Controlled Vesicle Self-Assembly in Continuous

Two Phase Flow Microfluidic Channels

      This application claims priority to U.S. Provisional Application No. 60/525,355, filed November 26, 2003.

      Methods for the formation of liposomes that encapsulate reagents in a continuous 2-phase flow microfluidic network with precision control of size, for example, from 100 nm to 300 nm, by manipulation of liquid flow rates are described.   By creating a solvent-aqueous interfacial region in a microfluidic format that is homogenous and controllable on the length scale of a liposome, fine control of liposome size and polydispersity can be achieved.  Traditional liposome preparation methods are based on mixing of bulk phases, leading to inhomogeneous chemical and/or mechanical conditions during formation; hence liposomes prepared by those methods are often polydisperse in size and lamellarity.  

      There are a growing number of applications for nanoscale particles in biology that include interrogating (see, for example: E. J. Park, M. Brasuel, C. Behrend, Anal. Chem. 75, 3784 (2003); A. S. Arbab, L. A. Bashaw, B. R. Miller BR, Transplantation  76, 1123 (2003); M. E. Akerman, W. C. W. Chan, P. Laakkonen, Proc. Natl. Acad. Sci. U.S.A. 99, 12617 (2002); M. Bruchez, M. Moronne, P. Gin P, Science 281, 2013 (1998); W. C. W. Chan, S. M. Nie, Science 281, 2016 (1998); B. Dubertret, P. Skourides, D. J. Norris, Science 298, 1759 (2002)), perturbing (see, for example: H. E. Sparrer, A. Santoso, F. C. Szoka, Science 289, 595 (2000); and I. Koltover, T. Salditt, J. O. Radler, Science 281, 78 (1998)) and stimulating (see, for example: A. K. Salem, P. C. Searson, K. W. Leong, Nat. Mater. 2, 668 (2003)) the cellular environment.  The design and production of nanometer scale objects, such as quantum dots, colloidal particles, and vesicles, can be accomplished in bulk either by chemical synthesis or self-assembly processes.  In the cellular factory, chemical synthesis and self-assembly processes are exquisitely controlled by the closely-regulated local environment to ensure the reproducible production of nanometer-scale components such as proteins and vesicles.   In bulk production methods, the local environment is not well controlled leading to significant chemical fluctuations, or electrical, mechanical perturbations that often result in inhomogeneous populations of nanoparticles. 

      Liposomes (see, e.g., A. D. Bangham, M. M. Standish, J. C. Watkins, J. Mol. Biol. 13, 238 (1965)) are one example of nanoparticles that have been used for a wide variety of biological applications including targeted drug delivery and DNA transfection (see, e.g.: G. Gregoriadis, Liposome Technology Volume 3; Targeted Drug Delivery and Biological Interactions (CRC Press, Boca Raton, 1983); and D. D. Lasic, D. Papahadjopoulos, Science 267, 1275 (1995)).  Liposomes are cellular mimetics composed of a lipid bilayer membrane that encapsulates and sequesters species inside from species residing outside the membrane.  Of critical importance to the successful implementation of liposomes in vivo is the ability to control the liposome size and size distribution, as size influences the clearance rate from the body and ultimately determines the drug dosage.  Conventional modes of liposome preparation require the mixing of two or more phases, typically liquid-liquid or liquid-solid, resulting in the spontaneous self-assembly of the lipid mixture into a spherical bilayer membrane (see, e.g.: G. Gregoriadis, H. da Silva, A. T. Florence, Int. J. Pharm. 65, 235 (1990); F. C. Szoka, D. Papahadjopoulos, Proc. Natl. Acad. Sci. U.S.A. 75, 4194 (1978); C. Pidgeon, S. McNeely, T. Schmidt, Biochem. 26, 17 (1987); H. Hauser, Biochim. Biophys. Res. Commun. 45, 1049 (1971); S. Batzri, E. D. Korn, Biochem. Biophys. Acta. 298, 1015 (1973); T. H. Fischer, D. D. Lasic, Mol. Cryst. Liq. Cryst. Lett. 102, 144 (1984); H. Kikuchi, H. Yamauchi, S. Hirota, Chem. Pharm. Bull. 39, 1522 (1991); A. Wagner, K. Uhl-Vorauer, G. Kreismayer, J. Lip. Res. 12, 259 (2002); T. S. Aurora, W. Li, H. Z. Cummins, Biochimica et Biophysica Acta 820, 250 (1985); P. L. Luisi, P. Walde, Giant Vesicles (John Wiley & Sons, Chichester, 2000)). These self-assembly processes typically occur in a system with a characteristic length on the order of centimeters, resulting in chemical and/or mechanical conditions that are highly heterogeneous on the length scale of a liposome.  Thus, a given liposome may experience any one of many different sets of mechanical and chemical conditions during its self-assembly, often leading to liposome preparations with large polydispersity with respect to size and lamellarity.

      To best mimic biological systems, it is desirable to create environments that are controll