Since the recent clinical approval of siRNA-based drugs and COVID-19 mRNA vaccines, the potential of RNA therapeutics for patient healthcare has become widely accepted. Lipid nanoparticles (LNPs) are currently the most advanced nanocarriers for RNA packaging and delivery. Nevertheless, the intracellular delivery efficiency of state-of-the-art LNPs remains relatively low and safety and immunogenicity concerns with synthetic lipid components persist, altogether rationalizing the exploration of alternative LNP compositions. In addition, there is an interest in exploiting LNP technology for simultaneous encapsulation of small molecule drugs and RNA in a single nanocarrier. Here, we describe how well-known tricyclic cationic amphiphilic drugs (CADs) can be repurposed as both structural and functional components of lipid-based NPs for mRNA formulation, further referred to as CADosomes. We demonstrate that selected CADs, such as tricyclic antidepressants and antihistamines, self-assemble with the widely-used helper lipid DOPE to form cationic lipid vesicles for subsequent mRNA complexation and delivery, without the need for prior lipophilic derivatization. Selected CADosomes enabled efficient mRNA delivery in various in vitro cell models, including easy-to-transfect cancer cells (e.g. human cervical carcinoma HeLa cell line) as well as hard-to-transfect primary cells (e.g. primary bovine corneal epithelial cells), outperforming commercially available cationic liposomes and state-of-the-art LNPs. In addition, using the antidepressant nortriptyline as a model compound, we show that CADs can maintain their pharmacological activity upon CADosome incorporation. Furthermore, in vivo proof-of-concept was obtained, demonstrating CADosome-mediated mRNA delivery in the corneal epithelial cells of rabbit eyes, which could pave the way for future applications in ophthalmology. Based on our results, the co-formulation of CADs, helper lipids and mRNA into lipid-based nanocarriers is proposed as a versatile and straightforward approach for the rational development of drug combination therapies.

自从最近基于 siRNA 的药物和 COVID-19 mRNA 疫苗获得临床批准以来，RNA 疗法在患者医疗保健中的潜力已被广泛接受。脂质纳米粒（LNPs）是目前最先进的用于RNA包装和递送的纳米载体。尽管如此，最先进的 LNP 的细胞内递送效率仍然相对较低，并且对合成脂质成分的安全性和免疫原性问题仍然存在，从而使替代 LNP 组合物的探索合理化。此外，利用 LNP 技术将小分子药物和 RNA 同时封装在单个纳米载体中也引起了人们的兴趣。在这里，我们描述了众所周知的三环阳离子两亲药物 (CAD) 如何重新用作基于脂质的 NPs 的结构和功能成分，用于 mRNA 制剂，进一步称为 CADosomes。我们证明，选定的 CAD，如三环类抗抑郁药和抗组胺药，与广泛使用的辅助脂质 DOPE 自组装形成阳离子脂质囊泡，用于随后的 mRNA 复合和递送，无需事先进行亲脂衍生。选定的 CADosomes 在各种情况下实现了高效的 mRNA 递送体外细胞模型，包括易于转染的癌细胞（例如人宫颈癌 HeLa 细胞系）以及难以转染的原代细胞（例如原代牛角膜上皮细胞），其性能优于市售的阳离子脂质体和 state of - 最先进的 LNP。此外，使用抗抑郁药去甲替林作为模型化合物，我们表明 CAD 可以在 CADosome 掺入后保持其药理活性。此外，在体内获得了概念验证，证明了兔眼角膜上皮细胞中 CADosome 介导的 mRNA 传递，这可能为未来在眼科中的应用铺平道路。基于我们的研究结果，建议将 CAD、辅助脂质和 mRNA 共同配制到基于脂质的纳米载体中，作为合理开发药物联合疗法的一种通用且直接的方法。