Pacheco-Torres Jesus, Penet Marie-France, Krishnamachary Balaji, Mironchik Yelena, Chen Zhihang, Bhujwalla Zaver M
Division of Cancer Imaging Research, The Russell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University School of Medicine, Baltimore, MD, United States.
Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD, United States.
Front Oncol. 2021 Feb 25;10:614365. doi: 10.3389/fonc.2020.614365. eCollection 2020.
The inhibition of immune checkpoints such as programmed cell death ligand-1 (PD-L1/CD274) with antibodies is providing novel opportunities to expose cancer cells to the immune system. Antibody based checkpoint blockade can, however, result in serious autoimmune complications because normal tissues also express immune checkpoints. As sequence-specific gene-silencing agents, the availability of siRNA has significantly expanded the specificity and range of "druggable" targets making them promising agents for precision medicine in cancer. Here, we have demonstrated the ability of a novel biodegradable dextran based theranostic nanoparticle (NP) to deliver siRNA downregulating PD-L1 in tumors. Optical imaging highlighted the importance of NP delivery and accumulation in tumors to achieve effective downregulation with siRNA NPs, and demonstrated low delivery and accumulation in several PD-L1 expressing normal tissues.
The dextran scaffold was functionalized with small molecules containing amine groups through acetal bonds. The NP was decorated with a Cy5.5 NIR probe allowing visualization of NP delivery, accumulation, and biodistribution. MDA-MB-231 triple negative human breast cancer cells were inoculated orthotopically or subcutaneously to achieve differences in vascular delivery in the tumors. Molecular characterization of PD-L1 mRNA and protein expression in cancer cells and tumors was performed with qRT-PCR and immunoblot analysis.
The PD-L1 siRNA dextran NPs effectively downregulated PD-L1 in MDA-MB-231 cells. We identified a significant correlation between NP delivery and accumulation, and the extent of PD-L1 downregulation, with imaging. The size of the NP of ~ 20 nm allowed delivery through leaky tumor vasculature but not through the vasculature of high PD-L1 expressing normal tissue such as the spleen and lungs.
Here we have demonstrated, for the first time, the feasibility of downregulating PD-L1 in tumors using siRNA delivered with a biodegradable dextran polymer that was decorated with an imaging reporter. Our data demonstrate the importance of tumor NP delivery and accumulation in achieving effective downregulation, highlighting the importance of imaging in siRNA NP delivery. Effective delivery of these siRNA carrying NPs in the tumor but not in normal tissues may mitigate some of the side-effects of immune checkpoint inhibitors by sparing PD-L1 inhibition in these tissues.
使用抗体抑制程序性细胞死亡配体1（PD-L1/CD274）等免疫检查点，为将癌细胞暴露于免疫系统提供了新的机会。然而，基于抗体的检查点阻断可能会导致严重的自身免疫并发症，因为正常组织也表达免疫检查点。作为序列特异性基因沉默剂，siRNA的可用性显著扩展了“可药物化”靶点的特异性和范围，使其成为癌症精准医学中有前景的药物。在此，我们展示了一种新型的基于可生物降解葡聚糖的诊疗纳米颗粒（NP）递送siRNA下调肿瘤中PD-L1的能力。光学成像突出了NP在肿瘤中的递送和积累对于用siRNA NPs实现有效下调的重要性，并证明在几个表达PD-L1的正常组织中递送和积累较低。
通过缩醛键用含胺基团的小分子对葡聚糖支架进行功能化。NP用Cy5.5近红外探针修饰，以便观察NP的递送、积累和生物分布。将MDA-MB-231三阴性人乳腺癌细胞原位或皮下接种，以实现肿瘤血管递送的差异。用qRT-PCR和免疫印迹分析对癌细胞和肿瘤中PD-L1 mRNA和蛋白表达进行分子表征。
PD-L1 siRNA葡聚糖NP有效下调了MDA-MB-231细胞中的PD-L1。我们通过成像确定了NP递送和积累与PD-L1下调程度之间存在显著相关性。约20 nm大小的NP能够通过渗漏的肿瘤血管进行递送，但不能通过高表达PD-L1的正常组织（如脾脏和肺）的血管进行递送。
在此我们首次证明了使用携带成像报告分子的可生物降解葡聚糖聚合物递送siRNA下调肿瘤中PD-L1的可行性。我们的数据证明了肿瘤NP递送和积累对于实现有效下调的重要性，突出了成像在siRNA NP递送中的重要性。这些携带siRNA的NP在肿瘤中而非正常组织中的有效递送，可能通过避免对这些组织中PD-L1的抑制来减轻免疫检查点抑制剂的一些副作用。
