Ultrasound-controlled Cancer Immunotherapy using DNA Nanostructures

本项目旨在开发一种新型的先进药物输送系统（DDS），用于癌症免疫治疗。该DDS将对超声作出响应。超声波是触发器的选择，因为它比其他类型的刺激有许多优点，例如更深的穿透能力和最小的侵入性。待递送的药物是短的单链DNA寡聚体，称为CpG寡脱氧核苷酸（CpG ODN）。这些ODN被某些类型的免疫细胞（即抗原呈递细胞）中的Toll样受体9（TLR9）识别，其进而激活T细胞以变得具有细胞毒性并发挥抗癌活性以消除肿瘤细胞。为此，有三个关键目标。首先是DDS的制作。携带和保护ODN的载体基于由单链DNA（ssDNA）的长聚合物组成的水凝胶，其可以被工程化以具有多种功能。ssDNA将使用称为滚环扩增（RCA）的过程酶促产生。该产品经过液体结晶过程，产生独特的花形3D结构，直径在纳米和微米尺寸范围内。这些纳米花具有与ODN杂交和结合的能力，并将它们保持在非活性状态，直到被超声激活。超声波在溶液中产生空化气泡，其破裂并施加导致聚合物碎裂的剪切力。在这一阶段，我们的目标是优化DDS的结合能力和对不同频率和功率范围内的超声的响应。其次，将对DDS进行体外和离体试验。对于体外测试，我们将使用基因工程报告细胞系，其在TLR家族蛋白中仅表达TLR9。在超声处理前，DDS将在其非激活状态下进行测试，然后在超声处理后，在其激活状态下进行测试。之后，将使用从人类供体血液样本中分离的初级免疫细胞（如巨噬细胞和树突状细胞）对该系统进行体外测试。这一阶段的目的是证明这些细胞被超声触发DDS激活，然后它们激活T细胞（也从人血液样品中分离）的能力，这反过来又产生所需的抗癌活性。最后，我们的目的是评估DDS在体内动物模型（即小鼠B16黑色素瘤模型）中的性能。我们将研究两种给药途径（皮下和肿瘤内），并在肿瘤细胞注射后第7天和第14天给予两个剂量。在某些时间点，将通过分析血液样本和采集的脾脏来评价DDS性能。这将在两种状态下进行：给药后的非活性DDS和肿瘤部位暴露于超声后的活化DDS。最后，将在治疗期间测量和评估肿瘤大小。

This project aims to develop a novel advanced drug delivery system (DDS), to be used for cancer immunotherapy. This DDS will be responsive to ultrasound. Ultrasound is the trigger of choice due to many advantages over other types of stimuli, such as deeper penetration ability and minimal invasiveness. The drugs to be delivered are short single stranded DNA oligomers, named CpG oligodeoxynucleotides (CpG ODNs). These ODNs are recognized by Toll-like Receptor 9 (TLR9) in certain types of immune cells (i.e. antigen presenting cells), which in turn activate T-cells to become cytotoxic and exert anticancer activity to eliminate tumour cells. Towards this end, there are three key objectives. First is the fabrication of the DDS. The vehicle that will carry and protect the ODNs is based on a hydrogel composed of long polymers of single stranded DNA (ssDNA), which can be engineered to have multiple functionalities. The ssDNA will be produced enzymatically using a process known as Rolling Circle Amplification (RCA). The product undergoes a process of liquid crystallization that yields unique 3D structures in the shape of flowers, with diameters in the nano- and the micrometre size range. Those nanoflowers have the capacity to hybridize and bind to ODNs and hold them in the inactive state until being activated by ultrasound. Ultrasound produces cavitation bubbles in solution, which collapse and exert shear forces that result in polymer fragmentation. At this stage, we aim to optimize the DDS binding capacity and responsiveness to ultrasound in different frequency and power ranges. Second, the DDS will be tested in-vitro and ex-vivo. For in-vitro testing, we will use a genetically engineered reporter cell line that expresses TLR9 exclusively among the TLR family proteins. The DDS will be tested in its inactive state prior to sonication, and then in its activated state after sonication. After that, the system will be tested ex-vivo, using primary immune cells isolated from human donors’ blood samples, such as macrophages and dendritic cells. The aim at this stage is to demonstrate the activation of these cells by the ultrasound-triggered DDS, then their capability of activating T-cells (also isolated from human blood samples), which in turn produce the desired anticancer activity. Finally, we aim to evaluate the DDS performance in animal models in-vivo (i.e. mice B16 melanoma model). We will study two routes of administration (subcutaneous and intra-tumour), and two doses will be administered at days 7 and 14 after tumour cell injection. At certain time points, the DDS performance will be evaluated by analysis of blood samples and harvested spleens. This will be done for the two states: the inactive DDS after administration, and the activated DDS after exposure to ultrasound at the tumour site. Finally, the tumour size will be measured and evaluated during treatment.