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 solventaqueous 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 closelyregulated 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 

 1 
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 

controllable on the dimension of the particle itself to elicit fine control of nanometer scale 

 2 
synthesis and self-assembly processes. As an aspect of the invention, it was discovered 

that several characteristics of microfluidic systems provide the ability to accomplish 

process control at this level. First, in these microfluidic systems, interfacial forces 

dominate and bulk inertial forces are typically negligible, leading to enhanced heat and 

diffusional mass transfer properties. Second, the laminar flow conditions in microfluidic 

channels can be used to create a well-defined and predictable interfacial region between 

two fluids. This characteristic has in fact been used to focus fluid streams 

hydrodynamically to submicrometer dimensional scales for rapid mixing (see, e.g., J. B. 

Knight, A. Vishwanath, J. P. Brody, Phys. Rev. Lett. 80, 3863 (1998)) and patterning 

(e.g.: P. J. A. Kenis. R. F. Ismagilov, G. M. Whitesides, Science 285, 83 (1999)). These 

properties of microfluidics allow control of chemical processes on nanometer length 

scales that were previously difficult to access experimentally. 

 According to the invention for the formation of liposomes in microfluidic 

systems, the characteristics of fluidic flow in a micrometer-scale channel can be used to 

precisely control the distribution of chemical conditions and mechanical forces so that 

they are constant on a length scale equivalent to that of a liposome. Hence, forming 

liposomes in a micrometer-scale flow field results in more homogenous conditions during 

liposome self-assembly and resultant liposome populations that are more uniform in size, 

hence of low polydispersity. Theoretical analysis of the laminar flow field engendered by 

microchannel flow when three microfluidic channels intersect show that the distribution 

of chemical species within the microfluidic network is constant and predictable (see, e.g., 

Figure 1a, further discussed below). 

 Thus, the invention includes methods for producing a liposome-containing 

composition, which includes: providing a solvent stream of a composition of lipids or 

lipid-forming materials dissolved in a solvent through a central microchannel having a 

hydrodynamic diameter of 100 μm or less, preferably 70 μm or less; and impinging on 

said solvent stream through at least one side microchannel at least one aqueous stream of 

an aqueous composition which hydrodynamically focuses the solvent stream and forms a 

stream within the central microchannel having an interfacial region where the solvent 

stream and the at least one aqueous stream diffuse into each other to provide conditions 

such that liposomes self-assemble from the lipids or lipid-forming materials. In a 

 3 
preferred embodiment, at least two aqueous streams provided by at least two side 

microchannels, which at least partially oppose one another, impinge on the solvent stream 

to hydrodynamically focus it. More preferably, two aqueous streams provided by two 

side microchannels each at a 90o or less angle to the solvent stream, and opposite one 

another, impinge on the solvent stream to hydrodynamically focus it. However, other 

means of providing one or more side streams to focus the solvent stream and provide the 

interfacial region for liposome formation may be used. 

 For collection of the formed liposomes, it can be useful to provide, downstream of 

the region where the liposomes form, at least one side outlet microchannel for removing 

non-liposome materials from the central microchannel. The formed liposomes will flow 

in the central microchannel, typically as a tightly focused stream in the center area of the 

channel, owing to the low Reynold’s number laminar flow typical of microfluidics and to 

the low diffusion coefficient of the formed liposomes. The outlet can be simply provided 

by one or more branching side microchannels. Material outside the diameter of the tightly 

focused stream of formed liposomes will be removed by such outlet channels facilitating 

isolation of a liposome suspension formed in the center of the central microchannel. In a 

preferred embodiment, non-liposome materials are removed from the central 

microchannel by two side outlet microchannels angled at 90o or more to the central 

microchannel and on opposite sides thereof. Other means for separating the formed 

liposomes can also be used. 

 Although any devices which provide the necessary microfluidics can be used to 

carry out the method, preferably, the device for carrying out the method is in the form of 

a silicon wafer containing the microchannels in the desired pattern to provide the central 

microchannel, side microchannels and outlet microchannels, as desired. Known 

photolithography techniques can be used to pattern microchannels in silicon materials. 

For example, the microchannels may be fabricated in the silicon chip with a transparent 

wafer, e.g., glass, cover using a two-step photolithography process in combination with 

an anisotropic wet-chemical etch (TMAH), followed by anodic bonding. In a first 

photolithographic step, through-holes for fluidic access to the device are patterned in the 

back of the silicon chip. In a second step, the microchannel pattern is formed on the front 

of the chip. After the patterning is complete, the silicon chips are cleaned, and a wet 

 4 
oxide layer (e.g., a 500 nm thick SiO2 film) is grown on the surface. Finally, the channels 

are sealed by a covering using an anodic bonding technique: for example, a 0.5mm thick 

borosilicate (Pyrex) wafer is brought into contact with the front of the silicon chip, and 

the silicon/Pyrex sandwich is heated to, e.g., 400C, with an applied voltage. 

Modifications may be made to these photolithography, etching and anodic bonding 

methods according to the knowledge in these arts. 

 In one embodiment, the microchannels are formed in a material transparent on at 

least one side to allow observation of the microchannel, e.g., with the glass cover 

construction discussed above. This allows observation of the flow and liposome 

formation in the microchannels. For this purpose, it can be advantageous to provide a 

fluorescing material in the solvent stream and/or the aqueous streams to allow fluorescent 

observation and/or imaging of the liposome formation through the transparent cover. 

 The microchannels are preferably etched on the surface of a silicon wafer to a 

width and depth of 10 to 200 μm, more preferably 20 to 160 μm. The entire length of the 

central microchannel is preferably about 10 to 30 mm. The fluid through-holes are etched 

preferably of the same width as the microchannels or larger on the back-side of the wafer 

and aligned to the microchannel network for input and output ports. The dimensions of 

the entire silicon chip with cover device are preferably about 20 mm to 50 mm long and 

15 mm to 30 mm wide with a thickness of 0.7 mm to 1 mm. 

 The flow of the solvent and aqueous streams through the device is preferably 

accomplished by pumping of the streams into the central and side microchannels, 

respectively. However, other fluid driving methods, such as electrically driven pumping 

could be used. In a preferred embodiment, pumping is accomplished by using gastight 

glass syringes interfaced to the microchannel network through capillary tubing and 

capillary connectors that are bonded to the fluid through-holes etched in the silicon wafer. 

Programmable syringe pumps can be used to control the fluid flow rates by computer, for 

example, using a LabVIEW software interface. Fine control of the respective flow rates is 

advantageous for providing fine control over the size of the resulting liposomes. 

Preferably, the central microchannel is provided with a flow rate of the solvent phase of 

 5 
about 1 to 12 mm/s and the side microchannels, when two are provided, of about 10 to 

100 mm/s. 

 When the two liquid phases come into contact, the solvent phase and aqueous 

phase rapidly diffuse into one another. The flow rates of the solvent and aqueous streams 

can be adjusted to control the degree of hydrodynamic focusing and ultimately the 

liposome size. The lipids self-assemble where the concentration of the solvent phase 

containing the lipid or lipid-forming materials and the aqueous composition is at a critical 

condition where lipids are no longer soluble and thus self-assemble into liposomes. The 

formed liposomes remain in the center of the microchannel because: (i) liposomes formed 

along the interfacial region follow stream lines and are directed to collect at the center 

point in the channel; and (ii) at this point the solvent has diluted to a concentration where 

it can no longer solubilize any fraction of the lipid. 

 One can control the liposome size by altering the ratio of the flow rate in the side 

inlet channels compared to the center inlet channel. As the bulk liquid flow rate in the 

center channel downstream of the first cross increases, the magnitude of the shear stresses 

applied to the liposomes as they self assemble also increases. This results in a decrease in 

both the mean and range (polydispersity) of liposome diameter. Thus, by tuning of the 

flow rates in the microfluidic channels, the physical characteristics of the resultant 

liposome preparation can be readily controlled, for example, over the range of 100 nm to 

300 nm diameter. Further, the liposome preparations are more monodisperse in size than 

liposomes prepared by traditional bulk methods. Although not intending to be bound by 

this theory, it is believed that this results from the precise control of the flow conditions 

that are achieved in the microchannel format. In bulk systems, as the solvent mixes into 

the aqueous phase, it does so in an uncontrolled fashion, resulting in different sections of 

solvent diluting to their critical concentration for lipid solubilization under different 

applied mechanical shear stresses. This heterogeneity results in different liposome sizes, 

hence a more polydisperse sample. However, by reducing the length scale of the fluidic 

system, and taking advantage of low Reynolds number, laminar flow in a microfluidic 

network facilitates more controlled fluidic mixing on the length scale of the liposome. 

Specifically, the solvent reaches its critical concentration under the same applied shear 

stress, resulting in a more monodisperse population of liposomes. 

 6 
A useful characteristic of liposomes is their ability to encapsulate (or perhaps 

excapsulate) ionic molecules from a surrounding aqueous medium. Thus, the invention 

includes embodiments wherein a reagent is included in the composition of lipids or lipidforming materials and/or in the aqueous composition and at least a portion of the reagent 

is encapsulated (or excapsulated) in the liposomes. Examples of reagents which may be 

encapsulated in liposomes as part of the above-described methods include small 

molecules (for example, drugs, fluorescent molecules, amino acids) and large molecules 

(for example, proteins, peptides, DNA and RNA). 

 The lipid or lipid-forming materials used in the central microchannel to carry out 

the invention include all known materials for liposome formation. Examples of useful 

materials include combinations of phospholipid molecules and cholesterol. Particularly 

preferred are combinations of dimyristoylphosphatidylcholine, cholesterol, and 

dicetylphosphate. These materials are provided in a solvent which will dissolve the lipid 

or lipid-forming materials. The solvent must also be water miscible in order to diffuse 

into the aqueous composition. Examples of useful solvents include alcohols, such as 

isopropanol, methanol or ethanol.. The lipids or lipid-forming materials are preferably 

provided in the solvent in a concentration of approximately 10mM. 

 The aqueous composition is preferably an aqueous buffer solution, particularly 

preferably a phosphate-buffered saline solution, phosphate buffer, TRIS buffer or HEPES 

buffer. 

 By changing the length scale of the fluidics in which lipids self assemble into 

liposomes and simultaneously manipulating both the length scale and the shear forces 

applied to the vesicles upon formation, the invention advantageously provides fine 

control of liposome size and homogeneity. Particularly, liposome-containing 

compositions with liposomes having a mean diameter from 100 nm to 300 nm and a size 

distribution of 15 to 20% can be produced using the described methods. Microfluidics 

allows adjustment of the flow fields precisely using the simple principle of hydrodynamic 

focusing. This is a fundamental change in the way in which liposome vesicles are formed, 

thus enabling the production of monodisperse populations without the need for 

subsequent processing steps to modify liposome size. 

 7 
The liposome self-assembly method described here can be used to provide 

liposomes for applications in on-demand drug encapsulation and delivery and is readily 

scaled up using microfluidics with the development of multiplexed multichannel systems. 

We also predict that the synthesis and self-assembly of nanoscale particles for other 

applications in nanotechnology will also greatly benefit from adaptations of this 

approach. 

 The entire disclosure of all applications, patents and publications, cited herein and 

of U.S. Provisional Application No. 60/525,335, to which this application claims priority, 

is incorporated by reference herein. 

Brief Description of the Drawings

 Figure 1(a) provides a schematic of the self-assembly liposome formation process 

in a microfluidic channel. The color contours are generated by modeling of the flow field 

using Navier-Stokes convection and Stoke-Einstein diffusion analysis and represent the 

concentration ratio of isopropanol to the aqueous buffer. The lipid vesicles are formed 

where the concentration ratio of alcohol to buffer meets a critical condition when the 

lipids are no longer soluble in water, determined to be at 40 wt %. Figure 1(b) provides a 

fluorescence micrograph of a microchannel network with input streams on the left side 

and output streams on the right side. Figure 1(c) details the hydrodynamic focusing of the 

isopropanol stream at the first cross and figure 1(d) details the liposome collection and 

excess buffer extraction streams at the second cross. 

 Figure 2(a) shows a three-dimensional color contour map of the fluorescence 

intensity of DiIC18 at the hydrodynamically focused injection region of liposome 

formation. The intensity exhibits a ridge of increased fluorescence at the alcohol-aqueous 

buffer interface owing to increased efficiency of DiIC18 fluorescence efficiency due to 

intercolation of the dye into the lipid bilayer. Figure 2(b) shows the liposome mean 

diameter and standard deviation measured by light scattering vs. fluid flow velocity in 

center channel (The mean and standard deviation of the diameter of each of the liposome 

formulations is characterized by light scattering measurements (N4MD, Coulter 

Electronics Ltd.). For these measurements, a 0.1 wt% of Triton X-100 surfactant (Sigma) 

was added to the liposome formulations to reduce possible aggregation.). The 

 8 
isopropanol flow velocity is maintained at 2.4 mm/s while the flow velocity of each 

buffer channel is varied from 2.4 mm/s to 59.8 mm/s. The fluid flow velocity in the 

center channel on the x-axis is the sum of the flow velocities in each of the three input 

streams. 

 Figure 3 provides optical micrographs of the fluid flow fields for the liposome 

self-assembly process. Flow profiles are imaged in the microchannel network with a 

fluorescence microscope (Axioplan 2, Carl Zeiss) using a halogen lamp as an excitation 

source with appropriate filters for excitation and detection for DiIC18 (excitation 540 nm 

± 12.5 nm; beam splitter 565 nm; emission 605 nm ± 27.5 nm) and carboxyfluorescein 

(excitation 470 nm ± 20 nm; beam splitter 510 nm; emission 515 nm) and digitized using 

a CCD camera coupled to the top port of the microscope. Figure 3(a) is a white light 

image showing the hydrodynamic focusing of the isopropoanol injected into the aqueous 

buffer and its diffusion downstream; 3(b) is a fluorescence micrograph of DiIC18 

exhibiting its confinement within the liposome stream which has a much lower diffusion 

constant than isoproponal; and 3(c) is a fluorescent micrograph of water soluble 

carboxyfluorescein dye in the aqueous stream. 

 Figure 4 provides two-color confocal fluorescence imaging of a single liposome 

formed in flow conditions of 0.5 μL/min flow rate of the isopropanol stream and 20 

μL/min flow rate of the aqueous buffer streams. Two-color fluorescence imaging of 

liposomes showing analyte encapsulation were obtained using a confocal fluorescence 

imaging system (LSM 5 Pascal, Carl Zeiss) with a 40x objective and 10x digital zoom. 

The DiIC18 liposome image was measured with a laser excitation of 543 nm, through a 

543 nm beam splitter, and emission was detected through a 560 nm low pass filter. The 

carboxyfluorescein liposome image was measured with a laser excitation of 488 nm 

passing through a 488 nm beam splitter, and emission was detected through a 530 nm low 

pass filter. The flow conditions in this example produce a liposome mean diameter of 133 

nm and standard deviation of 26 nm determined by light scattering measurements. Figure 

4(a) is a fluorescence image for DiIC18 that exhibits intercolation of the dye into the lipid 

bilayer; and 4(b) is a fluorescence image for carboxyfluorescein (CF) that is encapsulated 

in the liposome’s aqueous interior. 

 9 
Without further elaboration, it is believed that one skilled in the art can, using the 

preceding description, utilize the present invention to its fullest extent. The following 

preferred specific embodiments are, therefore, to be construed as merely illustrative, and 

not limitative of the remainder of the disclosure in any way whatsoever. 

 In the foregoing and in the following examples, all temperatures are set forth 

uncorrected in degrees Celsius and, all parts and percentages are by weight, unless 

otherwise indicated. 

Examples

 A stream of lipid tincture is hydrodynamically focused at a microchannel cross 

junction between two aqueous buffer streams each impinging on the lipid stream at 90o. 

 The lipid tincture contains dimyristoylphosphatidylcholine (DMPC) and 

cholesterol in a molar ratio of 1:1 diluted in chloroform solvent (all from Aldrich) with 

1 wt% of 1,1’-dioctadecyl-3,3,3’,3’-tetramethilindodicarbocyanine perchlorate (DiIC18) 

added for fluorescent imaging. The chloroform solvent was evaporated under a stream of 

nitrogen at room temperature to form a lipid film on the bottom of a test tube. The test 

tube was then placed in a vacuum desiccator for at least 24 h to ensure dryness. The dried 

lipid mixture was resolubilized with 500 μL dry isopropanol yielding a 10 mM 

concentration of lipid solution. 

 To provide the microchannel cross junction, a microchannel network (200 μm 

channel width) was anisotropically etched on the surface of a silicon wafer (<100> 

orientation, 75 mm diameter, 0.3 mm thickness, from Virginia Semiconductor) to a depth 

of 40 μm through a photolithographically patterned thermal SiO2 (100 nm) hard mask 

using tetramethylammonium hydroxide (TMAH, Alfa Aesar) (1:2 dilution 25 % w/w 

aqueous TMAH solution, 80 oC). The same procedure was followed to etch fluid 

through-holes on the back-side of the wafer that were aligned to the microchannel 

network in the front side. Following this, all surface oxide was removed (6 % buffered 

HF etch) and then re-oxidized to form a 100 nm thick SiO2 film encapsulating the silicon 

 10 
substrate. Finally, a glass cover wafer (75 mm diameter 0.1 mm thickness, Corning 7740) 

was anodically bonded to the front surface of the silicon wafer using a 580 V applied 

potential and heated to 400oC to seal the microchannel network. 

 Following a procedure analogous to the bulk procedure that is described in L. 

Locascio-Brown, A. L. Plant, V. Horvath, Anal. Chem. 62, 2587 (1990), isopropyl 

alcohol containing the dissolved lipids is flowed through the center inlet channel, and an 

aqueous phosphate buffered saline solution flows through each of the two side inlet 

channels. Reagent transfers can be accomplished using gastight glass syringes interfaced 

to the microchannel network through capillary tubing (PEEK Tubing, Upchurch 

Scientific) and capillary connectors (Nanoports, Upchurch Scientific) that are bonded to 

the fluid through-holes etched in the silicon wafer. Programmable syringe pumps 

(Harvard Apparatus) can be used to control the fluid flow rates by computer, for example, 

using a LabVIEW software interface. Phosphate buffered saline solution (10 mM 

phosphate, 27 mM potassium chloride, 137 mM sodium chloride, pH = 7.4) is used as the 

hydration buffer. For encapsulation experiments, a 1 mM carboxyfluorescein solution is 

prepared in this buffer. The liposome formulations (100 μL sample size) are collected at 

each flow condition in polycarbonate cuvets. After collection, 1 mL of phosphate 

buffered saline solution (10 mM) is added to each formulation and sealed for further 

characterization. 

 When the two liquid phases come into contact, the isopropanol rapidly diffuses 

into the aqueous phase and vice versa. The flow rates of the alcohol and buffer channels 

can be adjusted to control the degree of hydrodynamic focusing and ultimately the 

liposome size. The lipids self-assemble where the concentration of alcohol and buffer 

mixture is at a critical condition where lipids are no longer soluble and thus self-assemble 

into liposomes. Finite element modeling of this 2-phase flow process schematically 

presents the general approach to microfluidic self-assembly of liposomes in a 

microfluidic network (see Figure 1a). A fluorescence image of the device used in these 

experiments is shown in Figure 1b, with a close-up view in Figure 1c of the 

hydrodynamically focused isopropanol stream where liposome formation occurs. 

Immediately downstream of the first cross intersection, the fluorescent intensity of the 

center stream increases sharply indicating the formation of the lipids into liposomes (the 

 11 
quantum efficiency of the fluorescent dye in this experiment (DiIC18) increases 

dramatically upon incorporation into a lipid membrane). The liposomes then flow to the 

second cross intersection as a tightly focused stream owing to the low Reynold’s number 

laminar flow typical of microfluidics and the low diffusion coefficient of liposomes. At 

this second cross (Figure 1d) excess buffer is removed via the two side outlet channels 

and the liposome suspension is collected via the center outlet channel. 

 The liposomes form (as is manifested by the increased fluorescence of the 

DiIC18) along the boundary between the isopropanol and water, as can be seen in Figure 

2a. A ridge of increased fluorescence is clearly visible as the aqueous streams focus the 

isopropanol. Fluorescence intensity increases to its maximum value immediately 

downstream of the minimum width of the isopropanol stream indicating the highest 

concentration of liposomes. Two effects lead to the high liposome concentration here in 

the system: (i) liposomes formed along the interfacial region follow stream lines and are 

directed to collect at the center point in the channel; and (ii) at this point the alcohol has 

diluted to a concentration where it can no longer solubilize any fraction of the lipid. 

According to two-dimensional modeling of this flow field with Navier-Stokes convection 

and Stoke-Einstein diffusion analysis, this increase in fluorescence intensity corresponds 

to an isopropanol concentration of approximately 40 wt%. 

 One can control the liposome size by altering the ratio of the flow rate in the side 

inlet channels compared to the center inlet channel. As the bulk liquid flow rate in the 

center channel downstream of the first cross increases, the magnitude of the shear stresses 

applied to the liposomes as they self assemble also increases. This results in a decrease in 

both the mean and range (polydispersity) of liposome diameter, as can be seen in Figure 

2b. Thus, by tuning of the flow rates in the microfluidic channel, the physical 

characteristics of the resultant liposome preparation can be readily controlled, for 

example, over the range of 100 nm to 300 nm. Further, the liposome preparations are 

more monodisperse in size than liposomes prepared by traditional bulk methods. 

Although not intending to be bound by this theory, it is believed that this results from the 

precise control of the flow conditions that are achieved in the microchannel format. In 

bulk systems, as the isopropanol mixes into the aqueous media, it does so in an 

uncontrolled fashion, resulting in different sections of alcohol diluting to their critical 

 12 
concentration for lipid solubilization under different applied mechanical shear stresses. 

This heterogeneity results in different liposome sizes, hence a more polydisperse sample 

(M. C. Woodle, D. Papahadjopoulos, Meth. Enzymol. 171, 193 (1989)). However, by 

reducing the length scale of the fluidic system, and taking advantage of low Reynolds 

number, laminar flow in a microfluidic network facilitates more controlled fluidic 

diffusion-based mixing on the length scale of the liposome. Specifically, the alcohol 

reaches its critical concentration under the same applied shear stress, resulting in a more 

monodisperse population of liposomes. 

 A useful characteristic of liposomes is their ability to encapsulate (or perhaps 

excapsulate) ionic molecules from a surrounding aqueous medium. Thus, functional 

characterization of liposomes self-assembled in a microfluidic channel is of interest. 

Figure 3 shows three different images of the same microfluidic flow field. In panel (a), 

the microchannel network is viewed with transmitted light; immediately apparent is the 

visible refractive index change at the interface between the alcohol and aqueous phases 

that dissipates as the two phases interdiffuse. Panel (b) shows the microfluidic network 

under fluorescent imaging conditions appropriate to visualize DiIC18 that is present as 1 

wt% of the lipid fraction in the alcohol phase. The hydrodynamic focusing of this stream 

is clearly visible in this micrograph. It is also interesting that the DiIC18 stream does not 

get progressively wider as the alcohol-aqueous boundary does as it migrates down the 

microchannel. This is due to the incorporation of the low molecular weight DiIC18 into 

liposomes, which have a very low diffusion coefficient; and therefore diffuse minimally 

in the radial direction on the time frame of the liquid flowing through the microchannel. 

Panel (c) shows the microfluidic network under fluorescent imaging conditions 

appropriate to visualize carboxyfluorescein dye, which is present in the aqueous stream at 

a concentration of 1 mM. Here the aqueous streams can be seen focusing the alcohol 

stream. Careful inspection of this panel shows that the aqueous carboxyfluorescein dye 

does diffuse into the center region, showing that the carboxyfluorescein that is not 

encapsulated in liposomes still diffuses freely within the microfluidic channel. 

 Liposomes that self assemble in the microchannel presented in Figure 3 should 

then have an aqueous interior that contains carboxyfluorescein surrounded by a lipid 

membrane with DiIC18. Thus, two-color fluorescent imaging of a liposome should reveal 

 13 
the interior and membrane portions of the assembly. Figure 4 shows a single liposome 

collected from this microfluidic flow network and nonspecifically adhered to a glass 

coverslip. Panel (a) shows the liposome with a fluorescence microscopy appropriate to 

visualize the DiIC18 incorporated into the membrane, while panel (b) shows the same 

liposome visualized under the same magnification with fluorescence conditions to 

visualize carboxyfluorescein. The two images coincide in their spatial position and are 

stable over the time frame of several minutes as would be expected if the object in view 

is a contiguous membrane that encapsulates a continuous aqueous interior. 

 The preceding examples can be repeated with similar success by substituting the 

generically or specifically described reactants and/or operating conditions of this 

invention for those used in the preceding examples. 

 From the foregoing description, one skilled in the art can easily ascertain the 

essential characteristics of this invention and, without departing from the spirit and scope 

thereof, can make various changes and modifications of the invention to adapt it to 

various usages and conditions. 

 14 
We claim: 

1. A method for producing a liposome-containing composition, which comprises: 

 providing a solvent stream of a composition of lipids or lipid-forming materials 

dissolved in a solvent through a central microchannel having a hydrodynamic diameter of 

100 μm or less, and 

 impinging on said solvent stream through at least one side microchannel at least 

one aqueous stream of an aqueous composition which hydrodynamically focuses the 

solvent stream and forms a stream within the central microchannel having an interfacial 

region where the solvent stream and the at least one aqueous stream diffuse into each 

other providing conditions where liposomes self-assemble from the lipids or lipidforming materials. 

2. The method of claim 1, wherein a reagent is included in the composition of lipids 

or lipid-forming materials and/or in the aqueous composition and at least a portion of said 

reagent is encapsulated in the liposomes. 

3. The method of claim 1, wherein the liposome-containing composition contains 

liposomes having a mean diameter of from 100 nm to 300 nm and a size distribution of 

less than 20%. 

4. The method of claim 1, wherein at least two aqueous streams are provided by at 

least two side microchannels, which at least partially oppose one another, and which 

impinge on the solvent stream to hydrodynamically focus it. 

5. The method of claim 1, wherein two aqueous streams are provided by two side 

microchannels each at a 90o angle or less to the solvent stream, and opposite one another, 

which impinge on the solvent stream to hydrodynamically focus it. 

6. The method of claim 1, wherein, downstream of the region where the liposomes 

form, non-liposome materials are removed from the central microchannel by at least one 

side outlet microchannel. 

7. The method of claim 7, wherein non-liposome materials are removed from the 

central microchannel by two side outlet microchannels angled at 90o or more to the 

central microchannel and on opposite sides thereof. 

 15 
8. The method of claim 1, wherein the lipid or lipid-forming materials are selected 

from: a combination of phospholipid molecules and cholesterol. 

9. The method of claim 1, wherein the lipid or lipid-forming materials are selected 

from: a combination of dimyristoylphosphatidylcholine and cholesterol. 

10. The method of claim 1, wherein the solvent is isopropanol, ethanol or methanol. 

11. The method of claim 1, wherein the aqueous composition is an aqueous buffer 

solution. 

12. The method of claim 1, wherein the aqueous composition is a phosphate-buffered 

saline solution, a phosphate buffer, a HEPES buffer, or a TRIS buffer. 

13. The method of claim 1, wherein the microchannels are formed in a material 

transparent on at least one side to allow observation of the microchannel. 

14. The method of claim 13, wherein a fluorescing material is provided in the solvent 

stream and/or the aqueous stream to allow fluorescent observation and/or imaging of the 

liposome formation. 

15. The method of claim 1, wherein the microchannels are formed in a silicon wafer. 

16. The method of claim 1, wherein the solvent stream and aqueous stream(s) are 

pumped into the microchannels under computer-controlled flow rates. 

 16 
 17 

ABSTRACT 

 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 solventaqueous 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. 
(a) 

Figure 1 
1 Normalized intensity 

(a) 

0

198 m 

564 m 

0

50

0

0

0

0

0

350

0.0 20.0 40.0 60.0 80.0 100.0 120.0 140.0

(b) 

10

15

20

25

30

Li

posome Mean Diameter (nm) 

Fluid Flow Velocity (mm/s)

Figure 2 
(a)

(b)

(c)

Figure 3 
Figure 4