From the outside in: Modular bespoke artificial cells for drug discovery

我建议通过建立人工细胞作为生物分析工具来量化进出细胞的运输，从而在药物发现过程的早期测试方式上创造一个范式转变。我的研究提出了一种方法，可以自下而上地构建定制的人造细胞，其中包含主要的基于膜的细胞成分和细胞器，以研究分子运输。通过允许同时量化被动和主动分子传输，这有可能成为第一个可以用来回答药物发现中这一基本问题的技术。在过去的五年里，我的研究小组开发了一种独特的新方法，通过在微流体平台上构建定制的模仿人类的人造细胞膜，来量化分子如何进入细胞。我们开发了微流控技术，允许基于由人类肠道细胞中发现的主要磷脂制成的液滴界面双层(DIB)形成细胞大小的人造细胞膜。我们使用该平台创建了一种新型的药动学模型，并表明DIBS在预测分子吸收方面比商业体外技术提高了三倍。由于人们对哪些不同类型的脂质可用于构建DIBS知之甚少，因此我们研究了温度对这些基于脂质的人造细胞膜形成的影响，以使我们能够从广泛的磷脂中创建更多的仿生DIBS。然后，我们构建了不对称的DIBS，以模拟组成细胞膜的分子的结构特征。我们量化了分子的运输如何受到膜分子不对称性变化的影响，潜在地突出了一种新的化疗耐药机制。我们的工作表明，我们定制的人造膜将使我们能够发现细胞生物学的新方面，以及它们如何影响分子运输。在接下来的五年里，我们将把剩余的基于膜的成分添加到我们的人造细胞中。我们将利用这些系统对不同的膜成分对分子被动膜通透性的影响进行基础研究。这些包括脂域的形成，以及性别对通透性的影响。然后，我们将插入人膜转运蛋白，以能够量化主动转运。第三，由于细胞中的每个隔室由膜划定，我们将使用脂质体在基于液滴界面双层(DIB)的人工细胞内模拟这些隔膜。最后，我们将开发检测方法，使我们能够准确地量化药物在分隔的人工细胞的每个位置的转运动力学。这项研究将为该领域提供一种新的技术，以预测分子运输是通过被动机制还是主动机制发生的，这是药物发现中的一个基本科学问题。这将使加拿大能够更快、更便宜地开发药物。

I propose to create a paradigm shift in the way that drugs are tested early in the discovery process by building artificial cells as bio-analytical tools to quantify transport into and out of cells. My research proposes a way to build bespoke artificial cells from the bottom up that contain the major membrane-based cellular components and organelles to study molecular transport. By allowing both passive and active molecular transport to be quantified at the same time, this has the potential to be the first technique that can be used to answer this fundamental question in drug discovery. Over the last five years my research group have developed a unique new method to quantify how molecules enter cells by building custom-made human-mimetic artificial cell membranes on a microfluidic platform. We developed microfluidic technologies that allow the formation of cell-sized artificial cell membranes based on droplet interface bilayers (DIBs) made from the main phospholipids found in human intestinal cells. We used the platform to create a new type of pharmacokinetic compartment model and show that DIBs have a threefold improvement in the prediction of molecular absorption than the commercial in vitro technique. Since little is known about what different types of lipids can be used to build DIBs, we then investigated the effect of temperature on the formation of these lipid-based artificial cell membranes to enable us to create more biomimetic DIBs from a wide range of phospholipids. Then we built asymmetric DIBs to mimic a structural feature of the molecules that compose the cell membrane. We quantified how the transport of molecules is affected by the changes the molecular asymmetry of membranes, potentially highlighting a new mechanism for chemoresistance. Our work suggests that our bespoke artificial membranes will allow us to discover new aspects of cell biology and how they affect molecular transport. In the next five years, we will add the remaining membrane-based components to our artificial cells. We will use these systems to perform fundamental studies into the effect that different membrane components have on molecular passive membrane permeability. These include lipid domain formation, and the effect of sex on permeability. Then, we will insert human membrane transport proteins to be able to quantify active transport. Thirdly, since each compartment in a cell is delineated by a membrane, we will mimic these inside our droplet interface bilayer (DIB)-based artificial cells using liposomes. Finally, we will develop detection methods that allow us to quantify the kinetics of drug transport accurately in each location of the compartmentalised artificial cells. This research will significantly contribute to the field by providing a new technology to predict whether molecular transport occurs via passive or active mechanisms, which is a fundamental scientific question in drug discovery. This will allow Canada to develop drugs faster and cheaper.