Non-viral nitric oxide-based gene therapy improves perfusion and liposomal doxorubicin sonopermeation in neuroblastoma models.
Non-viral nitric oxide-based gene therapy improves perfusion and liposomal doxorubicin sonopermeation in neuroblastoma models.
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DOI：
10.7150/thno.81700
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发表时间：
2023
期刊：
影响因子：
12.4
通讯作者：
Sirsi SR
中科院分区：
文献类型：
--
作者：
Bellary A;Nowak C;Iwanicki I;Flores-Guzman F;Wu L;Kandel JJ;Laetsch TW;Bleris L;Hernandez SL;Sirsi SR
神经母细胞瘤（NB）是一种儿科恶性肿瘤，占癌症相关儿童死亡率的15%。高风险NB需要积极的放化疗方案，导致显著的脱靶毒性。尽管有这种侵入性治疗，许多患者要么复发，要么没有充分的反应。最近的研究表明，改善肿瘤灌注可以增强肿瘤组织内的药物积累和分布，潜在地增强治疗效果而不造成全身毒性。因此，在治疗之前瞬时增加肿瘤灌注的方法可能有助于对抗这种疾病。在这里，我们展示了基因治疗的使用，使诱导型一氧化氮合酶（iNOS）的表达仅在肿瘤空间，使用聚焦超声靶向。NOS催化产生一氧化氮（NO）的反应，一氧化氮是一种有效的内源性血管扩张剂。本研究报告了一种靶向非病毒图像引导平台的开发，该平台将iNOS表达质粒DNA（pDNA）递送至包裹肿瘤血管的血管内皮细胞。转染后，纵向定量对比增强超声（qCEUS）成像显示肿瘤灌注增加超过72小时，这归因于肿瘤内iNOS表达升高。研究方法：为了构建基因递送载体，使用阳离子超声响应剂（称为“微泡”）在循环中携带pDNA，并使用聚焦超声（FUS）能量在体内转染肿瘤血管内皮细胞。随后进行脂质体阿霉素（L-DOX）治疗。使用qCEUS成像纵向监测转染后肿瘤反应，以确定血容量和灌注速率的相对变化。治疗后，进行肿瘤的离体分析以检查与iNOS表达相关的生物效应。结果如下：通过将FUS疗法与阳离子超声造影剂（UCA）相结合，我们实现了编码iNOS酶的pDNA的选择性肿瘤内转染。虽然是暂时的，但表达程度足以诱导肿瘤灌注的显著增加，明显增强化疗有效负荷并延长原位异种移植模型中的存活时间。结论：我们已经证明了一种新的靶向非病毒基因治疗策略能够增强肿瘤灌注并改善L-DOX向NB异种移植物的递送。虽然我们的研究结果表明，短暂增加肿瘤灌注改善脂质体包封的化疗药物的摄取和分布，我们预计，我们的iNOS基因递送模式也可以显着改善放射和免疫治疗，通过增加放射增敏剂和免疫调节剂的交付，可能改善目前的NB治疗没有伴随的不良反应。我们的研究结果进一步表明，qCEUS成像可以有效地监测体内肿瘤灌注的变化，从而确定理想的治疗时间点。
Neuroblastoma (NB) is a pediatric malignancy that accounts for 15% of cancer-related childhood mortality. High-risk NB requires an aggressive chemoradiotherapy regimen that causes significant off-target toxicity. Despite this invasive treatment, many patients either relapse or do not respond adequately. Recent studies suggest that improving tumor perfusion can enhance drug accumulation and distribution within the tumor tissue, potentially augmenting treatment effects without inflicting systemic toxicity. Accordingly, methods that transiently increase tumor perfusion prior to treatment may help combat this disease. Here, we show the use of gene therapy to confer inducible nitric oxide synthase (iNOS) expression solely in the tumor space, using focused ultrasound targeting. NOS catalyzes the reaction that generates nitric oxide (NO), a potent endogenous vasodilator. This study reports the development of a targeted non-viral image-guided platform to deliver iNOS-expressing plasmid DNA (pDNA) to vascular endothelial cells encasing tumor blood vessels. Following transfection, longitudinal quantitative contrast-enhanced ultrasound (qCEUS) imaging revealed an increase in tumor perfusion over 72 h, attributed to elevated intratumoral iNOS expression. Methods: To construct a gene delivery vector, cationic ultrasound-responsive agents (known as “microbubbles”) were employed to carry pDNA in circulation and transfect tumor vascular endothelial cells in vivo using focused ultrasound (FUS) energy. This was followed by liposomal doxorubicin (L-DOX) treatment. The post-transfection tumor response was monitored longitudinally using qCEUS imaging to determine relative changes in blood volumes and perfusion rates. After therapy, ex vivo analysis of tumors was performed to examine the bioeffects associated with iNOS expression. Results: By combining FUS therapy with cationic ultrasound contrast agents (UCAs), we achieved selective intratumoral transfection of pDNA encoding the iNOS enzyme. While transitory, the degree of expression was sufficient to induce significant increases in tumoral perfusion, to appreciably enhance the chemotherapeutic payload and to extend survival time in an orthotopic xenograft model. Conclusion: We have demonstrated the ability of a novel targeted non-viral gene therapy strategy to enhance tumor perfusion and improve L-DOX delivery to NB xenografts. While our results demonstrate that transiently increasing tumor perfusion improves liposome-encapsulated chemotherapeutic uptake and distribution, we expect that our iNOS gene delivery paradigm can also significantly improve radio and immunotherapies by increasing the delivery of radiosensitizers and immunomodulators, potentially improving upon current NB treatment without concomitant adverse effects. Our findings further suggest that qCEUS imaging can effectively monitor changes in tumor perfusion in vivo, allowing the identification of an ideal time-point to administer therapy.
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