Development of active loading technologies for encapsulating highly charged molecules into liposomes

在接下来的5年里，我将专注于开发新的主动加载技术，将高电荷分子封装到脂质体中，以改善它们的传递。我将重点介绍三种类型的高电荷药物：带正电的小分子、带负电的小分子和带负电的大分子。我将从每个类别中选择一个模型药物，并为每个类别开发一种新的主动加载技术。假设：离子配对剂将中和化合物的电荷，以增加其膜的通透性，并将其加载到脂质体中。*目标1：开发一种主动负载方法，将带正电荷的小分子包裹到脂质体中。我们的模型药物是庆大霉素(GEN)，它有5个正电荷的氨基。我们将开发和优化一种新的制备脂质体GEN的方法，并将其在小鼠体内的药代动力学(PK)与游离GEN和被动加载制备的脂质体GEN进行比较。目的2：建立一种将带负电荷的小分子包裹到脂质体中的主动加载方法。我们的模型药物是氯屈膦酸盐(CLO)，它有两个带负电荷的磷酸基团。脂质体CLO被用来耗尽动物组织中的巨噬细胞以产生研究模型。我们将开发和优化一种制备脂质体CLO的新工艺，并将其与游离CLO和被动负载制备的脂质体CLO在小鼠体内的巨噬细胞清除活性进行比较。*目标3：开发一种主动加载方法，将带负电荷的大分子包裹到脂质体中。我们的模型药物是用于研究基因功能的小干扰核糖核酸(SiRNA)。我们将开发一种离子配对的主动负载方法来制备脂质体siRNA。我们将评估脂质体siRNA与血液成分和细胞的相互作用及其在荷瘤小鼠的生物分布(BD)，并与游离siRNA和阳离子脂质体-siRNA复合体进行比较。短期目标：开发主动加载技术将GEN、CLO和siRNA包裹到脂质体中，以改善它们的递送。长期目标：开发一种平台技术，将各种类型的高电荷药物加载到脂质体中，以改善其PK和BD，从而提高生物利用度，并有可能创造出有效的体内研究工具或药物产品。该项目将为HQP提供全面的培训，包括药物配方设计、制造和表征、体外细胞和分子分析以及体内PK/BD研究。这些知识和技能在制药领域的学术界和工业界非常受欢迎，在我的项目中培训的HQP对这些技术性工作具有很强的竞争力。我已经发表了25篇关于脂质体技术的论文和专利，并向业界授权了2项技术和2项临床试验中的产品。该项目将产生具有很高商业化价值的脂质体工程新技术。**

Over the next 5 years I will focus on developing new active loading technologies to encapsulate highly charged molecules into liposomes to improve their delivery. I will focus on three types of highly charged drugs: positively charged small molecules, negatively charged small molecules and negatively charged macromolecules. I will select one model drug from each class and develop a new active loading technology for each. Hypothesis: An ion-pairing agent will neutralize the charge of a compound to increase its membrane permeability and drive the loading into liposomes.******Obj 1: Develop an active loading method to encapsulate a positively charged small molecule into liposomes. Our model drug is gentamicin (GEN) that has 5 positively charged amino groups. We will develop and optimize a new loading method to prepare liposomal GEN, and will compare its pharmacokinetics (PK) in mice with free GEN and liposomal GEN prepared with passive loading.******Obj 2: Develop an active loading method to encapsulate a negatively charged small molecule into liposomes. Our model drug is clodronate (CLO) that has 2 negatively charged phosphate groups. Liposomal CLO is used to deplete tissue macrophages in animals to generate research models. We will develop and optimize a new loading technology to prepare liposomal CLO, and will compare its macrophage depleting activity in mice with free CLO and liposomal CLO prepared with passive loading. ******Obj 3: Develop an active loading method to encapsulate a negatively charged macromolecule into liposomes. Our model drug is small interfering ribonucleic acid (siRNA) used to study gene function. We will develop an ion-pairing active loading method to fabricate liposomal siRNA. We will assess the interaction of the liposomal siRNA with blood components and cells and its biodistribution (BD) in tumor-bearing mice, and compare with free siRNA and cationic liposome-siRNA complex.******Short term goal: Develop active loading technologies to encapsulate GEN, CLO and siRNA into liposomes to improve their delivery. Long term goal: Develop a platform technology to load various types of highly charged drugs into liposomes to improve their PK and BD for increased bioavailability, with potential to create effective in vivo research tools or pharmaceutical products. This program will offer comprehensive training for HQP, including pharmaceutical formulation design, fabrication and characterization, in vitro cell based and molecular assays and in vivo PK/BD studies. These knowledge and skills are highly sought after in academia and industry in the pharmaceutical area, and HQP trained in my program have been highly competitive for these skilled jobs. I have published >25 papers and patents with liposomal technologies and licensed 2 technologies to industry with 2 products in clinical trials. This program will yield new liposomal engineering technologies with high commercialization values.**