U.S. patent application number 16/184869 was filed with the patent office on 2019-03-14 for device and method for formulating particles at small volumes.
This patent application is currently assigned to The University of British Columbia. The applicant listed for this patent is The University of British Columbia. Invention is credited to Aysha Ansari, Timothy Leaver, Kevin Ou, Euan Ramsay, Robert James Taylor, Colin Walsh, Andre Wild.
Application Number | 20190076372 16/184869 |
Document ID | / |
Family ID | 52828708 |
Filed Date | 2019-03-14 |
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United States Patent
Application |
20190076372 |
Kind Code |
A1 |
Walsh; Colin ; et
al. |
March 14, 2019 |
DEVICE AND METHOD FOR FORMULATING PARTICLES AT SMALL VOLUMES
Abstract
Methods and devices for making particles at small volumes.
Inventors: |
Walsh; Colin; (Belmont,
CA) ; Wild; Andre; (Vancouver, CA) ; Taylor;
Robert James; (Vancouver, CA) ; Leaver; Timothy;
(Delta, CA) ; Ou; Kevin; (Toronto, CA) ;
Ramsay; Euan; (Vancouver, CA) ; Ansari; Aysha;
(Calgary, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of British Columbia |
Vancouver |
|
CA |
|
|
Assignee: |
The University of British
Columbia
Vancouver
CA
|
Family ID: |
52828708 |
Appl. No.: |
16/184869 |
Filed: |
November 8, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15029993 |
Apr 15, 2016 |
10159652 |
|
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PCT/US14/60961 |
Oct 16, 2014 |
|
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16184869 |
|
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61891758 |
Oct 16, 2013 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01F 13/0062 20130101;
B01F 3/08 20130101; B01F 5/02 20130101; B01F 3/0811 20130101; B01J
2/06 20130101; A61K 9/5123 20130101; A61P 43/00 20180101; B01F
3/0861 20130101; A61K 9/5192 20130101; B01F 2215/0032 20130101;
A61K 31/7105 20130101; B01F 3/0807 20130101; B01F 13/1022
20130101 |
International
Class: |
A61K 9/51 20060101
A61K009/51; B01F 5/02 20060101 B01F005/02; B01F 3/08 20060101
B01F003/08; B01F 13/00 20060101 B01F013/00; B01F 13/10 20060101
B01F013/10; B01J 2/06 20060101 B01J002/06; A61K 31/7105 20060101
A61K031/7105 |
Claims
1. A method for making particles, comprising: (a) introducing a
first stream comprising a first solvent into a channel; wherein the
channel has a first region adapted for flowing one or more streams
introduced into the channel and a second region for mixing the
contents of the one or more streams; and wherein the first solvent
comprises a therapeutic material and optionally one or more
particle-forming materials; (b) introducing a second stream
comprising one or more particle-forming materials and optionally a
therapeutic material in a second solvent into the channel to
provide first and second streams and wherein the first and second
solvents are not the same; (c) flowing the one or more first
streams and the one or more second streams from the first region of
the channel into the second region of the channel such that the one
or more first streams and the one or more second streams arrive at
the second region for mixing at substantially the same time; and
(d) mixing the contents of the one or more first streams and the
one or more second streams in the second region of the channel to
provide a third stream comprising particles.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/029,993, filed Apr. 15, 2016, which is the national stage of
International Application No. PCT/US2014/060961, filed Oct. 16,
2014, which claims the benefit of U.S. Application No. 61/891,758,
filed Oct. 16, 2013. Each application is incorporated herein by
reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to manufacturing particles,
and devices and methods for formulating the particles at small
volumes.
BACKGROUND OF THE INVENTION
[0003] Particles are important class of materials in medicine and
other applications. Particles exist at nanometer or micrometer
sizes and are used in a wide range of applications, including
pharmaceuticals, medical devices, research tools, cosmetics, paints
and inks, industrial applications, as well as others. For example,
a major challenge for many active pharmaceutical ingredients
(therapeutic materials) is the inability to deliver adequate
concentrations to target cells to elicit a biological affect.
Certain therapeutic materials, including many chemotherapeutic
materials, are toxic and cannot be administered systemically at
doses that are required to have an affect on a disease, while
others, including many biologics like oligonucleotide therapeutic
materials, are unable to cross cell membranes to access their site
of action. Polymers, lipids and other materials offer a promising
solution for encapsulating therapeutic materials and transporting
them to diseased cells and tissues in particles. Such particles can
increase a therapeutic material's therapeutic index by reducing
toxicity through shielding the therapeutic material from healthy
tissues, increasing the therapeutic material effectiveness through
targeting diseased tissue, and by enabling the active delivery of
therapeutic materials to their site of action.
[0004] A variety of methods have been developed to manufacture
particles. These methods include self-assembly, precipitation, and
homogenization. Various devices, including microfluidic devices
have demonstrated the ability to controllably and rapidly mix
fluids in continuous flow formats with precise control over
temperature, residence times, and solute concentrations.
Microfluidics has proven applications for the synthesis of
inorganic nanoparticles and microparticles, and can outperform
macroscale systems in large-scale production of particles. Droplet
techniques have been applied to produce monodisperse microparticles
for therapeutic material delivery or to produce large vesicles for
the encapsulation of cells, proteins, or other biomolecules.
Hydrodynamic flow focusing, a common microfluidic technique to
provide rapid mixing of reagents, has been used to create
monodisperse lipid particles of controlled size. This technique has
also proven useful in the production of polymer particles where
smaller, more monodisperse particles were obtained, with higher
encapsulation of small molecules as compared to bulk production
methods. Turbulent mixers, including T, W, or Y mixers with channel
dimensions >0.1 mm have been successfully used for the
manufacture of microparticles and nanoparticles.
[0005] Despite the availability of methods of manufacture for
particle systems, the manufacture of high quality particles at
small scales (<1 mL) remains at challenge due to the
difficulties of mixing very small volumes together effectively and
the wastage of fluids, or fluidic "dead volume," in the devices and
in connections to the devices. The present invention seeks to
fulfill this need and provides further related advantages.
SUMMARY OF THE INVENTION
[0006] In one aspect of the invention, methods for making particles
are provided.
[0007] In one embodiment, the method comprises:
[0008] (a) introducing a first stream comprising a first solvent
into a channel; wherein the channel has a first region adapted for
flowing one or more streams introduced into the channel and a
second region for mixing the contents of the one or more streams;
and wherein the first solvent comprises a therapeutic material and
optionally one or more particle-forming materials;
[0009] (b) introducing a second stream comprising one or more
particle-forming materials and optionally a therapeutic material in
a second solvent into the channel to provide first and second
streams and wherein the first and second solvents are not the
same;
[0010] (c) flowing the one or more first streams and the one or
more second streams from the first region of the channel into the
second region of the channel such that the one or more first
streams and the one or more second streams arrive at the second
region for mixing at substantially the same time; and
[0011] (d) mixing the contents of the one or more first streams and
the one or more second streams in the second region of the channel
to provide a third stream comprising particles.
[0012] In another embodiment, the method comprises:
[0013] (a) introducing a stream comprising a first solvent into a
channel; wherein the channel has a first region adapted for flowing
one or more streams introduced into the channel; and
[0014] (b) conducting the first stream through the channel and into
a reservoir comprising a second solvent,
[0015] wherein conducting the first stream into the reservoir
comprises mixing the contents of the first stream with the contents
of the reservoir to provide particles.
[0016] In another aspect, the invention provides devices for making
particles.
[0017] In one embodiment, the device comprises:
[0018] (a) a first well for receiving a first solution comprising a
first solvent;
[0019] (b) a first channel in fluid communication with the first
well;
[0020] (c) a second well for receiving a second solution comprising
a second solvent;
[0021] (d) a second channel in fluid communication with the second
well;
[0022] (e) a third channel for receiving first and second streams
flowed from the first and second wells through the first and second
channels, respectively, wherein the third channel has a first
region adapted for flowing the first and second streams introduced
into the channel and a second region adapted for mixing the
contents of the first and second streams to provide a third stream
comprising particles; and
[0023] (f) a third well for receiving the third stream comprising
particles.
[0024] In one embodiment, the device comprises:
[0025] (a) a first well for receiving a first solution comprising a
first solvent;
[0026] (b) a first channel in fluid communication with the first
well; and
[0027] (c) a second well for receiving a second solution comprising
a second solvent, wherein the second well further receives a first
stream flowed from the first well through the first channel, and
wherein the second well is adapted for mixing the contents of the
first stream and second solution in the second well to provide a
third solution comprising particles.
DESCRIPTION OF THE DRAWINGS
[0028] The foregoing aspects and many of the attendant advantages
of this invention will become more readily appreciated as the same
become better understood by reference to the following detailed
description, when taken in conjunction with the accompanying
drawings.
[0029] FIG. 1 is a schematic illustration of the challenges of
manufacturing particles at small volumes. The illustration includes
(a) requirements for timing fluidic mixing to maximize the yield of
manufactured particles; (b) areas for fluidic waste.
[0030] FIG. 2 is a schematic illustration of a representative
device and method of the invention for preparing particles at small
volumes: a device that uses a combination of input and output
reservoirs (wells) to control flow rates and flow timing. In this
device, input wells are used to contain input fluids. Channel
impedances are used to determine the relative flow rates between
flows from the inputs. An outlet well is added. In certain
embodiments, a backpressure or stopper is applied to the outlet
well to stop fluidic movement from the inputs due to the weight of
fluids in the input wells or other phenomena, prior to a pressure
applied to the inputs. In certain embodiments, a backpressure is
achieved by adding fluid to the outlet well prior to adding fluids
to the input wells. In this case fluids with the lowest surface
tension are added last because these are the fluids which move
through the chip at the highest rate. The input fluids are then
added into the input reservoirs and the inputs are pressurized to
create fluid flow. Flow rates of the different flows are controlled
by the impedances of the channels from the inputs to the mixer
chamber. The flows can be timed to reach the mixer at a similar
time by pressurizing the input wells simultaneously. In certain
embodiments, the device is purged of remaining fluid by applying
fluid (gas or liquid) to the inputs and flowed through the mixers
following nanoparticle manufacture.
[0031] FIG. 3 is an example of a representative device illustrated
in the schematic of FIG. 2. This device has two inlet wells (one
for an aqueous phase and one for an ethanol/lipid phase) and one
outlet well. In practice, a dilution buffer is loaded into the
outlet well, this buffer adds backpressure at the output of the
device and lowers the ethanol concentration of the final product
which stabilizes the particles. Aqueous reagents and lipids in
ethanol are loaded into the input wells, a manifold is then clamped
oven the inlet wells and pressurized using a syringe or other
mechanism. See FIG. 8. The pressurization pushes the reagents in
the inlet wells through the mixer (e.g., a staggered herringbone
mixer) and into the outlet well. The formulated particles are then
recovered using a pipette. The shown device is designed to have a
flow ratio of 3 parts aqueous to 1 part ethanol, which is achieved
with different channel lengths leading from the input wells to them
mixer. In this case, the ratio of 2.5:1 is used and this takes into
account the desired flow ratio and the viscosity difference between
the input reagents.
[0032] FIG. 4 is a schematic illustration of a representative
device and method of the invention for preparing particles at small
volumes: a device that flows a first stream of solvent (input wells
1 through n) into a second solvent contained in the outlet
reservoir (dilution well). Mixing of the first stream with the
contents of the outlet reservoir can occur through various
mechanisms including (i) convection flows occurring by introducing
the first stream into the reservoir and (ii) active mixing of the
combined fluids as the first stream is introduced into the
reservoir.
[0033] FIG. 5 is an example of a representative device illustrated
in the schematic of FIG. 4. The device has a single input well for
a lipid/ethanol solution and an outlet well into which an aqueous
solution is loaded. The device has a large number of microchannels
leading into the outlet well, the impedance of microchannels is
high compared to the channel feeding them. This is necessary for an
even distribution of fluid. After the reagents are loaded, the
inlet well is pressurized. The fluid in the inlet well flows
through the microchannels and into the output well. The fluid is
mixed by convection and by air bubbles flowing into the outlet
well.
[0034] FIG. 6 is a schematic illustration of a representative
device and method of the invention for preparing particles at small
volumes: a device using valves either at the inlets or outlet to
time the introduction of fluidic flows into the mixing chamber.
[0035] FIG. 7 is an image of a representative device of the
invention illustrated schematically in FIGS. 2 and 3.
[0036] FIG. 8 is an image of the representative device shown in
FIG. 7 further including a pressure activated manifold.
[0037] FIG. 9 is an image of the representative device shown in
FIG. 7 further including a clamping device and pressure-activated
manifold.
[0038] FIG. 10 is an image of a disposable device representative of
the device described in FIGS. 2 and 3. FIG. 10A shows the device
plus manifold and FIG. 10B shows the manifold covering the inlet
wells of the device. The manifold allows for an empty syringe to be
attached and pushing down on the syringe plunger forces the fluids
through the mixing device.
[0039] FIG. 11 compares PTEN Knockdown by siRNA-LNPs synthesized
using NanoAssemblr and Zero Dead Volume Chip.
[0040] FIG. 12 compares levels of GFP expression on treatment with
NanoAssemblr and Zero Dead Volume chip formulations.
DETAILED DESCRIPTION OF THE INVENTION
[0041] The present invention provides methods and devices for
manufacturing particles at small volumes.
[0042] In one aspect, the invention provides methods for making
particles that include a therapeutic material.
[0043] In another aspect, the invention provides devices for making
particles that include a therapeutic material.
[0044] In other aspects, the invention provides methods and devices
for making lipid nanoparticles, liposome particles, emulsions, or
other lipid-containing particles.
[0045] In other aspects, the invention provides methods and devices
for making lipid nanoparticles, liposome particles, emulsions, or
other lipid-containing particles that contain a therapeutic
material.
[0046] In further aspects, the invention provides methods and
devices for making polymer particles.
[0047] In other aspects, the invention provides methods and devices
for making polymer particles containing a therapeutic material.
[0048] In other aspects, the invention provides methods and devices
for making particles made by a combination of lipid, polymer,
protein, nucleic acid, and other materials.
[0049] In another aspect, the invention provides methods and
devices for making particles containing polymers, natural polymers,
synthetic polymers, synthetic copolymers, semi-synthetic polymers,
polymer conjugates, polymer-therapeutic material conjugate,
polymer-drug conjugate.
[0050] In a further aspect, the invention provides methods and
devices for manufacturing particles containing a research reagent
at small volumes.
[0051] In other aspects, the invention provides methods and devices
for making lipid nanoparticles, liposome particles, emulsions, or
other lipid-containing particles that contain a research
reagent.
[0052] In a further aspect of the invention, particles made by the
methods and/or devices of the invention are provided.
Methods for Making Particles at Small Volumes
[0053] In one aspect, the invention provides a method for making
particles at small volumes. As used herein, the term "small volume"
refers to volumes less than 2 mL and, in certain embodiments,
volumes less than 1 mL. The methods of the invention provide
particles in volumes in the tens of microliters (e.g., 50, 100,
150, 200, 250, 300, 400, 450, 500, 550, 600, 650, 700, 750, 800,
850, 900, 950 .mu.L). Small volume refers to capability of the
devices and methods of the invention to prepare nanoparticles
without materials loss. For example, the devices and methods of the
invention are capable of manufacturing 100 uL of nanoparticles with
no material loss: the volumes of particle-forming materials (e.g.,
lipids) and therapeutic materials (e.g., RNA) added to the device
are about 20 uL each (the remainder of the volume represents the
diluting buffer in the additional (e.g., third well of the device)
as shown in FIGS. 2 and 3.
[0054] In one embodiment, the method for making particles
comprises:
[0055] (a) introducing a first stream comprising a first solvent
into a channel; wherein the channel has a first region adapted for
flowing one or more streams introduced into the channel and a
second region for mixing the contents of the one or more streams;
and wherein the first solvent comprises a therapeutic material and
optionally one or more particle-forming materials;
[0056] (b) introducing a second stream comprising one or more
particle-forming materials and optionally a therapeutic material in
a second solvent into the channel to provide first and second
streams and wherein the first and second solvents are not the
same;
[0057] (c) flowing the one or more first streams and the one or
more second streams from the first region of the channel into the
second region of the channel such that the one or more first
streams and the one or more second streams arrive at the second
region for mixing at substantially the same time; and
[0058] (d) mixing the contents of the one or more first streams and
the one or more second streams in the second region of the channel
to provide a third stream comprising particles.
[0059] In the above method, dead volume is minimized and production
of particles in small volumes is maximized by combining the first
and second streams at substantially the same time prior to mixing.
By this method, the mixed volume containing particles comprising
the components of each of the first and second streams is
minimized.
[0060] In one embodiment, one stream (e.g., second stream
comprising particle-forming materials in a second solvent such as
ethanol) is introduced into the channel in a continuous manner and
the flowing stream is interrupted by the introduction of a second
stream (e.g., a discrete volume of a first stream comprising
therapeutic material) so as to create a plug of a combined volume
of the first and second streams. The combined volume is then mixed
to provide particles in the combined volume. In this method, the
combined volume is preceded and then followed by the second stream.
In this method, the relatively valuable first stream comprising the
therapeutic material is limiting in the context of therapeutic
material-containing particle formation and the second stream
comprising the particle-forming materials is used in excess.
[0061] In the methods of the invention, the streams to be combined
(i.e., first and second stream) are not the same. The composition
of each stream can vary and, in certain embodiments, each may
include both therapeutic materials and particle-forming materials.
It will be appreciated that the composition of each stream is such
that particle formation does not occur until the streams are mixed.
As further described below, the solvents for the first and second
streams are miscible and particles are produced on their mixing. As
described herein, the methods and device of the invention are
particularly useful for making therapeutic material-containing
particles in general, and therapeutic material-containing particles
in small volumes in particular.
[0062] In certain embodiments, the above method further includes
one or more of the following features: [0063] (i) flowing the one
or more first streams and the one or more second streams from the
first region of the channel into the second region of the channel
at defined flow ratios established by predetermined pressure drops
across one or more of the flow channels, by application of
predetermined pressure to one or more of the flow channels, or by a
combination of both (see impedances illustrated in FIGS. 2-5);
[0064] (ii) flowing a fluid (gas or liquid) into the one or more
first streams and/or the one or more second streams after or during
making the particles to expel the first and second streams from the
from the channels; [0065] (iii) applying a backpressure to the one
or more first streams and the one or more second streams sufficient
to prevent flow (due to gravity, wicking, or capillary action) into
the channels until a predetermined forward pressure is achieved to
flow the first stream into the first channel and the second stream
into the second channel; [0066] (iv) establishing a backpressure
sufficient to prevent flow (due to gravity, wicking, or capillary
action) into the first and second channels by physically blocking
the output channel until a predetermined forward pressure is
achieved to flow the first stream into the first channel and the
second stream into the second channel; or [0067] (v) using input or
output valves in the system to ensure the timing of the flows of
the one or more first streams and the one or more second streams
from the first region of the channel into the second region (e.g.,
the first channel further comprising a first input valve effective
to time flow of the first stream into the first channel, the second
channel further comprising a second input valve effective to time
flow of the second stream into the second channel, and/or an output
channel further comprising an output valve effective to time flow
of the first and/or second streams into the first and second
streams, respectively. See, for example, FIG. 6.
[0068] In certain embodiments of the methods, the time that either
the first stream or the second stream enters the second region of
the channel without the other is minimized and the mixing of fluids
together is maximized. Timing of the fluid flow may be achieved
using valves, pressure, impedance matching, or any other methods to
achieve the timing.
[0069] In certain embodiments of the above methods, the contents of
the first and second streams can be mixed by chaotic advection,
turbulent mixing, jetting, vortex methods, and stirring. Mixing may
be achieved by an active mixing device or passive mixing device.
The mixing may occur in a continuous flow format or in defined
volume format. The mixing may be achieved using a microfluidic
mixer, including a herringbone mixer, zig-zag mixer, micro-jet
mixer, micro-vortex mixer, tesla mixer, tear drop mixer, bubble
mixer, acoustic streaming. The mixing may be achieved using a
macroscopic mixer, including a T-mixer, Y-mixer, W-mixer, and
mixing tubes.
[0070] In certain embodiments of the above methods, mixing the
contents of the one or more first streams and the one or more
second streams comprises varying the concentration or relative
mixing rates of the one or more first streams and the one or more
second streams. Differing flow rations may be enabled by either
differential pressure applied to the flows, differential pressure
drops across the flow channels, differential channel impedances, or
combination therein, applied to the first and second streams.
Differential impedances of the channels through varying the channel
heights, widths, lengths, or surface properties, may be used to
achieve different flow rates. Fluidic surface tensions,
viscosities, and other surface properties of the flows in the one
or more first streams and the one or more second streams may be
used or considered to achieve different flow rates.
[0071] In certain embodiments of the above methods, after or during
manufacture of particles, flowing into the one or more first
streams and the one or more second streams from the first region of
the channel into the second region of the channel a fluid or gas to
expel the first stream and second streams. The first and second
channel may be fully purged or partially purged under this method.
Gases such as air, nitrogen, argon or others may be used. Liquids
including water, aqueous buffer, ethanol, oils, or any other liquid
may be used.
[0072] In certain embodiments of the above methods, backpressures
are applied to ensure the flows of the one or more first streams
and the one or more second streams from the first region of the
channel into the second region is limited until an initial desired
input pressure is achieved. This may be achieved by applying
pressure to the outlet channels, negative pressures to the input
channels. This may be achieved by loading an outlet reservoir with
fluid that may or may not be required in the final particle
solution.
[0073] In certain embodiments of the above methods, the fluids are
introduced into the device in ways that minimize fluidic waste.
This may be achieved by pipetting fluids into the device, pipetting
fluids out of the device, connecting the device to syringes.
[0074] In another embodiment, the invention provides a method for
making particles comprising:
[0075] (a) introducing a stream comprising a first solvent into a
channel; wherein the channel has a first region adapted for flowing
one or more streams introduced into the channel; and
[0076] (b) conducting the first stream through the channel and into
a reservoir comprising a second solvent,
[0077] wherein conducting the first stream into the reservoir
comprises mixing the contents of the first stream with the contents
of the reservoir to provide particles.
[0078] This embodiment is illustrated in FIGS. 4 and 5.
[0079] In this embodiment, the stream and the reservoir first and
second streams are as in the method described above. The first and
second solvents are not the same and are miscible. The stream and
the reservoir are not the same and each may include a therapeutic
material and particle-forming materials. In one embodiment, the
stream comprises a first solvent (ethanol) and particle-forming
materials and the reservoir comprises a second solvent (aqueous)
and a therapeutic material. In another embodiment, the stream
comprises a first solvent (aqueous) and a therapeutic material and
the reservoir comprises a second solvent (ethanol) and
particle-forming materials.
[0080] In certain embodiments of this embodiment of the method, the
method further includes one or more of features (i)-(v) described
above.
Devices for Making Particles at Small Volumes
[0081] In another aspect, the invention provides devices for
producing particles at small volumes. In certain embodiments, the
devices are useful for carrying out the methods of the
invention.
[0082] In one embodiment, the device includes:
[0083] (a) a first well for receiving a first solution comprising a
first solvent;
[0084] (b) a first channel in fluid communication with the first
well;
[0085] (c) a second well for receiving a second solution comprising
a second solvent;
[0086] (d) a second channel in fluid communication with the second
well;
[0087] (e) a third channel for receiving first and second streams
flowed from the first and second wells through the first and second
channels, respectively, wherein the third channel has a first
region adapted for flowing the first and second streams introduced
into the channel and a second region adapted for mixing the
contents of the first and second streams to provide a third stream
comprising particles; and
[0088] (f) a third well for receiving the third stream comprising
particles.
[0089] This embodiment is illustrated in FIGS. 2, 3, and 6-8.
[0090] It will be appreciated that devices of the invention can
include one or more first wells, one or more first channels, one or
more second wells, one or more second channels, one or more third
channels, and one or more third wells.
[0091] In one embodiment, the device further includes means for
diluting the third stream to provide a diluted stream comprising
stabilized particles.
[0092] In another embodiment, the device includes:
[0093] (a) a first well for receiving a first solution comprising a
first solvent;
[0094] (b) a first channel in fluid communication with the first
well; and
[0095] (c) a second well for receiving a second solution comprising
a second solvent, wherein the second well further receives a first
stream flowed from the first well through the first channel, and
wherein the second well is adapted for mixing the contents of the
first stream and second solution in the second well to provide a
third solution comprising particles.
[0096] This embodiment is illustrated in FIGS. 4 and 5.
[0097] It will be appreciated that devices of the invention can
include one or more first wells, one or more first channels, and
one or more second wells.
[0098] In certain embodiments, the devices of the invention are a
macrofluidic or microfluidic device. In certain embodiments, the
first and second streams can be mixed by chaotic advection,
turbulent mixing, jetting, vortex methods, bubble mixing, micro
acoustic streaming, stirring, or other mixing methods. Mixing may
be achieved by an active mixing device or passive mixing device.
The mixing may occur in a continuous flow format or in defined
volume format. The mixing may be achieved using a microfluidic
mixer, including a herringbone mixer, zig-zag mixer, micro-jet
mixer, or micro-vortex mixer. The mixing may be achieved using a
macroscopic mixer, including a T-mixer, Y-mixer, or W-mixer.
[0099] In certain embodiments, the device of the invention is a
microfluidic device including one or more microchannels (i.e., a
channel having its greatest dimension less than 1 millimeter). In
one embodiment, the microchannel has a hydrodynamic diameter from
about 20 to about 400 .mu.m. In certain embodiments, the
microchannel has two regions: a first region for receiving and
flowing at least two streams (e.g., one or more first streams and
one or more second streams) into the device. The contents of the
first and second streams are mixed in the microchannel's second
region. In one embodiment, the second region of the microchannel
has a principal flow direction and one or more surfaces having at
least one groove or protrusion defined therein, the groove or
protrusion having an orientation that forms an angle with the
principal direction (e.g., a staggered herringbone mixer), as
described in U.S. Patent Application Publication No. 2004/0262223,
expressly incorporated herein by reference in its entirety. In one
embodiment, the second region of the microchannel comprises
bas-relief structures. To achieve maximal mixing rates, it is
advantageous to avoid undue fluidic resistance prior to the mixing
region. Thus, one embodiment of the invention is a device in which
non-microfluidic channels, having dimensions greater than 1000
microns, are used to deliver the fluids to a single mixing
channel.
[0100] In certain embodiments mixing of the first and second
streams can also be accomplished with means for varying the
concentration and relative flow rates of the first and second
streams. Differing flow rations may be enabled by either
differential pressure applied to the flows, differential pressure
drops across the flow channels, differential channel impedances, or
combination therein, applied to the first and second streams.
Differential impedances of the channels through varying the channel
heights, widths, lengths, or surface properties, may be used to
achieve different flow rates. Fluidic surface tensions,
viscosities, and other surface properties of the flows in the one
or more first streams and the one or more second streams may be
used or considered to achieve different flow rates.
[0101] In certain embodiments, the device further includes means
for complete or partial purging of the system to minimize the waste
volume. After or during manufacture of particles, the device is
able to be flown into the one or more first streams and the one or
more second streams from the first region of the channel into the
second region of the channel a fluid or gas to expel the first
stream and second streams. The first and second channel may be
fully purged or partially purged under this method. Gasses such as
air, nitrogen, argon or others may be used. Liquids including
water, aqueous buffer, ethanol, oils, or any other liquid may be
used.
[0102] In certain embodiments, the device enables backpressures to
be applied to ensure the flows of the one or more first streams and
the one or more second streams from the first region of the channel
into the second region is limited until an initial desired input
pressure is achieved. This may be achieved by applying pressure to
the outlet channels, negative pressures to the input channels. This
may be achieved by loading an outlet reservoir with fluid that may
or may not be required in the final particle solution.
[0103] In certain embodiments, the device is designed such that
fluids are introduced into the device in ways that minimize fluidic
waste. This may be achieved by pipetting fluids into the device,
pipetting fluids out of the device, connecting the device to
syringes, or other methods.
[0104] In certain embodiments, the device is microfluidic and
produced by soft lithography, the replica molding of
microfabricated masters in elastomer. The device has two inlets,
one for each of the solutions prepared above, and one outlet. The
microfluidic device was produced by soft lithography, the replica
molding of microfabricated masters in elastomer. In one example,
the device features are 200 .mu.m wide and approximately 70 .mu.m
high mixing channel with herringbone structures formed by
approximately 25 .mu.m high and 50 .mu.m thick features on the roof
of the channel. The device was sealed using an oxygen plasma
treatment to a 75.times.25.times.1.5 mm glass slide. Other
examples, include devices with widths and associated relative
dimensions that are smaller (120 .mu.m wide) or larger (300 .mu.m
wide). Input and output ports are drilled into the device.
[0105] In a second embodiment, the device is microfluidic and
produced from a hard thermoplastic such as cyclic olefin copolymer.
A negative tool is machined using a CNC mill and devices formed
using injection molding. Channel dimensions are preserved with the
addition of a draft angle ranging between 1.degree. and 5.degree.
on vertical surfaces. Molded pieces are sealed to a blank substrate
using a variety of techniques, including but not limited to:
lamination, solvent welding, heat pressing and combinations
thereof. Bonded devices are annealed to remove residual stresses
from the production processes. Once formed, devices are installed
and used in the custom instrument in the same way as elastomer
devices.
[0106] To achieve maximal mixing rates it is advantageous to avoid
undue fluidic resistance prior to the mixing region. Thus one
embodiment of the invention is a device in which non-microfluidic
channels, having dimensions greater than 1000 microns, are used to
deliver fluids to a single mixing channel. This device for
producing particles includes:
[0107] (a) a single inlet channel for receiving a first solution
comprising solvent and none or some solution and a second solution
comprising particle components in a second solvent; and
[0108] (b) a second region adapted for mixing the contents of the
first and second streams to provide a third stream comprising
particles.
[0109] In such an embodiment, the first and second streams are
introduced into the channel by a single inlet or by one or two
channels not having micro-dimensions, for example, a channel or
channels having dimensions greater than 1000 .mu.m (e.g., 1500 or
2000 .mu.m or larger). These channels may be introduced to the
inlet channel using adjacent or concentric macrosized channels.
[0110] In the description above directed to devices of the
invention, the compositions of the solvents and streams are as
described above for the methods of the invention.
[0111] In certain embodiments, the device includes the components
described herein and may include additional components. In these
embodiments, the device "comprises" the specified components. In
other embodiments, the device includes the components described
herein and may include additional components that do not alter the
characteristics of the devices (e.g., do not include components
that alter the inventive aspects of the device). In these
embodiments, the device "consists essentially of" the specified
components. In further embodiments, the device includes only the
components described herein and no others. In these embodiments,
the device "consists of" the specified components.
Particles Produced using the Methods and Devices
[0112] In a further aspect of the invention, particles made by the
methods and/or devices of the invention are provided.
[0113] In certain embodiments of the above methods and devices, the
methods and devices are used to manufacture particles that are
<100 nm in diameter. In certain embodiments of the above methods
and devices, the methods and devices are used to manufacture
particles that are >100 nm and <1000 nm in diameter. In
certain embodiments of the above methods and devices, the methods
and devices are used to manufacture particles that are >1000 nm
in diameter.
[0114] In the above methods, particles are formed from one or more
solutions, streams, or reservoirs that include particle-forming
materials. In addition to particle-forming materials, the methods
utilize solutions, streams, and reservoirs that include any
combination of zero, one or more lipid components; zero, one or
more polymer components; zero, one or more protein components;
zero, one or more oligonucleotide components; or zero, one or more
lipid components.
[0115] In certain embodiments, the first solvent (e.g., therapeutic
material-containing solution) may include aqueous buffers, for
example citrate and acetate buffers, or organic solvents, for
example aqueous ethanol, 1,4-dioxane, tetrahydrofuran, acetone,
dimethyl sulfoxide, dimethylformamide, acids, and alcohols, and
acetonitrile 90%. Molecular components of the particles may or may
not be contained in the first stream.
[0116] In certain embodiments, the second solvent is miscible with
the first solvent. Suitable solvents include aqueous buffers, for
example citrate and acetate buffers, or organic solvents, for
example, aqueous ethanol, 1,4-dioxane, tetrahydrofuran, acetone,
dimethyl sulfoxide, dimethylformamide, acids, and alcohols, and
acetonitrile 90%.
[0117] In certain embodiments, the particles are formed in a
microfluidic process that utilizes relatively rapid mixing and high
flow rates. The rapid mixing provides particles having the
advantageous properties including size, homogeneity, encapsulation
efficiency. Mixing rates used in the practice of the methods of the
invention range from about 100 .mu.sec to about 20 msec.
Representative mixing rates include from about 0.5 to about 20
msec.
[0118] In one application of the present invention the methods and
devices are used for making lipid particles containing a bioactive
agent. In the methods and devices, a first stream comprising an
polynucleic acid in a first solvent and a second stream comprising
lipid particle-forming materials in a second solvent are introduced
into a channel having a first region adapted for receiving and
flowing the streams introduced therein and a second region for
mixing the contents of the two streams to provide a third stream
comprising lipid particles with encapsulated therapeutic agent.
[0119] In one aspect, the invention provides a method for making
lipid particles containing a therapeutic agent. In one embodiment,
the method includes
[0120] (a) introducing a first stream comprising a polynucleic acid
in a first solvent into a channel; wherein the channel has a first
region adapted for flowing one or more streams introduced into the
channel and a second region for mixing the contents of the one or
more streams;
[0121] (b) introducing a second stream comprising lipid
particle-forming materials in a second solvent in the channel to
provide first and second streams flowing, wherein the lipid
particle-forming materials comprise an ionizable lipid, and wherein
the first and second solvents are not the same;
[0122] (c) flowing the one or more first streams and the one or
more second streams from the first region of the channel into the
second region of the channel; and
[0123] (d) mixing of the contents of the one or more first streams
and the one or more second streams flowing in the second region of
the channel to provide a third stream comprising lipid particles
with encapsulated polynucleic acids.
[0124] In certain embodiments of this embodiment, the method
further includes one or more of features (i)-(v) described
above.
[0125] The contents of the first and second streams can be mixed by
chaotic advection. In one embodiment, mixing the contents of the
one or more first streams and the one or more second streams
comprises varying the concentration or relative mixing rates of the
one or more first streams and the one or more second streams.
[0126] To stabilize the third stream containing the lipid particles
with encapsulated polynucleic acids, the method can further include
comprising diluting the third stream with an aqueous buffer. In one
embodiment, diluting the third stream includes flowing the third
stream and an aqueous buffer into a second mixing structure. In
another embodiment, the aqueous buffer comprising lipid particles
with encapsulated polynucleic acids is dialyzed to reduce the
amount of the second solvent.
[0127] The first stream includes a polynucleic acid in a first
solvent. Suitable first solvents include solvents in which the
polynucleic acids are soluble and that are miscible with the second
solvent. Suitable first solvents include aqueous buffers.
Representative first solvents include citrate and acetate
buffers.
[0128] The second stream includes lipid particle-forming materials
in a second solvent. Suitable second solvents include solvents in
which the ionizable lipids are soluble and that are miscible with
the first solvent. Suitable second solvents include aqueous
alcohols. Representative second solvents include aqueous ethanol
90%.
[0129] The methods of the invention have a polynucleic acid
encapsulation efficiency is from about 60% to about 100%. In
certain embodiments, the polynucleic acid encapsulation efficiency
is about 100%.
[0130] In a further aspect, the invention provides lipid particles
made by the methods and/or devices of the invention. The lipid
particles of the invention have a diameter from about 30 to about
200 nm. In one embodiment, the lipid particles have a diameter of
about 80 nm.
[0131] Advantageously, the lipid particles include from about 1 to
about 5 mole percent PEG-lipid, PEG-based surfactant, or other
stabilizing agent. In one embodiment, the lipid particles include
about 1.5 mole percent PEG-lipid. In one embodiment, the lipid
particles include about 1-10 mole percent surfactants. In one
embodiment, the lipid particles include about 2.5 mole percent
stabilizing agent, like a surfactant.
Definitions
Lipid Nanoparticles
[0132] In one aspect, the invention provides lipid nanoparticles
containing anionic macromolecule(s). The lipid nanoparticles
include one or more cationic lipids, one or more second lipids, and
one or more nucleic acids.
Cationic Lipids
[0133] The lipid nanoparticles include a cationic lipid. As used
herein, the term "cationic lipid" refers to a lipid that is
cationic or becomes cationic (protonated) as the pH is lowered
below the pK of the ionizable group of the lipid, but is
progressively more neutral at higher pH values. At pH values below
the pK, the lipid is then able to associate with negatively charged
nucleic acids (e.g., oligonucleotides). As used herein, the term
"cationic lipid" includes zwitterionic lipids that assume a
positive charge on pH decrease.
[0134] The term "cationic lipid" refers to any of a number of lipid
species which carry a net positive charge at a selective pH, such
as physiological pH. Such lipids include, but are not limited to,
N,N-dioleyl-N,N-dimethylammonium chloride (DODAC);
N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA);
N,N-distearyl-N,N-dimethylammonium bromide (DDAB);
N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTAP); 3-(N--(N',N'-dimethylaminoethane)-carbamoyl)cholesterol
(DC-Chol); and
N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
bromide (DMRIE). Additionally, a number of commercial preparations
of cationic lipids are available which can be used in the present
invention. These include, for example, LIPOFECTIN.RTM.
(commercially available cationic liposomes comprising DOTMA and
1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), from GIBCO/BRL, Grand
Island, N.Y.); LIPOFECTAMINE.RTM. (commercially available cationic
liposomes comprising
N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethy-
l-ammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL);
and TRANSFECTAM.RTM. (commercially available cationic lipids
comprising dioctadecylamidoglycylcarboxyspermine (DOGS) in ethanol
from Promega Corp., Madison, Wis.). The following lipids are
cationic and have a positive charge at below physiological pH:
DODAP, DODMA, DMDMA, 1,2-dilinoleyloxy-N,N-dimethylaminopropane
(DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),
1,17-bis(2-octylcyclopropyl)heptadecan-9-yl 4-(dimethylamino)
butanoate.
[0135] In one embodiment, the cationic lipid is an amino lipid.
Suitable amino lipids useful in the invention include those
described in WO 2012/016184, incorporated herein by reference in
its entirety. Representative amino lipids include
1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),
1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA),
1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP),
1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S -DMA),
1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),
1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt
(DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride
salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane
(DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP),
3-(N,N-dioleylamino)-1,2-propanediou (DOAP),
1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane
(DLin-EG-DMA), and
2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane
(DLin-K-DMA).
[0136] Suitable amino lipids include those having the formula:
##STR00001##
[0137] wherein R.sub.1 and R.sub.2 are either the same or different
and independently optionally substituted C10-C24 alkyl, optionally
substituted C10-C24 alkenyl, optionally substituted C10-C24
alkynyl, or optionally substituted C10-C24 acyl;
[0138] R.sub.3 and R.sub.4 are either the same or different and
independently optionally substituted C1-C6 alkyl, optionally
substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl
or R.sub.3 and R.sub.4 may join to form an optionally substituted
heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms
chosen from nitrogen and oxygen;
[0139] R.sub.5 is either absent or present and when present is
hydrogen or C1-C6 alkyl;
[0140] m, n, and p are either the same or different and
independently either 0 or 1 with the proviso that m, n, and p are
not simultaneously 0;
[0141] q is 0, 1, 2, 3, or 4; and
[0142] Y and Z are either the same or different and independently
O, S, or NH.
[0143] In another embodiment, the cationic lipid has the
formula:
##STR00002##
[0144] or a pharmaceutically acceptable salt thereof, wherein:
[0145] R.sub.1 and R.sub.2 are each independently H, alkyl,
alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and
heterocyclyl,
[0146] wherein each of alkyl, alkenyl, alkynyl, cycloalkyl, aryl,
heteroaryl, and heterocyclyl is optionally substituted by H; halo;
hydroxy; cyano; oxo; C.sub.1-C.sub.6 alkyl optionally substituted
by halo, hydroxy, or alkoxy;
[0147] or R.sub.1 and R.sub.2 are taken together with the N atom to
which they are both attached to form a 3-8 member heteroaryl or
heterocyclyl; wherein each of the heteroaryl and heterocyclyl is
optionally substituted by H; halo; hydroxy; cyano; oxo; nitro;
C.sub.1-C.sub.6 alkyl optionally substituted by halo, hydroxyl, or
alkoxy;
[0148] R.sub.3 is absent, H, alkyl, alkenyl, alkynyl, cycloalkyl,
aryl, heteroaryl, or heterocyclyl;
[0149] R.sub.4 and R.sub.5 are each independently H, alkyl,
alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or
heterocyclyl;
[0150] wherein each of alkyl, alkenyl, alkynyl, cycloalkyl, aryl,
heteroaryl, and heterocyclyl is optionally substituted by H; halo;
hydroxy; cyano; oxo; C.sub.1-C.sub.6 alkyl optionally substituted
by halo, hydroxy, or alkoxy;
[0151] X is --O--, --S--, --NR.sub.4--, --S--S--, --OC(.dbd.O)--,
--C(.dbd.O)O--, --OC(.dbd.O)O--, --NR.sub.4C(.dbd.O)--,
C(.dbd.O)NR.sub.4--, --NR.sub.4C(.dbd.O)O--,
--OC(.dbd.O)NR.sub.4--, --NR.sub.4C(.dbd.O)NR.sub.4--,
--NR.sub.4C(.dbd.S)O--, OC(.dbd.S)NR.sub.4--,
--NR.sub.4C(.dbd.S)NR.sub.4--, --CR.sub.4R.sub.5--;
[0152] Y and Z are independently C.sub.10 to C.sub.30 groups having
the formula
L.sub.1--(CR.sub.6R.sub.7).sub..alpha.--[L.sub.2--(CR.sub.6R.sub.-
7).sub..beta.].sub..gamma.--L.sub.3--R.sub.8, wherein
[0153] L.sub.1 is a bond, --(CR.sub.6R.sub.7)--, --O--, --CO--,
--NR.sub.8--, --S--, or a combination thereof;
[0154] each R.sub.6 and R.sub.7, independently, is H; halo;
hydroxyl, cyano; C.sub.1-C.sub.6 alkyl optionally substituted by
halo, hydroxyl, or alkoxy:
[0155] L.sub.2 is a bond, --(CR.sub.6R.sub.7)--, --O--, --CO--,
--NR.sub.8--, --S--,
##STR00003##
or a combination thereof, or has the formula
##STR00004##
[0156] wherein b, c, and d are each independently 0, 1, 2, or 3,
given the sum of b, c, and d is at least 1 and no greater than 8;
and R.sub.9 and R.sub.10 are each independently R.sub.7, or
adjacent R.sub.9 and R.sub.10, taken together, are optionally a
bond;
[0157] L.sub.3 is a bond, --(CR.sub.6R.sub.7)--, --O--, --CO--,
--NR.sub.8--, --S--,
##STR00005##
or a combination thereof
[0158] R.sub.8 is independently H; halo; hydroxy; cyano; C1-C6
alkyl optionally substituted by halo, hydroxy, or alkoxy; aryl;
heteroaryl; or heterocyclyl; or R.sub.8 has the formula:
##STR00006##
[0159] a is 0, 1, 2, 3, or 4;
[0160] .alpha. is 0-6;
[0161] each .beta., independently, is 0-6;
[0162] .gamma. is 0-6.
[0163] Other suitable cationic lipids include cationic lipids,
which carry a net positive charge at about physiological pH, in
addition to those specifically described above,
N,N-dioleyl-N,N-dimethylammonium chloride (DODAC);
N-(2,3-dioleyloxy)propyl-N,N--N-triethylammonium chloride (DOTMA);
N,N-distearyl-N,N-dimethylammonium bromide (DDAB);
N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTAP); 1,2-dioleyloxy-3-trimethylaminopropane chloride salt
(DOTAP.Cl);
3.beta.-(N--(N',N'-dimethylaminoethane)carbamoyl)cholesterol
(DC-Chol),
N-(1-(2,3-dioleoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethy-
lammoniumtrifluoracetate (DOSPA),
dioctadecylamidoglycylcarboxyspermine (DOGS),
1,2-dioleoyl-3-dimethylammonium propane (DODAP),
N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and
N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
bromide (DMRIE). Additionally, a number of commercial preparations
of cationic lipids can be used, such as, e.g., LIPOFECTIN.RTM.
(including DOTMA and DOPE, available from GIBCO/BRL), and
LIPOFECTAMINE.RTM. (comprising DOSPA and DOPE, available from
GIBCO/BRL).
[0164] The cationic lipid is present in the particle in an amount
from about 30 to about 95 mole percent. In one embodiment, the
cationic lipid is present in an amount from about 30 to about 70
mole percent. In one embodiment, the cationic lipid is present in
an amount from about 40 to about 60 mole percent.
Neutral Lipids
[0165] In certain embodiments, the particle includes one or more
neutral lipids.
[0166] The term "lipid" refers to a group of organic compounds that
are esters of fatty acids and are characterized by being insoluble
in water but soluble in many organic solvents. Lipids are usually
divided in at least three classes: (1) "simple lipids" which
include fats and oils as well as waxes; (2) "compound lipids" which
include phospholipids and glycolipids; and (3) "derived lipids"
such as steroids.
[0167] The term "neutral lipid" refers to any one of a number of
lipid species that exist in either an uncharged or neutral
zwitterionic form at physiological pH. Representative neutral
lipids include diacylphosphatidylcholines,
diacylphosphatidylethanolamines, ceramides, sphingomyelins,
dihydrosphingomyelins, cephalins, and cerebrosides.
[0168] Exemplary lipids include, for example,
distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine
(DOPC), dipalmitoylphosphatidylcholine (DPPC),
dioleoylphosphatidylglycerol (DOPG),
dipalmitoylphosphatidylglycerol (DPPG),
dioleoyl-phosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoyl-phosphatidylethanolamine (POPE) and
dioleoyl-phosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),
dipalmitoylphosphatidyl ethanolamine (DPPE),
dimyristoylphosphoethanolamine (DMPE),
distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE,
16-O-dimethyl PE, 18-1-trans PE,
1-stearioyl-2-oleoyl-phosphatidyethanol amine (SOPE), and
1,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE).
[0169] In one embodiment, the neutral lipid is
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).
Sterols
[0170] In certain embodiments, the particle includes one or more
sterols.
[0171] The term "sterol" refers to a subgroup of steroids also
known as steroid alcohols. Sterols are usually divided into two
classes: (1) plant sterols also known as "phytosterols" and (2)
animal sterols also known as "zoosterols."
[0172] Exemplary sterols include, for example, campesterol,
sitosterol, stigmasterol, ergosterol, and cholesterol. In one
embodiment, the sterol is cholesterol.
Surfactants
[0173] In certain embodiments, the particle includes one or more
surfactants.
[0174] The term surfactant as used herein, refers to non-ionic,
amphipathic compounds that contain both hydrophobic groups and
hydrophilic groups.
[0175] In one embodiment, a surfactant is represented by the
formula
##STR00007##
[0176] wherein
[0177] R.sub.1 is H, C.sub.1-C.sub.6 alkyl;
[0178] X is --O--, --S--, --NR.sub.2--, --S--S--, --OC(.dbd.O)--,
--C(.dbd.O)O--, --OC(.dbd.O)O--, --NR.sub.2C(.dbd.O)--,
C(.dbd.O)NR.sub.2--, --NR.sub.2C(.dbd.O)O--,
--OC(.dbd.O)NR.sub.2--, --NR.sub.2C(.dbd.O)NR.sub.2--,
--NR.sub.2C(.dbd.S)O--, OC(.dbd.S)NR.sub.2--,
--NR.sub.2C(.dbd.S)NR.sub.2--, --CR.sub.2R.sub.3--;
[0179] R.sub.2 and R.sub.3 are each independently H, alkyl,
alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or
heterocyclyl;
[0180] wherein each of alkyl, alkenyl, alkynyl, cycloalkyl, aryl,
heteroaryl, and heterocyclyl is optionally substituted by H; halo;
hydroxy; cyano; oxo; C.sub.1-C.sub.6 alkyl optionally substituted
by halo, hydroxy, or alkoxy;
[0181] Y is a C.sub.10 to C.sub.40 group having the formula
L.sub.1--(CR.sub.4R.sub.5).sub..alpha.--[L.sub.2--(CR.sub.4R.sub.5).sub..-
beta.].sub..gamma.--L.sub.3--R.sub.6, wherein:
[0182] L.sub.1 is a bond, --(CR.sub.4R.sub.5)--, --O--, --CO--,
--NR.sub.2--, --S--, or a combination thereof; each R.sub.4 and
R.sub.5, independently, is H; halo; hydroxyl, cyano;
C.sub.1-C.sub.6 alkyl optionally substituted by halo, hydroxyl, or
alkoxy;
[0183] L.sub.2 and L.sub.3 each, independently, are a bond,
--(CR.sub.4R.sub.5)--, --O--, --CO--, --NR.sub.2--, --S--
##STR00008##
or a combination thereof;
[0184] R.sub.6 is independently H; halo; hydroxy; cyano;
C.sub.1-C.sub.6 alkyl optionally substituted by halo, hydroxy, or
alkoxy; aryl; heteroaryl; or heterocyclyl; or R.sub.6 has the
formula:
##STR00009##
[0185] a is 2-100;
[0186] .alpha. is 0-6;
[0187] each .beta., independently, is 0-6;
[0188] .gamma. is 0-6.
[0189] In another embodiment, a surfactant is represented by the
formula
##STR00010##
[0190] wherein:
[0191] x=1 to 50;
[0192] y=1 to 50; and
[0193] z=1 to 50.
[0194] In another embodiment, a surfactant is represented by the
formula
##STR00011##
wherein:
[0195] x=1 to 50;
[0196] y=1 to 50; and
[0197] z=1 to 50.
[0198] In certain embodiments, the surfactant is selected from the
group consisting of polyoxyethylene alkyl ethers, polyoxyethylene
alkyl esters, diblock co-polymers and triblock co-polymers.
Suitable surfactants include polyoxyethylene (20) oleyl ether,
polyoxyethylene (23) lauryl ether, polyoxyethylene (40) stearate,
poly(propylene glycol).sub.11 -block- poly(ethylene glycol).sub.16
-block- poly(propylene glycol).sub.11, poly(propylene
glycol).sub.12 -block- poly(ethylene glycol).sub.28 -block-
poly(propylene glycol).sub.12.
[0199] In certain embodiments, the surfactant is present in the
particle in an amount from about 0.1 to about 20 mole percent. In
one embodiment, the surfactant is present in an amount from about
0.5 to about 10 mole percent. In one embodiment, the surfactant is
present in the lipid nanoparticle in about 2 mole percent.
[0200] In one embodiment, the surfactant is polyoxyethylene (20)
oleyl ether.
[0201] In one embodiment, the surfactant is polyoxyethylene (40)
stearate.
Anionic Macromolecules
[0202] The lipid nanoparticles of the present invention are useful
for the systemic or local delivery of anionic macromolecules.
[0203] As used herein, the term "anionic macromolecule" refers to a
macromolecule that is anionic or becomes anionic (deprotonated) as
the pH is increased above the pK of the ionizable group of the
macromolecule, but is progressively more neutral at lower pH
values. At pH values above the pK, the macromolecule is then able
to associate with positively charged lipids (e.g., cationic
lipids). As used herein, the term "anionic macromolecule" includes
zwitterionic macromolecules that assume a negative charge on pH
increase.
[0204] The term "anionic macromolecule" refers to any of a number
of species which carry a net negative charge at a selective pH,
such as physiological pH. Such macromolecules include, but are not
limited to, nucleic acids, proteins, peptides and
carbohydrates.
Nucleic Acids
[0205] The lipid nanoparticles of the present invention are useful
for the systemic or local delivery of nucleic acids.
[0206] As used herein, the term "nucleic acid" is meant to include
any oligonucleotide or polynucleotide. Fragments containing up to
50 nucleotides are generally termed oligonucleotides, and longer
fragments are called polynucleotides. In particular embodiments,
oligonucleotides of the present invention are 20-50 nucleotides in
length. In the context of this invention, the terms
"polynucleotide" and "oligonucleotide" refer to a polymer or
oligomer of nucleotide or nucleoside monomers consisting of
naturally occurring bases, sugars and intersugar (backbone)
linkages. The terms "polynucleotide" and "oligonucleotide" also
includes polymers or oligomers comprising non-naturally occurring
monomers, or portions thereof, which function similarly. Such
modified or substituted oligonucleotides are often preferred over
native forms because of properties such as, for example, enhanced
cellular uptake and increased stability in the presence of
nucleases. Oligonucleotides are classified as
deoxyribooligonucleotides or ribooligonucleotides. A
deoxyribooligonucleotide consists of a 5-carbon sugar called
deoxyribose joined covalently to phosphate at the 5' and 3' carbons
of this sugar to form an alternating, unbranched polymer. A
ribooligonucleotide consists of a similar repeating structure where
the 5-carbon sugar is ribose. The nucleic acid that is present in a
lipid nanoparticle according to this invention includes any form of
nucleic acid that is known. The nucleic acids used herein can be
single-stranded DNA or RNA, or double-stranded DNA or RNA, or
DNA-RNA hybrids. Examples of double-stranded DNA include structural
genes, genes including control and termination regions, and
self-replicating systems such as viral or plasmid DNA. Examples of
double-stranded RNA include siRNA and other RNA interference
reagents. Single-stranded nucleic acids include antisense
oligonucleotides, ribozymes, microRNA, mRNA and triplex-forming
oligonucleotides.
[0207] In one embodiment, the polynucleic acid is an antisense
oligonucleotide. In certain embodiments, the nucleic acid is an
antisense nucleic acid, a ribozyme, tRNA, snRNA, siRNA, shRNA,
ncRNA, miRNA, mRNA, pre-condensed DNA, or an aptamer.
[0208] The term "nucleic acids" also refers to ribonucleotides,
deoxynucleotides, modified ribonucleotides, modified
deoxyribonucleotides, modified phosphate-sugar-backbone
oligonucleotides, other nucleotides, nucleotide analogs, and
combinations thereof, and can be single stranded, double stranded,
or contain portions of both double stranded and single stranded
sequence, as appropriate.
[0209] The term "nucleotide," as used herein, generically
encompasses the following terms, which are defined below:
nucleotide base, nucleoside, nucleotide analog, and universal
nucleotide.
[0210] The term "nucleotide base," as used herein, refers to a
substituted or unsubstituted parent aromatic ring or rings. In some
embodiments, the aromatic ring or rings contain at least one
nitrogen atom. In some embodiments, the nucleotide base is capable
of forming Watson-Crick and/or Hoogsteen hydrogen bonds with an
appropriately complementary nucleotide base. Exemplary nucleotide
bases and analogs thereof include, but are not limited to, purines
such as 2-aminopurine, 2,6-diaminopurine, adenine (A),
ethenoadenine, N6-2-isopentenyladenine (6iA),
N6-2-isopentenyl-2-methylthioadenine (2 ms6iA), N6-methyladenine,
guanine (G), isoguanine, N2-dimethylguanine (dmG), 7-methylguanine
(7mG), 2-thiopyrimidine, 6-thioguanine (6sG) hypoxanthine and
O6-methylguanine; 7-deaza-purines such as 7-deazaadenine
(7-deaza-A) and 7-deazaguanine (7-deaza-G); pyrimidines such as
cytosine (C), 5-propynylcytosine, isocytosine, thymine (T),
4-thiothymine (4sT), 5,6-dihydrothymine, O4-methylthymine, uracil
(U), 4-thiouracil (4sU) and 5,6-dihydrouracil (dihydrouracil; D);
indoles such as nitroindole and 4-methylindole; pyrroles such as
nitropyrrole; nebularine; base (Y). In some embodiments, nucleotide
bases are universal nucleotide bases. Additional exemplary
nucleotide bases can be found in Fasman, 1989, Practical Handbook
of Biochemistry and Molecular Biology, pp. 385-394, CRC Press, Boca
Raton, Fla., and the references cited therein. Further examples of
universal bases can be found, for example, in Loakes, N.A.R. 2001,
29:2437-2447 and Seela N.A.R. 2000, 28:3224-3232.
[0211] The term "nucleoside," as used herein, refers to a compound
having a nucleotide base covalently linked to the C-1' carbon of a
pentose sugar. In some embodiments, the linkage is via a
heteroaromatic ring nitrogen. Typical pentose sugars include, but
are not limited to, those pentoses in which one or more of the
carbon atoms are each independently substituted with one or more of
the same or different --R, --OR, --NRR or halogen groups, where
each R is independently hydrogen, (C1-C6) alkyl or (C5-C14) aryl.
The pentose sugar may be saturated or unsaturated. Exemplary
pentose sugars and analogs thereof include, but are not limited to,
ribose, 2'-deoxyribose, 2'-(C1-C6)alkoxyribose,
2'-(C5-C14)aryloxyribose, 2',3-dideoxyribose,
2',3'-didehydroribose, 2'-deoxy-3'-haloribose,
2'-deoxy-3'-fluororibose, 2'-deoxy-3'-chlororibose,
2'-deoxy-3'-aminoribose, 2'-deoxy-3'-(C1-C6)alkylribose,
2'-deoxy-3'-(C1-C6)alkoxyribose and
2'-deoxy-3'-(C5-C14)aryloxyribose. Also see, e.g., 2'-O-methyl,
4'-.alpha.-anomeric nucleotides, 1'-alpha-anomeric nucleotides
(Asseline (1991) Nucl. Acids Res. 19:4067-74), 2'-4'- and
3'-4'-linked and other "locked" or "LNA," bicyclic sugar
modifications (WO 98/22489; WO 98/39352; WO 99/14226). "LNA" or
"locked nucleic acid" is a DNA analogue that is conformationally
locked such that the ribose ring is constrained by a methylene
linkage between the 2'-oxygen and the 3'- or 4'-carbon. The
conformation restriction imposed by the linkage often increases
binding affinity for complementary sequences and increases the
thermal stability of such duplexes.
[0212] Sugars include modifications at the 2'- or 3'-position such
as methoxy, ethoxy, allyloxy, isopropoxy, butoxy, isobutoxy,
methoxyethyl, alkoxy, phenoxy, azido, amino, alkylamino, fluoro,
chloro and bromo. Nucleosides and nucleotides include the natural D
configurational isomer (D-form), as well as the L configurational
isomer (L-form) (Beigelman, U.S. Pat. No. 6,251,666; Chu, U.S. Pat.
No. 5,753,789; Shudo, EP0540742; Garbesi (1993) Nucl. Acids Res.
21:4159-65; Fujimori (1990) J. Amer. Chem. Soc. 112:7435; Urata,
(1993) Nucleic Acids Symposium Ser. No. 29:69-70). When the
nucleobase is purine, e.g., A or G, the ribose sugar is attached to
the N9-position of the nucleobase. When the nucleobase is
pyrimidine, e.g., C, T or U, the pentose sugar is attached to the
N1-position of the nucleobase (Kornberg and Baker, (1992) DNA
Replication, 2nd Ed., Freeman, San Francisco, Calif.).
[0213] One or more of the pentose carbons of a nucleoside may be
substituted with a phosphate ester. In some embodiments, the
phosphate ester is attached to the 3'- or 5'-carbon of the pentose.
In some embodiments, the nucleosides are those in which the
nucleotide base is a purine, a 7-deazapurine, a pyrimidine, a
universal nucleotide base, a specific nucleotide base, or an analog
thereof.
[0214] The term "nucleotide analog," as used herein, refers to
embodiments in which the pentose sugar and/or the nucleotide base
and/or one or more of the phosphate esters of a nucleoside may be
replaced with its respective analog. In some embodiments, exemplary
pentose sugar analogs are those described above. In some
embodiments, the nucleotide analogs have a nucleotide base analog
as described above. In some embodiments, exemplary phosphate ester
analogs include, but are not limited to, alkylphosphonates,
methylphosphonates, phosphoramidates, phosphotriesters,
phosphorothioates, phosphorodithioates, phosphoroselenoates,
phosphorodiselenoates, phosphoroanilothioates, phosphoroanilidates,
phosphoroamidates, boronophosphates, and may include associated
counterions. Other nucleic acid analogs and bases include for
example intercalating nucleic acids (INAs, as described in
Christensen and Pedersen, 2002), and AEGIS bases (Eragen, U.S. Pat.
No. 5,432,272). Additional descriptions of various nucleic acid
analogs can also be found for example in (Beaucage et al.,
Tetrahedron 49(10):1925 (1993) and references therein; Letsinger,
J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem.
81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986);
Sawai et al., Chem. Lett. 805 (1984), Letsinger et al., J. Am.
Chem. Soc. 110:4470 (1988); and Pauwels et al., ChemicaScripta
26:141 (1986)), phosphorothioate (Mag et al., Nucleic Acids Res.
19:1437 (1991); and U.S. Pat. No. 5,644,048. Other nucleic analogs
comprise phosphorodithioates (Briu et al., J. Am. Chem. Soc.
111:2321 (1989)), O-methylphosphoroamidite linkages (see Eckstein,
Oligonucleotides and Analogues: A Practical Approach, Oxford
University Press), those with positive backbones (Denpcy et al.,
Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones
(U.S. Pat. Nos. 5,386,023; 5,386,023; 5,637,684; 5,602,240;
5,216,141; and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed.
English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470
(1988); Letsinger et al., Nucleoside & Nucleotide 13:1597
(194): Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate
Modifications in Antisense Research," Ed. Y. S. Sanghui and P. Dan
Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett.
4:395 (1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994);
Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones,
including those described in U.S. Pat. Nos. 5,235,033 and
5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,
"Carbohydrate Modifications in Antisense Research," Ed. Y. S.
Sanghui and P. Dan Cook. Nucleic acids containing one or more
carbocyclic sugars are also included within the definition of
nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp.
169-176). Several nucleic acid analogs are also described in Rawls,
C & E News Jun. 2, 1997, page 35.
[0215] The term "universal nucleotide base" or "universal base," as
used herein, refers to an aromatic ring moiety, which may or may
not contain nitrogen atoms. In some embodiments, a universal base
may be covalently attached to the C-1' carbon of a pentose sugar to
make a universal nucleotide. In some embodiments, a universal
nucleotide base does not hydrogen bond specifically with another
nucleotide base. In some embodiments, a universal nucleotide base
hydrogen bonds with nucleotide base, up to and including all
nucleotide bases in a particular target polynucleotide. In some
embodiments, a nucleotide base may interact with adjacent
nucleotide bases on the same nucleic acid strand by hydrophobic
stacking. Universal nucleotides include, but are not limited to,
deoxy-7-azaindole triphosphate (d7AITP), deoxyisocarbostyril
triphosphate (dICSTP), deoxypropynylisocarbostyril triphosphate
(dPICSTP), deoxymethyl-7-azaindole triphosphate (dM7AITP),
deoxyImPy triphosphate (dImPyTP), deoxyPP triphosphate (dPPTP), or
deoxypropynyl-7-azaindole triphosphate (dP7AITP). Further examples
of such universal bases can be found, inter alia, in Published U.S.
application Ser. No. 10/290,672, and U.S. Pat. No. 6,433,134.
[0216] As used herein, the terms "polynucleotide" and
"oligonucleotide" are used interchangeably and mean single-stranded
and double-stranded polymers of nucleotide monomers, including
2'-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked by
internucleotidephosphodiester bond linkages, e.g., 3'-5' and 2'-5',
inverted linkages, e.g., 3'-3' and 5'-5', branched structures, or
internucleotide analogs. Polynucleotides have associated counter
ions, such as H.sup.+, NH.sub.4.sup.+, trialkylammonium, Mg.sup.2+,
Na.sup.+, and the like. A polynucleotide may be composed entirely
of deoxyribonucleotides, entirely of ribonucleotides, or chimeric
compositions thereof. Polynucleotides may be comprised of
internucleotide, nucleobase and/or sugar analogs. Polynucleotides
typically range in size from a few monomeric units, e.g., 3-40 when
they are more commonly frequently referred to in the art as
oligonucleotides, to several thousands of monomeric nucleotide
units. Unless denoted otherwise, whenever a polynucleotide sequence
is represented, it will be understood that the nucleotides are in
5' to 3' order from left to right and that "A" denotes
deoxyadenosine, "C" denotes deoxycytosine, "G" denotes
deoxyguanosine, and "T" denotes thymidine, unless otherwise
noted.
[0217] As used herein, "nucleobase" means those naturally occurring
and those non-naturally occurring heterocyclic moieties commonly
known to those who utilize nucleic acid technology or utilize
peptide nucleic acid technology to thereby generate polymers that
can sequence specifically bind to nucleic acids. Non-limiting
examples of suitable nucleobases include: adenine, cytosine,
guanine, thymine, uracil, 5-propynyl-uracil,
2-thio-5-propynyl-uracil, 5-methylcytosine, pseudoisocytosine,
2-thiouracil and 2-thiothymine, 2-aminopurine,
N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine,
N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) and
N8-(7-deaza-8-aza-adenine). Other non-limiting examples of suitable
nucleobase include those nucleobases illustrated in FIGS. 2(A) and
2(B) of Buchardt et al. (WO92/20702 or WO92/20703).
[0218] As used herein, "nucleobase sequence" means any segment, or
aggregate of two or more segments (e.g. the aggregate nucleobase
sequence of two or more oligomer blocks), of a polymer that
comprises nucleobase-containing subunits. Non-limiting examples of
suitable polymers or polymers segments include
oligodeoxynucleotides (e.g. DNA), oligoribonucleotides (e.g. RNA),
peptide nucleic acids (PNA), PNA chimeras, PNA combination
oligomers, nucleic acid analogs and/or nucleic acid mimics.
[0219] As used herein, "polynucleobase strand" means a complete
single polymer strand comprising nucleobase subunits. For example,
a single nucleic acid strand of a double stranded nucleic acid is a
polynucleobase strand.
[0220] As used herein, "nucleic acid" is a nucleobase
sequence-containing polymer, or polymer segment, having a backbone
formed from nucleotides, or analogs thereof.
[0221] Preferred nucleic acids are DNA and RNA.
[0222] As used herein, nucleic acids may also refer to "peptide
nucleic acid" or "PNA" means any oligomer or polymer segment (e.g.,
block oligomer) comprising two or more PNA subunits (residues), but
not nucleic acid subunits (or analogs thereof), including, but not
limited to, any of the oligomer or polymer segments referred to or
claimed as peptide nucleic acids in U.S. Pat. Nos. 5,539,082;
5,527,675; 5,623,049; 5,714,331; 5,718,262; 5,736,336; 5,773,571;
5,766,855; 5,786,461; 5,837,459; 5,891,625; 5,972,610; 5,986,053;
and 6,107,470; all of which are herein incorporated by reference.
The term "peptide nucleic acid" or "PNA" shall also apply to any
oligomer or polymer segment comprising two or more subunits of
those nucleic acid mimics described in the following publications:
Lagriffoul et al., Bioorganic & Medicinal Chemistry Letters,
4:1081-1082 (1994); Petersen et al., Bioorganic & Medicinal
Chemistry Letters, 6:793-796 (1996); Diderichsen et al., Tett.
Lett. 37:475-478 (1996); Fujii et al., Bioorg. Med. Chem. Lett.
7:637-627 (1997); Jordan et al., Bioorg. Med. Chem. Lett. 7:687-690
(1997); Krotz et al., Tett. Lett. 36:6941-6944 (1995); Lagriffoul
et al., Bioorg. Med. Chem. Lett. 4:1081-1082 (1994); Diederichsen,
U., Bioorganic & Medicinal Chemistry Letters, 7:1743-1746
(1997); Lowe et al., J. Chem. Soc. Perkin Trans. 1, (1997)
1:539-546; Lowe et al., J. Chem. Soc. Perkin Trans. 11:547-554
(1997); Lowe et al., J. Chem. Soc. Perkin Trans. 11:555-560 (1997);
Howarth et al., J. Org. Chem. 62:5441-5450 (1997); Altmann, K-H et
al., Bioorganic & Medicinal Chemistry Letters, 7:1119-1122
(1997); Diederichsen, U., Bioorganic & Med. Chem. Lett.,
8:165-168 (1998); Diederichsen et al., Angew. Chem. Int. Ed.,
37:302-305 (1998); Cantin et al., Tett. Lett., 38:4211-4214 (1997);
Ciapetti et al., Tetrahedron, 53:1167-1176 (1997); Lagriffoule et
al., Chem. Eur. J., 3:912-919 (1997); Kumar et al., Organic Letters
3(9):1269-1272 (2001); and the Peptide-Based Nucleic Acid Mimics
(PENAMS) of Shah et al. as disclosed in WO96/04000.
[0223] The lipid nanoparticle of the invention differs from other
similarly constituted materials by its morphology and characterized
as having a substantially solid core. A lipid nanoparticle having a
substantially solid core is a particle that does not have extended
aqueous regions on the interior and that has an interior that is
primarily lipid. In one embodiment, an extended region is a
continuous aqueous region with a volume greater than half the
particle volume. In a second embodiment, an extended aqueous region
is more than 25% of the particle volume. The extent of internal
aqueous regions may be determined by electron microscopy and appear
as regions of low electron density. Further, because the interior
of the solid core nanoparticle is primarily lipid, the aqueous
content of the particle (the "trapped volume") per lipid
constituting the particle is less than that expected for a
unilamellar bilayer lipid vesicle with the same radius. In one
embodiment, the trapped volume is less than 50% of that expected
for a unilamellar bilayer vesicle with the same radius. In a second
embodiment, the trapped volume is less than 25% of that expected
for a unilamellar bilayer vesicle of the same size. In a third
embodiment, the trapped volume is less than 20% of the total volume
of the particle. In one embodiment, the trapped volume per lipid is
less than 2 microliter per micromole lipid. In another embodiment
the trapped volume is less than 1 microliter per micromole lipid.
In addition, while the trapped volume per lipid increases
substantially for a bilayer lipid vesicle as the radius of the
vesicle is increased, the trapped volume per lipid does not
increase substantially as the radius of solid core nanoparticles is
increased. In one embodiment, the trapped volume per lipid
increases by less than 50% as the mean size is increased from a
diameter of 20 nm to a diameter of 100 nm. In a second embodiment,
the trapped volume per lipid increases by less than 25% as the mean
size is increased from a diameter of 20 nm to a diameter of 100 nm.
The trapped volume can be measured employing a variety of
techniques described in the literature. Because solid core systems
contain lipid inside the particle, the total number of particles of
a given radius generated per mole of lipid is less than expected
for bilayer vesicle systems. The number of particles generated per
mol of lipid can be measured by fluorescence techniques amongst
others.
[0224] The lipid nanoparticles of the invention can also be
characterized by electron microscopy. The particles of the
invention having a substantially solid core have an electron dense
core as seen by electron microscopy. Electron dense is defined such
that area-averaged electron density of the interior 50% of the
projected area of a solid core particle (as seen in a 2-D cryo EM
image) is not less than x % (x=20%, 40%, 60%) of the maximum
electron density at the periphery of the particle. Electron density
is calculated as the absolute value of the difference in image
intensity of the region of interest from the background intensity
in a region containing no nanoparticle.
Therapeutic Material
[0225] As used herein, the term "therapeutic material" is defined
as a substance intended to furnish pharmacological activity or to
otherwise have direct effect in the diagnosis, cure, mitigation,
treatment or prevention of disease, or to have direct effect in
restoring, correcting or modifying physiological functions.
Therapeutic materials include but are not limited to small molecule
drugs, nucleic acids, proteins, peptides, polysaccharides,
inorganic ions and radionuclides.
Research Reagent
[0226] As used herein, the term "research reagent" is defined as a
substance intended to furnish a defined activity or to otherwise
have direct influence on the biological effect of cells, tissues or
organs. Research Reagents include but are not limited to small
molecule organic compounds (e.g., organic compounds having
molecular weights less than 800 g/mole, or less than 500 g/mole),
nucleic acids, proteins, peptides, polysaccharides, inorganic ions
and radionuclides. Examples of nucleic acid Research Reagents
include but are not limited to antisense oligonucleotides,
ribozymes, microRNA, mRNA, ribozyme, tRNA, snRNA, siRNA, shRNA,
ncRNA, miRNA, mRNA, pre-condensed DNA, pDNA or an aptamer. Nucleic
acid Research Reagents are used to silence genes (with for example
siRNA), express genes (with for example mRNA), edit genomes (with
for example CRISPR/Cas9).
Polymers
[0227] As used herein, the term "polymer" refers to compounds of
usually high molecular weight built up chiefly or completely from a
large number of similar units bonded together. Such polymers
include any of numerous natural, synthetic and semi-synthetic
polymers.
Natural Polymers
[0228] The term "natural polymer" refers to any number of polymer
species derived from nature. Such polymers include, but are not
limited to the polysaccharides, cellulose, chitin, and
alginate.
Synthetic Polymers
[0229] The term "synthetic polymer" refers to any number of
synthetic polymer species not found in nature. Such synthetic
polymers include, but are not limited to, synthetic homopolymers
and synthetic copolymers. Synthetic homopolymers include, but are
not limited to, polyethylene glycol, polylactide, polyglycolide,
polyacrylates, polymethacrylates, poly-.epsilon.-caprolactone,
polyorthoesters, polyanhydrides, polylysine, and polyethyleneimine.
"Synthetic copolymer" refers to any number of synthetic polymer
species made up of two or more synthetic homopolymer subunits. Such
synthetic copolymers include, but are not limited to,
poly(lactide-co-glycolide), poly(lactide)-poly(ethylene glycol),
poly(lactide-co-glycolide)-poly(ethylene glycol), and
poly(.epsilon.-caprolactone)-poly(ethylene glycol).
Semi-Synthetic Polymers
[0230] The term "semi-synthetic polymer" refers to any number of
polymers derived by the chemical or enzymatic treatment of natural
polymers. Such polymers include, but are not limited to,
carboxymethylcellulose, acetylated carboxymethylcellulose,
cyclodextrin, chitosan, and gelatin.
Polymer Conjugate
[0231] As used herein, the term "polymer conjugate" refers to a
compound prepared by covalently, or non-covalently conjugating one
or more molecular species to a polymer. Such polymer conjugates
include, but are not limited to, polymer-therapeutic material
conjugates.
Polymer-Therapeutic Material Conjugate
[0232] As used herein, the term "polymer-therapeutic material
conjugate" refers to a polymer conjugate where one or more of the
conjugated molecular species is a therapeutic material. Such
polymer-therapeutic material conjugates include, but are not
limited to, polymer-drug conjugates.
Polymer-Drug Conjugate
[0233] As used herein, the term "polymer-drug conjugate" refers to
any number of polymer species conjugated to any number of drug
species. Such polymer drug conjugates include, but are not limited
to, acetyl methylcellulose-polyethylene glycol-docetaxel.
[0234] As noted above, the nanoparticles of the invention are
composed of particle-forming materials. Particle-forming materials
include, among other components, lipids and polymers as described
herein.
[0235] The following example is provided for the purpose of
illustrating, not limiting, the invention.
EXAMPLE
Materials
[0236] 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) was
purchased from Avanti Polar Lipids (Alabaster, Ala., USA),
cholesterol was obtained from Sigma (St Louis, Mo., USA),
1,17-bis(2-octylcyclopropyl)heptadecan-9-yl
4-(dimethylamino)butanoate (CL, for example, cationic lipid) was
synthesized by Avanti Polar Lipids (Alabaster, Ala., USA), and
polyethylene glycol-dimyristoyl propylamine (PEG-c-DMA) was
synthesized by the Center for Drug Research and Development
(Vancouver, BC, Canada). A 21-mer duplex siRNA was used for
encapsulation in LNP systems.
Representative Preparation of siRNA-LNP Systems at Small
Volumes
[0237] CL, DSPC, cholesterol, and PEG-lipid were first solubilized
in ethanol at a molar ratio of 50:10:38.5:1.5 and total lipid
concentration of 30.5 mg/mL to give the ethanol lipid solution. The
siRNA was solubilized in a 25 mM acetate, pH=4.0 buffer at a
concentration of 0.927 mg/mL to give the aqueous siRNA solution. A
target siRNA/lipid ratio of 0.09 (wt/wt) was used. 40 .mu.L of PBS
was pipetted into the outlet well of the device. 30 .mu.L of the
aqueous siRNA solution was pipetted into the siRNA inlet well. 10
.mu.L of the ethanol lipid solution was pipetted into the lipid
inlet well. A manifold was then clamped over the inlet wells and
pressurized using a Luer-lock syringe. Pressurization pushes the
reagents in the inlet wells through the device and into the outlet
well, where they are immediately diluted at a ratio of 1:1 by the
PBS that is preloaded in the outlet well. The sample volume of 80
.mu.L is recovered by pipetting out of the outlet well and further
diluted at a ratio of 1:1 with 80 .mu.L of PBS.
[0238] The following protocol is with reference to FIGS. 3, 7, 9
and 10
Low Dead Volume Device Protocol (160 .mu.L Formulation)
[0239] 1. Add 40 .mu.L of dilution buffer (1.times. PBS) to the
outlet port.
[0240] 2. Add 30 .mu.L of aqueous stock (with siRNA) to the inlet
port marked "aqueous."
[0241] 3. Add 10 .mu.L of lipid stock to the inlet port marked
"lipid."
[0242] 4. Next, place the chip in the clamping device with the
manifold on top of the chip, so that both the inlet ports are
positioned inside the O-ring (place the manifold using bars on the
clamping device as a guide to ensure the same).
[0243] 5. Carefully lower the clamping block so that it sits on the
manifold and push the lever towards the chip in order to secure the
chip in place as well as seal the inlet ports within the O-ring of
the manifold.
[0244] 6. Fill a 3 mL syringe with about 2 mL of air and fix it
onto the Luer lock port on the top side of the manifold..
[0245] 7. Push the plunger rapidly.
[0246] 8. Collect the formulation from the outlet port.
[0247] 9. Add 80 .mu.L of dilution buffer (1.times. PBS) to the
formulation and pipette up and down a few times to ensure good
mixing.
Washing the Device
[0248] 1. Add 40 .mu.L of distilled water and ethanol to the inlet
ports marked "aqueous" and "lipid," respectively.
[0249] 2. Fix the chip onto the manifold and pressurize with 2 mL
of air. Remove the waste from the outlet port.
[0250] 3. Repeat the above until the chip is clear and free of any
deposits.
[0251] 4. Push air through the chip (without any liquid) to expel
of the remaining fluid inside the chip.
[0252] 5. Blot out all three ports with a Kimwipe.
[0253] 6. Leave the chip to dry at room temperature (takes around
1.5 to 2 hours).
[0254] The manufactured nanoparticles were cationic
lipid:DSPC:Cholesterol:PEG-Lipid (50:10:38.5:1.5) encapsulating a
21-nucleotide duplex siRNA. The final volume of the nanoparticle
solution was 160 .mu.L.
Representative Preparation of mRNA-LNP Systems at Small Volumes
[0255] The process described above for siRNA-LNP systems can be
adapted for preparation of mRNA-LNP. Essentially, the process is
identical except that the mRNA was solubilized in a 75 mM acetate,
pH=4.0 buffer and the (+/-) charge ratio, as expressed in the ratio
of positive amino groups to negative phosphate groups, is increased
from 3:1 to 8:1.
LNP Characterization
[0256] Particle size was determined by dynamic light scattering
using a Malvern Zetasizer NanoZS (Malvern Instruments, Westboro,
Mass., USA). Intensity-weighted distribution data was used, and the
average of two independent measurements was used for each sample.
Encapsulation efficiency (% EE) was determined using the Quant-iT
RiboGreen RNA Assay Kit (Life Technologies, Carlsbad, Calif., USA)
from the ratio of fluorescence signal of the sample in the absence
and presence of the LNP lysing detergent Triton X-100.
Encapsulation efficiency was calculated using the formula:
% EE=1-(F.sub.-Triton)/(F.sub.+Triton)
where:
[0257] F.sub.-Triton=Fluorescence signal in the absence of Triton
X-100
[0258] F.sub.+Triton=Fluorescence signal in the presence of Triton
X-100
All reported results are reported as the average of three (3)
independent experiments.
[0259] Particle size, particle polydispersity, and percent of
encapsulated active agent for the production of lipid nanoparticles
prepared as described above using the device of FIG. 3 are
summarized in Table 1.
TABLE-US-00001 TABLE 1 Lipid Nanoparticle Characteristics. Mixer
Channel Width Size (nm) PDI Encapsulation Efficiency 200 .mu.m 94.1
0.11 90.60%
In Vitro Testing of ZDV Formulations
[0260] The siRNA-lipid nanoparticles (siRNA-LNPs) synthesized using
a representative small volume microfluidic device of the invention
(Zero Dead Volume Chip) were compared with those prepared using the
NanoAssemblr (a fluidic device for making nanoparticles
commercially available from Precision NanoSystems, Vancouver,
British Columbia, Canada) by testing them in vitro on a rat E18
cortical neuron culture (co-cultured with glia and astrocytes). The
cells were transfected on DIV 13 (days in vitro) at a dose of 100
ng of PTEN siRNA per ml of cell culture media. The knockdown of
PTEN gene expression was then analysed at days 3 and 8 by RT-qPCR
(with Actin .beta. acting as a reference gene). The level of PTEN
knockdown for both siRNA-LNP formulations was similar as shown in
FIG. 11.
[0261] The GFP mRNA-LNPs synthesized using the small volume
microfluidic device (zero dead volume device) were compared with
those prepared using the NanoAssemblr by testing them in vitro on a
rat E18 cortical neuron culture (co-cultured with glia and
astrocytes). The cells were transfected on DIV 13 (days in vitro)
at a dose of 500 ng of GFP mRNA per ml of cell culture media. The
expression of GFP was analyzed on day 3 by flow cytometry. The
levels GFP expression for both the NanoAssemblr and Zero Dead
Volume Chip mRNA-LNP formulations were observed to be similar as
can be seen in FIG. 12.
[0262] While the preferred embodiment of the invention has been
illustrated and described, it will be appreciated that various
changes can be made therein without departing from the spirit and
scope of the invention.
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