Targeted drug delivery to neurons and glia using light- and field-sensitive microcapsules

靶向给药在实验和转化生物医学研究中一直是一个高度优先的问题。最近在生物纳米技术方面的突破为这一领域打开了新的机会，促使生物学家、医生和企业界迅速增长的兴趣。这一领域的一个主要挑战是开发将不同功能合并到一个工程实体中的交付系统。这种系统应能够将生物活性物质运送到目标地点，并以空间和时间受控方式释放它们。我们一直在关注3D微胶囊系统，因为它们能够包含广泛的化学物质，可调节的膜渗透性和对不同刺激(pH、温度、光、渗透压等)的敏感性，所有这些都结合在一个实体中，最近引起了人们的强烈关注。这种纳米技术已被仔细调整，以适应特定的细胞系统和组织，允许受控的货物释放、有效载荷保护、空间操作以及与活组织的完全兼容。所有这些功能原则上都可以通过我们目前开发的封装技术来实现。除了本身的药物输送，我们的微米大小的胶囊可以为在有限的空间中模拟生化过程提供一个很好的实验模型，例如模拟细胞细胞器。事实上，当这些胶囊具有特定的膜特性时，可以作为长期的细胞内报告程序或酶反应装置，可以嵌入组织或细胞中。尽管取得了这些突破，但迄今为止还没有人尝试将这种方法应用于神经细胞或脑组织的研究。将荧光染料、毒素、受体配体或蛋白质材料输送到靶神经或神经胶质细胞一直是神经生物学和神经学研究中的关键因素。然而，到目前为止，在空间和时间上以高度受控的方式实现这种传递是可能的，只有使用微注射或电穿孔等侵入性的“一次”方法。因此，拟议的项目提供了一个独特的机会，可以将多功能封装的方法学突破与将这种方法应用于神经组织的明确实验(以及潜在的翻译)需求结合起来。伦敦大学学院的团队是英国首批将膜片钳电生理学与双光子激发成像结合在一起的团队之一，后来又将双光子植入有组织的脑组织，积累了突触和大脑回路的生理学和生物物理学方面的国际公认的专业知识。QMUL小组最近展示了使用聚电解质多层微胶囊在细胞内传递各种有效载荷，以及使用可见光和红外激光辐射远程释放货物。因此，这两个团队在测试和实施这项新技术方面都处于独特的地位，这可能会给生物医学研究带来革命性的变化，也为临床应用提供了无数机会。因此，我们计划开发合成微囊的方案，优化为在神经元胶质细胞培养和有组织的脑组织中成功传递、跟踪和激光触发的开放。我们将优化靶细胞和细胞群体内外的包膜有效载荷释放的效率、动态范围和动力学。我们将在神经生物学环境中通过实时监测药物应用对常见神经生物学任务的生理影响来测试所提出的方法：(A)神经递质的细胞外递送，(B)神经元内的靶向配体递送，(B)神经胶质内的靶向毒素递送。实现我们的目标不仅将为神经生物学实验探索的新维度铺平道路，也将为药物输送的临床相关应用奠定基础。

Targeted drug delivery has been a high-priority issue in both experimental and translational biomedical research. Recent breakthroughs in bionanotechnology have opened new opportunities in this area, prompting a rapidly growing interest among biologists, medical practitioners and business community. A major challenge in this area has been to develop delivery systems that would incorporate different functionalities in one engineered entity. Such system should enable to carry bioactive substances to a target site and release them in a space- and time-controlled fashion. We have been focusing on 3D microcapsule systems that have recently attracted intense attention owing to their ability to contain a wide range of chemicals, the adjustable membrane permeability and sensitivity to different stimuli (pH, temperature, light, osmolarity, etc), all combined in one entity. This nano-technology has be carefully adapted for particular cell systems and tissues, by allowing controlled cargo release, payload protection, manipulation in space and full compatibility with live tissue. All these functions can be achieved in principle by the encapsulation technology have we have developed to date. In addition to drug delivery per se, our micron-sized capsules could provide an excellent experimental model for mimicking biochemical processes in a confined space, such as imitating cell organelles. Indeed, when equipped with specific membrane properties these capsules can serve as a long-term intracellular reporter or enzymatic reactor which can be embedded in tissues or cells. Notwithstanding these breakthroughs, there have been no attempts to date to adapt this methodology to studies of nerve cells or brain tissue. Delivery of fluorescent dyes, toxins, receptor ligands, or protein material to target nerve or glial cells has been a critical element in both neurobiological and neurological research. However, achieving this delivery in a highly controlled manner in space and time has hitherto been possible only using invasive, 'one-shot' methods such as microinjection or electroporation. The proposed project provides therefore a unique opportunity to combine a methodological breakthrough in multifunctional encapsulation with a clear experimental (and potentially translational) demand for adapting this approach to nervous tissue. The UCL group was among the first in the UK to combine patch-clamp electrophysiology with two-photon excitation imaging and later with two-photon uncaging in organised brain tissue, accumulating internationally recognised expertise in physiology and biophysics of synapses and brain circuits. The QMUL group has recently demonstrated intracellular delivery of various payloads using polyelectrolyte multilayer microcapsules and remote cargo release using visible and infrared laser radiation. Both teams are therefore uniquely positioned for testing and implementation of this new technique, which could potentially revolutionise bio-medical research also offering numerous opportunities for clinical applications. We therefore plan to develop protocols of synthetic microcapsulation optimised for successful delivery, tracking and laser-triggered opening in neuron-glial cultures and organised brain tissue. We will optimise efficiency, dynamic range and kinetics of capsular payload release inside and outside target cells and cell populations. We will test the proposed methodology in neurobiological environment, by monitoring physiological effects of drug application in real time for common neurobiological tasks: (a) extracellular delivery of neurotransmitter, (b) targeted ligand delivery inside neurons, (b) targeted toxin delivery inside glia. Achieving our objectives will not only pave the way for a new dimension of experimental probing in neurobiology, but also lay the foundation for clinically relevant applications in drug delivery.