Targeted Nanosyringe Delivery of miRNA Analogs to Subcutaneous Adipocytes for Treatment of Obesity

摘要 人类肥胖已经达到了大流行的程度，影响到了所有年龄和社会经济群体， 超过15亿超重的成年人，其中至少有5亿人肥胖。1目前的医学和行为 肥胖症的治疗未能实现其长期治疗目标。因此，迫切需要 寻找治疗肥胖症的新方法。在这种情况下，一种药理作用的可能性 将皮下脂肪中的白色脂肪组织转换为棕色脂肪组织以燃烧卡路里 对于肥胖的临床治疗很有吸引力，动物模型已经令人信服地证明了这一点。 MicroRNAs(MiRNAs)代表了人类疾病的下一波突破性治疗。激动剂 MiRNAs(Agomir)和拮抗剂(Antagomir)被开发用于治疗各种人类疾病。为 例如，miR-122抗凝剂正在进行血脂异常和丙型肝炎的临床测试，miR-34抗凝剂正在进行临床试验 肺部和其他实体肿瘤的临床检测。 为了解决基于miRNA的肥胖症治疗的非靶点效应和长期安全性这一关键问题， 它们的靶向传递到脂肪细胞的胞浆(miRNAs的作用部位)是必不可少的。这样做的目的是 该项目将完成一个用于交付的工作原型，并在体内展示创新的 以皮下脂肪细胞为靶点的microRNA治疗人类肥胖。 我们提出了一种治疗性纳米载体，它能够分子靶向白色脂肪细胞，传递 MiRNA类似物，并利用光触发它们进入细胞质。我们的激光引发纳米注射器(LINS) MiRNA传递平台基于液体全氟碳(PFC)核心，与miRNA类似物和 一种近红外吸收染料。使用靶向适配子可以结合的脂类来稳定表面。 LINS通过与白色脂肪细胞表面生物标记物结合来发挥作用。使用短脉冲激光进行照射 导致染料吸收并触发PFC核心的汽化事件，其作用类似于纳米注射器， 瞬间打开细胞膜，将miRNA直接输送到细胞质中。我们之前已经 证明了LINS平台在没有miRNA负载的情况下是生物相容的，并且需要光来 脉冲功率因数远低于ANSI的安全激光通量限制。爆发事件是温和的和高度瞬变的， 使细胞存活并完好无损。这种按需将纳米材料输送到细胞质的创新工具可能具有 肥胖症治疗药物本地化的范式转换效应。 我们将通过针对白色脂肪组织(WAT)和 提供一种治疗性的miRNA类似物。MiRNA加载和释放，确认传递到胞浆， 将对粒子稳定性和细胞靶向性进行评估。总之，LINS平台具有令人难以置信的多功能性， 能够靶向WAT、miRNA传递和局部治疗。 1 The，G.B.D.O.C.等人。年全球、区域和国家超重和肥胖率 1980-2013年儿童和成人：系统分析。《柳叶刀》(英国伦敦)384,766-781， DOI：10.1016/S0140-6736(14)60460-8(2014年)。 纳米杂化公司的专利和保密性质

ABSTRACT Human obesity has reached pandemic proportions, affecting all ages and socioeconomic groups, with more than 1.5 billion overweight adults and at least 500 million of them obese.1 Current medical and behavioral treatments of obesity fail to achieve their long-term therapeutic goals. Consequently, there is a pressing need for novel therapeutic approaches to obesity treatment. In this context, the possibility of a pharmacological switch (“Browning Effect”) from white to brown adipose tissue in subcutaneous fat to burn calories is highly attractive for the clinical management of obesity, as it has been convincingly demonstrated in animal models. MicroRNAs (miRNAs) represent the next wave of breakthrough therapies for human diseases. Agonists (agomirs) and antagonists (antagomirs) of miRNAs are developed to treat various human diseases. For instance, miR-122 antagomirs are in clinical testing for dyslipidemia and hepatitis C and miR-34 agomirs are in clinical testing for lung and other solid tumors. To address the critical issue of off-target effects and long-term safety of miRNA-based therapies for obesity, their targeted delivery to the cytosol of adipocytes (the action site of miRNAs) is essential. The objective of this project is to complete a working prototype for delivery and demonstrate in vivo efficacy for an innovative microRNA-based therapy of human obesity targeting subcutaneous adipocytes. We propose a therapeutic nanocarrier that is capable of molecularly targeting white adipocyte cells, delivering miRNA analogs, and triggering their transit into the cytosol using light. Our Laser Initiated Nanosyringe (LINs) miRNA delivery platform is based on a liquid perfluorocarbon (PFC) core co-loaded with a miRNA analog and a near infrared absorbing dye. The surface is stabilized using lipids to which targeting aptamers can be bound. LINs works by binding to a white adipocyte cell surface biomarker. Irradiation using short pulses of laser light causes absorption by the dye and triggers a vaporization event of the PFC core that acts like a nanosyringe, transiently opening the cell membrane and delivering miRNA directly to the cytosol. We have previously demonstrated that the LINs platform, without miRNA loading, is biocompatible and that the light needed to burst the PFC is well below ANSI safe laser fluence limits. The bursting event is gentle and highly transient, leaving the cell viable and intact. This innovative tool for nano-delivery to the cytosol on demand could have a paradigm shifting effect on localized delivery of therapeutics for obesity treatment. We will demonstrate the efficacy of LINs as a platform technology by targeting white adipose tissue (WAT) and delivering a therapeutic miRNA analog. MiRNA loading and release, confirmation of delivery to the cytosol, particle stability and cell targeting will be evaluated. In summary, the LINs platform is incredibly versatile, enabling targeting of WAT, miRNA delivery, and localized therapy. 1 The, G. B. D. O. C. et al. Global, regional and national prevalence of overweight and obesity in children and adults 1980-2013: A systematic analysis. Lancet (London, England) 384, 766-781, doi:10.1016/S0140-6736(14)60460-8 (2014). Proprietary and Confidential Property of NanoHybrids Inc