文献：Targeted nanobody complex enhanced photodynamic therapy for lung cancer by overcoming tumor microenvironment

文献链接：https://pubmed.ncbi.nlm.nih.gov/33292202/

作者：Qing Zhang, Lian Wu, Shaozheng Liu, Qingjie Chen, Lingpeng Zeng, Xuezhong Chen and Qing Zhang

相关产品：mPEG-SS-PLGA-SH（甲氧基聚乙二醇-二硫键-聚乳酸-羟基乙酸共聚物-巯基）

原文摘要：

Background: To investigate the efcacy of a PLGA-based nanobody complex in photodynamic therapy (PDT) and NIR-II imaging in A549 tumor hypoxic model.

Method: IR1048-MZ was frstly synthesized by conjugating a nitro imidazole group to IR1048. IR1048-MZ and Cat were then encapsulated in PLGA-SH solution. Anti-EGFR-Nanobody was also expressed and purifed, and fnally Anti-EGFR-Nanobody@PLGA-IR1048MZ-Cat (Nb@IC-NPs) nanobody complex was obtained based on the formation of desulfde bond between PLGA-SH and Anti-EGFR-Nanobody. Size distribution and morphology were characterized by TEM and DLS. Spectrum of Nb@IC-NPs towards NTR was measured by UV and fuorescence, while the particle’s selective response was studied using fuorescence. The uptake of Nb@IC-NPs in A549 cells was observed by fow cytometry and CLSM. In the meantime, its’ catalytic ability that decomposes H2O2 both extra-and intra-cellular was observed by fuorescence and CLSM. In vitro photodynamic toxicity of Nb@IC-NPs was examined by MTT, Live/Dead Cell Staining, Flow Cytometry and Apoptosis Assay. Tumor-bearing model was constructed to observe a semi-quantitative fuorescent distribution and the possibility of NIR-II fuorescence/photoacoustic (PA) imaging. Efect of Nb@IC-NPs on enhancing A549 tumor hypoxia and expression profle of HIF-1α was investigated in the presence of NIR. An A549 tumor metastasis model was also constructed to confrm the complex’ potential to destroy primary tumor, inhibit lung metastasis, and prolong mice’ survival. Lastly, impact of Nb@IC-NPs on mice’ main organs and blood indices was observed.

Results: Nb@IC-NPs was successfully fabricated with good homogeneity. The fuorescent absorbance of Nb@IC-NPs showed a linear relationship with the concentration of NTR, and a higher concentration of NTR corresponded to a stronger photoacoustic signal. In addition, Nb@IC-NPs showed a stable selectivity toward NTR. Our results also suggested a high efcient uptake of Nb@IC-NPs in A549 cells, which was more efcient than IC-NPs and IR1048-MZ alone. In vitro assays confrmed the efects of Nb@IC-NPs on catalytic O2 generation even in hypoxic cells. The cell viability was upregulated with the nanocomplex at the absence of the laser, whereas it was dramatically declined with laser treatment that excited at 980 nm. Nb@IC-NPs achieved tumor hypoxia NIR-II/PA imaging through assisting A549 gathering. When NIR was applied, Nb@IC-NPs can signifcantly relieve A549 cellular/tumor hypoxia by generating more reactive oxygen species (ROS), which in turn helps lower the expression level of HIF-1α. In summary, Nb@IC-NPs based PDT can efciently decimate A549 primary tumor, inhibit metastatic lung cancer, and prolong the lifespan of the mice under tolerable dosage. At last, in vivo toxicity tests of the nanocomplex showed its biosafety to the main organs and normal blood indices values.

Conclusion: Nb@IC-NPs improves tumor hypoxia through catalytic reaction and lowers the expression level of HIF-1α. It achieves tumor PA imaging through intensifed NIR-II fuorescence signal that caused by response of the complex to the lesion’s nitroreductase (NTR). Nb@IC-NPs based PDT can efciently kill A549 primary tumor, inhibit a lung metastasis, as well as prolong mice’ survival cycle.

mPEG-SS-PLGA-SH：mPEG（甲氧基聚乙二醇）：具有良好的亲水性。其甲氧基端使 PEG 链段相对稳定，并且可以调节共聚物整体的亲水性程度。SS（二硫键）：二硫键是一种重要的化学键，它在生物体内的氧化还原环境中可以发生断裂和重建。在 mPEG - SS - PLGA - SH 结构中，二硫键起到了连接 mPEG 和 PLGA - SH 的作用，同时赋予了材料在特定环境下的可降解性。PLGA（聚（乳酸 - 羟基乙酸）：由乳酸和羟基乙酸单体缩聚而成的生物可降解聚酯。其性质会因乳酸和羟基乙酸的比例不同而变化，如降解速度、机械性能等。SH（巯基）：巯基是一种具有较高反应活性的官能团。它可以用于进一步的化学反应，如与其他含有双键、环氧基等活性基团的分子发生反应，从而对材料进行功能化修饰。基于mPEG-SS-PLGA-SH的性能，抗-EGFR-纳米体@PLGA-IR1048MZ-Cat（Nb@IC-NPs）纳米体复合物的合成如下：

图：合成及作用示意

Nb@IC-NPs的制备：

将IR1048-MZ和Cat溶解在DMSO中，然后与含有DMF的mPEGSS-PLGA-SH溶液混合。将整个溶液滴加入到水中，直到搅拌后形成PLGA-IR1048MZ-Cat纳米体（IC-NPs）。将IC-NPs和5,5‘-Dithio双（2-硝基苯甲酸）（DTNB）一起在室温下孵育，以激活mPEG-SS-PLGA-SH中的-SH基团。然后将活化的纳米体离心，用PBS洗涤。另一方面，Nb（PEG）用α-盐酸氨基乙硫醇处理，并由AKTA系统使用脱盐柱（HiTrapTM）纯化。将Te纯化的Nb（PEG）与活化的IC-NPs以一定的比例混合。孵育后，通过离心去除Te多余的Nb。最终产物（Nb@IC-NPs）被重悬，并用PBS洗涤。

图：表征图像

结论：

该文献成功制备基于mPEG-SS-PLGA-SH合成的抗-EGFR-纳米体@PLGA-IR1048MZ-Cat（Nb@IC-NPs）纳米体复合物。该物质具有良好的均匀性，对NTR表现出稳定的选择性。基于Nb@IC-NPs的PDT可以有效地 kill A549原发tumour，抑制转移性肺cancer，并在耐受剂量下延长小鼠生存周期。最后，纳米复合物Nb@IC-NPs的体内poison性试验表明其具有生物安全性。