The utility of liposomes as functional nanoparticles for biological and biomedical applications is presently limited by the bulk production methods used for their manufacture. For example, although considerable progress has been made towards the commercialization of liposomes as drug delivery vehicles, existing production methods result in polydisperse formulations that exhibit variations in drug encapsulation levels, blood clearance rates, and cell uptake, with negative consequences for drug efficacy and toxicity. Our goal here is to develop a fundamental understanding of the physical processes which drive liposome self-assembly in a new microfluidic process, based on the hydrodynamic focusing of two miscible solvent streams. By taking advantage of the unique interactions that occur at the submicron boundary between the solvent streams, small and uniform unilamellar liposomes may be generated in a simple integrated microfluidic chip. We propose a combined computational and experimental effort to improve our understanding of the liposome selfassembly process within the microfluidic system, and apply this understanding to optimize the process for the integrated and in-line formation of functionalized immunoliposomes.
Intellectual Merit:
The proposed effort is expected to result in three specific advances in the nanoparticle arena, namely (1) theoretical and direct experimental evaluation leading to an improved understanding of the liposome formation process, made possible by the ability of the microfluidic system to precisely specify chemical and molecular distributions within a well-defined laminar mixing zone, (2) a multi-scale model coupling the underlying physics with system-level parameters including channel geometries and flow conditions, and (3) application of this model to demonstrate fully integrated system for the on-demand production of immunoliposomes with minimal polydispersity, and with diameters that may be dynamically tuned by the simple adjustment of on-chip flow conditions. Thus the combined computational and experimental approach will impact our fundamental understanding of the liposome self-assembly process while also leading to the development of a unique and novel tool for controlled liposome and immunoliposome production.
Broader Impact:
The ability to generate liposomes with tunable and narrowly distributed diameters over a wide size range has important implications for and range of biological and biomedical applications. The high throughput process is directly scalable to large volume production of encapsulated drugs, together with in-line decoration of liposomes with antibodies or other ligands for targeted drug delivery. As a result of these features, the method offers great promise as a simple and low-cost approach to the production of personalized drug preparations in point-of-care settings. Beyond drug delivery, homogeneous liposomes are also of great value for application to immunoassays, where controlled signal amplification can only occur when the liposome populations exhibit a narrow size distribution [1]. In this application, fluorescent encapsulants within the immunoliposomes provide signal amplification for each antibody-antigen interaction, enabling highly sensitive detection. The microfluidic system itself offers a potential base for future development of an integrated immunosensor platform employing on-demand formation of immunoliposomes. The technology will find application in a range of biosensing systems, including portable and ultrasensitive quantitative diagnostic tests. Finally, the project will contribute to the education of next-generation students at the graduate, undergraduate, and K-12 levels. The project will provide interdisciplinary training of one Ph.D. Bioengineering student who will receive training across the fields of bioengineering, mechanical engineering, and chemistry, providing a solid bridge across these disciplines. Undergraduate students will also be recruited to particulate in the research project through an established NSF sponsored Molecular and Cellular Bioengineering REU Program, and local high school seniors will participate in selected experimental aspects of the project through an established internship program.
Liposomes are biologically-inspired particles consisting of a two molecule thick membrane of lipids containing a water-filled core. The utility of liposomes as functional nanoparticles for biological and biomedical applications is presently limited by the bulk production methods used for their manufacture. For example, although considerable progress has been made towards the commercialization of liposomes as drug delivery vehicles, existing production methods result in formulations that exhibit wide variations in size, drug encapsulation levels, blood clearance rates, and cell uptake, with negative consequences for drug efficacy and toxicity. In this project we have explored the physical processes which drive liposome self assembly in a new microfluidic process which offers a unique approach to improving nanoparticle preparation. Key outcomes of the effort include: (1) A continuous-flow method for creating functionalized vesicles incorporating polymers and molecules capable of selective anchoring on target tissues. (2) Rapid active encorporate drug molecules into liposome cores with efficiency exceeding that of conventional bulk scale processes in a fraction of the time. (3) Greatly increased liposome production rates enabled by improved numerical models of the process (4) New insights into the central relationships between nanoparticle size and cell uptake which have been previously hidden due to the large size variations of conventional liposomes (5) Demonstration that small and uniform populations of liposomes can transport intact through skin, opening the door to applications in transdermal nanoparticle delivery of targeted liposomal drugs.
