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

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.

Targeted drug delivery has been a high-priority issue in both experimental and translational biomedical research. We propose to develop a novel methodology of highly-targeted, time-controlled delivery of signalling molecules into nerve and glial cells. To achieve this, we will exploit a recent breakthrough in bionanotechnology (co-authored by one of the Applicants) focusing on fabrication of microscopic biocapsules with pre-designed properties and intra-lumen load. Firstly, we will develop protocols of synthetic microcapsulation to optimise geometry and mechanical properties of microcapsules for successful delivery in cultures and organised tissue. Secondly, we will adjust microcapsule sensitivity to magnetic field and light (using established nanoparticle insertion technologies) to enable capsule navigation and laser-triggered payload release in situ. Thirdly, we will establish experimental protocols for loading, tracking and laser-triggered opening of microcapsules in neuron-glia cultures and acute brain slices using established one- and two-photon excitation laser scanning microscopy techniques. Specialised hardware will be designed to navigate magnetic field-sensitive microcapsules in cultures and slices under a microscope. Fourthly, we will combine these methods with patch-clamp electrophysiology and fluorescence imaging to optimise efficiency, dynamic range and kinetics of capsular payload release. Finally, 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 the proposal's 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.

Developing novel ways of targeted drug delivery is an area of intense interest among biomedical researchers, nanobiotechnology experts and medical practitioners. If successful, the present project will therefore benefit a wide range of academic, clinical and technology-oriented research fields as well as related industries and commercial developers. 1. Beneficiaries in research, development and application of nanobiotechnology products: Establishing material-related methodologies, experimental protocols and procedures for designing, fabrication, function-enabling and handling of purpose-built microcapsules compatible with live tissue will have a strong impact on both academic and commercial research and development. Advances in this area of nanobiotechnology will benefit a wide range of biomedical applications dealing with the interface between purpose-designed materials and living tissue. 2. Beneficiaries in areas of academic neuroscience and cell biology research: Delivering microcapsulated cargo of signalling molecules to individual nerve and glial cells will help to provide conceptual breakthroughs to the experimental design in various areas of neuroscience and cell biology. At present, targeted micro-application of signalling molecules or specific ligands is restricted either to a volumetric micro-injection, a single cell held in whole-cell mode, or local photolytic uncaging. Volume injection methods do not confine the desired pharmacological action to an individual cell, whole-cell filling is technically unfeasible to apply repeatedly or across a sample of cells in one experiment, and uncaging has been efficient for a limited number of molecules. These deficiencies will be overcome by the proposed method in which individual microcapsules could be activated by light in individual cells in a space- and time-controlled manner. This methodology opens therefore a new dimension for cutting-edge academic research in basic and applied neurosciences. 3. Beneficiaries in clinical applications and pharmaceutical industry: The methodology we are developing deals with novel ways of delivering pharmacological agents to cellular populations or specific organs for the purposes of therapeutic intervention. The microcapsule delivery system should provide previously unattainable accuracy and time control over drug transport and time-controlled activation benefiting both translational research and pharmaceutical industry. In terms of outcome, our research will provide essential information, test results and feasibility assessment required for successful development of related methodologies in the commercial environment.