U.S. patent application number 17/513738 was filed with the patent office on 2022-09-15 for nucleic acid-containing lipid particles and related methods.
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 Nathan M. Belliveau, Pieter R. Cullis, Carl Lars Genghis Hansen, Jens Huft.
Application Number | 20220290185 17/513738 |
Document ID | / |
Family ID | 1000006374430 |
Filed Date | 2022-09-15 |
United States Patent
Application |
20220290185 |
Kind Code |
A1 |
Cullis; Pieter R. ; et
al. |
September 15, 2022 |
NUCLEIC ACID-CONTAINING LIPID PARTICLES AND RELATED METHODS
Abstract
Lipid particles containing a nucleic acid, devices and methods
for making the lipid particles, and methods for using the lipid
particles.
Inventors: |
Cullis; Pieter R.;
(Vancouver, CA) ; Belliveau; Nathan M.; (Weymouth,
CA) ; Hansen; Carl Lars Genghis; (Vancouver, CA)
; Huft; Jens; (Vancouver, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of British Columbia |
Vancouver |
|
CA |
|
|
Assignee: |
The University of British
Columbia
Vancouver
CA
|
Family ID: |
1000006374430 |
Appl. No.: |
17/513738 |
Filed: |
October 28, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16049872 |
Jul 31, 2018 |
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17513738 |
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15687218 |
Aug 25, 2017 |
10041091 |
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16049872 |
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13464690 |
May 4, 2012 |
9758795 |
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15687218 |
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PCT/CA2010/001766 |
Nov 4, 2010 |
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13464690 |
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61280510 |
Nov 4, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2320/32 20130101;
C12N 15/113 20130101; C12N 15/111 20130101; A61K 9/146 20130101;
A61K 31/7088 20130101; A61K 9/145 20130101; A61K 9/1272 20130101;
C12N 2310/14 20130101; A61K 47/22 20130101; C12N 15/88
20130101 |
International
Class: |
C12N 15/88 20060101
C12N015/88; A61K 9/127 20060101 A61K009/127; A61K 31/7088 20060101
A61K031/7088; A61K 47/22 20060101 A61K047/22; C12N 15/11 20060101
C12N015/11; A61K 9/14 20060101 A61K009/14; C12N 15/113 20060101
C12N015/113 |
Claims
1. A method for introducing a polynucleic acid into a cell,
comprising contacting a cell with a lipid particle, comprising an
electron dense core comprising a polynucleic acid and an ionizable
lipid, the core coated with one or more polar lipids comprising a
PEG-lipid.
2. The method of claim 1, wherein the ionizable lipid is an amino
lipid.
3. The method of claim 1, wherein the ionizable lipid is a
dilinoleyl amino lipid.
4. The method of claim 1, wherein the ionizable lipid is selected
from the group consisting of DODAC, DOTMA, DDAB, DOTAP, DOTAP.Cl,
DC-Chol, DOSPA, DOGS, DOPE, DODAP, DODMA, and DMRIE.
5. The method of claim 1, wherein the ionizable lipid has the
formula: ##STR00003## wherein R.sub.1 and R.sub.2 are either the
same or different and independently optionally substituted
C.sub.10-C.sub.24 alkyl, optionally substituted C.sub.10-C.sub.24
alkenyl, optionally substituted C.sub.10-C.sub.24 alkynyl, or
optionally substituted C.sub.10-C.sub.24 acyl; R.sub.3 and R.sub.4
are either the same or different and independently optionally
substituted C.sub.1-C.sub.6 alkyl, optionally substituted
C.sub.2-C.sub.6 alkenyl, or optionally substituted C.sub.2-C.sub.6
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; R.sub.5 is either
absent or present and when present is hydrogen or C.sub.1-C.sub.6
alkyl; 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; q is 0, 1 , 2, 3, or 4; and Y and Z are
either the same or different and independently O, S, or NH.
6. The method of claim 1, wherein the PEG-lipid is selected from
the group consisting of PEG-modified phosphatidylethanolamines,
PEG-modified phosphatidic acids, PEG-modified ceramides,
PEG-modified dialkylamines, PEG-modified diacylglycerols, and
PEG-modified dialkylglycerols.
7. The method of claim 1, wherein the PEG-lipid is selected from
the group consisting of PEG-c-DMA, PEG-c-DOMG, and PEG-s-DMG.
8. The method of claim 1, comprising from about 1 to about 5 mole
percent PEG-lipid.
9. The method of claim 1, comprising about 1 mole percent
PEG-lipid.
10. The method of claim 1, wherein the polar lipids are selected
from the group consisting of neutral lipids and sterols.
11. The method of claim 1, wherein the polar lipids comprise a
neutral lipid selected from the group consisting of
diacylphosphatidylcholines, diacylphosphatidylethanolamines,
ceramides, sphingomyelins, dihydrosphingomyelins, cephalins, and
cerebrosides.
12. The method of claim 1, wherein the polar lipids comprise a
neutral lipid selected from DSPC and DOPC.
13. The method claim 1, wherein the polar lipids comprise a
sterol.
14. The method of claim 1, wherein the polynucleic acid is a DNA,
an RNA, a locked nucleic acid, a nucleic acid analog, or a plasmid
capable of expressing a DNA or an RNA.
15. The method of claim 1, wherein the polynucleic acid is ssDNA or
dsDNA.
16. The method of claim 1, wherein the polynucleic acid is siRNA or
microRNA.
17. The method particle of claim 1, wherein the polynucleic acid is
an oligonucleotide.
18. The method of claim 1, wherein the polynucleic acid is an
antisense oligonucleotide.
19. The method of claim 1, wherein the particle has a diameter from
about 30 nm to about 200 nm.
20. The method of claim 1, wherein the particle has a diameter of
about 80 nm.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 16/049,872, filed Jul. 31, 2018, which is a continuation of
U.S. application Ser. No. 15/687,218, filed Aug. 25, 2017 (now U.S.
Pat. No. 10,041,091), which is a continuation of U.S. application
Ser. No. 13/464,690, filed May 4, 2012 (now U.S. Pat. No.
9,758,795), which is a continuation of International Application
No. PCT/CA2010/001766, filed Nov. 4, 2010, which claims the benefit
of U.S. Provisional Application No. 61/280,510, filed Nov. 4, 2009,
each of which is expressly incorporated herein by reference in its
entirety.
STATEMENT REGARDING SEQUENCE LISTING
[0002] The sequence listing associated with this application is
provided in text format in lieu of a paper copy and is hereby
incorporated by reference into the specification. The name of the
text file containing the sequence listing is 64113_Seq_Final.txt.
The text file is 1.02 KB; was created on Aug. 24, 2017; and is
being submitted via EFS-Web with the filing of the
specification.
BACKGROUND OF THE INVENTION
[0003] Lipid nanoparticles (LNP) are the most clinically advanced
drug delivery systems, with seven LNP-based drugs having received
regulatory approval. These approved drugs contain small molecules
such as anticancer drugs and exhibit improved efficacy and/or
reduced toxicity compared to the "free" drug. LNP carrier
technology has also been applied to delivery of "genetic" drugs
such as plasmids for expression of therapeutic proteins or small
interfering RNA (siRNA) oligonucleotides (OGN) for silencing genes
contributing to disease progression. Devising methods for efficient
in vivo delivery of siRNA OGN and other genetic drugs is the major
problem impeding the revolutionary potential of these agents as
therapeutics.
[0004] Recent advances in LNP technology and the design of the
cationic lipids required for encapsulation and delivery of genetic
drugs highlight the potential of LNP systems to solve the in vivo
delivery problem. LNP-siRNA systems have been shown to induce
silencing of therapeutically relevant target genes in animal
models, including non-human primates following intravenous (i.v.)
injection and are currently under evaluation in several clinical
trials.
[0005] A variety of methods have been developed to formulate LNP
systems containing genetic drugs. These methods include mixing
preformed LNP with OGN in the presence of ethanol or mixing lipid
dissolved in ethanol with an aqueous media containing OGN and
result in LNP with diameters of 100 nm or less and OGN
encapsulation efficiencies of 65-95%. Both of these methods rely on
the presence of cationic lipid to achieve encapsulation of OGN and
poly(ethylene glycol) (PEG) lipids to inhibit aggregation and the
formation of large structures. The properties of the LNP systems
produced, including size and OGN encapsulation efficiency, are
sensitive to a variety of formulation parameters such as ionic
strength, lipid and ethanol concentration, pH, OGN concentration
and mixing rates. In general, parameters such as the relative lipid
and OGN concentrations at the time of mixing, as well as the mixing
rates are difficult to control using current formulation
procedures, resulting in variability in the characteristics of LNP
produced, both within and between preparations.
[0006] Microfluidic devices provide an ability to controllably and
rapidly mix fluids at the nanoliter scale with precise control over
temperature, residence times, and solute concentrations. Controlled
and rapid microfluidic mixing has been previously applied in the
synthesis of inorganic nanoparticles and microparticles, and can
outperform macroscale systems in large scale production of
nanoparticles. Microfluidic two-phase droplet techniques have been
applied to produce monodisperse polymeric microparticles for drug
delivery or to produce large vesicles for the encapsulation of
cells, proteins, or other biomolecules. The use of hydrodynamic
flow focusing, a common microfluidic technique to provide rapid
mixing of reagents, to create monodisperse liposomes of controlled
size has been demonstrated. This technique has also proven useful
in the production of polymeric nanoparticles where smaller, more
monodisperse particles were obtained, with higher encapsulation of
small molecules as compared to bulk production methods.
[0007] Despite advances in the development of methods for LNP
systems containing genetic drugs, a need exists for devices and
methods for preparing lipid nanoparticles containing therapeutic
materials, as well as improved lipid nanoparticles containing
therapeutic materials. The present invention seeks to fulfill this
need and provides further related advantages.
SUMMARY OF THE INVENTION
[0008] In one aspect, the invention provides lipid particles
comprising nucleic acids.
[0009] In one embodiment, the lipid particle comprises (a) one or
more cationic lipids, (b) one or more second lipids, and (c) one or
more nucleic acids, wherein the lipid particle comprises a
substantially solid core, as defined herein.
[0010] In one embodiment, the cationic lipid is DLin-KC2-DMA. In
certain embodiments, the particle comprises from about 30 to about
95 mole percent cationic lipid.
[0011] In one embodiment, the second lipid is PEG-c-DMA. In one
embodiment, the second lipid is
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In certain
embodiments, the particle comprises from about 1 to about 10 mole
percent second lipid.
[0012] The nucleic acid can be a DNA, a RNA, a locked nucleic acid,
a nucleic acid analog, or a plasmid capable of expressing a DNA or
an RNA.
[0013] In another embodiment, the lipid particle comprises (a) one
or more cationic lipids, (b) one or more neutral lipids, (c) one or
more PEG-lipids, (d) one or more sterols; and (e) one or more
nucleic acids, wherein the lipid particle comprises a substantially
solid core, as defined herein. In one embodiment, the cationic
lipid is DLin-KC2-DMA. In one embodiment, the PEG-lipid is
PEG-c-DMA. In one embodiment, the neutral lipid is
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In one
embodiment, the sterol is cholesterol. In one embodiment, nucleic
acid is an siRNA.
[0014] In a further embodiment, the lipid particle consists of one
or more cationic lipids, and one or more nucleic acids. In one
embodiment, the lipid particle comprises a substantially solid
core, as defined herein. In one embodiment, the cationic lipid is
DLin-KC2-DMA. In one embodiment, the nucleic acid is an siRNA.
[0015] In other aspects, the invention provides methods for using
the lipid particles.
[0016] In one embodiment, the invention provides a method for
administering a nucleic acid to a subject, comprising administering
a lipid particle of the invention to a subject in need thereof.
[0017] In one embodiment, the invention provides a method for
introducing a nucleic acid into a cell, comprising contacting a
cell with the lipid particle of the invention.
[0018] In one embodiment, the invention provides a method for
modulating the expression of a target polynucleotide or
polypeptide, comprising contacting a cell with the lipid particle
of the invention, wherein the nucleic acid capable of modulating
the expression of a target polynucleotide or polypeptide.
[0019] In one embodiment, the invention provides a method of
treating a disease or disorder characterized by overexpression of a
polypeptide in a subject, comprising administering to the subject
the lipid particle of the invention, wherein the nucleic acid
capable of silencing or decreasing the expression of the
polypeptide.
[0020] In other aspect, the invention provides a method for making
lipid particles.
[0021] In one embodiment, the invention provides a method for
making lipid particles containing a nucleic acid, comprising:
[0022] (a) introducing a first stream comprising a nucleic acid in
a first solvent into a microfluidic device; wherein the device has
a first region adapted for flowing one or more streams introduced
into the device and a second region for mixing the contents of the
one or more streams with a microfluidic mixer;
[0023] (b) introducing a second stream comprising lipid
particle-forming materials in a second solvent into the device to
provide first and second streams flowing under laminar flow
conditions, wherein the device has a first region adapted for
flowing one or more streams introduced into the microchannel and a
second region for mixing the contents of the one or more streams,
wherein the lipid particle-forming materials comprise a cationic
lipid, and wherein the first and second solvents are not the
same;
[0024] (c) flowing the one or more first streams and the one or
more second streams from the first region of the device into the
second region of the device; and
[0025] (d) mixing of the contents of the one or more first streams
and the one or more second streams flowing under laminar flow
conditions in the second region of the device to provide a third
stream comprising lipid nanoparticles with encapsulated nucleic
acid.
[0026] In another embodiment, the invention provides a method for
making lipid particles containing a nucleic acid, comprising:
[0027] (a) introducing a first stream comprising a nucleic acid in
a first solvent into a channel; wherein the device 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;
[0028] (b) introducing a second stream comprising lipid
particle-forming materials in a second solvent; 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;
[0029] (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, while maintaining a physical
separation of the two streams, wherein the one or more first
streams and the one or more second streams do not mix until
arriving at the second region of the channel; and
[0030] (d) mixing of the contents of the one or more first streams
and the one or more second streams flowing under laminar flow
conditions in the second region of the microchannel to provide a
third stream comprising lipid nanoparticles with encapsulated
nucleic acids.
[0031] 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.
[0032] In certain embodiments of the above methods, the methods
further comprise diluting the third stream with an aqueous buffer.
In certain embodiments, diluting the third stream comprises flowing
the third stream and an aqueous buffer into a second mixing
structure.
[0033] In certain embodiments of the above methods, the methods
further comprise dialyzing the aqueous buffer comprising lipid
particles with encapsulated nucleic acids to reduce the amount of
the second solvent.
[0034] In certain embodiments of the above methods, the first
solvent is an aqueous buffer. In certain embodiments of the above
methods, the second solvent is an aqueous alcohol.
[0035] In certain embodiments of the above methods, mixing the
contents of the first and second streams comprises chaotic
advection. In certain embodiments of the above methods, mixing the
contents of the first and second streams comprises mixing with a
micromixer.
[0036] In certain embodiments of the above methods, the nucleic
acid encapsulation efficiency is from about 90 to about 100%.
[0037] In certain embodiments of the above methods, mixing of the
one or more first streams and the one or more second streams is
prevented in the first region by a barrier. In certain embodiments,
the barrier is a channel wall, sheath fluid, or concentric
tubing.
[0038] In another aspect of the invention, devices for making lipid
particles are provided. In one embodiment, the invention provides a
device for producing a lipid particle encapsulating a nucleic acid,
comprising:
[0039] (a) a first inlet for receiving a first solution comprising
a nucleic acid in a first solvent;
[0040] (b) a first inlet microchannel in fluid communication with
the first inlet to provide a first stream comprising the nucleic
acid in the first solvent;
[0041] (c) a second inlet for receiving a second solution
comprising lipid particle-forming materials in a second
solvent;
[0042] (d) a second inlet microchannel in fluid communication with
the second inlet to provide a second stream comprising the lipid
particle-forming materials in the second solvent; and
[0043] (e) a third microchannel for receiving the first and second
streams, wherein the third microchannel has a first region adapted
for flowing the first and second streams introduced into the
microchannel under laminar flow conditions and a second region
adapted for mixing the contents of the first and second streams to
provide a third stream comprising lipid particles with encapsulated
nucleic acid.
[0044] In one embodiment, the device further comprises means for
diluting the third stream to provide a diluted stream comprising
stabilized lipid particles with encapsulated nucleic acid. In
certain embodiments, the means for diluting the third stream
comprises a micromixer.
[0045] In one embodiment, the microchannel has a hydrodynamic
diameter from about 20 to about 300 um.
[0046] In one embodiment, the second region of the microchannel
comprises bas-relief structures. 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. In one embodiment, the
second region comprises a micromixer.
[0047] In certain embodiments, the device further comprises means
for varying the flow rates of the first and second streams.
[0048] In certain embodiments, the device further comprises a
barrier effective to physically separate the one or more first
streams from the one or more second streams in the first
region.
DESCRIPTION OF THE DRAWINGS
[0049] 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.
[0050] FIG. 1 is a schematic illustration of a representative
fluidic device of the invention.
[0051] FIG. 2 is a schematic illustration of a representative
fluidic device of the invention that is an elaboration of the
device illustrated in FIG. 1.
[0052] FIG. 3 is a schematic illustration of a representative
fluidic device of the invention that is an elaboration of the
device illustrated in FIG. 2.
[0053] FIG. 4 is a schematic illustration of a representative
fluidic device and method of the invention.
[0054] FIG. 5 is a schematic illustration of a representative array
of the invention comprising ten of the fluidic devices illustrated
in FIG. 4.
[0055] FIG. 6 is a schematic illustration of a representative
fluidic device of the invention.
[0056] FIG. 7 is a schematic illustration of a representative array
of the invention comprising ten of the representative fluidic
devices illustrated in FIG. 6.
[0057] FIG. 8 is a schematic illustration of a representative
fluidic device of the invention having three inlets and a single
outlet (device 800 includes mixing channel 810).
[0058] FIG. 9 is a schematic illustration of a representative
fluidic device of the invention having two inlets and a single
outlet (device 900 includes mixing channel 910).
[0059] FIG. 10 is a schematic illustration of a representative
fluidic device of the invention having a multiplicity (n) of serial
inlets and a single outlet (device 1000 includes mixing channels
1010a, 1010b, 1010c, and 1010d).
[0060] FIG. 11 is a schematic illustration of a representative
fluidic device of the invention having three inlets and a single
outlet (device 1100 includes mixing channels 1110a, 1110b, and
1110c).
[0061] FIG. 12 is a schematic illustration of a representative
fluidic device of the invention having seven inlets and a single
outlet (device 1200 includes mixing channels 1210a, 1210b, 1210c,
and 1210d).
[0062] FIG. 13 is a schematic illustration of a representative
fluidic device of the invention having a multilaminate mixer
(device 1300 includes mixing channel 1310).
[0063] FIG. 14 is a close-up view of the multilaminate mixer
illustrated in FIG. 14.
[0064] FIG. 15A is a schematic illustration of a representative
microfluidic (MF) method of the invention for making lipid
nanoparticles (LNP): Lipid-ethanol and siRNA-aqueous solutions are
pumped into inlets of a microfluidic mixing device; herringbone
features in the device induce chaotic advection of the laminar
stream and cause the lipid species to rapidly mix with the aqueous
stream and form lipid nanoparticles. The mixing channel is 200
.mu.m wide and 79 .mu.m high. The herringbone structures are 31
.mu.m high and 50 .mu.m thick.
[0065] FIG. 15B is a schematic illustration of a preformed vesicle
(PFV) method for making lipid nanoparticles (LNP): (a) a
lipid-ethanol solution is added to an aqueous solution, pH 4.0,
resulting in the formation of vesicle type particles; (b) extrusion
through 80 nm polycarbonate membrane (Nuclepore) at room
temperature using a Lipex Extruder provides a more uniform particle
distribution; and (c) addition of siRNA solution while vortexing
and incubation at 35.degree. C. for 30 minutes promotes
encapsulation of siRNA.
[0066] FIGS. 16A-16C illustrate the influence of flow rate in
microfluidic device on mixing and LNP particle size. Two 10 .mu.M
fluorescein (fluorescent at pH 8.8, non-fluorescent at pH 5.15)
solutions mix to produce completely fluorescent solution.
[0067] FIG. 16A compares the extent of mixing (%) as determined by
mean fluorescent intensity along channel width as a function of
with mixing time (msec) calculated from average fluid velocity and
travel length (0.2, 0.8, 1.4, and 2 mL/min). FIGS. 16B and 16C
compare mean particle diameter for LNP composed of
DLin-KC2-DMA/DSPC/Cholesterol/PEG-c-DMA at mole ratios of
40:11.5:47.5:1, siRNA-total lipid ratio 0.06 wt/wt, with 10 mM
lipid-ethanol phase mixed with 25 mM acetate buffer, pH 4,
containing siRNA. FIG. 16B compares mean particle diameter (nm) for
LNP as a function of flow rate (mL/min). FIG. 16C compares mean
particle diameter (nm) for LNP as a function of ethanol/aqueous
flow rate ratio. Error bars represent standard deviation of the
mean particle diameter as measured by dynamic light scattering.
[0068] FIG. 17 illustrates the influence of lipid concentration on
LNP particle size by comparing mean particle diameter (nm) as a
function of lipid concentration in ethanol (mM). Increasing the
lipid concentration results in an increase in mean particle
diameter. The total lipid content in the ethanol phase being mixing
in the microfluidic chip was varied from 10 mM to 50 mM. LNP
composed of Dlin-KC2-DMA/DSPC/Cholesterol/PEG-c-DMA at mole ratios
of 40:11.5:47.5:1, siRNA-total lipid ratio 0.06 wt/wt. Total flow
rate inside microfluidic mixer was maintained at 2 ml/min Error
bars represent standard deviation of the mean particle diameter as
measured by dynamic light scattering.
[0069] FIGS. 18A and 18B illustrate the influence of PEG-lipid and
cationic lipid on LNP systems. FIG. 18A compares mean particle
diameter (nm) as a function of PEG-c-DMA content (mol % in LNP) for
LNP prepared by the PFV and MF methods. The PEG-lipid was varied
from 1 mol % to 10 mol % in the LNP composition. Modification of
PEG-lipid content was compensated by adjustment of cholesterol
content. LNP were composed of
Dlin-KC2-DMA/DSPC/Cholesterol/PEG-c-DMA at mole ratios of
40:11.5:47.5:1 (-x):1 (+x), (where x=1 to 9), siRNA-total lipid
ratio 0.06 wt/wt. FIG. 18B compares mean particle diameter (nm) as
a function of DLin-KC2-DMA content (mol %) for LNP prepared by the
PFV and MF methods. The cationic lipid was varied from 40 mol % to
70 mol %. PEG-c-DMA was kept constant at 1 mol % and a 0.25 molar
ratio was maintained with DSPC-cholesterol. Total flow rate inside
microfluidic mixer was maintained at 2 ml/min 10 mM lipid-ethanol
phase mixed with 25 mM acetate buffer, pH 4, containing siRNA.
Error bars represent standard deviation of the mean particle
diameter as measured by dynamic light scattering.
[0070] FIG. 19 illustrates the influence of siRNA/Lipid ratio on
particle size and encapsulation by comparing mean particle diameter
(nm) and encapsulation (%) as a function of siRNA/lipid ratio
(wt/wt) (also expressed as nucleotide/phosphate (N/P).
[0071] Encapsulation determined by separation of LNP suspension
from free siRNA using an anionic exchange spin column LNP were
composed of Dlin-KC2-DMA/DSPC/Cholesterol/PEG-c-DMA at mole ratios
of 40:11.5:47.5:1, siRNA-total lipid ratio 0.06 wt/wt. Total flow
rate inside microfluidic mixer was maintained at 2 ml/min 10 mM
lipid-ethanol phase mixed with 25 mM acetate buffer, pH 4,
containing siRNA. Error bars represent standard deviation of the
mean particle diameter as measured by dynamic light scattering.
[0072] FIGS. 20A and 20B illustrate the morphology of PEG-lipid and
cationic lipid LNP systems prepared by the microfluidic mixer using
Cryo-Transmission Electron Microscopy (TEM). LNP were imaged at 29K
magnification by Cryo-TEM.
[0073] FIG. 20A is an image of empty LNP composed of
Dlin-KC2-DMA/DSPC/Cholesterol/PEG-c-DMA at mole ratios of
40:11.5:47.5:1. FIG. 20B is an image of siRNA loaded LNP composed
of Dlin-KC2-DMA/DSPC/Cholesterol/PEG-c-DMA at mole ratios of
40:11.5:47.5:1, siRNA-total lipid ratio 0.06 wt/wt. Formulation was
performed using the microfluidic mixer at 20 mM lipid in the
ethanol phase. Loaded LNP-siRNA and empty particles containing 1
mol % PEG-c-DOMG exhibited identical morphology and are very
homogeneous in structure. Scale bar represents 100 nm.
[0074] FIG. 21 illustrates in vivo silencing activity of
microfluidic produced LNP in Factor VII Mouse Model by comparing
relative FVII Protein Level (%) as a function of siRNA dosage
(mg/kg) varying DLin-KC2-DMA content in the LNP from 40 mol % to 60
mol %. Formulation of LNP containing 1 mol % PEG-c-DOMG and 60 mol
% DLin-KC2-DMA provide FVII silencing similar to that previously
reported using alternative approaches. Gene silencing progressively
improves for LNP containing DLin-KC2-DMA over the range from 40 mol
% to 60 mol %. Systemic injection of LNP-siRNA to mice was
performed by tail vein injection (n=3 per dose level). Blood
collection was performed after 24 hrs post-injection and factor VII
levels were determined by colorimetric assay. LNP
DSPC-to-Cholesterol ratio was kept at 0.2 wt/wt and contained 1 mol
% PEG-c-DOMG. LNP siRNA-to-lipid ratio was 0.06 wt/wt.
[0075] FIGS. 22A-22C illustrate cryo electron microscopy of lipid
nanoparticles prepared by the microfluidics method. Empty lipid
nanoparticles prepared by microfluidics (40% DLinKC2-DMA, 11.5%
DSPC, 47.5% cholesterol, 1% PEG-c-DMA) showed an electron dense
interior indicating solid core structure (FIG. 22A). Samples
composed with POPC showed a less dense interior correlating with
aqueous core vesicles (FIG. 22B). Systems containing POPC/triolein
which have a hydrophobic core of triolein surrounded by a monolayer
of POPC showed an electron dense interior similar to sample A (FIG.
22C).
[0076] FIG. 23 illustrates limit size LNP prepared with
DLinKC2-DMA/PEG-lipid system (90/10, mol/mol) using microfluidic
mixing by comparing mean particle diameter (nm) as a function of
ethanol/aqueous flow rate ratio for LNP were produced in the
presence of siRNA (N/P=1) and without siRNA present (No siRNA).
Formulation was performed using a 10 mM lipid-ethanol phase mixed
with 25 mM acetate buffer, pH 4. The particle size was determined
by dynamic light scattering and number-weighted mean diameters are
reported.
[0077] FIGS. 24A-24C illustrate .sup.31P NMR of siRNA encapsulated
in 50% DLinKC2-DMA, 45% cholesterol, and 5% PEG-c-DMA using
microfluidic mixing. DSPC was omitted to avoid conflicting
phosphorus signal arising from the phospholipid. .sup.31P signal
from the siRNA cannot be detected for intact LNP (FIG. 24A) or
after the addition of 150 mM ammonium acetate (FIG. 24B). Signal
can only be detected after the addition of 1% SDS to solubilize the
particle (FIG. 24C).
[0078] FIG. 25 is an electrophoretic gel illustrates the results of
an RNase protection assay. siRNA was encapsulated using either the
microfluidic method (MF) or the PFV approach, or left
unencapsulated. Triton X-100 was added to completely solubilize and
lyse the lipid particles. Gel electrophoresis was performed on 20%
native polyacrylamide gel and siRNA visualized by staining with
CYBR-Safe.
[0079] FIG. 26 illustrates the results of a lipid mixing fusion
assay represented as percent lipid mixing as a function of time
(seconds). To assess the amount of exposed cationic lipid present
in the outermost layer of the LNP, three LNP systems were prepared:
in the absence of siRNA (No siRNA), at N/P=4, and N/P=1. Lipid
assay was performed at pH 5.5 to ensure nearly complete ionization
of the cationic lipid, and the reaction was initiated by injecting
the LNP into the cuvette containing highly anionic
DOPS/NBD-PE/Rh-PE (98:1:1 molar ratio) vesicles.
[0080] FIG. 27 is a schematic representation of the solid core LNP
siRNA system formed by microfluidic mixing in accordance with the
method of the invention.
[0081] FIGS. 28A and 28B illustrate mean particle diameter (nm) and
zeta potential (mV), respectively, as a function of sequential
lipid nanoparticle composition prepared using the microfluidic
mixer.
[0082] FIG. 29 is a schematic representation of a representative
device and method of the invention for the sequential assembly of
lipid nanoparticles.
[0083] FIG. 30 is a schematic representation of a representative
device and method of the invention.
[0084] FIG. 31 is a schematic representation of a representative
device and method of the invention.
[0085] FIG. 32 is a schematic representation of a representative
device and method of the invention.
[0086] FIG. 33 is a schematic representation of a representative
device and method of the invention.
[0087] FIGS. 34A and 34B compare cryo-transmission electron
microscopy images of two LNPs. LNP produced by the microfluidic
method of the invention result in small spherical particles. As
shown in FIG. 34A LNP composed of pure DOPC produced by the
microfluidics procedure are very small "limit size" vesicles in the
range of 30-50 nm diameter. The vesicles have an interior with
lower electron density consistent with a bilayer shell surrounding
an inner aqueous core. Interestingly, LNP consisting of
DOPE/DOPC/PEG-lipid produced by the microfluidics procedure of the
invention (FIG. 34B) exhibit somewhat larger sizes where the
majority of the particles have an electron dense inner core. This
could be consistent with the formation of solid core particles
where lipids with the lowest solubility in water (e.g., DOPE)
condense out to form nucleation points that are subsequently coated
by more polar lipids such as DOPC and PEG-lipid. Such a process may
be occurring for the cationic lipids and cationic lipid-polynucleic
acid systems in the lipid mixtures employed for polynucleic acid
entrapment.
DETAILED DESCRIPTION OF THE INVENTION
[0088] The present invention provides lipid particles containing a
therapeutic agent, methods and devices for making the lipid
particles containing a therapeutic agent, and methods for
delivering a therapeutic agent using the lipid particles.
[0089] Lipid Particles
[0090] In one aspect, the invention provides lipid particles
containing a therapeutic agent. The lipid particles include one or
more cationic lipids, one or more second lipids, and one or more
nucleic acids.
[0091] Cationic lipid. The lipid particles 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.
[0092] 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, NY); LIPOFECTAMINE.RTM. (commercially available cationic
liposomes comprising N-(1-(2,3
-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium
trifluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL); and
TRANSFECTAM.RTM. (commercially available cationic lipids comprising
dioctadecylamidoglycyl carboxyspermine (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).
[0093] The lipid particle-forming materials include an ionizable
lipid. As used herein, the term "ionizable lipid" refers to a lipid
that 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 polynucleic acids
(e.g., oligonucleotides). In one embodiment, the ionizable lipid is
an amino lipid.
[0094] In one embodiment, the cationic lipid is an amino lipid.
Suitable amino lipids useful in the invention include those
described in WO 2009/096558, 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),
l-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-propanedio (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).
[0095] Suitable amino lipids include those having the formula:
##STR00001##
[0096] wherein R.sub.1 and R.sub.2 are either the same or different
and independently optionally substituted C.sub.10-C.sub.24 alkyl,
optionally substituted C.sub.10-C.sub.24 alkenyl, optionally
substituted C.sub.10-C.sub.24 alkynyl, or optionally substituted
C.sub.10-C.sub.24 acyl;
[0097] R.sub.3 and R.sub.4 are either the same or different and
independently optionally substituted C.sub.1-C.sub.6 alkyl,
optionally substituted C.sub.2-C.sub.6 alkenyl, or optionally
substituted C.sub.2-C.sub.6 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;
[0098] R.sub.5 is either absent or present and when present is
hydrogen or C.sub.1-C.sub.6 alkyl;
[0099] 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;
[0100] q is0, 1,2, 3,or4; and
[0101] Y and Z are either the same or different and independently
O, S, or NH.
[0102] In one embodiment, R.sub.1 and R.sub.2 are each linoleyl,
and the amino lipid is a dilinoleyl amino lipid. In one embodiment,
the amino lipid is a dilinoleyl amino lipid.
[0103] A representative useful dilinoleyl amino lipid has the
formula:
##STR00002##
[0104] wherein n is 0, 1, 2, 3, or 4.
[0105] In one embodiment, the cationic lipid is a DLin-K-DMA. In
one embodiment, the cationic lipid is DLin-KC2-DMA (DLin-K-DMA
above, wherein n is 2).
[0106] 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-dimeth-
ylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl
carboxyspermine (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
(including DOTMA and DOPE, available from GIBCO/BRL), and
LIPOFECTAMINE (comprising DOSPA and DOPE, available from
GIBCO/BRL).
[0107] The cationic lipid is present in the lipid particle in an
amount from about 30 to about 95 mole percent. In one embodiment,
the cationic lipid is present in the lipid particle in an amount
from about 30 to about 70 mole percent. In one embodiment, the
cationic lipid is present in the lipid particle in an amount from
about 40 to about 60 mole percent.
[0108] In one embodiment, the lipid particle includes ("consists
of") only of one or more cationic lipids and one or more nucleic
acids. The preparation and characterization of a lipid particle of
the invention consisting of a cationic lipid and a nucleic acid is
described in Example 5.
[0109] Other suitable ionizable lipids include DODAC, DOTMA, DDAB,
DOTAP, DOTAP.Cl, DC-Chol, DOSPA, DOGS, DOPE, DODAP, DODMA, DODMA,
and DMRIE, among others. See, for example, lipids described in WO
2009/096558, expressly incorporated herein by reference in its
entirety.
[0110] In addition to the ionizable lipid, the second stream
includes one or more other lipid particle-forming materials.
Representative lipid particle-forming materials include
polyethylene glycol-lipids, neutral lipids, and sterols.
[0111] Second lipids. In certain embodiments, the lipid particles
include one or more second lipids. Suitable second lipids stabilize
the formation of particles during their formation.
[0112] 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.
[0113] Suitable stabilizing lipids include neutral lipids and
anionic lipids. Neutral Lipid. 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.
[0114] 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),
dipalmitoyl phosphatidyl ethanolamine (DPPE),
dimyristoylphosphoethanolamine (DMPE),
distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE,
16-O-dimethyl PE, 18-1-trans PE,
1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), and
1,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE).
[0115] In one embodiment, the neutral lipid is
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).
[0116] Anionic Lipid. The term "anionic lipid" refers to any lipid
that is negatively charged at physiological pH. These lipids
include phosphatidylglycerol, cardiolipin,
diacylphosphatidylserine, diacylphosphatidic acid,
N-dodecanoylphosphatidylethanol-amines,
N-succinylphosphatidylethanolamines,
N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,
palmitoyloleyolphosphatidylglycerol (POPG), and other anionic
modifying groups joined to neutral lipids.
[0117] Other suitable lipids include glycolipids (e.g.,
monosialoganglioside GM.sub.1). Other suitable second lipids
include sterols, such as cholesterol.
[0118] Polyethylene glycol-lipids. In certain embodiments, the
second lipid is a polyethylene glycol-lipid. Suitable polyethylene
glycol-lipids include PEG-modified phosphatidylethanolamine,
PEG-modified phosphatidic acid, PEG-modified ceramides (e.g.,
PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified
diacylglycerols, PEG-modified dialkylglycerols. Representative
polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and
PEG-s-DMG. In one embodiment, the polyethylene glycol-lipid is
N-[(methoxy poly(ethylene
glycol).sub.2000)carbamy]1-1,2-dimyristyloxlpropyl-3-amine
(PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is
PEG-c-DOMG).
[0119] Representative polyethylene glycol-lipids include
PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic
acid, PEG-modified ceramides, PEG-modified dialkylamines,
PEG-modified diacylglycerols, PEG-modified dialkylglycerols,
PEG-C-DOMG, PEG-DMA, and PEG-s-DMG. See, for example, PEG-lipids
described in WO 2009/096558, expressly incorporated herein by
reference in its entirety.
[0120] Advantageously, the lipid particles include from about 1 to
about 5 mole percent PEG-lipid. In one embodiment, the lipid
particles include about 1 mole percent PEG-lipid.
[0121] In certain embodiments, the second lipid is present in the
lipid particle in an amount from about 1 to about 10 mole percent.
In one embodiment, the second lipid is present in the lipid
particle in an amount from about 1 to about 5 mole percent. In one
embodiment, the second lipid is present in the lipid particle in
about 1 mole percent.
[0122] Nucleic Acids. The lipid particles of the present invention
are useful for the systemic or local delivery of nucleic acids. As
described herein, the nucleic acid is incorporated into the lipid
particle during its formation.
[0123] 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 particle 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, and triplex-forming
oligonucleotides. 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, pre-condensed DNA, or an aptamer.
[0124] The method of the invention provides lipid particles
containing a therapeutic agent, such as a genetic material (e.g., a
polynucleic acid). As used herein, the term "polynucleic acid"
refers to a DNA, a RNA, a locked nucleic acid (LNA), or other
nucleic acid analog known in the art, or a plasmid capable of
expressing a DNA or a RNA. In one embodiment, the polynucleic acid
is an oligonucleotide. The polynucleic acid may be a ssDNA or a
dsDNA, an siRNA or a microRNA. In one embodiment, the polynucleic
acid is an antisense oligonucleotide.
[0125] 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.
[0126] The term "nucleotide," as used herein, generically
encompasses the following terms, which are defined below:
nucleotide base, nucleoside, nucleotide analog, and universal
nucleotide.
[0127] 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 (2ms6iA), N6-methyladenine,
guanine (G), isoguanine, N2-dimethylguanine (dmG), 7-methylguanine
(7mG), 2-thiopyrimidine, 6-thioguanine (6sG) hypoxanthine and
06-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.
[0128] 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'-0-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.
[0129] 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, 2.sup.nd Ed., Freeman, San Francisco, Calif.).
[0130] 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.
[0131] 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., Chemica Scripta
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-methylphophoroamidite 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.
[0132] 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),
deoxylmPy 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. 10/290672, and U.S. Pat. No. 6,433,134.
[0133] 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
internucleotide phosphodiester 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+, NH4+, trialkylammonium, Mg2+,
Na+, and the like. A polynucleotide may be composed entirely of
deoxyribonucleotides, entirely of ribonucleotides, or chimeric
mixtures 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.
[0134] 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).
[0135] 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.
[0136] 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.
[0137] As used herein, "nucleic acid" is a nucleobase
sequence-containing polymer, or polymer segment, having a backbone
formed from nucleotides, or analogs thereof.
[0138] Preferred nucleic acids are DNA and RNA.
[0139] 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.
[0140] Lipid Particle Characteristics
[0141] Morphology. The lipid particle of the invention differs from
other similarly constituted materials by its morphology and
characterized as having a substantially solid core. A lipid
particle 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.
[0142] The lipid particles 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.
[0143] The results presented in this work demonstrate that a
microfluidic device containing a staggered herringbone mixer can be
used to generate LNP with a variety of lipid compositions, can be
used to efficiently encapsulate OGN. There are three aspects of
these results that are of particular interest. The first concerns
the mechanisms whereby LNP and LNP OGN systems are formed using the
microfluidics device, the second the advantages of the
microfluidics approach as compared to previously available
procedures and third the potential improvements that can be made to
this process.
[0144] With regard to the mechanism(s) whereby the microfluidics
process allows the formation of LNP and LNP containing OGN, two
points of interest concern the mechanism whereby LNP of 100 nm size
or smaller are formed and the mechanism whereby OGN can be
encapsulated to levels approaching 100%. With regard to formation
of LNP the rate of mixing is clearly the important parameter. Rapid
mixing of the ethanol-lipid solution with aqueous buffer results in
an increased polarity of the medium to some critical value where
the dissolved lipids come out of solution and form bilayers. Rapid
mixing results in high supersaturation of lipid unimers throughout
the mixing volume, and consequently a rapid and homogeneous
nucleation of nanoparticles. Higher flow rates increase the rate of
supersaturation and thus result in higher nucleation rates.
Increased nucleation and growth of nanoparticles depletes the
surrounding liquid of free lipid, limiting subsequent growth by the
aggregation of free lipid. This proposed mechanism is consistent
with the observation that lower concentrations of the lipid in
ethanol (reduced free lipid) result in smaller LNP and that higher
flow rates, causing a faster and more homogeneous approach to
supersaturation, lead to formation of smaller LNP.
[0145] The observation that LNP OGN systems formulated by the
microfluidics technique exhibit OGN encapsulation efficiencies
approaching 100% is difficult to understand in the absence of
further morphological and other studies. Cryo-TEM studies on the
LNP OGN produced by microfluidics show that the interior of the LNP
is electron dense, suggestive of a solid core of lipid and OGN.
This would suggest an encapsulation process whereby cationic lipid
and OGN associates and serves as a nucleus for subsequent coating
by DSPC and PEG-lipid. In any event, the ability of the
microfluidics formulation process to allow encapsulation
efficiencies for antisense and siRNA OGN approaching 100%
independent of nucleic acid composition is a major advantage of the
procedure.
[0146] Particle size. The lipid particle of the invention has a
diameter (mean particle diameter) from about 15 to about 300 nm. In
some embodiments, the lipid particle has a diameter of about 300 nm
or less, 250 nm or less, 200 nm or less, 150 nm or less, 100 nm or
less, or 50 nm or less. In one embodiment, the lipid particle has a
diameter from about 15 to about 100 nm. These particles generally
exhibit increased circulatory lifetime in vivo compared to large
particles. In one embodiment, the lipid particle has a diameter
from about 15 to about 50 nm. These particles are capable of
advantageously escaping the vascular system. In one embodiment, the
lipid particle has a diameter from about 15 to about 20 nm. These
particles near the limit size for particles that contain a nucleic
acid; such particles may include a single polynucleotide (e.g.,
siRNA).
[0147] 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.
[0148] The lipid particles of the invention are substantially
homogeneous in their size distribution. In certain embodiments, the
lipid particles of the invention have a mean particle diameter
standard deviation of from about 65 to about 25%. In one
embodiment, the lipid particles of the invention have a mean
particle diameter standard deviation of about 60, 50, 40, 35, or
30%.
[0149] Encapsulation efficiency. The lipid particles of the
invention can be further distinguished by the encapsulation
efficiency. As described below, the lipid particles of the
invention are prepared by a process by which nearly 100% of the
nucleic acid used in the formation process is encapsulated in the
particles. In one embodiment, the lipid particles are prepared by a
process by which from about 90 to about 95% of the nucleic acid
used in the formation process is encapsulated in the particles.
[0150] Microfluidic Methods for Making Lipid Particles
[0151] In one aspect, the invention provides a method for making
lipid particles containing a therapeutic agent. In one embodiment,
the method includes
[0152] (a) introducing a first stream comprising a therapeutic
agent (e.g., polynucleic acid) in a first solvent into a
microchannel; wherein the microchannel has a first region adapted
for flowing one or more streams introduced into the microchannel
and a second region for mixing the contents of the one or more
streams;
[0153] (b) introducing a second stream comprising lipid
particle-forming materials in a second solvent in the microchannel
to provide first and second streams flowing under laminar flow
conditions, wherein the lipid particle-forming materials comprise
an ionizable lipid, and wherein the first and second solvents are
not the same;
[0154] (c) flowing the one or more first streams and the one or
more second streams from the first region of the microchannel into
the second region of the microchannel; and
[0155] (d) mixing of the contents of the one or more first streams
and the one or more second streams flowing under laminar flow
conditions in the second region of the microchannel to provide a
third stream comprising lipid particles with encapsulated
therapeutic agents.
[0156] 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. In
the above embodiment, unlike known methods, the method does not
include a dilution after mixing.
[0157] To further stabilize the third stream containing the lipid
particles with encapsulated therapeutic agents, the method can, but
need not 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 therapeutic agents is
dialyzed to reduce the amount of the second solvent.
[0158] The first stream includes a therapeutic agent in a first
solvent. Suitable first solvents include solvents in which the
therapeutic agents are soluble and that are miscible with the
second solvent. Suitable first solvents include aqueous buffers.
Representative first solvents include citrate and acetate
buffers.
[0159] 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 1,4-dioxane,
tetrahydrofuran, acetone, acetonitrile, dimethyl sulfoxide,
dimethylformamide, acids, and alcohols. Representative second
solvents include aqueous ethanol 90%.
[0160] The methods of the invention are distinguished from other
microfluidic mixing methods in several ways. Whereas certain known
methods require an equal or substantially equal proportion of
aqueous and organic solvents (i.e., 1:1), the method of the
invention generally utilizes a solvent ratio of aqueous to organic
that exceeds 1:1. In certain embodiments, the solvent ratio of
aqueous to organic is about 2:1. In certain embodiments, the
solvent ratio of aqueous to organic is about 3:1. In certain
embodiments, the solvent ratio of aqueous to organic is about 4:1.
In certain other embodiments, the solvent ratio of aqueous to
organic is about 5:1, about 10:1, about 50:1, about 100:1, or
greater.
[0161] The lipid particles of the invention are advantageously
formed in a microfluidic process that utilizes relatively rapid
mixing and high flow rates. The rapid mixing provides lipid
particles having the advantageous properties noted above including
size, homogeneity, encapsulation efficiency. Mixing rates used in
the practice of the method of the invention range from about 100
.mu.sec to about 10 msec. Representative mixing rates include from
about 1 to about 5 msec. Whereas hydrodynamic flow focusing methods
operate at relatively low flow rates (e.g., 5 to 100 .mu.L/minute)
with relatively low lipid volumes, the method of the invention
operates at relatively high flow rates and relatively high lipid
volumes. In certain embodiments, for methods that incorporate a
single mixing region (i.e., mixer), the flow rate is about 1
mL/min. For methods of the invention that utilize mixer arrays
(e.g., 10 mixers), flow rates of 40 mL/minute are employed (for 100
mixers, flow rate 400 mL/min). Thus, the methods of the invention
can be readily scaled to provide quantities of lipid particles
necessary for demanding production requirements. Coupled with the
advantageous particle size and homogeneity and encapsulation
efficiencies realized, the method of the invention overcomes
disadvantages of known microfluidic methods for producing the lipid
particles. One advantage of the methods of the invention for making
the lipid particles is that the methods are scalable, which means
that the methods do not change on scaling and that there is
excellent correspondence on scaling.
[0162] Microfluidic Devices for Making Lipid Particles
[0163] In another aspect, the invention provides devices for
producing a lipid particle encapsulating a nucleic acid. In one
embodiment the device includes:
[0164] (a) a first inlet for receiving a first solution comprising
a nucleic acid in a first solvent;
[0165] (b) a first inlet microchannel in fluid communication with
the first inlet to provide a first stream comprising the nucleic
acid in the first solvent;
[0166] (c) a second inlet for receiving a second solution
comprising lipid particle-forming materials in a second
solvent;
[0167] (d) a second inlet microchannel in fluid communication with
the second inlet to provide a second stream comprising the lipid
particle-forming materials in the second solvent;
[0168] (e) a third microchannel for receiving the first and second
streams, wherein the third microchannel has a first region adapted
for flowing the first and second streams introduced into the
microchannel under laminar flow conditions and a second region
adapted for mixing the contents of the first and second streams to
provide a third stream comprising lipid particles with encapsulated
nucleic acid.
[0169] In one embodiment, the device further includes means for
diluting the third stream to provide a diluted stream comprising
stabilized lipid particles with encapsulated therapeutic agent.
[0170] 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 300
.mu.m. As noted above, 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) under laminar
flow conditions. 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. 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.
[0171] In other aspects of the invention, the first and second
streams are mixed with other micromixers. Suitable micromixers
include droplet mixers, T-mixers, zigzag mixers, multilaminate
mixers, or other active mixers.
[0172] 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.
[0173] In another embodiment, the device for producing a lipid
particle encapsulating a nucleic acid includes microchannel for
receiving the first and second streams, wherein the microchannel
has a first region adapted for flowing the first and second streams
introduced into the microchannel under laminar flow conditions and
a second region adapted for mixing the contents of the first and
second streams to provide a third stream comprising lipid particles
with encapsulated therapeutic agent. In this embodiment, the first
and second stream are introduced into the microchannel by means
other than first and second microchannels as noted above.
[0174] 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
a lipid particle encapsulating a nucleic acid includes:
[0175] (a) a single inlet microchannel for receiving both a first
solution comprising a nucleic acid in a first solvent and a second
solution comprising lipid particle-forming materials in a second
solvent;
[0176] (b) a second region adapted for mixing the contents of the
first and second streams to provide a third stream comprising lipid
particles with encapsulated nucleic acid.
[0177] In such an embodiment, the first and second streams are
introduced into the microchannel 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 microchannel using adjacent or concentric macrosized
channels.
[0178] FIG. 1 is a schematic illustration of a representative
fluidic device of the invention. Referring to FIG. 1, device 100
includes Region A for receiving a first stream comprising a
therapeutic agent in a first solvent and Region B for receiving a
stream comprising lipid particle-forming materials in a second
solvent. First and second streams are introduced into Region C
flowing under laminal flow conditions, to Region D where rapid
mixing occurs, and then to Region E where the final product, lipid
particles containing therapeutic agent, exit the device.
[0179] FIG. 2 is a schematic illustration of a representative
fluidic device of the invention that is an elaboration of the
device and method illustrates in FIG. 1. Referring to FIG. 2,
device 200 includes Region A for receiving a first stream
comprising a therapeutic agent in a first solvent into a
microchannel, wherein the microchannel has a first region adapted
for flowing one or more streams (A-a) are introduced (A-b) and
mixed (A-c); Region B for receiving a second stream comprising
lipid particle-forming materials in a second solvent, wherein the
microchannel has a first region adapted for flowing one or more
streams (B-a) are introduced (B-b) and mixed (B-c); Region C
introduces the flows of Region A and Region B under laminal flow
conditions (C-a) and rapidly mixed (C-b); and Region D, where the
formulation is ready for further processing such as dilution, pH
adjustment, or other events required for nanoparticle synthesis, or
where the final product, lipid particles containing therapeutic
agent, exit the device
[0180] FIG. 3 is a schematic illustration of a representative
fluidic device of the invention that is an elaboration of the
device and method illustrated in FIG. 2. Referring to FIG. 3,
device 300 includes Region A for receiving a first stream
comprising a therapeutic agent in a first solvent into a
microchannel, wherein the microchannel has a first region adapted
for flowing one or more streams (A-a) are introduced (A-b) and
mixed (A-c); Region B for receiving a second stream comprising
lipid particle-forming materials in a second solvent, wherein the
microchannel has a first region adapted for flowing one or more
streams (B-a) are introduced (B-b) and mixed (B-c); Region C
introduces the flows of Region A and Region B under laminal flow
conditions (C-a) and rapidly mixed (C-b); Region D for receiving a
third stream comprising of any number of materials including
further particle-forming materials, dilution, pH adjustments, or
other events required for nanoparticle synthesis; Region E
introduces the flows of Region C and Region D under laminal flow
conditions (E-a) and rapidly mixed (E-b); Region F, where the
formulation is ready for further processing like dilution, pH
adjustments, or other events required for nanoparticle synthesis,
or where the final product, lipid particles containing therapeutic
agent, exit the device.
[0181] FIG. 4 is a schematic illustration of another representative
fluidic device (400) of the invention. FIG. 5 is a schematic
illustration of a representative array of the representative
fluidic device illustrated in FIG. 4.
[0182] FIG. 6 is a schematic illustration of another representative
fluidic device (600) of the invention. Referring to FIG. 6, device
600 includes mixing channels 610a, 610b, and 610c. FIG. 7 is a
schematic illustration of a representative array of the
representative fluidic device illustrated in FIG. 6.
[0183] The formation of nanoparticles on microfluidic devices is
limited by the reagent volumes that participate in the mixing event
and the limited backpressure that devices can withstand before
leakage occurs. Single elements of the herringbone or multilaminate
mixer achieve a 100-1000 fold increase in flow rate compared to
droplet or flow focusing approaches. In order to achieve production
scale throughput, multiple mixer elements can be arrayed. In one
embodiment each reagent is distributed to the individual mixer
elements using a low impedance bus channel. If the impedance of the
bus channel is negligible compared to the impedance of the mixer
element, the individual flow rates at the inlet of each mixer are
identical. As multiple mixer elements are operated in parallel, the
impedance of the system decreases resulting in a higher volumetric
throughput. This has the advantage that the mixing characteristics
that are observed using a single mixer element can be maintained in
a mixer array. In one embodiment, mixing in each mixer array
element is achieved by introducing multiple streams into a
microchannel. In this case the streams will mix by diffusion. The
width of the streamlines may be varied by controlling the relative
flow rates through the injection channels (e.g., by adjusting the
dimensions of these channels (FIG. 5). In another embodiment,
mixing is achieved by chaotic advection (staggered herringbone
mixer, SHM). As shown in FIG. 7, each mixer element of the array
may consist of a series of mixers. By adding elements to each array
subset additional functionalities can be integrated in-line on the
microfluidic device. Such functionalities may include on-chip
dilution, dialysis, pH adjustments or other events that require
interlaced streamlines, streams sharing the same channel or streams
that are separated from each other by a porous material. In one
embodiment, 10 mM POPC is dissolved in 100% ethanol and mixed with
phosphate buffered saline (PBS), pH 7.4 in the first mixer element
of each array subset. The LNPs that are formed after mixing are
stabilized by diluting the mixture by a factor of 2 with PBS.
[0184] Table 1 compares the size distribution of particles formed
on a single mixer and a mixer array consisting of ten individual
mixers. The total flow rate through a single mixer may be 4 ml/min
with a mixing ratio of 50:50 at each intersection. The volumetric
throughput can be increased ten-fold by operating ten mixers in
parallel resulting in a total volumetric flow rate of 40 ml/min
While the throughput of the array is amenable to production scale
synthesis, LNP dimensions are maintained.
TABLE-US-00001 TABLE 1 Size distribution of particles formed on a
single mixer and a mixer array consisting of ten individual mixers.
Diameter (nm) Std. Deviation (nm) Chi squared Single Mixer
Intensity 73.0 37.8 1.58 Volume 62.8 32.5 Number 25.7 13.3 Mixer
Array Intensity 72.1 35.1 1.08 Volume 62.7 32.4 Number 27.0
13.7
[0185] Any combination of any number of parallel reagent inlets,
sequential mixing chambers, and branching architectures can be used
to optimize the nanoparticle formulation process. This has the
advantage that different formulation process can be precisely
controlled and multiple steps of the nanoparticle formulation
process can be integrated. Examples include, but are not limited
to, the following: (a) the introduction of two or more inlets
consisting of combinations of distinct (FIG. 8) or the same (FIG.
9) reagents to allow for the independent input control (uses
include independent control of the flow rates of the input
reagents, varying the ratios between input reagents, and others;
(b) two or more mixers in sequence to allow for the sequential
addition of nanoparticle reagents or formulation processing steps
(FIG. 10) (uses include the addition of input reagents in sequence
for the controlled bottom up assembly of nanoparticles, the
integration of formulation processes like dilution, pH adjustments,
or other events required for nanoparticle synthesis, and others; or
(c) any combination of inputs, mixing chambers, and branching
architectures, FIG. 11 and FIG. 12 illustrate two step and three
step mixers with a varying number of parallel reagent inputs and
branching microfluidic structures (uses include the integration of
multiple steps of the formulation process that includes on chip
mixing of nanoparticle reagents, nanoparticle nucleation and
growth, on-chip dilution, dialysis, pH adjustments or other events
required for nanoparticle synthesis.
[0186] FIG. 13 is a schematic illustration of a representative
fluidic device of the invention having a multilaminate mixer.
Referring to FIG. 13, device 1300 includes mixing channels 1310.
FIG. 14 is a close-up view of the multilaminate mixer illustrated
in FIG. 14.
[0187] As described above, methods of making lipid
micro/nanoparticles have been conventionally "top down" approaches
where larger structures are formed by dispersion of lipids in
water, followed by disruption of the multilamellar vesicles (micron
size range) through polycarbonate filters with a pore size such as
100 nm or alternatively, using tip sonication.
[0188] One aspect lacking in such batch processes is the ability to
precisely control the structure and assembly of each lipid mixture
constituent. This is especially important if certain constituents
are easily degraded if exposed to their external environment, or if
certain ligands must reside on the exterior of the particle for
targeting purposes. For example, with therapeutics it may be
important to first produce a particle that has a net positive or
negative surface charge to associate a certain therapeutic drug.
Further processing may then be needed to complete the assembly, by
encapsulating such a particle with other lipid material or to
modify its surface characteristics. This may for example include
addition of lipids to produce a net neutral particle, or addition
of targeting molecules that must reside on the exterior of the
particle for functional purposes.
[0189] In one embodiment, the method for making lipid nanoparticles
includes sequential assembly and growth of lipid nanoparticles
through charge association, and further, can provide encapsulation
of therapeutic small interfering RNA (siRNA). This method can be
used to completely alter the surface charge characteristics from
net positive to net negative and vice versa.
[0190] A lipid nanoparticle encapsulating siRNA (-ive) was prepared
with a charge ratio of about 2 (+ive/-ive). The lipids included 90
mol % DLin-KC2-DMA (+ive) and 10 mol % PEG-c-DMA. The resulting
particle was 23 nm in diameter (FIG. 28A) and had a positive zeta
potential of about 7 mV (FIG. 28B). The anionic lipid was then
incorporation in 4-fold excess to the cationic lipid via
microfluidic mixing. This led to an increase in particle size to 33
nm and a shift to a negative zeta potential of -14 mV. Further
incorporation of additional cationic lipid (in 4-fold excess to the
previous DOPS) and then incorporation of DOPS led to a continued
increase in particle size and alteration between net positive and
net negative zeta potentials.
[0191] The results were obtained by mixing in a single microfluidic
mixer, recovered, and then re-injected into the micromixer to add
the next lipid component. However, a single microfluidic device
could be designed to produce such particles in a continuous manner
(FIG. 29).
[0192] The following devices minimize the fluidic impedances and
the interaction between the lipid and aqueous fluids prior to
entering the micromixer.
[0193] FIG. 30 is a schematic representation of a representative
device 3000 and method of the invention. Referring to FIG. 30,
device 3000 includes Region A, where a first stream comprising a
polynucleic acid in a first solvent into a channel of large width
(>2 mm) and Region B, where a stream comprising lipid
particle-forming materials in a second solvent into a channel of
large width (>2 mm). The streams are introduced into Region C
where rapid mixing occurs in a micromixer and then ultimately to
Region D, the final product.
[0194] FIG. 31 is a schematic representation of a representative
device and method of the invention. Referring to FIG. 31, device
3100 includes Region A, where a first stream comprising a
polynucleic acid in a first solvent into a channel of large width
(>2 mm) and Region B, where a stream comprising lipid
particle-forming materials in a second solvent into a channel of
large width (>2 mm). The streams are introduced into Region C
where rapid mixing occurs in a micromixer and then ultimately to
Region D, the final product.
[0195] FIG. 32 is a schematic representation of a representative
device and method of the invention. Referring to FIG. 32, device
3200 includes Region A, where a first stream comprising a
polynucleic acid in a first solvent into a channel of large width
(>2 mm); Region B, where a second stream comprising the first
solvent to act as sheath fluid for the flow of Region A; Region C,
where a stream comprising lipid particle-forming materials in a
second solvent into a channel of large width (>2 mm); and Region
D, where a second stream comprising the second solvent to act as
sheath fluid for the flow of Region C. The streams are introduced
into Region E where rapid mixing occurs in a micromixer and then
ultimately to Region F, the final product. The dotted lines
represent fluidic interfaces.
[0196] FIG. 33 is a schematic representation of a representative
device and method of the invention. Referring to FIG. 33, device
3300 includes Region A, where 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 flown
concentrically into a channel, are introduced into Region B where
rapid mixing occurs in a micromixer and then ultimately to Region C
where we have the final product. The two fluids in Region A may be
separated by a physical barrier or by sheath fluid as demonstrated
in the cross-sectional view.
[0197] Method for Delivering Therapeutic Agents Using Lipid
Particles
[0198] The lipid particles of the present invention may be used to
deliver a therapeutic agent to a cell, in vitro or in vivo. In
particular embodiments, the therapeutic agent is a nucleic acid,
which is delivered to a cell using nucleic acid-lipid particles of
the present invention. The methods and compositions may be readily
adapted for the delivery of any suitable therapeutic agent for the
treatment of any disease or disorder that would benefit from such
treatment.
[0199] In certain embodiments, the present invention provides
methods for introducing a nucleic acid into a cell. Preferred
nucleic acids for introduction into cells are siRNA, miRNA,
immune-stimulating oligonucleotides, plasmids, antisense and
ribozymes. These methods may be carried out by contacting the
particles or compositions of the present invention with the cells
for a period of time sufficient for intracellular delivery to
occur.
[0200] Typical applications include using well known procedures to
provide intracellular delivery of siRNA to knock down or silence
specific cellular targets. Alternatively, applications include
delivery of DNA or mRNA sequences that code for therapeutically
useful polypeptides. In this manner, therapy is provided for
genetic diseases by supplying deficient or absent gene products.
Methods of the present invention may be practiced in vitro, ex
vivo, or in vivo. For example, the compositions of the present
invention can also be used for deliver of nucleic acids to cells in
vivo, using methods which are known to those of skill in the
art.
[0201] The delivery of siRNA by a lipid particle of the invention
and its effectiveness in silencing gene expression is described
below.
[0202] For in vivo administration, the pharmaceutical compositions
are preferably administered parenterally (e.g., intraarticularly,
intravenously, intraperitoneally, subcutaneously, or
intramuscularly). In particular embodiments, the pharmaceutical
compositions are administered intravenously or intraperitoneally by
a bolus injection. Other routes of administration include topical
(skin, eyes, mucus membranes), oral, pulmonary, intranasal,
sublingual, rectal, and vaginal.
[0203] In one embodiment, the present invention provides a method
of modulating the expression of a target polynucleotide or
polypeptide. These methods generally comprise contacting a cell
with a lipid particle of the present invention that is associated
with a nucleic acid capable of modulating the expression of a
target polynucleotide or polypeptide. As used herein, the term
"modulating" refers to altering the expression of a target
polynucleotide or polypeptide. Modulating can mean increasing or
enhancing, or it can mean decreasing or reducing.
[0204] In related embodiments, the present invention provides a
method of treating a disease or disorder characterized by
overexpression of a polypeptide in a subject, comprising providing
to the subject a pharmaceutical composition of the present
invention, wherein the therapeutic agent is selected from an siRNA,
a microRNA, an antisense oligonucleotide, and a plasmid capable of
expressing an siRNA, a microRNA, or an antisense oligonucleotide,
and wherein the siRNA, microRNA, or antisense RNA comprises a
polynucleotide that specifically binds to a polynucleotide that
encodes the polypeptide, or a complement thereof.
[0205] In a further aspect, the invention provides a pharmaceutical
composition comprising a lipid particle of the invention and a
pharmaceutically acceptable carrier or diluent. Representative
pharmaceutically acceptable carriers or diluents include solutions
for intravenous injection (e.g., saline or dextrose). The
composition can take the form of a cream, ointment, gel,
suspension, or emulsion.
[0206] The following is a description of a representative LNP
system, device and method for making the LNP system, and method for
using a LNP for delivering therapeutic agents.
[0207] Rapid microfluidic mixing allows production of monodisperse
lipid nanoparticles. Formulation of lipid nanoparticles was
performed by rapidly mixing a lipid-ethanol solution with an
aqueous buffer inside a microfluidic mixer (FIG. 15B) designed to
induce chaotic advection and provide a controlled mixing
environment at intermediate Reynolds number (24<Re<240). The
microfluidic channel contains herringbones that generate a chaotic
flow by changing the orientation of herringbone structures between
half cycles, causing a periodic change in the centers of local
rotational and extensional flow.
[0208] To determine mixing performance inside the device, the pH
sensitivity of fluorescein was used where two 10 .mu.M fluorescein
streams were mixed, one fluorescent at pH 8.88 and the other
non-fluorescent at pH 5.15. The channel length required for mixing
to occur (extent of mixing >95%) was found to be between 0.8 cm
and 1.0 cm. This resulted in mixing times of approximately 45 ms,
10 ms, and 5 ms and 3 ms for flow rates of 0.1 ml/min, 0.4 ml/min,
0.7 ml/min and 1.0 ml/min, respectively. The small difference in
mixing length is expected in a chaotic flow, which grows only
logarithmically with Peclet number (Pe=Ul/D where U is the fluid
velocity, 1 is the cross-sectional channel length, and D is the
diffusivity of the molecule).
[0209] The following representative formulations include an
ionizable cationic lipid, DLin-KC2-DMA, having an apparent pKa of
6.7 rendering the lipid suitable for encapsulation of siRNA at low
pH and providing a near neutral cationic surface charge density at
physiological pH. Using this LNP-siRNA scheme as a model system,
the effect of flow rate on LNP formation was determined. As the
mixing time dramatically decreases with increased flow rate, the
speed at which lipids are introduced into the aqueous phase was
expected to influence their final size and dispersity. Using
identical flow rates, from 0.1 ml/min to 1 ml/min per channel, FIG.
16B shows the mean particle diameter of LNP-siRNA systems produced
by the microfluidic mixer. The buffer contained siRNA to yield a
siRNA/total lipid ratio of 0.06 (wt/wt) and the LNP mixture was
diluted directly into buffer to reduce ethanol content to
approximately 22 vol %. Particle size decreased significantly when
increasing total flow rate from 0.2 ml/min to 2 ml/min. Particle
size was largest under a flow rate of 0.2 ml/min and the LNP
reached a limit size of approximately 40 nm as determined from the
number-weighted particle diameter. Alternatively, the mixing time
was also adjusted by changing the ratio of the ethanol and aqueous
streams. Increasing the flow rate of the aqueous stream in effect
provides a quicker dilution of the lipids with the aqueous stream.
With the lipid-ethanol stream kept constant at 0.5 ml/min, an
increase in the aqueous flow rate resulted in a decrease in
particle size (FIG. 16C). The substantial drop in particle size,
from about 70 nm to 35 nm, with a three-fold increase in the
aqueous flow rate highlights the importance in rapidly reducing the
ethanol content.
[0210] Because these LNP are expected to form spontaneously as the
lipids encounter a more aqueous environment, it was also important
to explore the effect of lipid concentration. As lipid
concentration is increased, the amount of lipids available to
incorporate into a LNP would be expected to increase or otherwise
form additional particles. This was monitored as the lipid
concentration was increased from 10 mM to 50 mM in the ethanol
stream. An increase in mean particle diameter from about 40 nm to
70 nm was observed following this increase in lipid concentration
(FIG. 17).
[0211] Rapid microfluidic mixing provides a broad formulation range
of LNP-siRNA systems. While recent improvements of the cationic
lipid have advanced LNP potency several fold, it has also become
apparent that further improvements can be provided via optimization
of the LNP composition. In particular, it can influence their
bilayer-destabilizing capacity and endosomolytic potential or may
influence their circulation behavior at physiological pH. For
example, formulations with less PEG-lipid and increased cationic
lipid have shown dramatic improvements in in vivo efficacy of LNP
systems targeting liver hepatocytes. This was observed in a recent
report for a mouse Factor VII model, which provided a further
five-fold reduction in ED50 in the optimized LNP. Although the
PEG-lipid is necessary for particulate stability, it can also
diminish the membrane-destabilizing property of these LNP systems.
With the preformed vesicle (PFV) method, difficulties have been
encountered when attempting to produce LNP systems with less than 5
mol % PEG-lipid; this is presumably due to less PEG content on the
exterior of the vesicles which increases fusion between LNPs.
Further, the incubation step necessary for reorganization of
preformed lipid particles and encapsulation of siRNA requires
ethanol solutions in the range of 30% (v/v). This increased lipid
fluidity can promote instability and lead to additional aggregation
and fusion of the preformed lipid particles.
[0212] Using PEG-c-DMA, the ability of the microfluidic (MF) method
(fast mixing times and short residence prior to dilution of the LNP
below 25% ethanol (v/v)) to produce LNP-siRNA systems with varying
PEG-lipid content was explored. An initial composition of
DLin-KC2-DMA, DSPC, cholesterol, and PEG-c-DMA (40: 11.5: 38.5: 10
mol/mol) was used with a siRNA/total lipid ratio of 0.06 (wt/wt).
Additional cholesterol was used to compensate for the decreased
amount of PEG-c-DMA. Titration of PEG-c-DMA to 2 mol % led to only
a minor increase in particle size using the microfluidic approach.
Further decrease to 1 mol % PEG led to an increase in diameter from
about 20 nm to about 40 nm (FIG. 18A). In contrast, the mean
particle diameter using the PFV method showed a constant increase
in particle diameter, from 20 nm to 70 nm, as PEG-lipid content was
decreased to 1 mol %. In addition to producing LNP with low amounts
of PEG-lipid, it is of interest to be able to vary the amount of
cationic lipid. As DLin-KC2-DMA was increased from 40 mol % to 70
mol %, a general increase in particle size was observed, from about
40 nm to 70 nm, for those produced by the microfluidic approach
(FIG. 18B).
[0213] Self assembly in a microfluidic device can produce LNP with
near complete encapsulation. In producing LNP-siRNA systems, a
robust process necessarily will provide high percent encapsulation
of the OGN product. siRNA encapsulation was evaluated by varying
the siRNA/total lipid ratio from 0.01 to 0.2 (wt/wt) using the
LNP-siRNA formulation with 1 mol % PEG. LNP formulations achieved
percent encapsulation approaching 100 percent over this range (FIG.
19). Upon reaching a siRNA/total lipid ratio of 0.21 (wt/wt),
corresponding to a charge balance between the cationic lipid and
anionic siRNA (N/P=1), encapsulation was observed to diminish (data
not shown). This later trend was expected due to insufficient
cationic charge required to complex the siRNA and encapsulate in
the LNP.
[0214] Morphology. LNP produced by the microfluidic and preformed
vesicle methods were visualized with cryo-TEM. Particle sizes of
the LNP were similar to that measured by dynamic light scattering.
LNP-siRNA systems containing
DLin-KC2-DMA/DSPC/Cholesterol/PEG-c-DOMG at 40/11.5/47.5/1 mol %
with siRNA-to-lipid ratio of 0.06 wt/wt are shown in FIG. 20A. In
addition, empty LNP samples of the same composition are shown in
FIG. 20B. The particles produced are predominately spherical and
homogeneous in size. LNP formulated with the preformed approach and
of identical composition was also imaged. These shared similar
features with the microfluidic LNP, though other features such as
coffee-bean structures were also observed. These LNP were also
larger in size, as expected from the dynamic light scattering
results.
[0215] LNP siRNA systems produced by microfluidics can be highly
potent gene silencing agents in vivo. The ability of LNP siRNA
systems to induce gene silencing in vivo following i.v. injection
was investigated using the mouse Factor VII model. Formulations
containing Dlin-KC2-DMA/DSPC/Cholesterol/PEG-c-DOMG with a
siRNA-to-lipid ratio of 0.06 (w/w) were created using the
microfluidic approach. Administration of the LNP-siRNA was by tail
vein injection. The cationic lipid, DLin-KC2-DMA, was varied from
30 mol % to 60 mol % while keeping the DSPC-to-Cholesterol ratio at
0.2 wt/wt. Increasing the cationic lipid content in the LNP
resulted in a progressive improvement in FVII silencing. The best
performing LNP contained 60 mol % DLin-KC2-DMA, resulting in an
effective dose for 50% FVII silencing at about 0.03 mg/kg (FIG.
21). It is interesting to note that further increase to 70 mol %
led to no observable improvement in efficacy over the 60 mol %
Dlin-KC2-DMA LNP.
[0216] The results demonstrate that a microfluidic device
containing a staggered herringbone mixer can be used to generate
LNP with a variety of lipid compositions, can be used to
efficiently encapsulate OGN such as siRNA and that the LNP siRNA
systems produced exhibit excellent gene silencing capabilities both
in vitro and in vivo.
[0217] The microfluidics device and system of the invention allow
for the formation of LNP and LNP containing OGN of 100 nm size or
smaller and provide OGN encapsulation 100%. With regard to
formation of LNP, the rate and ratio of mixing are clearly the
important parameters. Rapid mixing of the ethanol-lipid solution
with aqueous buffer results in an increased polarity of the medium
that reduces the solubility of dissolved lipids, causing them to
precipitate out of solution and form nanoparticles. Rapid mixing
causes the solution to quickly achieve a state of high
supersaturation of lipid unimers throughout the entire mixing
volume, resulting in the rapid and homogeneous nucleation of
nanoparticles. Increased nucleation and growth of nanoparticles
depletes the surrounding liquid of free lipid, thereby limiting
subsequent growth by the aggregation of free lipid. This proposed
mechanism is consistent with the observation that lower
concentrations of the lipid in ethanol (reduced free lipid) result
in smaller LNP (see FIG. 17), that higher flow rates, causing a
faster and more homogeneous approach to supersaturation, lead to
formation of smaller LNP, and that increasing the relative ratio of
the aqueous to organic solvent components also results in smaller
particles (FIG. 17).
[0218] LNP OGN systems of the invention formulated by the
microfluidics method exhibit OGN encapsulation efficiencies
approaching 100%. Previous cryo-TEM studies using the PFV technique
for antisense OGN have revealed the presence of small multilamellar
vesicles leading to the possibility that encapsulation involves OGN
adsorption to a preformed vesicle which serves as a nucleation
point for association with additional preformed vesicles that wrap
around the original vesicle. In contrast, cryo-TEM studies of the
LNP OGN produced by the microfluidics method show that the majority
of the LNP systems are "solid core" structures and suggest that a
different mechanism of OGN encapsulation is operative. In
particular, these structures are consistent with the association of
siRNA with cationic lipid monomers prior to or simultaneously with
nanoparticle assembly. The ability of the microfluidics method to
facilitate encapsulation efficiencies for antisense and siRNA OGN
approaching 100% independent of nucleic acid composition is a major
advantage over previously reported methods.
[0219] The microfluidics method provides advantages over three
alternative LNP synthesis techniques including the classical
extrusion procedure for producing LNP, the preformed vesicle
method, and the spontaneous vesicle formation methods for OGN
encapsulation. The microfluidics method provides LNP in the 100 nm
size range or smaller and, when cationic lipid is present, allows
LNP to be formed with low levels of stabilizing PEG-lipid.
Disadvantages of the microfluidics method relate to the need to
remove ethanol after preparation, the fact that certain lipids are
relatively insoluble in ethanol, and potential scalability issues.
The microfluidics method offers advantages in encapsulation
efficiencies, the use of high cationic lipid contents and low
PEG-lipid levels that are difficult to employ using the PFV
process, removal of the need to generate preformed vesicles, and
the ability to produce small scale batches using as little as 150
.mu.g of oligonucleotide with little loss due to the small dead
volume (1 .mu.l) of the apparatus.
[0220] Advantages of the microfluidics method as compared to the
SVF "T tube" procedure for generating LNP systems loaded with OGN
are similar to those indicated for the PFV process, with the
exception that preformed vesicles are not required. The aperture of
the T tube is approximately 1.5 mm in diameter, requiring high flow
rates (>1 ml/s) to achieve the velocities required for rapid
mixing to occur. The micromixer allows LNP OGN formulation to occur
under well defined, reproducible conditions at much lower flow
rates and reduced losses due to dead volumes, allowing more
straightforward preparation of small-scale batches for LNP
optimization and in vitro testing.
[0221] LNP OGN systems can be scaled up. Although a device that has
a maximum flow rate of 1 ml/min may be insufficient, a single
microfluidics chip may contain 10 or more micromixers in to achieve
total flow rates of about 10 mL/min. Given the relatively
inexpensive nature of this technology it is practical that a number
of such chips to be used in parallel, potentially allowing flow
rates of 100 ml/min or higher from a single bench-top instrument.
Furthermore, upstream fluid handling could easily be incorporated
into such a device to allow for precise programmable formulations
from multiple components, a feature that would be highly
advantageous in the screening and optimization of synthesis
formulations and parameters.
[0222] Solid Core LNP
[0223] Certain models of LNP siRNA formulations suggest a bilayer
vesicle structure of the LNP with siRNA on the inside in an aqueous
interior. However, a number of observations suggest that such
models are incorrect, at least for LNP siRNA systems generated by
the microfluidic mixing approach. For example, cryo-electron
microscopy of LNP siRNA systems produced by microfluidic mixing
indicates the presence of electron-dense cores rather than the
aqueous cores consistent with vesicular structure. As noted above,
formulation of LNP siRNA systems can routinely result in siRNA
encapsulation efficiencies approaching 100%, an observation that is
not consistent with bilayer structures where maximum encapsulation
efficiencies of 50% might be expected.
[0224] The structure of LNP siRNA systems was evaluated employing a
variety of physical and enzymatic assays. The results obtained
indicate that these LNP siRNA systems have a solid core interior of
consisting of siRNA monomers complexed with cationic lipid as well
as lipid organized in inverted micellar or related structures.
[0225] LNP systems exhibit electron dense solid core structure as
indicated by cryo EM in the presence and absence of encapsulated
siRNA. LNP systems produced by microfluidic mixing exhibit electron
dense cores as visualized by cryo EM, consistent with a solid
cores, in contrast to the aqueous core structures suggested for LNP
siRNA systems created by alternative methods. This was confirmed as
shown in FIG. 22A for an LNP siRNA formulation consisting of
DLin-KC2-DMA/DSPC/Chol/PEG-lipid (40/11.5/47.5/1; mol/mol)
containing siRNA at a 0.06 siRNA/lipid (wt/wt) content, which
corresponds to a negative charge (on the siRNA) to positive charge
(on the fully protonated cationic lipid) N/P ratio of 4. As a
result approximately 75% of the cationic lipid is not complexed to
siRNA in the LNP. The solid core electron dense structure contrasts
with the less dense interior of a vesicle system composed of POPC
(FIG. 22B) and is visually similar to the electron dense interior
of a POPC/triolein (POPC/TO) LNP (FIG. 22C). POPC/TO LNP produced
by microfluidic mixing consist of a hydrophobic core of TO
surrounded by a monolayer of POPC.
[0226] An interesting feature of FIG. 22A is that 75% of the
ionizable cationic lipid is not complexed to siRNA, but the LNP
siRNA particle as a whole exhibits a solid core interior. This
suggests that the cationic lipid may contribute to the solid core
interior even when it is not complexed to siRNA. LNP systems with
the same lipid composition but no siRNA were formulated employing
the microfluidics process and characterized by cryo EM. As shown in
FIG. 22B, the electron dense core was observed in the absence of
siRNA, indicating that ionizable cationic lipids such as
DLin-KC2-DMA, possibly in combination with DSPC and cholesterol,
can adopt non-lamellar electron dense structures in the LNP
interior.
[0227] LNP structures exhibit limit sizes indicating that ionizable
cationic lipid forms inverted micellar structures in the LNP
interior. The contribution of the cationic lipid to the electron
dense LNP core raises the question of what the molecular structure
of such LNP systems may be. It is logical to propose that the
cationic lipid, in association with a counter-ion, adopts an
inverted structure such as an inverted micelle, consistent with the
propensity of these lipids for inverted structures such as the
hexagonal H.sub.II phase in mixtures with anionic lipids. In turn,
this would suggest that LNP systems composed of pure cationic lipid
should exhibit limit sizes with diameters in the range of 10 nm,
which is essentially the thickness of two bilayers surrounding an
inverted micelle interior with diameter 2-3 nm. The diameter of the
aqueous channels found for phosphatidylethanolamine in the H.sub.II
phase is 2.6 nm. The microfluidics formulation process provides
fast mixing kinetics that drive the generation of limit size
systems for LNP systems. The limit size that could be achieved for
a DLin-KC2-DMA/PEG-lipid system (90/10, mol/mol) was evaluated. As
shown in FIG. 23, measurements by dynamic light scattering on these
LNP formed by the microfluidic method confirm that the particle
size is approximately 10 nm in diameter, a finding that is not
consistent with a significant aqueous core or trapped volume.
[0228] A related question concerns the structure of the cationic
lipid-siRNA complex. Again, it is logical to suppose that it
consists of a distorted inverted micelle of cationic lipid
surrounding the siRNA oligonucleotide. In turn, this would suggest
a limit size in the range of 15-20 nm, assuming that the siRNA
contained in this inverted micelle is surrounded by an interior
monolayer of cationic lipid and then an outer monolayer of
remaining lipid and that the dimensions of the siRNA are 2.6 nm in
diameter and 4.8 nm in length. In order to determine whether this
is consistent with experiment, the limit size of LNP siRNA systems
consisting of DLin-KC2-DMA and PEG-lipid (90/10; mol/mol) at high
levels of siRNA corresponding to an N/P ratio of one was
determined. As shown in FIG. 23, the inclusion of siRNA resulted in
a limit-size systems of approximately 21 nm diameter, consistent
with hypothesis.
[0229] Encapsulated siRNA is immobilized in the LNP. If the siRNA
is complexed to cationic lipid and localized in a solid core inside
the LNP it would be expected to be less mobile than if freely
tumbling in the aqueous interior of a bilayer vesicle system. The
mobility of the siRNA can be probed using .sup.31P NMR techniques.
In particular, it would be expected that limited motional averaging
would be possible for complexed siRNA, leading to very broad "solid
state" .sup.31P NMR resonances due to the large chemical shift
anisotropy of the phosphate phosphorus. Under the conditions
employed, such resonances would not be detectable. If, on the other
hand, the siRNA is able to freely tumble in an aqueous environment,
rapid motional averaging would be expected to lead to narrow,
readily detectable, .sup.31P NMR spectra. In order to eliminate
complications arising from .sup.31P NMR signals arising from the
phospholipid phosphorus, DSPC was omitted from formulations of LNP
to test this hypothesis. As shown in FIG. 24A, for LNP siRNA
systems with lipid composition DLin-KC2-DMA/Chol/PEG-lipid (50/45/5
mol %) and containing siRNA (0.06 siRNA/lipid; wt/wt), no .sup.31P
NMR signal is observable for the encapsulated siRNA, consistent
with immobilization within the LNP core. If the detergent sodium
dodecyl sulphate is added (1%) to solubilize the LNP and release
the encapsulated siRNA then a narrow .sup.31P NMR signal is
detected as shown in FIG. 24C.
[0230] Encapsulated siRNA is fully protected from degradation by
external RNase A. A test of the internalization of siRNA is that if
siRNA is sequestered in the LNP core they should be fully protected
from degradation by externally added RNase. LNP siRNA systems with
the lipid composition DLin-KC2-DMA/DSPC/Chol/PEG-lipid (40/11/44/5
mol %) were incubated with RNase A to determine whether
encapsulated siRNA could be digested. As shown in the gel presented
in FIG. 25, the free siRNA is degraded, while the siRNA associated
within the LNP particles made by the microfluidic method is
completely protected (FIG. 25 arrow). As also shown in FIG. 25,
addition of the detergent Triton X-100 to the LNP results in
dissolution of the LNP, release of the siRNA, and degradation in
the presence of RNase.
[0231] Encapsulated siRNA is complexed with internalized cationic
lipid. The solid core of the LNP siRNA systems consists of
encapsulated siRNA complexed to cationic lipid and the remaining
lipid (cationic lipid, cholesterol and PEG-lipid) is either present
in the core in inverted micellar or similar structures, or resident
on the LNP exterior. For high siRNA contents, where essentially all
of the cationic lipid is complexed with internalized siRNA, it
would be expected that little cationic lipid would be localized on
the LNP exterior. A fluorescence resonance energy transfer (FRET)
assay was developed to determine external cationic lipid. The assay
required the preparation of negatively charged vesicular LNP
composed of dioleoylphosphatidylserine (DOPS) that contained the
FRET pair, NBD-PE/Rh-PE at high (self-quenching) concentrations.
The negatively charged DOPS LNP were then incubated with LNP siRNA
systems consisting of DLin-KC2-DMA/DSPC/Chol/PEG-lipid
(40/11.5/47.5/1 mol %) at pH 5.5. The pKa of DLin-KC2-DMA is 6.7
and thus nearly all the DLin-KC2-DMA on the outside of the LNP will
be charged at pH 5.5, promoting an interaction and potentially
fusion with the negatively charged DOPS LNP. Fusion is reported as
an increase in the NBD-PE fluorescence at 535 nm as the NBD-PE and
Rh-PE probes become diluted following lipid mixing.
[0232] As shown in FIG. 26, when the LNP systems contained no siRNA
substantial fusion is observed consistent with a considerable
proportion of the DLin-KC2-DMA residing on the outer monolayer of
the LNP system. When the LNP systems contained siRNA at a siRNA to
lipid ratio of 0.06 (wt/wt), which corresponds to a positive
(cationic lipid) charge to negative (siRNA) N/P charge ratio of 4,
however, fusion was considerably reduced (FIG. 26), whereas for LNP
siRNA systems prepared with an N/P of 1, little or no fusion was
observed, indicating that little of no DLin-KC2-DMA was present on
the LNP siRNA exterior. This supports the hypothesis that high
siRNA content essentially all of the cationic lipid is complexed
with siRNA and sequestered in the LNP interior.
[0233] The results provide evidence that the interior of LNP siRNA
systems consist of a solid core composed of siRNA monomers
complexed to cationic lipids, as well as lipids arranged in
inverted micelle or related structures. These results imply a model
for LNP siRNA structure, provide a rationale for the high siRNA
encapsulation efficiencies that can be achieved and suggest methods
for manufacturing LNP siRNA systems with properties appropriate to
particular applications.
[0234] The model for LNP siRNA structure based on the results is
shown in FIG. 27. The model proposes that encapsulated siRNA
resides in a distorted inverted micelle surrounded by cationic
lipid, and that remaining lipid is organized in inverted micelles
surrounding anionic counterions and also makes up the outermost
monolayer.
[0235] The model provides an understanding of how siRNA
encapsulation efficiencies approaching 100% can be achieved during
the microfluidic mixing formulation process. This is a major
problem for siRNA encapsulation in bilayer systems because,
assuming the cationic lipid is equally distributed on both sides of
the bilayer, a maximum of 50% siRNA internalization would be
expected. The model points to ways in which LNP siRNA size,
composition, and surface charge may be readily modulated. With
regard to size, the limit size structure is clearly one that
contains one siRNA monomer per particle, suggesting a limit size of
approximately 15-20 nm. Such LNP siRNA particles are readily
achieved using microfluidic method of the invention. The limit size
LNP siRNA system consisting of a monomer of siRNA can be
potentially used as a building block to achieve LNP siRNA systems
of varying composition and surface charge using microfluidic mixing
technology. Rapid mixing of preformed limit size LNP siRNA with an
ethanol solution containing negatively charged lipids, for example,
may be expected to result in an interaction with excess cationic
lipids to produce internal inverted micellar core structures and a
negatively charged surface.
[0236] The lipid particles of the invention described herein
include (i.e., comprise) the components recited. In certain
embodiments, the particles of the invention include the recited
components and other additional components that do not affect the
characteristics of the particles (i.e., the particles consist
essentially of the recited components). Additional components that
affect the particles' characteristics include components such as
additional therapeutic agents that disadvantageously alter or
affect therapeutic profile and efficacy of the particles,
additional components that disadvantageously alter or affect the
ability of the particles to solubilize the recited therapeutic
agent components, and additional components that disadvantageously
alter or affect the ability of the particles to increase the
bioavailability of the recited therapeutic agent components. In
other embodiments, the particles of the invention include only
(i.e., consist of) the recited components.
[0237] The following examples are provided for the purpose of
illustrating, not limiting, the claimed invention.
EXAMPLES
[0238] Materials
[0239] 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),
1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3
-benzoxadiazol-4-yl) (NBD-PE),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine
rhodamine B sulfonyl) (Rh-PE) were obtained from Avanti Polar
Lipids (Alabaster, Ala.). 4-(2-Hydroxyethyl)
piperazine-1-ethanesulfonic acid (HEPES) and cholesterol was
obtained from Sigma (St Louis, Mo.). N-[(Methoxy poly(ethylene
glycol).sub.2000)carbamyl]-1,2-dimyristyloxlpropyl-3-amine
(PEG-C-DMA) was synthesized by AlCana Technologies.
2-(N-Morpholino)ethanesulfonic acid (MES) was obtained from BDH.
Ammonium acetate, sodium acetate and sodium chloride were obtained
from Fisher Scientific (Fair Lawn, N.J.). RNase A was obtained from
Applied Biosystems/Ambion (Austin, Tex.). Factor VII (FVII)
targeting, and low GC negative control siRNA were purchased from
Invitrogen (Carlsbad, Calif.). Factor VII siRNA: (SEQ ID NO: 1)
5'-GGAUCAUCUCAAGUCUUACTT-3' (FVII sense), and (SEQ ID NO: 2)
5'-GUAAGACUUGAGAUGAUCCTT-3' (FVII antisense). DLin-KC2-DMA was
obtained from AlCana Technologies Inc. (Vancouver, BC).
Example 1
[0240] Preparation of LNP Systems: Preformed Vesicle Method
[0241] In the example, the preparation of an LNP-siRNA system using
the preformed vesicle method is described.
[0242] LNP-siRNA systems were made using the preformed vesicle
method as depicted in FIG. 15A and as described in N. Maurer, K. F.
Wong, H. Stark, L. Louie, D. McIntosh, T. Wong, P. Scherrer, S.
Semple and P. R. Cullis, "Spontaneous Entrapment of Polynucleotides
Upon Electrostatic Interaction With Ethanol Destabilized Cationic
Liposomes: Formation of Small Multilamellar Liposomes," Biophys.
J., 80:2310-2326 (2001). Cationic lipid, DSPC, cholesterol and
PEG-lipid were first solubilized in ethanol at the appropriate
molar ratio. The lipid mixture was then added dropwise to an
aqueous buffer (citrate or acetate buffer, pH 4) while vortexing to
a final ethanol and lipid concentration of 30% (v/v). The hydrated
lipids were then extruded five times through two stacked 80 nm
pore-sized filters (Nuclepore) at room temperature using a Lipex
Extruder (Northern Lipids, Vancouver, Canada). The siRNA
(solubilized in an identical aqueous solution containing 30%
ethanol) was added to the vesicle suspension while mixing. A target
siRNA/lipid ratio of 0.06 (wt/wt) was generally used. This mixture
was incubated for 30 minutes at 35.degree. C. to allow vesicle
re-organization and encapsulation of the siRNA. The ethanol was
then removed and the external buffer replaced with
phosphate-buffered saline (PBS) by dialysis (12-14k MW cut-off,
Spectrum medical instruments) to 50 mM citrate buffer, pH 4.0 and
then dialysis to PBS, pH 7.4.
Example 2
[0243] Preparation of LNP Systems: Microfluidic Staggered
Herringbone Mixer
[0244] In the example, a representative LNP-siRNA system of the
invention using a microfluidic staggered herringbone mixer is
described.
[0245] LNP-siRNA preparation. Oligonucleotide (siRNA) solution was
prepared in 25 mM acetate buffer at pH 4.0. Depending on the
desired oligonucleotide-to-lipid ratio and formulation
concentration, solutions were prepared at a target concentration of
0.3 mg/ml to 1.9 mg/ml total lipid. A lipid solution containing
DLin-KC2-DMA, DSPC, cholesterol, and a PEG-lipid at the appropriate
molar ratio was prepared in ethanol and diluted with 25 mM acetate
buffer to achieve an ethanol concentration of 90% (v/v). FIG. 15B
is a schematic illustration of the microfluidic apparatus used in
this example. 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. The device features a 200
.mu.m wide and 79 .mu.m high mixing channel with herringbone
structures formed by 31 .mu.m high and 50 .mu.m thick features on
the roof of the channel. Fluidic connections were made with 1/32''
I.D., 3/32'' O.D. tubing that was attached to 21G1 needles for
connection with syringes. 1 ml syringes were generally used for
both inlet streams. A dual syringe pump (KD200, KD Scientific) was
used to control the flow rate through the device. The flow rate of
each stream was varied from 0.1 ml/min to 1 ml/min. The syringe
pump introduces the two solutions into the microfluidic device
(inlet a and inlet b in FIG. 15B), where they come into contact at
a Y-junction. Insignificant mixing occurs under laminar flow by
diffusion at this point, whereas the two solutions become mixed as
they pass along the herringbone structures.
[0246] Mixing occurs in these structures by chaotic advection,
causing the characteristic separation of laminate streams to become
increasingly small, thereby promoting rapid diffusion. This mixing
occurs on a millisecond time scale and results in the lipids being
transferred to a progressively more aqueous environment, reducing
their solubility and resulting in the spontaneous formation of LNP.
By including cationic lipids in the lipid composition, entrapment
of oligonucleotide species is obtained through association of the
positively charged lipid head group and negatively charged
oligonucleotide. Following mixing in the microfluidic device, the
LNP mixture was generally diluted into a glass vial containing two
volumes of stirred buffer. Ethanol is finally removed through
dialysis to 50 mM citrate buffer, pH 4.0 and then dialysis to PBS,
pH 7.4. Empty vesicles were similarly produced, with the
oligonucleotide absent from the buffer solution.
[0247] LNP Image Analysis. Mixing times were measured by
fluorescent imaging of the mixing of fluorescein solutions with
different pH values. Images were collected using an Olympus
inverted confocal microscope using a 10.times. objective and Kalman
filter mode with 2 scans per line. Twenty-five equally spaced
slices were taken along the height of the channel and combined to
determine total intensity profiles. For each position imaged, ten
adjacent rows of pixels along the flow direction were averaged to
obtain an intensity profile along the width of the channel and used
to determine the extent of mixing. Mixing experiments were
performed with two 10 .mu.M fluorescein solutions supplemented with
0.5 M NaCl to suppress the formation of a liquid junction potential
due to a large difference in sodium and phosphate ion
concentrations. One solution contained 14 mM phosphate buffer at pH
8.88, while the other contained 1 mM phosphate buffer at pH 5.15.
The increase in fluorescence of the solution initially at pH 5.15
will overwhelm the small drop in fluorescence in the basic
solution, resulting in an increase in total fluorescence intensity
by a factor of two. The extent of mixing was determined at
approximately 2.1 mm, 6.2 mm, and 10.1 mm along the channel length
using flow rates of the individual streams at 0.1 ml/min, 0.4
ml/min, 0.7 ml/min and 1.0 ml/min.
[0248] LNP Characterization. Particle size was determined by
dynamic light scattering using a Nicomp model 370 Submicron
Particle Sizer (Particle Sizing Systems, Santa Barbara, Calif.).
Number-weighted and intensity-weighted distribution data was used.
Lipid concentrations were verified by measuring total cholesterol
using the Cholesterol E enzymatic assay from Wako Chemicals USA
(Richmond, Va.). Removal of free siRNA was performed with VivaPureD
MiniH columns (Sartorius Stedim Biotech GmbH, Goettingen, Germany)
The eluents were then lysed in 75% ethanol and siRNA was quantified
by measuring absorbance at 260 nm. Encapsulation efficiency was
determined from the ratio of oligonucleotide before and after
removal of free oligonucleotide content, normalized to lipid
content.
[0249] LNP Cyro-Transmission Electron Microscopy. Samples were
prepared by applying 3.mu.L of PBS containing LNP at 20-40 mg/ml
total lipid to a standard electron microscopy grid with a
perforated carbon film. Excess liquid was removed by blotting with
a Vitrobot system (FEI, Hillsboro, Oreg.) and then plunge-freezing
the LNP suspension in liquid ethane to rapidly freeze the vesicles
in a thin film of amorphous ice. Images were taken under cryogenic
conditions at a magnification of 29K with an AMT HR CCD side mount
camera. Samples were loaded with a Gatan 70 degree cryo-transfer
holder in an FEI G20 Lab6 200kV TEM under low dose conditions with
an underfocus of 5-8 .mu.m to enhance image contrast.
[0250] In vivo Activity of LNP-siRNA for FVII activity. Six to
eight week old, female C57B1/6 mice were obtained from Charles
River Laboratories. LNP-siRNA containing Factor VII siRNA were
filtered through a 0.2 .mu.m filter and diluted to the required
concentrations in sterile phosphate buffered saline prior to use.
The formulations were administered intravenously via the lateral
tail vein at a volume of 10 ml/kg. After 24 h, animals were
anaesthetized with Ketamine/Xylazine and blood was collected by
cardiac puncture. Samples were processed to serum (Microtainer
Serum Separator Tubes; Becton Dickinson, N.J.) and tested
immediately or stored at -70.degree. C. for later analysis of serum
Factor VII levels. All procedures were performed in accordance with
local, state, and federal regulations as applicable and approved by
the Institutional Animal Care and Use Committee (IACUC).
[0251] Serum Factor VII levels were determined using the
colorimetric Biophen VII assay kit (Anaira). Control serum was
pooled and serially diluted (200%-3.125%) to produce a calibration
curve for calculation of FVII levels in treated animals
Appropriately diluted plasma samples from treated animals (n=3 per
dosage) and a saline control group (n=4) were analyzed using the
Biophen VII kit according to manufacturer's instructions. Analysis
was performed in 96-well, flat bottom, non-binding polystyrene
assay plates (Corning, Corning, N.Y.) and absorbance was measured
at 405 nm. Factor VII levels in treated animals were determined
from a calibration curve produced with the serially diluted control
serum.
Example 3
[0252] LNP Systems: Solid Core
[0253] In the example, a structure of a representative LNP-siRNA
system of the invention having a solid core is described.
[0254] Preparation of lipid nanoparticles. LNP were prepared by
mixing desired volumes of lipid stock solutions in ethanol with an
aqueous phase employing the micro-mixer described above. For the
encapsulation of siRNA, the desired amount of siRNA was mixed with
25 mM sodium acetate buffer at pH 4. Equal volumes of the
lipid/ethanol phase and the siRNA/aqueous phase were combined in a
micro-mixer containing a herring-bone structure to promote mixing.
The ethanol content was quickly diluted to 25% with sodium acetate
buffer upon leaving the micro-mixer. The flow rate through the
micro-mixing was regulated using a dual-syringe pump (Kd
Scientific). The lipid mixture then underwent a 4 hour dialysis in
50 mM MES/sodium citrate buffer (pH 6.7) followed by an overnight
dialysis in phosphate buffered saline (pH 7.4).
[0255] Cryo-EM. Samples were prepared by applying 3 .mu.L of PBS
containing LNP at 20-40 mg/ml total lipid to a standard electron
microscopy grid with a perforated carbon film. Excess liquid was
removed by blotting with a Vitrobot system (FEI, Hillsboro, Oreg.)
and then plunge-freezing the LNP suspension in liquid ethane to
rapidly freeze the vesicles in a thin film of amorphous, vitreous
ice. Images were taken under cryogenic conditions at a
magnification of 29K with an AMT HR CCD side mount camera. Samples
were loaded with a Gatan 70 degree cryo-transfer holder in an FEI
G20 Lab6 200kV TEM under low dose conditions with an underfocus of
5-8 um to enhance image contrast.
[0256] RNase protection assay. Factor VII siRNA was encapsulated
with 40% DLinKC2-DMA, 11% DSPC, 44% cholesterol and 5% PEG-c-DMA
using the microfluidics mixing method. 1 ug of siRNA was incubated
with 0.05 ug RNase A (Ambion, Austin, Tex.) in 50 uL of 20 mM HEPES
(pH 7.0) at 37.degree. C. for 1 hour. At the end of the incubation,
a 10 uL aliquot of the reaction mix was added to 30 uL FA dye
(deionized formamide, TBE, PBS, xylene cyanol, bromophenol blue,
yeast tRNA) to halt the RNase reaction. Gel electrophoresis was
performed using 20% native polyacrylamide gel and nucleic acids
were visualized by staining with CYBR-Safe (Invitrogen, Carlsbad,
Calif.).
[0257] .sup.31P-NMR studies. Proton decoupled .sup.31P NMR spectra
were obtained using a Bruker AVII 400 spectrometer operating at 162
MHz. Free induction decays (FID) corresponding to about 10.sup.4
scans were obtained with a 15 .mu.s, 55-degree pulse with a 1 s
interpulse delay and a spectral width of 64 kHz. An exponential
multiplication corresponding to 50 Hz of line broadening was
applied to the FID prior to Fourier transformation. The sample
temperature was regulated using a Bruker BVT 3200 temperature unit.
Measurements were performed at 25.degree. C.
[0258] FRET membrane fusion studies. Fusion between LNP siRNA
nanoparticles and anionic DOPS vesicles was assayed by a lipid
mixing assay employing fluorescence resonance energy transfer.
Labeled DOPS vesicles containing NBD-PE and Rh-PE (1 mol % each)
were prepared by direct re-hydration of lipid film with the
appropriate buffer followed by 10 extrusions through a 100 nm pore
size polycarbonate membrane using the Lipex Extruder. LNP comprised
of 40% DLinKC2-DMA, 11.5% DSPC, 47.5% cholesterol, 1% PEG-c-DMA
were prepared with siRNA-to-lipid ratio (D/L ratio, wt/wt) of 0,
0.06 and 0.24. A D/L=0.24 represents an equimolar ratio of positive
(cationic lipid) to negative (siRNA) charges (N/P=1). Lipid mixing
experiments were conducted. Labeled DOPS vesicles and unlabeled LNP
were mixed at a 1:2 mol ratio into a stirring cuvette containing 2
mL of 10 mM acetate, 10 mM MES, 10 mM HEPES, 130 mM NaCl
equilibrated to pH 5.5. Fluorescence of NBD-PE was monitored using
465 nm excitation, and 535 nm emission using an LS-55 Perkin Elmer
fluorometer using a 1.times.1 cm cuvette under continuous low speed
stirring. Lipid mixing was monitored for approximately 10 min,
after which 20 .mu.L of 10% Triton X-100 was added to disrupt all
lipid vesicles, representing infinite probe dilution. Lipid mixing
as a percentage of infinite probe dilution was determined using the
equation: % lipid mixing=(F-F.sub.o)/(F.sub.max-F.sub.o).times.100,
where F is the fluorescence intensity at 535 nm during assay,
F.sub.o is the initial fluorescence intensity, and F.sub.max is the
maximum fluorescence intensity at infinite probe dilution after the
addition of Triton X-100.
Example 4
[0259] Sequential Assembly of Lipid Nanoparticles
[0260] In this example, a representative method of the invention,
sequential assembly, for making lipid nanoparticles is
described.
[0261] The oligonucleotide (siRNA) solution was prepared at 1.31
mg/ml in 25 mM acetate buffer at pH 4.0. The lipid mixture was
prepared to contain 90 mol % cationic lipid (DLin-KC2-DMA) and 10
mol % PEG-c-DMA (10 mM total lipid dissolved in ethanol). The two
solutions were mixed using the microfluidic mixer at a total flow
rate 2 ml/min and diluted 2-fold with 25 mM acetate buffer, pH 4.0,
to bring ethanol down to about 23 vol %, forming the initial or
core nanoparticle. Sequential assembly was performed by taking this
initial lipid particle suspension and mixing it with another lipid
solution containing an anionic lipid dioleoylphosphatidylserine
(DOPS) dissolved in methanol and further diluting to approximately
25 vol % solvent (methanol and ethanol). The second lipid, DOPS,
was added at about 4.times. molar excess to the cationic lipid. The
sequential assembly process was repeated by alternating between the
cationic lipid and anionic lipid.
[0262] Particle size was determined by dynamic light scattering
using a Malvern Zetasizer Nano-ZS (Malvern Instruments Ltd,
Malvern, Worcestershire, UK). Number-weighted distribution data was
used. Zeta potential which provides a measure of the surface charge
of the LNP systems was measured with the Malvern Zetasizer using
disposable capillary cells (DTS1060, Malvern Instruments Ltd.). The
LNP systems were diluted to approximately 0.3 mg/ml total lipid in
25 mM acetate buffer, pH 4.0.
Example 5
[0263] Preparation and Characteristics of a Representative Lipid
Particle
[0264] In this example, a representative lipid particle the
invention consisting only of a cationic lipid and a nucleic acid
(DLin-KC2-DMA -- siRNA), are described.
[0265] The siRNA solution was prepared at 0.38 mg/ml in 25 mM
acetate buffer, pH 4.0. The lipid solution was prepared to contain
DLin-KC2-DMA at a concentration of 10 mM in ethanol. The
siRNA-to-lipid ratio was 0.06 (wt/wt). Each solution was input into
the microfluidic mixer at equal flow rates and a total flow rate of
2 ml/min. The sample was further diluted with 25 mM acetate buffer,
pH 4.0, to bring ethanol content to 25 vol %.
[0266] Particle size was determined by dynamic light scattering
using a Nicomp model 370 Submicron Particle Sizer (Particle Sizing
Systems, Santa Barbara, Calif., USA). Sample measurement was
performed in 25 mM acetate and number-weighted distribution data
was used. The particles had a mean particle diameter of 14.2 nm, a
coefficient of variance of 0.487, and .chi..sup.2of 1.93.
[0267] While illustrative embodiments have 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.
Sequence CWU 1
1
2121DNAArtificial Sequencesyntheticmisc_feature(1)..(21)Sequence is
DNA/RNA hybrid 1ggaucaucuc aagucuuact t 21221DNAArtificial
Sequencesyntheticmisc_feature(1)..(21)Sequence is DNA/RNA hybrid
2guaagacuug agaugaucct t 21
* * * * *