufficient triplet energy is generated and an electron-rich environment is created to provide electrons to the excited PS, which is the key to the design of Type I ROS. Minimizing the energy level difference (ΔEst) between the singlet state and the triplet state is an effective way to accelerate the ISC to increase the triplet energy generation by creating a strong intramolecular charge transfer (ICT) state.Charge interactions are common in biological systems. The recently reported anion -π+AIEgens has good biocompatibility, hydrophilicity and subcellular targeting. Materials with this nature can meet the requirements of PDT.In addition to this advantage, the anion-π+ AIEgen has inherent advantages in generating electron-rich conditions from the anionic reducing agent used to excite the photosensitizer. Therefore, constructing an electron-rich anion -π+AIEGENS with strong ICT characteristics and the ability to inhibit KIC and promote KISC will enhance the trapping effect of excited triplet PSS on electrons and realize a radical ROS generator.

How to design the required molecules?

In order to reflect this design idea, the designer prepared anion-π+AIEgen from different construction units:

• the rotor-type reductant triphenylamine (TPA) and its methoxy-substituted derivative (MTPA) linked to benzo-2,1,3-thiadiozole (BZ)/naphtho[2,3-c][1,2,5]thiadiazole (NZ) moieties containing electron-rich heteroatoms (S, N) to serve as collaborative AIE-active donors to suppress nonradiative IC and activate radiative and ISC channels in the aggregate state;

• styrylpyridine cation as an acceptor to ensure regular ICT intensity for the excited triplet energy conversion;

• iodide anion and collaborative donors with strong reducibility to create electron-rich conditions in the aggregated microenvironment and provide electrons to excited PSs.

• The final experimental and theoretical results show that the electron-rich anion -π+AIEgens can generate more ICT, more free radical reactive oxygen species (ROS) and brighter fluorescence. Under normoxia/hypoxia environment, the new anion -π+PSS (AIE-PSS) can generate more oxygen radical ROS, which makes it have excellent in vivo and in vitro fluorescence imaging and photodynamic efficiency.The purpose of this research is to guide the design of typeⅠROS to overcome the problems of traditional PDT.

The specific process of the experiment

The production process and photophysical properties of the four compounds4,7-Dibromobenzo[c][1,2,5]thiadiazole (BrBZ) was purchased from commercial sources, acceptor 4,9-dibromonaphtho[2,3-c][1,2 , 5] Thiadiazole (BrNZ) is prepared according to previous literature.Based on the asymmetric design, the electron donor TPA and MTPA units are coupled with the acceptor through Suzuki carbon-carbon coupling to obtain intermediates (TBZBr, MTBZBr, TNZBr and MTNZBr) with an ideal yield of 64-82%.(4-Formylphenyl) boronic acid is further coupled to the other side of these intermediates to obtain an aldehyde-containing intermediate with a yield of 87-92%, which is further compared with the previously prepared 1,4-lutidine- 1- Onium iodide salt reaction to produce final products (TBZPy, MTBZPy, TNZPy and MTNZPy) in a yield of over 90%.All products were confirmed by 1H NMR spectroscopy, and the final products were further characterized by high-resolution mass spectrometry. First, the optical properties of the four compounds were studied by ultraviolet-visible spectroscopy and fluorescence spectroscopy.The maximum absorption peak of MTBZPy is located at 476 nm, which is longer than the maximum absorption peak of TBZPy (447 nm). This is because the stronger donor MTPA replaces TPA. This shows that the ICT effect has been improved after the enhancement of the electronic power.When NZ replaces the BZ unit, since the absorption wavelength (511nm) of TNZPy is longer than that of the BZ-based compound, the electron donating ability is further improved. MTNZPy shows the strongest ICT effect because its longest absorption wavelength is longer (528nm).In addition, in the THF solvent, the wavelength of the emission peaks of TNZPy (662nm) and MTNZPy (686nm) is longer than that of TBZPy (625nm) and MTBZPy (659nm), which confirms that the NZ-based fluorophore is better than the BZ-based fluorescence The group’s ICT is stronger. Therefore, the ability of these compounds to produce ICT follows the same pattern, as expected:TBZPy <MTBZPy <TNZPy <MTNZPyDensity functional theory (DFT) calculations show that the electron cloud of the highest occupied molecular orbitals (HOMOs) is mainly distributed on the TPA and MTPA units, slightly delocalized to the BZ and NZ blocks, while the lowest unoccupied molecular orbitals (LUMOs) are composed of benzene Vinylpyridine cation dominates. Therefore, the spatial separation of HOMO and LUMO confirms that the TBZ/MTBZ and TNZ/MTNZ blocks can be used as cooperative electron donors, because the electron-rich TPA/MTPA and BZ/NZ parts are combined to form a whole.In order to clearly compare the ICT properties of the compounds, their ground state and excited state properties in various solvents with different polarities were characterized.When the solvent polarity gradually increases, the maximum absorption band does not change significantly, indicating that the compound is not sensitive to the polar environment in the ground state.The excited state is highly sensitive to the polar environment, and a significant emission red shift can be observed. It is worth noting that MTNZPy exhibits atypical sensitivity to solvent polarity in the excited state, which is related to the almost perpendicular dihedral angle between HOMO and LUMO in its ground state.The dihedral angles of TBZPy and MTBZPy are 54.6° and 52.0°, respectively, while the dihedral angles of TNZPy and MTNZPy increase to 86.9° and 89.5°, respectively. Therefore, the emission red shift (65 and 66 nm) of BZ-based fluorophores is relatively wider than that of NZ-based fluorophores (39 and 10 nm), and is accompanied by an increase in solvent polarity. These obvious red shifts show typical ICT characteristics.In addition, the Lippert-Mataga model compares their ICT intensity by comparing the slopes of Stoke shift (va − vf) and environmental polarity (δf). The slope of TBZPy is 11,060cm-1, which is smaller than the slopes of MTBZPy (14,633cm-1) and TNZPy (22,321cm-1), indicating that the enhanced electron donating ability should correspond to the enhanced ICT intensity. In addition, their dipole moments (μ) were calculated on the M06-2X/6-31G(d, p) level. TNZPy shows a larger μ (30.4 D) than TBZPy (26.7 D) and MTBZPy (29.5 D), which is consistent with their experimental slope, while MTNZPy has the largest 33.5 D.This result shows that when the acceptor is the same, the order of the electron donating capacity of the cooperative donor is:TBZPy< MTBZPy< TNZPy< MTNZPy The optical properties of these four compounds were studied, and fluorescence showed weak fluorescence (0.03~1.1% ΦF) in dimethyl submetalmic scia (DMSO), but fluorescence enhancements after aggregation (2.2–9.5%) showed that they had potential AIEs. Take TNZPy as an example, it shows very weak fluorescence in DMSO, which may be attributed to the frequent intramolecular rotation of non-molecular channels, which have strong ICT and high energy consumption.When water is added as a bad solvent, the fluorescence intensity of the excited energy is significantly increased due to the limited rotation of the molecule and preventing the excited energy from being consumed through a non-radiation route, showing a typical AIE effect of TNZPy. The other three compounds show similar AIE behavior. All AIEgens show a shorter fluorescence life (1.70-13.78 ns) in solution and aggregate. Their Kr value in the aggregate state is much higher than the single molecular state, indicating that the accelerated radiation decay channel mainly responds to their AIE, due to the limitation of non-radiation decay channels. However, the combination form of KIC+ISC is also higher (and the KIC+ISC of the NZ group is significantly higher than that of the BZ-based material, which may help the KISC process of the NZ matrix to be more efficient and faster, because they have a stronger ability to produce ICT.

Light-T riggered ROS Generation

In order to determine "whether more ICT will lead to more free radical ROS generation", the fluorescent indicator 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA) was used to assess total ROS generation.Since the compound has a strong absorption in the visible light region, the easily available harmless white light is used as the radiation source to trigger the generation of ROS. When the four kinds of AIEgen are dissolved in DMSO, the efficiency of ROS generation is very low. However, the fluorescence of the H2DCF-DA solution containing AIE-PS nanoparticles was triggered, and its intensity increased rapidly within a short irradiation time (20s), reflecting the efficient and rapid generation of ROS. This confirms that aggregation promotes ISC and promotes the generation of ROS because aggregation inhibits non-radiative channels. Interestingly, the trend of total ROS production efficiency after the aggregation of AIEgens is related to the trend of ICT intensity, indicating that our view of "increasing ICT intensity to promote ROS production" is credible.The research continues to study in detail the production of reactive oxygen species, a common and proprietary 1O2 probe ABDA, which is used to selectively detect type II 1O2.Under light irradiation, the absorption peak of ABDA (blank group) at 378 nm decreased slightly, and the peaks of the two NZ-based AIE-PSS also decreased slightly, while the peaks of TBZPy and MTBZPy decreased rapidly, indicating that the BZ-based AIE -PSS produces 1O2 efficiently. Next, the 1O2 production of (RB)PS and the four compounds were compared. The two BZ-based AIE-PSS dyes have higher 1O2 efficiency than RB dyes, and TBZPy has higher 1O2 efficiency than MTBZPy.However, in contradiction with the above conclusion, the order of total ROS production is TBZPy<MTBZPy<TNZPy<MTNZPy, which contradicts the results of ABDA and SOSG indicators, so NZ-PSS may also produce type I (dominated by free radicals) ROS .According to previous studies, we used fluorescent probe DHR123 and reduced vitamin C (VC) as indicators to verify the generation of free radical ROS.The results showed that the fluorescence intensity of pure DHR123 solution changed slightly. After adding TBZPy and MTBZPy, the fluorescence intensity increased sharply and then stabilized. However, the strength of the AIE-PSS of the NZ group has steadily increased, and finally surpassed the AIE-PSS of the BZ group, which means that there are free radical ROS in the AIE-PSS based on the NZ group.The free radical scavenger VC verified that the increase in fluorescence intensity of DHR123 was indeed caused by free radical ROS. MTNZPy showed reduced emission intensity after adding VC, and the other two AIE-PSS (MTBZPy and TNZPy) showed similar intensity trends. However, adding Vc to the DHR123 solution containing TBZPy did not affect the fluorescence intensity of DHR123, which indicates that TBZPy mainly produces 1O2 free radicals.TBZPy and RB have the same emission intensity after adding VC scavenger, but the emission intensity of the other three (MTBZPy, TNZPy, and MTNZPy) is significantly reduced, indicating that MTBZPy can simultaneously produce 102 and free radical ROS, but with NZ- group The AIE-PSS mainly produces free radical ROS.The study can confirm that DHR123 probe reacts to 1O2 and free radical ROS, because commercial 1O2(RB) also reacts when it comes into contact with DHR123. The analysis of another fluorescent indicator, hydroxyphenylfluorescein, confirmed the significant free radical ROS generation in NZ-AIE-PSS, and showed that compared with BZ-based ROS generation, free radicals in NZ group AIE-PSS ROS generation efficiency is higher.In order to further provide solid evidence for the generation of free radical ROS by AIE-PSS based on NZ-groups, electron paramagnetic resonance (EPR) spectroscopy was used. Both BMPO and BMPO+AIEgen (TNZPy and MTNZPy) do not produce any EPR signal in the dark, and when BMPO+TNZPy/MTNZPy solution is irradiated, a clear EPR signal can be observed, which is related to the generation of paramagnetic radical species. Therefore, we can confirm that the low ICT TBZPy only produced 1o2. According to the results of the AIE-PSS of the MTBZPy and NZ groups, once the strength of ICT is enhanced by the enhanced electron-donating ability, the 1O2 species will be converted into free radical ROS. This is in line with the research put forward that "more ICT leads to more The production of free radical ROS" is consistent.

Triplet Excited State Characterization

In order to comprehensively study the effect of energy distribution on the efficiency of ROS generation by the four AIE-PSS, at the level of CAM-B3LYP/6-31G+(d, p), the four AIEs were studied using time-dependent density functional theory (TD-DFT) -PSS uses the triplet energy conversion of ISC.The ΔEst (S1/T1-2) between the low-excited state singlet state and the triplet state shows that in this series, as the ICT intensity goes from TBZPy to MTNZPy, the ΔEst of AIE-PSs decreases, which means that most of the excitation energy ISC channel can be consumed by this experiment with weak fluorescence spectrum NZ-based group is the same.MTBZPy exhibits a smaller ΔEst than TBZPy, resulting in ROS production greater than that of TBZPy, but still much lower than the ROS produced by the two NZ-based enzymes, which produce more efficient free radical ROS. The four kinds of AIE-PSS all show significant triplet states, and the change trend of the experimental ΔEst value is consistent with the theoretical prediction. MTNZPy has the smallest ΔEst (0.08eV) and should be able to respond to the highest ROS generation due to its efficient ISC. Therefore, the theoretical and experimental results support the view of "the electron-rich anion-π+PSS promotes the generation of ICT of free radicals".

Cell Imaging

Since the two NZ-based AIE-PSS are more efficient than BZ-based AIE-PSS in generating free radical active oxygen, we used these two NZ-based AIE-PSS in subsequent biological experiments. In view of its unique AIE characteristics, the study of in vitro fine fluorescence imaging was carried out.Dynamic light scattering (DLS) shows that the average hydrodynamic diameters of TNZPy and MTNZPy nano-aggregates are 43 nm and 90 nm, respectively. The smaller size distribution ensures that these nanoparticles can easily enter cells and have enhanced permeability and Retention. Then studied the process of AIE-PSS entering the cell.The detailed observation of the two AIE-PSS and HeLa cells incubated for different times (2, 4 and 6h) showed that both AIE-PSS gradually penetrated the cells and had obvious dynamic processes. In the first 2h, some red particle fluorescent spots can be observed around the membrane. As time goes by, the red fluorescent area gradually increases, and the primary particle fluorescent spots turn into red reticulated fluorescence, which diffuses in the entire cytoplasm and interacts with the cell. The background forms a good image contrast, confirming that AIE-PSS has successfully entered the cell. In addition, these AIE-PSS showed better organelle targeting to both mitochondria and lysosomes.

In Vitro PDT Performance under Normoxia and Hypoxia

The efficiency of PDT for cancer cells should be studied by standard MTT method and co-staining method for live/dead cells. The CCK-8 method was used to evaluate the dark toxicity of two AIE-PSS to HeLa and T24 cells, showing good biocompatibility and sufficient safety. Then, further evaluate the photodynamic efficiency (PDT) of tumor cells under normoxia and hypoxia.Under normoxia, when the incubation intensity of MTNZPy was 6μm, cancer cells were almost completely eliminated, but similar treatment results were achieved on TNZPy of higher intensity (8μm), which indicates that MTNZPy has better PDT performance than TNZPy.Under hypoxic conditions, tumor cells can be cultured in an 8% oxygen environment to simulate the in vitro hypoxic environment of tumors. It is reported that the oxygen content of tumor cells is about 0-4%.Studies have shown that under hypoxic conditions, both AIE-PSS maintain good PDT results. The PDT efficiency of MTNZPy is still higher than that of TNZPy because it generates higher free radical ROS. Live/dead cell co-staining experiments further proved the PDT results of these two AIE-PSS under normoxia and hypoxia. The two AIE-PSS maintained better PDT efficiency under hypoxic environment than under normoxia. Not only HeLa cells, but also T24 cells under normoxia and hypoxia maintained excellent lethality.

Therapeutic Mechanism

According to previous studies, ROS generated by PSS triggered by light can quickly destroy the biological functions of organelles, leading to apoptosis, necrosis and autophagy.This study found that these two AIE-PSS have dynamic and specific targeting to lysosomes and mitochondria. Whether these two kinds of AIE-PSS will cause the destruction of lysosome and mitochondrial organelles at the same time during PDT is still unclear.Since acridine orange (AO) will produce multi-color fluorescence changes when it comes into contact with different organelles, acridine orange (AO) can be used as an indicator of organelle integrity. PSS alone or in light-treated cells showed very strong granular AO fluorescence, indicating that the lysosomal compartment is still intact. When cells are incubated with AIE-PSS under light irradiation, AO fluorescence disappears, the integrity of lysosomes and mitochondria is severely damaged, and autophagic vesicles are formed. In addition, the cell nucleus also exhibited such phenomena as shrinkage, shrinkage, leakage of cytoplasmic components, and cell collapse.These results confirm that photodynamically triggered destruction of mitochondria and lysosomes improves the results of PDT through the coordinated destruction of organelles.

Live tumor fluorescence imaging and PDT performance

Because AIE-PSS has near-infrared (NIR) emission and ideal PDT performance in hypoxic environment, the study conducted small animal fluorescence imaging and solid tumor suppression experiments on T24 skin tumor nude mice.After intratumor injection of AIE-PSS, the tumor site can be clearly observed by fluorescent signal and distinguished from surrounding tissues. 24 hours after the injection, the tumor site was still clearly visible, indicating that AIE-PSS effectively preserved the tumor. The mice were sacrificed 24h after injection, and in vitro fluorescence images of mouse organs and tumor tissues were obtained. There is still a strong tumor fluorescence signal at the tumor site, but no fluorescence signal is detected in other organs such as liver, kidney, heart, spleen, lung, intestine, etc., indicating that AIE-PSS has good retention at the tumor site.Next, when the tumor volume reached about 50mm3, PDT treatment in vivo was performed 24 hours after intratumoral injection. The tumor volume in the control group increased significantly throughout the treatment. The growth of the tumor in the PBS treatment group was not affected by light, but the growth was slower than that in the control group, indicating that light had less inhibitory effect on the tumor. In addition, mice treated with TNZPy and MTNZPy alone showed uninhibited tumor growth, demonstrating the biological safety of these compounds. However, after 15 minutes of irradiation (0.15W cm-2), the growth of tumors in each treatment group was significantly inhibited, and the tumors shrank and scabled. The corresponding tumor photos confirm the anti-tumor effect of AIE-PSS.Importantly, during the entire PDT treatment, none of the mice showed abnormal changes in body weight. H&E stained section images showed that the morphology of the main organs did not change significantly after PDT treatment, but the differentiation of the tumor was significantly inhibited. These data indicate that the two AIE-PSS are biocompatible, can be used in in vivo systems, and have clinical therapeutic potential.