**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 controllable on the dimension of the particle itself to elicit
fine control of nanometer scale 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 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 oxide
layer (e.g., a 500 nm thick SiO~2~ 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 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.

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 lipid-forming 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.

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]{.underline}

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)

Figure 2(a) shows a three-dimensional color contour map of the
fluorescence intensity of DiIC~18~ 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 DiIC~18~ 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 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 DiIC~18~ (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 DiIC~18~ 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 DiIC~18~ 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.

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]{.underline}

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
(DiIC~18~) 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 SiO~2~ (100 nm) hard mask using
tetramethylammonium hydroxide (TMAH, Alfa Aesar) (1:2 dilution 25 % w/w
aqueous TMAH solution, 80 ^o^C). 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 SiO~2~ film encapsulating the silicon 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 400^o^C 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 quantum efficiency of the fluorescent dye in
this experiment (DiIC~18~) 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 DiIC~18~) 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 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 DiIC~18­~ 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 DiIC~18~ 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 DiIC~18~ 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 DiIC~18~. Thus, two-color
fluorescent imaging of a liposome should reveal 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 DiIC~18~
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.

**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
lipid-forming 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.

**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.

[ABSTRACT]{.underline}

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.

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