Deng Siyuan, Iscaro Alessandra, Zambito Giorgia, Mijiti Yimin, Minicucci Marco, Essand Magnus, Lowik Clemens, Muthana Munitta, Censi Roberta, Mezzanotte Laura, Di Martino Piera
School of Pharmacy, University of Camerino, Via S. Agostino 1, 62032 Camerino, Italy.
Medical School, University of Sheffield, Beech Hill Road, Sheffield S10 2RX, UK.
Nanomaterials (Basel). 2021 Jan 8;11(1):144. doi: 10.3390/nano11010144.
Oncolytic viruses (OVs) are emerging as promising and potential anti-cancer therapeutic agents, not only able to kill cancer cells directly by selective intracellular viral replication, but also to promote an immune response against tumor. Unfortunately, the bioavailability under systemic administration of OVs is limited because of undesired inactivation caused by host immune system and neutralizing antibodies in the bloodstream. To address this issue, a novel hyaluronic acid based redox responsive nanohydrogel was developed in this study as delivery system for OVs, with the aim to protect the OVs following systemic administration. The nanohydrogel was formulated by water in oil (W/O) nanoemulsion method and cross-linked by disulfide bonds derived from the thiol groups of synthesized thiolated hyaluronic acid. One DNA OV Ad[I/PPT-E1A] and one RNA OV Rigvir ECHO-7 were encapsulated into the developed nanohydrogel, respectively, in view of their potential of immunovirotherapy to treat cancers. The nanohydrogels showed particle size of approximately 300-400 nm and negative zeta potential of around -13 mV by dynamic light scattering (DLS). A uniform spherical shape of the nanohydrogel was observed under the scanning electron microscope (SEM) and transmission electron microscope (TEM), especially, the successfully loading of OV into nanohydrogel was revealed by TEM. The crosslinking between the hyaluronic acid chains was confirmed by the appearance of new peak assigned to disulfide bond in Raman spectrum. Furthermore, the redox responsive ability of the nanohydrogel was determined by incubating the nanohydrogel into phosphate buffered saline (PBS) pH 7.4 with 10 μM or 10 mM glutathione at 37 °C which stimulate the normal physiological environment (extracellular) or reductive environment (intracellular or tumoral). The relative turbidity of the sample was real time monitored by DLS which indicated that the nanohydrogel could rapidly degrade within 10 h in the reductive environment due to the cleavage of disulfide bonds, while maintaining the stability in the normal physiological environment after 5 days. Additionally, in vitro cytotoxicity assays demonstrated a good oncolytic activity of OVs-loaded nanohydrogel against the specific cancer cell lines. Overall, the results indicated that the developed nanohydrogel is a delivery system appropriate for viral drugs, due to its hydrophilic and porous nature, and also thanks to its capacity to maintain the stability and activity of encapsulated viruses. Thus, nanohydrogel can be considered as a promising candidate carrier for systemic administration of oncolytic immunovirotherapy.
溶瘤病毒正作为有前景且具潜力的抗癌治疗药物崭露头角，它不仅能够通过选择性细胞内病毒复制直接杀死癌细胞，还能促进针对肿瘤的免疫反应。不幸的是，由于宿主免疫系统和血液中的中和抗体导致的非预期失活，溶瘤病毒全身给药时的生物利用度有限。为解决这一问题，本研究开发了一种新型的基于透明质酸的氧化还原响应纳米水凝胶作为溶瘤病毒的递送系统，旨在保护全身给药后的溶瘤病毒。该纳米水凝胶通过油包水（W/O）纳米乳液法制备，并由合成的硫醇化透明质酸的硫醇基团衍生的二硫键交联。鉴于其免疫病毒疗法治疗癌症的潜力，将一种DNA溶瘤病毒Ad[I/PPT-E1A]和一种RNA溶瘤病毒Rigvir ECHO-7分别封装到所开发的纳米水凝胶中。通过动态光散射（DLS）测定，纳米水凝胶的粒径约为300 - 400 nm，zeta电位约为 - 13 mV。在扫描电子显微镜（SEM）和透射电子显微镜（TEM）下观察到纳米水凝胶呈均匀球形，特别是TEM显示溶瘤病毒成功负载到纳米水凝胶中。拉曼光谱中出现归属于二硫键的新峰，证实了透明质酸链之间的交联。此外，通过将纳米水凝胶在37℃下于pH 7.4的磷酸盐缓冲盐水（PBS）中与10 μM或10 mM谷胱甘肽孵育来测定纳米水凝胶的氧化还原响应能力，谷胱甘肽可模拟正常生理环境（细胞外）或还原环境（细胞内或肿瘤内）。通过DLS实时监测样品的相对浊度，结果表明纳米水凝胶在还原环境中由于二硫键的断裂可在10小时内迅速降解，而在正常生理环境中5天后仍保持稳定。此外，体外细胞毒性试验表明负载溶瘤病毒的纳米水凝胶对特定癌细胞系具有良好的溶瘤活性。总体而言，结果表明所开发的纳米水凝胶因其亲水性和多孔性，以及维持封装病毒稳定性和活性的能力，是一种适用于病毒药物的递送系统。因此，纳米水凝胶可被视为溶瘤免疫病毒疗法全身给药的有前景的候选载体。
