U.S. patent application number 15/689667 was filed with the patent office on 2018-01-25 for lipid nanoparticle compositions and methods of making and methods of using the same.
This patent application is currently assigned to Ohio State Innovation Foundation. The applicant listed for this patent is Ohio State Innovation Foundation. Invention is credited to Robert J. Lee.
Application Number | 20180021447 15/689667 |
Document ID | / |
Family ID | 48539454 |
Filed Date | 2018-01-25 |
United States Patent
Application |
20180021447 |
Kind Code |
A1 |
Lee; Robert J. |
January 25, 2018 |
Lipid Nanoparticle Compositions and Methods of Making and Methods
of Using the Same
Abstract
Lipid nanoparticle formulations, methods of making, and methods
of using same are disclosed.
Inventors: |
Lee; Robert J.; (Columbus,
OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ohio State Innovation Foundation |
Columbus |
OH |
US |
|
|
Assignee: |
Ohio State Innovation
Foundation
Columbus
OH
|
Family ID: |
48539454 |
Appl. No.: |
15/689667 |
Filed: |
August 29, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14403313 |
Nov 24, 2014 |
9750819 |
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PCT/US13/42458 |
May 23, 2013 |
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15689667 |
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61650729 |
May 23, 2012 |
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61784892 |
Mar 14, 2013 |
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Current U.S.
Class: |
424/450 ;
424/130.1; 424/94.64; 435/455; 514/19.3; 514/44A; 514/785 |
Current CPC
Class: |
C07K 16/00 20130101;
A61P 35/00 20180101; A61K 47/62 20170801; A61K 31/7088 20130101;
A61K 45/06 20130101; A61K 47/544 20170801; C12Y 304/21064 20130101;
C12N 15/113 20130101; C12N 15/1135 20130101; A61K 38/08 20130101;
A61K 9/127 20130101; C12N 15/1137 20130101; A61K 38/10 20130101;
A61K 9/1271 20130101; A61K 38/482 20130101; A61K 47/543 20170801;
A61K 47/549 20170801; A61K 47/6911 20170801; A61K 47/59 20170801;
A61K 47/643 20170801; C12N 2310/11 20130101; A61K 31/713 20130101;
A61K 47/6907 20170801; A61K 9/1272 20130101; A61K 48/00 20130101;
C12N 2310/14 20130101 |
International
Class: |
A61K 47/64 20060101
A61K047/64; C07K 16/00 20060101 C07K016/00; C12N 15/113 20060101
C12N015/113; A61K 9/127 20060101 A61K009/127; A61K 47/69 20060101
A61K047/69; A61K 31/713 20060101 A61K031/713; A61K 38/08 20060101
A61K038/08; A61K 38/10 20060101 A61K038/10; A61K 38/48 20060101
A61K038/48; A61K 45/06 20060101 A61K045/06; A61K 47/59 20060101
A61K047/59; A61K 31/7088 20060101 A61K031/7088 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under Grant
Numbers R01 CA135243, DK088076, and CA152969 awarded by the
National Institutes of Health. The government has certain rights in
the invention.
Claims
1. A lipid nanoparticle comprising a combination of tertiary and
quaternary amine-based cationic lipids, except not, wherein said
combination consists of the tertiary amine-cationic lipid present
at about 40.0 molar percent; wherein the tertiary amine-cationic
lipid comprises N-[1-(2, 3-dioleyloyx) propyl]-N--N--N-dimethyl
ammonium chloride (DODMA), and the concentration of the quaternary
amine-cationic lipid present at about 5.0 molar percent.
2. The lipid nanoparticle of claim 1, wherein the tertiary
amine-cationic lipids are chosen from DODAP, DODMA, DC-CHOL,
N,N-dimethylhexadecylamine, or combinations thereof.
3. The lipid nanoparticle of claim 1, wherein the quaternary
amine-cationic lipids are selected from DOTAP, DOTMA, DDAB, or
combinations thereof.
4. The lipid nanoparticle of claim 1, wherein the concentration of
the tertiary amine-cationic lipids is below 60.0 molar percent of
the total lipid content.
5. The lipid nanoparticle of claim 1, wherein the concentration of
quaternary amine-cationic lipids is below 20.0 molar percent of the
total lipid content.
6. The lipid nanoparticle of claim 1, wherein the nanoparticle
encapsulates molecules selected from nucleic acids, proteins,
polysaccharides, lipids, radioactive substances, therapeutic
agents, prodrugs, nutritional supplements, biomarkers, or
combinations thereof.
7. The lipid nanoparticle of claim 6, wherein the encapsulated
molecules comprise a nucleic acid selected from plasmid DNAs,
antisense oligonucleotides, miRs, anti-miRs, shRNAs, siRNAs, or
combinations thereof.
8. The lipid nanoparticle of claim 1, further comprising a cationic
polymer.
9. The lipid nanoparticle of claim 8, wherein the cationic polymer
is selected from the group consisting of: spermine, dispermine,
trispermine, tetraspermine, oligospermine, thermine, spermidine,
dispermidine, trispermidine, oligospermidine, putrescine,
polylysine, polyarginine, a polyethylenimine of branched or linear
type, and polyallylamine.
10. The lipid nanoparticle of claim 1, further comprising a
fusogenic peptide.
11. The lipid nanoparticle of claim 6, wherein the encapsulation
rate of therapeutic agents or nucleotides is 20% or higher.
12. The lipid nanoparticle of claim 1, wherein the lipid
nanoparticle has a diameter under 300 nm.
13. The lipid nanoparticle of claim 1, wherein the lipid
nanoparticle comprises the lipids DODMA and DOTMA in a molar ratio
selected from 45:0, 5:40, 15:30, 22.5:22.5, 30:15, or 40:5.
14. The lipid nanoparticle of claim 1, wherein the lipid
nanoparticle comprises the lipids DMHDA and DOTAP in a molar ratio
selected from 90:10, 70:30, 50:50, 30:70, or 10:90.
15. A lipid nanoparticle having a diameter of less than 300 nm and
comprising a peptide.
16. The lipid nanoparticle of claim 15, wherein the peptide is
selected from gramicidin A, B, C, D, or S; JTS-1; proteinase K
(PrK); trichorovin-Xlla; rabies virus glycoprotein; interleukin-1
.beta.; HIV-Tat; herpes simplex virus VP22 protein; and
combinations thereof.
17. The lipid nanoparticle of claim 15, wherein the peptide
comprises an antibiotic.
18. The lipid nanoparticle of claim 17, wherein the antibiotic is
selected from gramicidin A, B, C, D, or S.
19. The lipid nanoparticle of claim 15, wherein the peptide
consists essentially of a lipidated JTS-1 fusogenic peptide.
20. The lipid nanoparticle of claim 19, wherein the lapidated JTS-1
fusogenic peptide is present at about 0 to about 30 molar percent
of the total formulation.
21. The lipid nanoparticle of claim 15, further comprising
proteinase K.
22. The lipid nanoparticle of claim 15, wherein the proteinase K is
present at about 0 to about 30 molar percent of the total
formulation.
23. The lipid nanoparticle of claim 15, wherein the lipid
nanoparticle encapsulates molecules selected from nucleic acids,
proteins, polysaccharides, lipids, radioactive substances,
therapeutic agents, prodrugs, nutritional supplements, biomarkers,
or combinations thereof.
24. The lipid nanoparticle of claim 23, wherein the encapsulated
molecules comprise a nucleic acid selected from plasmid DNAs,
antisense oligonucleotides, miRs, anti-miRs, shRNAs, siRNAs, or
combinations thereof.
25. A lipid nanoparticle comprising a DNase- or RNase-degrading
agent.
26. The lipid nanoparticle of claim 25, wherein the DNase- or
RNase-degrading agent consists essentially of proteinase K.
27. The lipid nanoparticle of claim 25, wherein the nanoparticle
encapsulates molecules selected from nucleic acids, proteins,
polysaccharides, lipids, radioactive substances, therapeutic
agents, prodrugs, nutritional supplements, biomarkers, or
combinations thereof.
28. The lipid nanoparticle of claim 27, wherein the encapsulated
molecules comprise an oligonucleotide selected from pDNAs,
antisense oligonucleotides, miRs, anti-miRs, shRNAs, siRNAs, or
combinations thereof.
29. The lipid nanoparticle of claim 25, wherein the lipid
nanoparticle has a diameter under 300 nm.
30. A lipid nanoparticle having a diameter of less than 300 nm and
comprising a combination of two or more of: a mixture of tertiary
and quaternary amine-cationic head groups; an antibiotic; or a
DNase or RNase-degrading agent.
31. The lipid nanoparticle of claim 30, wherein the RNase or
RNase-degrading agent consists essentially of proteinase K.
32. The lipid nanoparticle of claim 30, wherein the antibiotic
comprises gramicidin A, B, C, D, or S.
33. A lipid nanoparticle of claim 1, further comprising a
polyethyleneglycol-lipid conjugate.
34. The lipid nanoparticle of claim 33, wherein the
polyethyleneglycol-lipid conjugate selected from polysorbate 80,
TPGS, mPEG-DSPE, PEG-DMG, DPPE-PEG or mPEG-DMPE.
35. The lipid nanoparticle of claim 33, wherein the
polyethyleneglycol-lipid is present at a concentration less than
about 10.0 molar percent.
36. A lipid nanoparticle of claim 33, further comprising
N,N-dimethylhexadecylamine.
37. The lipid nanoparticle of claim 36, wherein the
N,N-dimethylhexadecylamine is present at a concentration of less
than about 60.0 molar percent of the formulation.
38. A lipid nanoparticle of claim 36, further comprising a ligand
capable of binding to a target cell or a target molecule.
39. The lipid nanoparticle of claim 38, wherein the ligand is an
antibody or an antibody fragment.
40. The lipid nanoparticle of claim 38, wherein the ligand is
selected from cRGD, galatose-containing moieties, transferrin,
folate, low density lipoprotein, or epidermal growth factors.
41. A pharmaceutical composition comprising a lipid nanoparticle of
claim 1, and a pharmaceutically acceptable excipient.
42. The pharmaceutical composition of claim 41, wherein the
pharmaceutical composition is administered perorally,
intravenously, intraperitoneally, subcutaneously, or
transdermally.
43. The pharmaceutical composition of claim 41, wherein the
pharmaceutical composition is prepared as an orally administered
tablet.
44. The pharmaceutical composition of claim 41, wherein the
pharmaceutical composition is prepared as a sterile solution.
45. The pharmaceutical composition of claim 41, wherein the
pharmaceutical composition is prepared as a sterile suspension.
46. The pharmaceutical composition of claim 41, wherein the
pharmaceutical composition is prepared as a lyophilized powder.
47. The pharmaceutical composition of claim 41, wherein the
pharmaceutical composition is prepared as a suppository.
48. A method of diagnosing or treating a cancer or infectious
disease, the method comprising administering an effective amount of
a pharmaceutical composition of claim 41 to a patient in need
thereof.
49. The lipid nanoparticle of claim 1, wherein the lipid
nanoparticle encapsulates at least one molecule selected from
nucleic acids, chemotherapeutic agents, and combinations
thereof.
50. The lipid nanoparticle of claim 49, wherein the encapsulated
molecule comprises a nucleic acid selected from plasmid DNAs,
antisense oligonucleotides, miRs, anti-miRs, shRNAs, siRNAs, and
combinations thereof.
51. The lipid nanoparticle of claim 49, wherein the encapsulated
molecule comprises a therapeutic agent selected from:
antineoplastic agents, anti-infective agents, local anesthetics,
anti-allergics, antianemics, angiogenesis, inhibitors,
beta-adrenergic blockers, calcium channel antagonists,
anti-hypertensive agents, anti-depressants, anti-convulsants,
anti-bacterial, anti-fungal, anti-viral, anti-rheumatics,
anthelminithics, antiparasitic agents, corticosteroids, hormones,
hormone antagonists, immunomodulators, neurotransmitter
antagonists, anti-diabetic agents, anti-epileptics,
anti-hemmorhagics, anti-hypertonics, antiglaucoma agents,
immunomodulatory cytokines, sedatives, chemokines, vitamins,
toxins, narcotics, imaging agents, and combinations thereof.
52. The lipid nanoparticle of claim 49, wherein the encapsulated
molecule comprises a nucleic acid therapeutic agent.
53. The lipid nanoparticle of claim 52, wherein the nucleic acid
therapeutic agent is selected from: pDNA, siRNA, miRNA, anti-miRNA,
ASO, and combinations thereof.
54. The lipid nanoparticle of claim 52, wherein the nucleic acid
therapeutic agent is stabilized by modifications to substituent NA
base units and/or by modifying the ribose 2' position or
substituting phosphodiester linkers.
55. The lipid nanoparticle of claim 1, wherein the lipid
nanoparticle has a diameter under about 300 nm.
56. The lipid nanoparticle of claim 1, wherein the lipid
nanoparticle has a diameter under about 200 nm.
57. The lipid nanoparticle of claim 1, wherein the polymer is bound
to an external surface of the lipid via direct connection or via a
linker.
58. The lipid nanoparticle of claim 16, wherein the lipid
nanoparticle has an encapsulation efficiency of the molecule of at
least about 40%.
59. The lipid nanoparticle of claim 1, further including a
polyethylene glycol-conjugated lipid.
60. The lipid nanoparticle of claim 59, wherein the polyethylene
glycol-conjugated lipid comprises one or more of: polysorbate 80,
TPGS, DPPE-PEG, mPEG-DMPE, and mPEG-DSPE.
61. The lipid nanoparticle of claim 27, wherein the polyethylene
glycol-conjugated lipid is present at a concentration less than
about 15.0 molar percent.
62. The lipid nanoparticle of claim 1, further comprising a ligand
capable of binding to a target cell or a target molecule.
63. The lipid nanoparticle of claim 62, wherein the ligand is an
antibody or an antibody fragment.
64. The lipid nanoparticle of claim 62, wherein the ligand is
selected from cRGD, galatose-containing moieties, transferrin,
folate, low density lipoprotein, or epidermal growth factors.
65. A pharmaceutical composition comprising the lipid nanoparticle
of claim 1, and a pharmaceutically acceptable excipient.
66. The pharmaceutical composition of claim 65, wherein the
pharmaceutical composition is administered perorally,
intravenously, intraperitoneally, subcutaneously, or
transdermally.
67. The pharmaceutical composition of claim 65, wherein the
pharmaceutical composition is prepared as an orally administered
tablet, an inhalant, or a suppository.
68. The pharmaceutical composition of claim 65, wherein the
pharmaceutical composition is prepared as a sterile solution, a
sterile suspension, or a lyophilized powder.
69. A lipid nanoparticle comprising, an anti-miR-221 and SPLN-G20,
wherein SPLN-G20 comprises: DMHDA, DOTAP, GRAM, DOPE and TPGS at a
molar ratio of 40:5:20:30:5.
70. The lipid nanoparticle of claim 69, wherein the anti-miR-221
has the sequence:
5'-g.sub.sa.sub.saacccagcagacaaugu.sub.sa.sub.sg.sub.sc.sub.su--
Chol-3', SEQ ID NO. 1.
71. The lipid nanoparticle of claim 70, wherein the sequence
includes 2'-O-Methyl-modified oligonucleotides (lower case letters)
and phosphorothioate linkages (s subscript) to increase nuclease
stability of the oligonucleotides.
72. A composition comprising anti-miR-221 combined a lipid
nanoparticle.
73. The composition of claim 72, wherein the lipid nanoparticle
comprises SPLN-G20v1 and SPLN-G20v2, wherein SPLN-G20 version 1
(SPLN-G20v1) comprises DMHDA, DOTAP, GRAM, DOPE and TPGS at a molar
ratio of 40:5:20:30:5, and wherein SPLN-G20 (SPLN-G20v2) comprises
DODAP, DOTAP, GRAM, Soy PC (SPC) and TPGS at a molar ratio of
40:5:20:30:5.
74. The composition of claim 73, wherein the lipid nanoparticle
comprises SPLN-G20v1 and SPLN-G20v2 at an N:P of 15:1.
75. A method of treating a subject having, or suspected of having,
breast cancer, comprising administering an effective amount of the
lipid nanoparticle of claim 69.
76. A method of providing anti-oncogenic effect in a subject
suffered from breast cancer, in which miR-221 expression level is
higher in cancer cells of case subject relative to a control
subject, comprising administering to the subject an effective
amount of a composition of claim 69.
77. The method of claim 76, wherein the lipid nanoparticle
down-regulates miR-221 expression.
78. The method of claim 76, wherein the breast cancer comprises
triple negative breast cancer.
79. The method of claim 78, wherein the subject is a human.
80. A composition comprising anti-miR-155 combined with a lipid
nanoparticle.
81. The composition of claim 80, wherein the lipid nanoparticle
comprises Lac-GLN, wherein Lac-GLN comprises a lipophilic
asialoglycoprotein receptor (ASGR) targeting ligand composed of
lactobionic acid (LA), bearing a galactose moiety, and linked to a
phospholipid.
82. The composition of claim 80, further including gramicidin A
incorporated into the lipid nanoparticle.
83. The composition of claim 82, comprising DODAP, Lac-DOPE, DOPE,
DMG-PEG and gramicidin A at a molar ratio of 50:10:28:2:10.
84. The composition of claim 80, wherein the anti-miR-155 has the
sequence: 5'-A*C*CCCUAUCACGAUUAGCAUU*A*A-3', SEQ ID NO. 6.
85. The composition of claim 84, wherein the sequence contains
phosphorothioate linkages (*) and 2'-O-Methyl.
86. A method of treating a subject having, or suspected of having
liver cancer, comprising administering an effective amount of the
lipid nanoparticle of claim 80.
87. A method of providing anti-oncogenic effect in a subject
suffered from liver cancer, in which miR-155 expression level is
higher in cancer cells of case subject relative to a control
subject, comprising administering to the subject an effective
amount of the lipid nanoparticle of claim 80.
88. The method of claim 87, wherein the lipid nanoparticle
down-regulates C/EBP.beta. and FOXP3 genes.
89. The method of claim 86, wherein the subject is a human.
Description
RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. Ser.
No. 14/403,313 filed Nov. 24, 2014, now U.S. Pat. No. 9,750,819
issued Sep. 5, 207, which is a national stage application filed
under 35 USC .sctn.371 of international application
PCT/US2013/042458 filed May 23, 2013, which claims priority to U.S.
Provisional Application 61/650,729, filed May 23, 2012, and U.S.
Provisional Application 61/784,892, filed Mar. 14, 2013, the
disclosures of which are hereby incorporated by reference in their
entirety.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted via EFS-web and is hereby incorporated by
reference in its entirety. The ASCII copy, created on May 22, 2013,
is named 604_54848_SEQ_LIST_OSU-2013-246.txt, and is 3,490 bytes in
size.
TECHNICAL FIELD
[0004] The present disclosure pertains to lipid nanoparticles (LNs)
usable for the delivery of therapeutic compositions, including, but
not limited to nucleic acids (NAs).
BACKGROUND OF THE INVENTION
[0005] A liposome is a vesicle composed of one or more lipid
bilayers, capable of carrying hydrophilic molecules within an
aqueous core or hydrophobic molecules within its lipid bilayer(s).
As used herein, "Lipid nanoparticles" (LNs) is a general term to
described lipid-based particles in the submicron range. LNs can
have structural characteristics of liposomes and/or have
alternative non-bilayer types of structures. Drug delivery by LNs
via systemic route requires overcoming several physiological
barriers. The reticuloendothelial system (RES) is responsible for
clearance of LNs from the circulation. Once escaping the
vasculature and reaching the target cell, LNs are typically taken
up by endocytosis and must release the drug into the cytoplasm
prior to degradation within acidic endosome conditions.
[0006] In particular, the delivery of such nucleic acids (NAs),
including siRNA and other therapeutic oligonucleotides is a major
technical challenge that has limited their potential for clinical
translation.
[0007] The development of efficient delivery vehicles is a key to
clinical translation of oligonucleotide (ON) therapeutics. It is
desired that a LN formulation should be able to (1) protect the
drug from enzymatic degradation; (2) transverse the capillary
endothelium; (3) specifically reach the target cell type without
causing excessive immunoactivation or off-target cytotoxicity; (4)
promote endocytosis and endosomal release; and (5) form a stable
formulation with colloidal stability and long shelf-life.
SUMMARY OF THE INVENTION
[0008] Provided herein are lipid nanoparticles that can encapsulate
therapeutic oligonucleotides with high efficiency and fulfill
physical and biological criteria for efficacious delivery. In
certain embodiments, the lipid nanoparticles comprise a combination
of cationic lipids with tertiary and quaternary amine headgroups.
In certain embodiments, the lipid nanoparticles comprise small
peptides, such as gramicidin, in addition to lipids. In certain
embodiments, the lipid nanoparticles comprise an RNase- or
DNase-degrading agent, such as proteinease K. Combinations of these
embodiments are further provided. The incorporation of a
combination of quaternary and tertiary amine-cationic lipids
(QTsome), gramicidin (A, B, C, or D) (SPLN-G), and/or proteinase K
(PrKsome) increases the transfection efficiency of lipid
nanoparticle formulations.
[0009] In a first broad aspect, provided herein is a lipid
nanoparticle comprising a combination of tertiary and quaternary
amine-cationic lipids. In certain embodiments, the tertiary
amine-cationic lipids are chosen from DODAP, DODMA, DC-CHOL,
N,N-dimethylhexadecylamine, or combinations thereof. In certain
embodiments, the quaternary amine-cationic lipids are selected from
DOTAP, DOTMA, DDAB, or combinations thereof. In certain
embodiments, the concentration of the tertiary amine-cationic
lipids is below 50.0 molar percent of the total lipid content. In
certain embodiments, the concentration of quaternary amine-cationic
lipids is below 20.0 molar percent of the total lipid content. In
particular embodiments, the lipid nanoparticle comprises the lipids
DODMA and DOTMA in a molar ratio selected from 45:0, 5:40, 15:30,
22.5:22.5, 30:15, or 40:5. In certain embodiments, the lipid
nanoparticle comprises the lipids DMHDA and DOTAP in a molar ratio
selected from 90:10, 70:30, 50:50, 30:70, or 10:90.
[0010] In certain embodiments, the lipid nanoparticle encapsulates
molecules selected from nucleic acids, proteins, polysaccharides,
lipids, radioactive substances, therapeutic agents, prodrugs,
nutritional supplements, biomarkers, or combinations thereof. In
certain embodiments, the encapsulated molecules comprise a nucleic
acid selected from plasmid DNAs, antisense oligonucleotides, miRs,
anti-miRs, shRNAs, siRNAs, or combinations thereof. In certain
embodiments, the encapsulation rate of therapeutic agents or
nucleotides is 20% or higher.
[0011] In certain embodiments, the lipid nanoparticle further
comprises a cationic polymer. In particular embodiments, the
cationic polymer is selected from the group consisting of:
spermine, dispermine, trispermine, tetraspermine, oligospermine,
thermine, spermidine, dispermidine, trispermidine, oligospermidine,
putrescine, polylysine, polyarginine, a polyethylenimine of
branched or linear type, and polyallylamine.
[0012] In certain embodiments, the lipid nanoparticle further
comprises a fusogenic peptide.
[0013] In certain embodiments, the lipid nanoparticle has a
diameter under 300 nm.
[0014] In another broad aspect, there is provided herein a lipid
nanoparticle having a diameter of less than 300 nm and comprising a
peptide. In certain embodiments, the peptide is selected from
gramicidin A, B, C, D, or S; JTS-1; proteinase K (PrK);
trichorovin-Xlla; rabies virus glycoprotein; interleukin-1 .beta.;
HIV-Tat; herpes simplex virus VP22 protein; and combinations
thereof. In certain embodiments, the peptide comprises an
antibiotic. In particular embodiments, the antibiotic is selected
from gramicidin A, B, C, D, or S. In particular embodiments, the
peptide consists essentially of a lipidated JTS-1 fusogenic
peptide. In particular embodiments, the lapidated JTS-1 fusogenic
peptide is present at about 0 to about 30 molar percent of the
total formulation.
[0015] In certain embodiments, the lipid nanoparticle further
comprises proteinase K. Proteinase K can be present at from about 0
to about 30 molar percent of the total formulation.
[0016] In certain embodiments, the lipid nanoparticle encapsulates
molecules selected from nucleic acids, proteins, polysaccharides,
lipids, radioactive substances, therapeutic agents, prodrugs,
nutritional supplements, biomarkers, or combinations thereof. In
certain embodiments, the encapsulated molecules comprise a nucleic
acid selected from plasmid DNAs, antisense oligonucleotides, miRs,
anti-miRs, shRNAs, siRNAs, or combinations thereof.
[0017] In another broad aspect, provided herein is a lipid
nanoparticle comprising a DNase- or RNase-degrading agent. In
certain embodiments, the DNase- or RNase-degrading agent consists
essentially of proteinase K. In certain embodiments, the
nanoparticle encapsulates molecules selected from nucleic acids,
proteins, polysaccharides, lipids, radioactive substances,
therapeutic agents, prodrugs, nutritional supplements, biomarkers,
or combinations thereof. In particular embodiments, the
encapsulated molecules comprise an oligonucleotide selected from
pDNAs, antisense oligonucleotides, miRs, anti-miRs, shRNAs, siRNAs,
or combinations thereof. In certain embodiments, the lipid
nanoparticle has a diameter under 300 nm.
[0018] In another broad aspect, provided herein is a lipid
nanoparticle having a diameter of less than 300 nm and comprising a
combination of two or more of: a mixture of tertiary and quaternary
amine-cationic head groups; an antibiotic; and a DNase or
RNase-degrading agent. In certain embodiments, the RNase or
RNase-degrading agent consists essentially of proteinase K. In
certain embodiments, the antibiotic comprises gramicidin A, B, C,
D, or S.
[0019] Any of the lipid nanoparticle formulations described herein
may comprise a polyethyleneglycol-lipid conjugate. In certain
embodiments, the polyethyleneglycol-lipid conjugate selected from
polysorbate 80, TPGS, mPEG-DSPE, PEG-DMG. In certain embodiments,
the polyethyleneglycol-lipid is present at a concentration less
than about 10.0 molar percent. In certain embodiments, the lipid
nanoparticle further comprises N,N-dimethylhexadecylamine. In
particular embodiments, the N,N-dimethylhexadecylamine is present
at a concentration of less than about 60.0 molar percent of the
formulation.
[0020] In certain embodiments, the lipid nanoparticle further
comprises a ligand capable of binding to a target cell or a target
molecule. In certain embodiments, the ligand is an antibody or an
antibody fragment. In particular embodiments, the ligand is
selected from cRGD, galatose-containing moieties, transferrin,
folate, low density lipoprotein, or epidermal growth factors.
[0021] In another broad aspect, provided herein is a pharmaceutical
composition comprising a lipid nanoparticle as described herein and
a pharmaceutically acceptable excipient. In certain embodiments,
the pharmaceutical composition is administered perorally,
intravenously, intraperitoneally, subcutaneously, or transdermally.
In certain embodiments, the pharmaceutical composition is prepared
as an orally administered tablet, a sterile solution, a sterile
suspension, a lyophilized powder, or a suppository.
[0022] In another broad aspect, provided herein is a method of
diagnosing or treating a cancer or infectious disease. The method
comprises administering an effective amount of a pharmaceutical
composition as described herein to a patient in need thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The patent or application file may contain one or more
drawings executed in color and/or one or more photographs. Copies
of this patent or patent application publication with color
drawing(s) and/or photograph(s) will be provided by the Patent
Office upon request and payment of the necessary fee.
[0024] FIG. 1: Downregulation of luciferase expression in SK-HEP-1
cells. Cells expressing luciferase were treated with
luciferase-specific siRNA delivered by LNs comprising several
different combinations of tertiary amine (DODMA, DMA) and
quaternary amine (DOTMA, TMA), (QTsome). Lipofectamine 2000
(Lipo2000) is used as a positive control. Luciferase activity is
expressed as a percentage relative to untreated cells.
[0025] FIG. 2: Downregulation of Bcl-2 expression in KB cells by
G3139 in QTsome. QTsomes containing varying amounts of DMHDA were
evaluated. Lipo2000 is used as a positive control. Bcl-2 mRNA
expression relative to actin was determined by RT-PCR where
untreated KB cells served as a baseline for mRNA expression.
[0026] FIG. 3: Zeta potential of SPLN-G condensed with c-myb at a
N:P of 15:1.
[0027] FIG. 4: Viability of SK-HEP-1 cells treated with SPLN-G
under 0% and 20% serum conditions. Cell viabilities are expressed
as a percentage relative to the mean viability of the untreated
SK-HEP-1 cells.
[0028] FIG. 5: Downregulation of luciferase expression in SK-HEP-1
cells by luciferase-siRNA delivered using SPLN-G. Lipofectamine
2000 (Lipo2000) is used as a positive control. Luciferase activity
is expressed as a percentage relative to untreated cells.
[0029] FIG. 6: Downregulation of Bcl-2 expression in MCF-7 cells
using SPLN-G loaded with G3139, an ASO against Bcl-2. Lipo2000 is
used as a positive control Bcl-2 mRNA expression relative to actin
was determined by RT-PCR where untreated MCF-7 cells served as a
baseline for mRNA expression.
[0030] FIG. 7: Downregulation of Bcl-2 expression in KB cells using
SPLN-G loaded with G 3139, an ASO against Bcl-2. Lipo2000 is used
as a positive control. Bcl-2 mRNA expression relative to actin was
determined by RT-PCR where untreated KB cells served as a baseline
for mRNA expression.
[0031] FIG. 8: Effect of lactosylation on SPLN-G size and zeta
potential. These are targeting liver and liver cancer cells via the
asialoglycoprotein receptor (ASGR).
[0032] FIG. 9: Evaluation of asialoglycoprotein receptor (ASGR)
targeting and gramicidin in SPLN-G by luciferase assay in
luciferase-expressing SK-HEP-1 cells. Luciferase activity is
expressed as a percentage relative to untreated cells.
[0033] FIG. 10: Cellular trafficking of SPLN-G via confocal
microscopy. DAPI (blue) was used to stain the nuclei of cells.
siRNA-Cy3 (red) was used to track siRNA for internalization. The
overlay shows that a high percentage of siRNA is delivered to the
cytosol.
[0034] FIG. 11: Cellular uptake of non-targeted SPLN-G and
lactosylated SPLN-G (Lac-SPLN-G) encapsulating Cy3-siRNA via flow
cytometry.
[0035] FIG. 12: Downregulation of miR-155 in SK-HEP-1 cells by
Lac-SPLN-G-anti-miR-155. Scrambled control miRNA (SC) was used as a
negative control. miR-155 expression relative to RNU6B was
determined by RT-PCR where untreated SK-HEP-1 cells served as a
baseline for mRNA expression.
[0036] FIG. 13: Gel mobility shift analysis of Lac-SPLN-G-siRNA
complexes at varying lipid-to-siRNA (w/w) ratios.
[0037] FIG. 14: Downregulation of RNR R2 expression in KB cells
using PrKsome carrying LOR-1284, an siRNA targeting ribonucleotide
reductase RNR R2 subunit (purchased from Dharmacon). RNR R2 mRNA
expression relative to actin was determined by qRT-PCR where
untreated KB cells served as a baseline for mRNA expression.
[0038] FIGS. 15A-15C: Downregulation of RNR R2 expression levels
with PrKsome in varying serum conditions. FIG. 15A shows
serum-free; FIG. 15B shows 5% FBS. FIG. 15C shows 10% FBS. RNR R2
mRNA expression relative to actin was determined by real-time
RT-PCR where untreated KB cells served as a baseline for mRNA
expression.
[0039] FIG. 16: Comparative cell viability study of lipid
nanoparticle with and without PrK. Cell viabilities are expressed
as a percentage relative to the mean viability of the untreated KB
cells.
[0040] FIG. 17: Stability study of LOR-1284 siRNA in blood plasma.
PrKsome formulations were incubated in mouse plasma over a period
of 72 hours and the relative amount of siRNA remaining was
visualized by gel electrophoresis.
[0041] FIG. 18: Temperature dependent zeta potential of PrKsome
containing 1:0.3 siRNA: PrK (w/w).
[0042] FIG. 19: Temperature-dependent efficacy of RNR R2
downregulation by LOR-1284 siRNA delivered by PrKsome in 10% FBS
medium. RNR R2 mRNA expression relative to actin was determined by
real-time RT-PCR where untreated KB cells served as a baseline for
mRNA expression.
[0043] FIG. 20: QTsome mechanism of action.
[0044] FIG. 21: Depiction of SPLN-Gs.
[0045] FIG. 22: The proteinase K coating protects oligonucleotides
from DNase and RNase present in serum.
[0046] FIG. 23: Diagrams of SPLN-J with the JTS-1 fusogenic peptide
and a LN with proteinase K coating.
[0047] FIG. 24: Upregulation of p27/kip1 mRNA by SPLN-G20 loaded
with anti-miR-221 in MDA-MB-468 breast cancer cells. This shows
that SPLN-G20 is an effective vehicle for delivery of anti-miRs.
P27/Kip1 are targets of miR-221. This upregulation indicates
inhibition of miR-221 function.
[0048] FIG. 25: SPLN-G20 transfection in p27/Kip1 of BT-549
cells.
[0049] FIG. 26: SPLN-G20 transfection in ER.alpha. of BT-549
cells.
[0050] FIG. 27: FTIR spectra of Lac-DOPE (blue), DOPE (red), and
lactobionic acid (green).
[0051] FIGS. 28A-28B: Characterization of Lac-GLN: FIG. 28A shows
particle size and zeta potential of GLN with varying degrees of
lactosylation. Each value represented the mean.+-.SD of five
measurements. FIG. 28B shows Morphology of anti-miR-155-Lac-GLN by
TEM. Scale bar represents 100 nm.
[0052] FIGS. 29A-29B: Characterization of Lac-GLN: FIG. 29A)
Colloidal stability of Lac-GLB. Lac-GLN-anti-miR-155 was stored at
4.degree. C. or 25.degree. C. and particle size was measured over
time. Results are the mean of three separate experiments. Error
bars stand for standard deviation. FIG. 29B) Serum stability of
anti-miR-155-Lac-GLN. Anti-miR-155 alone or anti-miR-155-Lac-GLN
were mixed with 50% FBS at 37.degree. C. for 0 hr, 4 hr, and 12 hr.
Samples were then analyzed with gel electrophoresis.
[0053] FIG. 30: Physicochemical properties of anti-miR-155
containing GLN and Lac-GLN. Value are mean.+-.SD (n=5).
[0054] FIG. 31: Cellular uptake of Cy3-anti-miR-155 containing
Lac-GLN and other control formulations in HepG2 cells as determined
by confocal microscopy.
[0055] FIGS. 32A-32C: HepG2 cells were treated with GLN, Lac-GLN,
or Lac-GLN pre-incubated with 20 mM lactose and 1% BSA.
Fluorescence signals were measured on a FACSCalibur flow cytometer.
FIG. 32A shows HepG2 cells were treated with GLN or Lac-GLN. FIG.
32B and FIG. 32C show the effect of pre-incubation with 20 mM
lactose and 1% BSA on Lac-GLN, respectively. Results are shown in
the histogram with the X- and Y-axis indicating the fluorescence
intensity and the cell count, respectively.
[0056] FIGS. 33A-33C: In vitro delivery of Lac-GLN and other
formulations. FIG. 33A) In vitro delivery of Luci-siRNA containing
Lac-GLN and other control formulations. SK-HEP-1-cells were
transfected with luci-siRNA containing Lac-GLN at the concentration
of 100 nM for 4 hr, and luciferase gene expression was evaluated 48
hr post transfection. The results are the mean of four repeats.
Error bar stand for standard deviations. FIG. 33B) Cytotoxicity of
Lac-GLN from MTS analysis. Results represent the mean.+-.SD. FIG.
33C) hi vitro delivery of anti-miR-155 containing Lac-GLN and
control formulations. HepG2 cells were transfected with
anti-miR-155 containing Lac-GLN at the concentration of 100 nM for
4 hr, and miR-155 expression was evaluated 48 hr after
transfection. The results are the mean of three repeats. Error bars
stand for standard deviations.
[0057] FIGS. 34A-24B: In vitro evaluation of different
concentrations of anti-miR-155 treatments on miR-155 and target
gene expression. FIG. 34A shows evaluation of different
concentrations of anti-miR-155 treatments on miR-155 expression.
HepG2 cells were transfected with 100 nM or 200 nM anti-miR-155
containing Lipofectamine2000 and Lac-GLN for 4 hr, and miR-155
expression was evaluated 48 hr after transfection. Values represent
the mean.+-.SD (n=3). FIG. 34B shows evaluation of miR-155
targeting gene expressions. C/EBP.beta. and FOXP3 gene expression
were evaluated 48 hr after HepG2 cells were transfected with
positive control or Lac-GLN containing 100 nM and 200 nM
anti-miR-155. The results are the mean of three repeats. Error bars
stand for standard deviations.
[0058] FIGS. 35A-35B: Tissue distribution of Cy5-anti-miR-155
containing GLN and Lac-GLN. Heart, lung, spleen, kidney, and liver
were harvested from C57BL/6 mice 4 hr after intravenous
administration of Cy5-anti-miR-155 containing GLN or Lac-GLN. Cy5
fluorescence signals were measured by IVIS Imaging System.
[0059] FIGS. 36A and 36B: Confocal microscopy of Cy3-anti-miR-155
containing GLN and Lac-GLN in liver and other organs. Liver, lung,
and spleen were harvested from C57Bl/6 mice after 4 hr intravenous
administration of Cy3-anti-miR-155 containing GLN or Lac-GLN. Cye3
fluorescence signals were visualized on an Olympus FV1000 Filter
Confocal Microscope. In FIG. 36A, the red and yellow arrows
indicate the uptake of Cy4-anti-miR-155 by hepatocytes and Kupffer
cells, respectively.
[0060] FIGS. 37A-37C: In vivo evaluation of anti-miR-155 treatments
on miR-155 and target gene expressions. C57BL/6 mice were treated
with 1.5 mg/kg anti-miR-155 containing Lac-GLN and control
formulations. 4 hr after intravenous administration, liver tissues
were harvested and RNA was extracted. Each value represents the
mean.+-.SD of three measures. FIG. 37A shows the expression of
miR-155 was analyzed by real time RT-PCR. FIGS. 37B and 37C show
the expression of the mir-155 downstream targets, C/EBP.beta. and
FOXP3, were analyzed by real time RT-PCR. Each value represents the
mean.+-.SD of three measurements.
DETAILED DESCRIPTION OF THE INVENTION
[0061] Various embodiments are described herein in the context of
lipid nanoparticles. Those of ordinary skill in the art will
realize that the following detailed description of the embodiments
is illustrative only and not intended to be in any way limiting.
Other embodiments will readily suggest themselves to such skilled
persons having the benefit of this disclosure. Reference to an
"embodiment," "aspect," or "example" herein indicate that the
embodiments of the invention so described may include a particular
feature, structure, or characteristic, but not every embodiment
necessarily includes the particular feature, structure, or
characteristic. Further, repeated use of the phrase "in one
embodiment" does not necessarily refer to the same embodiment,
although it may.
[0062] Not all of the routine features of the implementations or
processes described herein are shown and described. It will, of
course, be appreciated that in the development of any such actual
implementation, numerous implementation-specific decisions will be
made in order to achieve the developer's specific goals, such as
compliance with application- and business-related constraints, and
that these specific goals will vary from one implementation to
another and from one developer to another. Moreover, it will be
appreciated that such a development effort might be complex and
time-consuming, but would nevertheless be a routine undertaking for
those of ordinary skill in the art having the benefit of this
disclosure.
GENERAL DESCRIPTION
[0063] Nucleic acid (NA)-based therapies are being developed to
promote or inhibit gene expression. As mutations in genes and
changes in miRNA profile are believed to be the underlying cause of
cancer and other diseases, NA-based agents can directly act upon
the underlying etiology, maximizing therapeutic potential.
Non-limiting examples of NA-based therapies include: plasmid DNA
(pDNA), small interfering RNA (siRNA), small hairpin RNA (shRNA),
microRNA (miR), mimic (mimetic), anti-miR/antagomiR/miR inhibitor,
and antisense oligonucleotide (ASO). Until the development of the
nanoparticle compositions described herein, the clinical
translation of NA-based therapies faced several obstacles in their
implementation since transporting NAs to their intracellular target
was particularly challenging and since NAs are relatively unstable
and subject to degradation by serum and cellular nucleases.
Further, the high negative charges of NAs made it impossible for
transport across the cell membrane, further limiting utility.
[0064] The LNs described herein provide a useful platform for the
delivery of both traditional therapeutic compounds and NA-based
therapies. Drugs formulated using LNs provide desirable
pharmacokinetic (PK) properties in vivo, such as increased blood
circulation time and increased accumulation at the site of solid
tumors due to enhanced permeability and retention (EPR) effect.
Moreover, in certain embodiments, the LNs may be surface-coated
with polyethylene glycol to reduce opsonization of LNs by serum
proteins and the resulting RES-mediated uptake, and/or coated with
cell-specific ligands to provide targeted drug delivery.
[0065] It is desired that the zeta potential of LNs not be
excessively positive or negative for systemic delivery. LNs with a
highly positive charge tend to interact non-specifically with
non-target cells, tissues, and circulating plasma proteins, and may
cause cytotoxicity. Alternatively, LNs with a highly negative
charge cannot effectively incorporate NAs, which are themselves
negatively charged, and may trigger rapid RES-mediated clearance,
reducing therapeutic efficacy. LNs with a neutral to moderate
charge are best suited for in vivo drug and gene delivery.
[0066] Provided herein are lipid nanoparticles (LNs) with improved
transfection activity. The lipid nanoparticles may either partition
hydrophobic molecules within the lipid membrane or encapsulate
water-soluble particles or molecules within the aqueous core. In
certain embodiments, the LN formulations comprise a mixture of
lipids, generally including a charged lipid and a neutral lipid,
and optionally further including a PEGylating lipid and/or
cholesterol. The LN formulations of the present disclosure may
combinations of quaternary and tertiary amines, peptides such as
gramicidin (A, B, C, D, or S), or RNase- or DNase-degrading agents
such as proteinase K. In certain embodiments, the lipid
nanoparticles are produced by combining cationic lipids with
quaternary amine headgroups and cationic lipids with tertiary amine
headgroups. In certain embodiment, the lipid nanoparticles are
small peptidic lipid nanoparticles (SPLN) and comprise a peptide
such as gramicidin or JTS1. In certain embodiments, the lipid
nanoparticles are coated with proteinase K, which enhances
transfection in the presence of serum. For ease of reference, the
SPLNs comprising gramicin are referred to herein as SPLN-Gs, the
SPLNs comprising JTS-1 peptide are referred to herein as SPLN-J,
and the lipid nanoparticles comprising proteinase K are referred to
herein as PrKsomes. Combinations of these different embodiments are
further provided. The LNs have a diameter of less than 300 nm, or
in particular embodiments between about 50 and about 200 nm. These
LNs show enhanced transfection and reduced cytotoxicity, especially
under high serum conditions found during systemic administration.
The LNs are applicable to a wide range of current therapeutic
agents and systems, serum stability, and targeted delivery, with
high transfection efficiency.
[0067] The term "lipid nanoparticle" as used herein refers to any
vesicles formed by one or more lipid components. The LN
formulations described herein may include cationic lipids. Cationic
lipids are lipids that carry a net positive charge at any
physiological pH. The positive charge is used for association with
negatively charged therapeutics such as ASOs via electrostatic
interaction.
[0068] Suitable cationic lipids include, but are not limited to:
3.beta.-[N--(N',N'-dimethylaminoethane)-carbamoyl]cholesterol
hydrochloride (DC-Chol); 1,2-dioleoyl-3-trimethylammonium-propane
(DOTAP); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP);
dimethyldioctadecylammonium bromide salt (DDAB);
1,2-dilauroyl-sn-glycero-3-ethylphosphocholine chloride (DL-EPC);
N-[1-(2, 3-dioleyloyx) propyl]-N--N--N-trimethyl ammonium chloride
(DOTMA); N-[1-(2, 3-dioleyloyx) propyl]-N--N--N-dimethyl ammonium
chloride (DODMA); N,N-dioctadecyl-N,N-dimethylammonium chloride
(DODAC);
N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethyl-
ammonium trifluoracetate (DOSPA);
1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl ammonium bromide
(DMRIE); dioctadecylamidoglycylspermine (DOGS); neutral lipids
conjugated to cationic modifying groups; and combinations thereof.
In addition, a number of cationic lipids in available preparations
could be used, such as LIPOFECTIN.RTM. (from GIBCO/BRL),
LIPOFECTAMINE.RTM. (from GIBCO/MRL), siPORT NEOFX.RTM. (from
Applied Biosystems), TRANSFECTAM.RTM. (from Promega), and
TRANSFECTIN.RTM. (from Bio-Rad Laboratories, Inc.). The skilled
practitioner will recognize that many more cationic lipids are
suitable for inclusion in the LN formulations. The cationic lipids
of the present disclosure may be present at concentrations ranging
from about 0 to about 80.0 molar percent of the lipids in the
formulation, or from about 5.0 to about 50.0 molar percent of the
formulation.
[0069] In certain embodiments, the LN formulations presently
disclosed may also include anionic lipids. Anionic lipids are
lipids that carry a net negative charge at physiological pH. These
anionic lipids, when combined with cationic lipids, are useful to
reduce the overall surface charge of LNs and introduce pH-dependent
disruption of the LN bilayer structure, facilitating nucleotide
release by inducing nonlamellar phases at acidic pH or induce
fusion with the cellular membrane.
[0070] Examples of suitable anionic lipids include, but are not
limited to: fatty acids such as oleic, linoleic, and linolenic
acids; cholesteryl hemisuccinate;
1,2-di-O-tetradecyl-sn-glycero-3-phospho-(1'-rac-glycerol) (Diether
PG); 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium
salt); 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (sodium salt);
1-hexadecanoyl,2-(9Z,12Z)-octadecadienoyl-sn-glycero-3-phosphate;
1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG);
dioleoylphosphatidic acid (DOPA); and
1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS); anionic
modifying groups conjugated to neutral lipids; and combinations
thereof. The anionic lipids of the present disclosure are present
at concentrations up to about 60.0 molar percent of the
formulation, or from about 5.0 to about 25.0 molar percent of the
formulation.
[0071] In certain embodiments, charged LNs are advantageous for
transfection, but off-target effects such as cytotoxicity and
RES-mediated uptake may occur. Hydrophilic molecules such as
polyethylene glycol (PEG) may be conjugated to a lipid anchor and
included in the LNs described herein to discourage LN aggregation
or interaction with membranes. Hydrophilic polymers may be
covalently bonded to lipid components or conjugated using
crosslinking agents to functional groups such as amines.
[0072] Suitable conjugates of hydrophilic polymers include, but are
not limited to: polyvinyl alcohol (PVA); polysorbate 80;
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG2000
(DSPE-PEG2000); D-alpha-tocopheryl polyethylene glycol 1000
succinate (TPGS); dimyristoylphosphatidylethanolamine-PEG2000
(DMPE-PEG2000); and dip almitoylphosphatidlyethanolamine-PEG2000
(DPPE-PEG2000). The hydrophilic polymer may be present at
concentrations ranging from about 0 to about 15.0 molar percent of
the formulation, or from about 5.0 to about 10.0 molar percent of
the formulation. The molecular weight of the PEG used is between
about 100 and about 10,000 Da, or from about 100 to about 2,000
Da.
[0073] The LNs described herein may further comprise neutral and/or
amphipathic lipids as helper lipids. These lipids are used to
stabilize the formulation, reduce elimination in vivo, or increase
transfection efficiency. The LNs may be formulated in a solution of
saccharides such as, but not limited to, glucose, sorbitol,
sucrose, maltose, trehalose, lactose, cellubiose, raffinose,
maltotriose, dextran, or combinations thereof, to promote
lyostability and cryostability.
[0074] Neutral lipids have zero net charge at physiological pH. One
or a combination of several neutral lipids may be included in any
LN formulation disclosed herein.
[0075] Suitable neutral lipids include, but are not limited to:
phosphatidylcholine (PC), phosphatidylethanolamine, ceramide,
cerebrosides, sphingomyelin, cephalin, cholesterol,
diacylglycerols, glycosylated diacylglycerols, prenols, lysosomal
PLA2 substrates, N-acylglycines, and combinations thereof.
[0076] Other suitable lipids include, but are not limited to:
phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine,
phosphatidylglycerol, phosphatidylcholine, and
lysophosphatidylethanolamine; sterols such as cholesterol,
demosterol, sitosterol, zymosterol, diosgenin, lanostenol,
stigmasterol, lathosterol, and dehydroepiandrosterone; and
sphingolipids such as sphingosines, ceramides, sphingomyelin,
gangliosides, glycosphingolipids, phosphosphingolipids,
phytoshingosine; and combinations thereof.
[0077] The LN formulations described herein may further comprise
fusogenic lipids or fusogenic coatings to promote membrane fusion.
Examples of suitable fusogenic lipids include, but are not limited
to, glyceryl mono-oleate, oleic acid, palmitoleic acid,
phosphatidic acid, phosphoinositol 4,5-bisphosphate (PIP.sub.2),
and combinations thereof.
[0078] The LN formulations described herein may further comprise
cationic lipids. The headgroups of such lipids may be primary,
secondary, tertiary, or quaternary amines in nature. In certain
embodiments, the LNs comprise a mixture of tertiary and quaternary
amines.
[0079] Suitable tertiary aminolipids include, but are not limited
to: DODAP; DODMA; N,N-dimethylhexadecylamine (DMHDA); and DC-CHOL.
Suitable quaternary aminolipids include, but are not limited to:
DOTAP, DOTMA, and DDAB. Combinations of multiple aminolipids,
particularly of tertiary and quaternary cationic lipids, are
beneficial towards LN delivery of therapeutic agents. Cationic
lipids may be present in concentrations up to about 60 molar
percent combined.
[0080] The LN formulations described here may further comprise
cationic polymers or conjugates of cationic polymers. Cationic
polymers or conjugates thereof may be used alone or in combination
with lipid nanocarriers.
[0081] Suitable cationic polymers include, but are not limited to:
polyethylenimine (PEI); pentaethylenehexamine (PEHA); spermine;
spermidine; poly(L-lysine); poly(amido amine) (PAMAM) dendrimers;
polypropyleneiminie dendrimers; poly(2-dimethylamino
ethyl)-methacrylate (pDMAEMA); chitosan; tris(2-aminoethyl)amine
and its methylated derivatives; and combinations thereof. Chain
length and branching are important considerations for the
implementation of polymeric delivery systems. High molecular weight
polymers such as PEI (MW 25,000) are used as transfection agents,
but suffer from cytotoxicity. Low molecular weight PEI (MW 600)
does not cause cytotoxicity, but is limited due to its inability to
facilitate stable condensation with nucleic acids.
[0082] Anionic polymers may be incorporated into the LN
formulations presently disclosed as well. Suitable anionic polymers
include, but are not limited to: poly(propylacrylic acid) (PPAA);
poly(glutamic acid) (PGA); alginates; dextrans; xanthans;
derivatized polymers; and combinations thereof.
[0083] In certain embodiments, the LN formulation includes
conjugates of polymers. The conjugates may be crosslinked to
targeting agents, lipophilic moieties, peptides, proteins, or other
molecules that increase the overall therapeutic efficacy.
[0084] Suitable crosslinking agents include, but are not limited
to: N-succinimidyl 3-[2-pyridyldithio]-propionate (SPDP); dimethyl
3,3'-dithiobispropionimidate (DTBP); dicyclohexylcarbodiimide
(DCC); diisopropyl carbodiimide (DIC);
1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC);
N-hydroxysulfosuccinimide (Sulfo-NHS); N'--N'-carbonyldiimidazole
(CDI); N-ethyl-5-phenylisoxazolium-3'sulfonate (Woodward's reagent
K); and combinations thereof.
[0085] The LN formulations may further comprise peptides and/or
proteins. Peptides and proteins, especially those derived from
bacteria and viruses or used as antibiotic agents, may aid in
membrane permeation. The peptides or proteins may be directly mixed
with lipids, covalently attached, or conjugated to lipid moieties
with crosslinking agents.
[0086] Suitable peptides and proteins include, but are not limited
to: gramicidin A, B, C, D, and S; HA2; JTS-1; proteinase K (PrK);
trichorovin-Xlla (TV-Xlla); rabies virus glycoprotein (RVG);
interleukin-1 .beta.; HIV-Tat; herpes simplex virus (HSV) VP22
protein; and combinations thereof. In certain embodiments, JTS-1
and/or gramicidin is used at about 0 to about 40 molar percent. In
certain embodiments, PrK at a concentration of about 0 to about 30
molar percent is applied by direct mixing with oligonucleotide or
conjugation to hexadecyl isothiocyanate for LN surface coating of
PrK.
[0087] The addition of targeting agents to the LN provides
increased efficacy over passive targeting approaches. Targeting
involves incorporation of specific targeting moieties such as, but
not limited to, ligands or antibodies against cell surface
receptors, peptides, lipoproteins, glycoproteins, hormones,
vitamins, antibodies, antibody fragments, prodrugs, and conjugates
or combinations of these moieties.
[0088] In certain embodiments, maximization of targeting efficiency
includes the surface coating of the LN with the appropriate
targeting moiety rather than encapsulation of the targeting agent.
This method optimizes interaction with cell surface receptors.
[0089] It is to be understood that targeting agents may be either
directly incorporated into the LN during synthesis or added in a
subsequent step. Functional groups on the targeting moiety as well
as specifications of the therapeutic application (e.g., degradable
linkage) dictate the appropriate means of incorporation into the
LN. Targeting moieties that do not have lipophilic regions cannot
insert into the lipid bilayer of the LN directly and require prior
conjugation to lipids before insertion or must form an
electrostatic complex with the LNs.
[0090] Also, under certain circumstances, a targeting ligand cannot
directly bind to a lipophilic anchor. In these circumstances, a
molecular bridge in the form of a crosslinking agent may be
utilized to facilitate the interaction. In certain embodiments, it
is advantageous to use a crosslinking agent if steric restrictions
of the anchored targeting moiety prevent sufficient interaction
with the intended physiological target. Additionally, if the
targeting moiety is only functional under certain orientations
(e.g., monoclonal antibody), linking to a lipid anchor via
crosslinking agent is beneficial. Traditional methods of
bioconjugation may be used to link targeting agents to LNs.
Reducible or hydrolysable linkages may be applied to prevent
accumulation of the formulation in vivo and subsequent
cytotoxicity.
[0091] Various methods of LN preparation are suitable to synthesize
the LNs of the present disclosure. For example, ethanol dilution,
freeze-thaw, thin film hydration, sonication, extrusion, high
pressure homogenization, detergent dialysis, microfluidization,
tangential flow diafiltration, sterile filtration, and/or
lyophilization may be utilized. Additionally, several methods may
be employed to decrease the size of the LNs. For example,
homogenization may be conducted on any devices suitable for lipid
homogenization such as an Avestin Emulsiflex C5.RTM. device.
Extrusion may be conducted on a Lipex Biomembrane extruder using a
polycarbonate membrane of appropriate pore size (0.05 to 0.2
.mu.m). Multiple particle size reduction cycles may be conducted to
minimize size variation within the sample. The resultant LNs may
then be passed through a size exclusion column such as Sepharose
CL4B or processed by tangential flow diafiltration to purify the
LNs.
[0092] Any embodiment of the LNs described herein may further
include ethanol in the preparation process. The incorporation of
about 30-50% ethanol in LN formulations destabilizes the lipid
bilayer and promotes electrostatic interactions among charged
moieties such as cationic lipids with anionic ASO and siRNA. LNs
prepared in high ethanol solution are diluted before
administration. Alternatively, ethanol may be removed by dialysis,
or diafiltration, which also removes non-encapsulated NA.
[0093] In certain embodiment, it is desirable that the LNs be
sterilized. This may be achieved by passing of the LNs through a
0.2 or 0.22 .mu.m sterile filter with or without
pre-filtration.
[0094] Physical characterization of the LNs can be carried through
many methods. Dynamic light scattering (DLS) or atomic force
microscopy (AFM) can be used to determine the average diameter and
its standard deviation. In certain embodiments, it is especially
desirable that the LNs have about a 200 nm diameter. Zeta potential
measurement via zeta potentiometer is useful in determining the
relative stability of particles. Both dynamic light scattering
analysis and zeta potential analysis may be conducted with diluted
samples in deionized water or appropriate buffer solution.
Cryogenic transmission electron microscopy (Cryo-TEM) and scanning
electron microscopy (SEM) may be used to determine the detailed
morphology of LNs.
[0095] The LNs described herein are stable under refrigeration for
several months. LNs requiring extended periods of time between
synthesis and administration may be lyophilized using standard
procedures. A cryoprotectant such as 10% sucrose may be added to
the LN suspension prior to freezing to maintain the integrity of
the formulation. Freeze drying loaded LN formulations is
recommended for long term stability.
[0096] Quaternary and Tertiary Amine-Cationic Lipids (QTsome)
[0097] While the physical characteristics of LNs promote enhanced
permeation and retention (EPR) in the fenestrated tumor
vasculature, endosomal escape remains a challenge for conventional
LN formulations. To this end, lipid nanoparticles comprising
positively charged quaternary or tertiary amine-based cationic
lipids for the complexation of nucleic acids have been developed.
Quaternary amine-based cationic lipids carry a permanent positive
charge and are capable of forming stable electrostatic complexes
with nucleic acids. Tertiary amine-cationic lipids, however, are
conditionally ioniziable and their positive charge is largely
regulated by pH. Provided herein are LNs comprising a combination
of quaternary and tertiary amine-cationic lipids (QTsomes), which
provides a mechanism by which therapeutic agents may be released
from LNs within the endosome. QTsomes are conditionally ionizable
and facilitate disruption of the lipid bilayer and oligonucleotide
endosomal release under the acidic conditions of the endosome.
Quaternary amino-catinoic lipids are permanently charged, ensuring
strong interaction between the lipids and the oligonucleotide,
thereby ensuring stability. The combination of tertiary and
quaternary cationic lipids provides an optimum pH response profile
that is not possible with each class of lipid individually. QTsomes
are more active than regular cationic liposomes in transfecting
cells.
[0098] QTsomes demonstrate greater transfection activity than
standard cationic lipid formulations. Fine tuning the balance
between quaternary and tertiary amine-cationic lipids allows for
the precise controlled release of nucleic acids into the cytosol.
In a particular embodiment, the use of particular release
parameters provides a technique whereby the activity of nucleic
acid-based therapeutics can be maximized. For example, it is noted
that tertiary amine-cationic lipids have pH-dependent ionization
profiles when used alone. Since a single lipid species may not
provide a desired level of control of LN charge characteristics, a
combination of a tertiary and a quaternary amine-cationic lipid can
be used, thus resulting in significantly improved activity of such
combinations in siRNA delivery.
[0099] FIG. 1 depicts the relative luciferase expression of
combinations of tertiary and quaternary cationic lipids. At a
Q-to-T amine-cationic lipid ratio of 5:40, over 85% downregulation
is demonstrated for luciferase siRNA transfection in HCC cells
expressing luciferase. FIG. 20 depicts the QTsome mechanism of
action. Under pH 7.4, quaternary amine-cationic lipids (QA-CLs)
maintain "+" charge to provide stability. Under pH 5.5, both QA-CLs
and tertiary amine-cationic lipids (TA-CLs) are charged, which
promotes endosome membrane interaction/disruption.
[0100] Small Peptidic Lipid Nanoparticles (SPLN)
[0101] Further provided herein are LNs comprising that are small
peptidic lipid nanoparticles (SPLNs). In certain embodiments, the
SPLNs comprise the antibiotic gramicidin. Described herein are
certain variants of gramicidin (A, B, C, D) that have not
previously been investigated as transfection agents. Though these
gramicidin subtypes share a conserved sequence of peptides with
gramicidine S, gramicidin A, B, C, and D form a beta-helix
structure while gramicidin forms a cyclic structure. Therefore,
gramicidins A-D are different from gramicidin S. Gramicidins
dimerize and form an ionophore and promote membrane fusion, which
promotes destabilization of the lipid bilayers of the endosome and
the LN. Consequently, SPLNs comprising gramicidin are ideal in
nucleic acid-based therapies. Incorporation of gramicidin A, B, C,
D, or S into LNs significantly increases the cellular transfection
efficiency of ASO and siRNA.
[0102] In certain embodiments, SPLNs utilizing gramicidin are
designated SPLN-G, followed by a number corresponding to the molar
percentage of gramicidin in the formulation. FIG. 21 depicts
SPLN-Gs.
[0103] Combining gramicidin A, B, C, D, or S into lipid
nanoparticle formulations increases the transfection efficiency of
ASO and siRNA formulations in the presence of serum. The gramicidin
assists permeabilization of endosome membrane bilayers to ODNs. In
contrast, transfection agents such as Lipofectamine.TM. 2000 show
markedly diminished transfection activity in the presence of serum.
SPLN-G with luciferase siRNA demonstrates low cytotoxicity and
greater transfection activity than the transfection agent
Lipofectamine 2000 (LF) in the presence of serum in HCC cells, as
shown in FIG. 5. Lipid nanoparticle formulations that do not show
reduction in transfection activity in the presence of serum are
advantageous as serum conditions best simulate those in the actual
patient, thus facilitating a better translation into clinical
study.
[0104] Further provided herein are SPLNs incorporating lapidated
JTS-1 fusogenic peptide. These SPLN-J particles show high
transfection activity, and have a high membrane fusion activity
that is triggered by pH-dependent conformational change of JTS-1
from a coil to a helix.
[0105] Proteinase K (PrK) (PrKsome)
[0106] Degradation of nucleotides in serum after administration is
a perpetual concern for nucleic acid-based therapies, even those
involving lipid or polymer carriers. Often, alterations to the
nucleotide, such as backbone and base pair modifications, are
conducted to better protect the nucleotide against degradation.
However, these modifications may result in reduced or off-target
activity of the drug. In order to overcome this problem, provided
herein are lipid nanoparticle formulations comprising a DNase- or
RNase-degrading agent. In particular embodiments, the DNase- or
RNase-degrading agent is proteinase K (PrK). The proteinase K
coating protects oligonucleotides from DNase and RNase present in
serum. This is depicted by FIG. 23. Proteinase K is able to protect
siRNA better than lipid nanoparticles without proteinase K.
Inclusion of proteinase K increases transfection efficiency in the
presence of serum without significant cytotoxicity in KB cells.
PrKsomes are highly versatile and applicable to both natural and
chemically modified oligonucleotides.
[0107] Proteinase K coatings can be incorporated into any
embodiment of LNs described here. By way of non-limiting example,
FIG. 24 depicts a SPLN-J with a proteinase K coating.
[0108] Applications
[0109] Depending on the application, the lipid nanoparticles
disclosed herein may be designed to favor characteristics such as
increased interaction with nucleic acids, increased serum
stability, lower RES-mediated uptake, targeted delivery, or pH
sensitive release within the endosome. Because of the varied nature
of LN formulations, any one of the several methods provided herein
may be applied to achieve a particular therapeutic aim. Cationic
lipids, anionic lipids, PEG-lipids, neutral lipids, fusogenic
lipids, aminolipids, cationic polymers, anionic polymers, polymer
conjugates, peptides, targeting moieties, and combinations thereof
may be applied to meet specific aims.
[0110] The lipid nanoparticles described herein can be used as
platforms for therapeutic delivery of oligonucleotide (ON)
therapeutics, such as siRNA, shRNA, miRNA, anti-miR, and antisense
ODN. These therapeutics are useful to manage a wide variety of
diseases such as various types of cancers, leukemias, viral
infections, and other diseases. For instance, targeting moieties
such as cyclic-RGD, folate, transferrin, or antibodies greatly
enhance activity by enabling targeted drug delivery. A number of
tumors overexpress receptors on their cell surface. Non-limiting
examples of suitable targeting moieties include transferrin (Tf),
folate, low density lipoprotein (LDL), and epidermal growth
factors. In addition, tumor vascular endothelium markers such as
alpha-v-beta-3 integrin and prostate-specific membrane antigen
(PSMA) are valuable as targets for targeted LNs. In certain
embodiments, LN formulations having particles measuring about 300
nm or less in diameter with a zeta potential of less than 50 mV and
an encapsulation efficiency of greater than 20.0% are useful for NA
delivery.
[0111] Implementation of embodiments of the LN formulations
described herein alone or in combination with one another
synergizes with current paradigms of lipid nanoparticle design.
[0112] A wide spectrum of therapeutic agents may be used in
conjunction with the LNs described herein. Non-limiting examples of
such therapeutic agents include antineoplastic agents,
anti-infective agents, local anesthetics, anti-allergics,
antianemics, angiogenesis, inhibitors, beta-adrenergic blockers,
calcium channel antagonists, anti-hypertensive agents,
anti-depressants, anti-convulsants, anti-bacterial, anti-fungal,
anti-viral, anti-rheumatics, anthelminithics, antiparasitic agents,
corticosteroids, hormones, hormone antagonists, immunomodulators,
neurotransmitter antagonists, anti-diabetic agents,
anti-epileptics, anti-hemmorhagics, anti-hypertonics, antiglaucoma
agents, immunomodulatory cytokines, sedatives, chemokines,
vitamins, toxins, narcotics, imaging agents, and combinations
thereof.
[0113] Nucleic acid-based therapeutic agents are highly applicable
to the LN formulations of the present disclosure. Examples of such
nucleic acid-based therapeutic agents include, but are not limited
to: pDNA, siRNA, miRNA, anti-miRNA, ASO, and combinations thereof.
To protect from serum nucleases and to stabilize the therapeutic
agent, modifications to the substituent nucleic acids and/or
phosphodiester linker can be made. Such modifications include, but
are not limited to: backbone modifications (e.g., phosphothioate
linkages); 2' modifications (e.g., 2'-O-methyl substituted bases);
zwitterionic modifications (6'-aminohexy modified ODNs); the
addition of a lipophilic moiety (e.g., fatty acids, cholesterol, or
cholesterol derivatives); and combinations thereof. The modified
sequences synergize with the LN formulations disclosed herein. For
example, addition of a 3'-cholesterol to an ODN supplies stability
to a LN complex by adding lipophilic interaction in a system
otherwise solely held together by electrostatic interaction. In
addition, this lipophilic addition promotes cell permeation by
localizing the ODN to the outer leaflet of the cell membrane.
Applying a peptide such as gramicidin or JTS-1 further promotes
cell permeation of the formulation due to its fusogenic properties.
Alternatively, addition of an enzyme such as proteinase K could
further aid the ODN in resisting degradation.
[0114] Depending on the therapeutic application, the LNs described
herein may be administered by the following methods: peroral,
parenteral, intravenous, intramuscular, subcutaneous,
intraperitoneal, transdermal, intratumoral, intraarterial,
systemic, or convection-enhanced delivery. In particular
embodiments, the LNs are delivered intravenously, intramuscularly,
subcutaneously, or intratumorally. Subsequent dosing with different
or similar LNs may occur using alternative routes of
administration.
[0115] Pharmaceutical compositions of the present disclosure
comprise an effective amount of a lipid nanoparticle formulation
disclosed herein, and/or additional agents, dissolved or dispersed
in a pharmaceutically acceptable carrier. The phrases
"pharmaceutical" or "pharmacologically acceptable" refers to
molecular entities and compositions that produce no adverse,
allergic or other untoward reaction when administered to an animal,
such as, for example, a human. The preparation of a pharmaceutical
composition that contains at least one compound or additional
active ingredient will be known to those of skill in the art in
light of the present disclosure, as exemplified by Remington's
Pharmaceutical Sciences, 2003, incorporated herein by reference.
Moreover, for animal (e.g., human) administration, it will be
understood that preparations should meet sterility, pyrogenicity,
general safety and purity standards as required by FDA Office of
Biological Standards.
[0116] A composition disclosed herein may comprise different types
of carriers depending on whether it is to be administered in solid,
liquid or aerosol form, and whether it need to be sterile for such
routes of administration as injection. Compositions disclosed
herein can be administered intravenously, intradermally,
transdermally, intrathecally, intraarterially, intraperitoneally,
intranasally, intravaginally, intrarectally, topically,
intramuscularly, subcutaneously, mucosally, in utero, orally,
topically, locally, via inhalation (e.g., aerosol inhalation), by
injection, by infusion, by continuous infusion, by localized
perfusion bathing target cells directly, via a catheter, via a
lavage, in cremes, in lipid compositions (e.g., liposomes), or by
other method or any combination of the forgoing as would be known
to one of ordinary skill in the art (see, for example, Remington's
Pharmaceutical Sciences, 2003, incorporated herein by
reference).
[0117] The actual dosage amount of a composition disclosed herein
administered to an animal or human patient can be determined by
physical and physiological factors such as body weight or surface
area, severity of condition, the type of disease being treated,
previous or concurrent therapeutic interventions, idiopathy of the
patient and on the route of administration. Depending upon the
dosage and the route of administration, the number of
administrations of a preferred dosage and/or an effective amount
may vary according to the response of the subject. The practitioner
responsible for administration will, in any event, determine the
concentration of active ingredient(s) in a composition and
appropriate dose(s) for the individual subject.
[0118] In certain embodiments, pharmaceutical compositions may
comprise, for example, at least about 0.1% of an active compound.
In other embodiments, an active compound may comprise between about
2% to about 75% of the weight of the unit, or between about 25% to
about 60%, for example, and any range derivable therein. Naturally,
the amount of active compound(s) in each therapeutically useful
composition may be prepared is such a way that a suitable dosage
will be obtained in any given unit dose of the compound. Factors
such as solubility, bioavailability, biological half-life, route of
administration, product shelf life, as well as other
pharmacological considerations will be contemplated by one skilled
in the art of preparing such pharmaceutical formulations, and as
such, a variety of dosages and treatment regimens may be
desirable.
[0119] In other non-limiting examples, a dose may also comprise
from about 1 microgram/kg/body weight, about 5 microgram/kg/body
weight, about 10 microgram/kg/body weight, about 50
microgram/kg/body weight, about 100 microgram/kg/body weight, about
200 microgram/kg/body weight, about 350 microgram/kg/body weight,
about 500 microgram/kg/body weight, about 1 milligram/kg/body
weight, about 5 milligram/kg/body weight, about 10
milligram/kg/body weight, about 50 milligram/kg/body weight, about
100 milligram/kg/body weight, about 200 milligram/kg/body weight,
about 350 milligram/kg/body weight, about 500 milligram/kg/body
weight, to about 1000 mg/kg/body weight or more per administration,
and any range derivable therein. In non-limiting examples of a
derivable range from the numbers listed herein, a range of about 5
mg/kg/body weight to about 100 mg/kg/body weight, about 5
microgram/kg/body weight to about 500 milligram/kg/body weight,
etc., can be administered, based on the numbers described
above.
[0120] In certain embodiments, a composition herein and/or
additional agents is formulated to be administered via an
alimentary route Alimentary routes include all possible routes of
administration in which the composition is in direct contact with
the alimentary tract. Specifically, the pharmaceutical compositions
disclosed herein may be administered orally, buccally, rectally, or
sublingually. As such, these compositions may be formulated with an
inert diluent or with an assimilable edible carrier.
[0121] In further embodiments, a composition described herein may
be administered via a parenteral route. As used herein, the term
"parenteral" includes routes that bypass the alimentary tract.
Specifically, the pharmaceutical compositions disclosed herein may
be administered, for example but not limited to, intravenously,
intradermally, intramuscularly, intraarterially, intrathecally,
subcutaneous, or intraperitoneally (U.S. Pat. Nos. 6,753,514,
6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363 are each
specifically incorporated herein by reference in their
entirety).
[0122] Solutions of the compositions disclosed herein as free bases
or pharmacologically acceptable salts may be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions may also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms. The
pharmaceutical forms suitable for injectable use include sterile
aqueous solutions or dispersions and sterile powders for the
extemporaneous preparation of sterile injectable solutions or
dispersions (U.S. Pat. No. 5,466,468, specifically incorporated
herein by reference in its entirety). In all cases the form must be
sterile and must be fluid to the extent that easy injectability
exists. It must be stable under the conditions of manufacture and
storage and must be preserved against the contaminating action of
microorganisms, such as bacteria and fungi. The carrier can be a
solvent or dispersion medium containing, for example, water,
ethanol, polyol (i.e., glycerol, propylene glycol, and liquid
polyethylene glycol, and the like), suitable mixtures thereof,
and/or vegetable oils. Proper fluidity may be maintained, for
example, by the use of a coating, such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. The prevention of the action of
microorganisms can be brought about by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars or
sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the use in the compositions of
agents delaying absorption such as, for example, aluminum
monostearate or gelatin.
[0123] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous, and
intraperitoneal administration. In this connection, sterile aqueous
media that can be employed will be known to those of skill in the
art in light of the present disclosure. For example, one dosage may
be dissolved in 1 ml of isotonic NaCl solution and either added to
1000 ml of hypodermoclysis fluid or injected at the proposed site
of infusion, (see for example, "Remington's Pharmaceutical
Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some
variation in dosage will necessarily occur depending on the
condition of the subject being treated. The person responsible for
administration will, in any event, determine the appropriate dose
for the individual subject. Moreover, for human administration,
preparations should meet sterility, pyrogenicity, general safety,
and purity standards as required by FDA Office of Biologics
standards.
[0124] Sterile injectable solutions are prepared by incorporating
the compositions in the required amount in the appropriate solvent
with various of the other ingredients enumerated above, as
required, followed by filter sterilization. Generally, dispersions
are prepared by incorporating the various sterilized compositions
into a sterile vehicle which contains the basic dispersion medium
and the required other ingredients from those enumerated above. In
the case of sterile powders for the preparation of sterile
injectable solutions, some methods of preparation are vacuum-drying
and freeze-drying techniques which yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof. A powdered composition is
combined with a liquid carrier such as, e.g., water or a saline
solution, with or without a stabilizing agent.
[0125] In other embodiments, the compositions may be formulated for
administration via various miscellaneous routes, for example,
topical (i.e., transdermal) administration, mucosal administration
(intranasal, vaginal, etc.) and/or via inhalation.
[0126] Pharmaceutical compositions for topical administration may
include the compositions formulated for a medicated application
such as an ointment, paste, cream or powder. Ointments include all
oleaginous, adsorption, emulsion and water-soluble based
compositions for topical application, while creams and lotions are
those compositions that include an emulsion base only. Topically
administered medications may contain a penetration enhancer to
facilitate adsorption of the active ingredients through the skin.
Suitable penetration enhancers include glycerin, alcohols, alkyl
methyl sulfoxides, pyrrolidones and luarocapram. Possible bases for
compositions for topical application include polyethylene glycol,
lanolin, cold cream and petrolatum as well as any other suitable
absorption, emulsion or water-soluble ointment base. Topical
preparations may also include emulsifiers, gelling agents, and
antimicrobial preservatives as necessary to preserve the
composition and provide for a homogenous mixture. Transdermal
administration of the compositions may also comprise the use of a
"patch." For example, the patch may supply one or more compositions
at a predetermined rate and in a continuous manner over a fixed
period of time.
[0127] In certain embodiments, the compositions may be delivered by
eye drops, intranasal sprays, inhalation, and/or other aerosol
delivery vehicles. Methods for delivering compositions directly to
the lungs via nasal aerosol sprays has been described in U.S. Pat.
Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein
by reference in their entirety). Likewise, the delivery of drugs
using intranasal microparticle resins (Takenaga et al., 1998) and
lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,
specifically incorporated herein by reference in its entirety) are
also well-known in the pharmaceutical arts and could be employed to
deliver the compositions described herein. Likewise, transmucosal
drug delivery in the form of a polytetrafluoroetheylene support
matrix is described in U.S. Pat. No. 5,780,045 (specifically
incorporated herein by reference in its entirety), and could be
employed to deliver the compositions described herein.
[0128] It is further envisioned the compositions disclosed herein
may be delivered via an aerosol. The term aerosol refers to a
colloidal system of finely divided solid or liquid particles
dispersed in a liquefied or pressurized gas propellant. The typical
aerosol for inhalation consists of a suspension of active
ingredients in liquid propellant or a mixture of liquid propellant
and a suitable solvent. Suitable propellants include hydrocarbons
and hydrocarbon ethers. Suitable containers will vary according to
the pressure requirements of the propellant. Administration of the
aerosol will vary according to subject's age, weight and the
severity and response of the symptoms.
EXAMPLES
[0129] Certain embodiments of the present invention are defined in
the Examples herein. It should be understood that these Examples,
while indicating preferred embodiments of the invention, are given
by way of illustration only. From the above discussion and these
Examples, one skilled in the art can ascertain the essential
characteristics of this invention, and without departing from the
spirit and scope thereof, can make various changes and
modifications of the invention to adapt it to various usages and
conditions.
Example 1
[0130] Lipid stock solutions were created by dissolving lipids in
100% ethanol. All lipids were obtained from Avanti Polar Lipids
(USA) or Sigma Aldrich (USA) and used without further purification.
Lipids (egg phosphatidylcholine:cholesterol:TPGS, 15:35:5) were
combined with varying concentrations of tertiary (DODMA) and
quaternary (DOTMA) amine (45:0, 5:40, 15:30, 22.5:22.5, 30:15,
40:5, 45:0; DODMA:DOTMA) in 1.0 mL vials. Additional ethanol was
added to reach a volume of 180 .mu.L. This was then combined with
420 .mu.L 10 mM citric acid buffer to reach a final concentration
of 30% ethanol. The formulations were combined with SILENCER
Firefly Luciferase (GL2+GL3) siRNA (Invitrogen) at an amine to
phosphate (N:P) ratio of 15:1. Formulations were allowed to
incubate for 15 minutes prior to dilution with serum-free DMEM
(GIBCO) to a total volume of 300 .mu.L. Lipofectamine 2000
(Invitrogen) was used as a positive control and combined with the
same amount of siRNA at the same N:P and diluted to the same total
volume.
[0131] SK-HEP-1 (hepatocellular carcinoma) cells expressing
luciferase, grown in DMEM medium at 37.degree. C. under 5% CO.sub.2
atmosphere, were plated 24 h prior to transfection at a density of
2.times.10.sup.4 cells per well in a 96-well plate. Cells were
grown to approximately 80% confluency and the serum containing
media was removed. Cells were transfected with 70 .mu.L
transfection media and treated for 4 h. Experiments were performed
with four replicates. After treatment was completed, cells were
washed with 1.times.PBS and serum-containing DMEM was restored. 48
h after treatment was completed, cells were analyzed for luciferase
expression by a Luciferase Assay Kit (Promega) per the
manufacturer's instructions. The results are shown in FIG. 1. The
most efficacious transfection activity was exhibited by the
formulation containing 5% DOTMA and 40% DODMA, showing over 85%
knockdown in luciferase expression.
Example 2
[0132] Lipid stock solutions were created by dissolving lipids in
100% ethanol. All lipids were obtained from Avanti Polar Lipids
(USA) or Sigma Aldrich (USA) and used without further purification.
Lipids (egg phosphatidylcholine:cholesterol:TPGS, 15:35:5) were
combined with varying concentrations of tertiary (DMHDA) and
quaternary (DOTMA) amine (90:10, 70:30, 50:50, 30:70, 10:90;
DMHDA:DOTMA) in 1.0 mL vials. Additional ethanol was added to reach
a volume of 180 .mu.L. This was then combined with 420 .mu.L 10 mM
citric acid buffer to reach a final concentration of 30% ethanol.
The formulations were combined with G3139 (Genasense) ODN at an
amine to phosphate ratio (N:P) of 15:1. Formulations were allowed
to incubate for 15 min prior to dilution with serum-free RPMI 1640
(Mediatech) to a total volume of 300 .mu.L. Lipofectamine 2000
(Invitrogen) was used as a positive control and combined with the
same amount of ODN at the same N:P and diluted to the same total
volume.
[0133] KB (a subline of HeLa) cells, grown in RPMI 1640 medium at
37.degree. C. under 5% CO.sub.2 atmosphere, were plated 24 h prior
to transfection at a density of 2.times.10.sup.4 cells per well in
a 96-well plate. Cells were grown to approximately 80% confluency
and the serum containing media was removed. Cells were transfected
with 70 .mu.L transfection media and treated for 4 h. Experiments
were performed with four replicates. After treatment was completed,
cells were washed with 1.times.PBS and serum-containing RPMI 1640
was restored. 48 h after treatment was completed, cells were
analyzed for Bcl-2 downregulation. Real-time polymerase chain
reaction (RT-PCR) was used to assess the downregulation of Bcl-2
relative to actin. As shown in FIG. 2, transfection with the
formulation containing DMHDA/DOTAP (90/10 molar ratio) results in
greater than 75% downregulation of Bcl-2 expression compared to
untransfected cells. Lipofectamine.TM., DMHDA alone, and DOTAP
alone served as controls. Formulations of DMHDA/DOTAP at
alternative ratios showed lower rates of transfection
efficiency.
Example 3
[0134] Various SPLN-G formulations were analyzed for transfection
efficacy: SPLN-G50 (DMHDA, DOTAP, GRAM, TPGS at a molar ratio of
40:5:50:5), SPLN-G35 (DMHDA, DOTAP, GRAM, DOPE, TPGS at a molar
ratio of 40:5:35:15:5), SPLN-G30 (DMHDA, DOTAP, GRAM, DOPE, TPGS at
a molar ratio of 40:5:30:20:5), SPLN-G20 (DMHDA, DOTAP, GRAM, DOPE,
TPGS at a molar ratio of 40:5:20:30:5), SPLN-G10 (DMHDA, DOTAP,
GRAM, DOPE, TPGSS at a molar ratio of 40:5:10:40:5), and SPLN-GO
(DMHDA, DOTAP, DOPE, TPGS at a molar ratio of 40:5:50:5). All
lipids were purchased from Avanti Polar Lipids (USA) or Sigma
Aldrich (USA) and used without further purification. Lipids and
peptides were dissolved in ethanol and combined at the appropriate
ratios to form LNs. Additional ethanol and citric acid buffer was
added to attain a final ethanol content of 30%. The 2.0 mg/mL LN
solution was combined with siRNA or ODN at an N:P of 15:1 and
allowed to react at room temperature for 15 min before further
dilution. Particle sizes of the formulation ranged between 100-300
nm. Zeta potential measurements (FIG. 3) of the formulation diluted
with deionized water and combined with c-myb ODN demonstrated zeta
potentials ranging between 5-15 mV for gramicidin-containing
formulations. This is in contrast to the positive control,
Lipofectamine 2000, which exhibited a zeta potential above 35
mV.
[0135] SK-HEP-1 cells, cultured in DMEM media (GIBCO) supplemented
with 10% FBS and 1% streptomycin/penicillin at 37.degree. C. under
5% CO.sub.2 atmosphere were grown to confluency and plated at a
density of 2.times.10.sup.4 cells per well in a 96-well plate.
Firefly Luciferase (GL2+GL3) siRNA was combined with the
formulations at N:P 15:1. Formulations were allowed to combine with
lipid formulations for 15 min at room temperature prior to dilution
with DMEM. Transfection efficiency was tested in both serum-free
and 20% serum conditions. Culture medium was removed and replaced
with 70 .mu.L transfection medium per well. Cells were treated for
4 h before washing three times with 1.times.PBS. 48 h after
treatment, cell viability (FIG. 4) and luciferase expression (FIG.
5) were analyzed by MTS assay and Luciferase Assay Kit,
respectively. Formulations containing 35% or less gramicidin
exhibited lower cytotoxicity and greater transfection efficiency
than Lipofectamine 2000 under high serum transfection
conditions.
[0136] Bcl-2 downregulation via ODN G3139 was investigated in MCF-7
(ER positive breast cancer) cells. Transfection occurred in the
presence of 20% serum RT-PCR (FIG. 6) was used to assess the
downregulation of Bcl-2 relative to actin. SLN-G20 exhibited
significant downregulation of Bcl-2 relative to Lipofectamine 2000.
This was repeated in KB cells with replicates (n=3), as seen in
FIG. 7.
Example 4
[0137] Lactosylated DOPE (L-DOPE) was formed by crosslinking
lactobionic acid with DOPE using EDC/NHS (1:5:10:5,
DOPE:LA:EDC:NHS). Lipids and peptide
(DODAP:DOTAP:L-DOPE:DMG-PEG:Gramicidin at a molar ratio of
45:5:5:10:28:2:10) were combined in 1.times.PBS. Other control
formulations were completed with L-DOPE (substituted with DOPE)
and/or gramicidin. The particle size (FIG. 8) of the formulated LNs
fell between 50 and 150 nm. The formulation displayed colloidal
stability over a 30 day period. Zeta potential (FIG. 8) of the LNs
ranged between -10 and 10 mV. Investigation of ODN loading
efficiency revealed over 75% condensation.
[0138] Luciferase assay was used to determine targeting efficiency
of LLNs in SK-HEP-1 cells expressing luciferase. SK-HEP-1 cells,
cultured in DMEM media (GIBCO) supplemented with 10% FBS and 1%
streptomycin/penicillin at 37.degree. C. under 5% CO.sub.2
atmosphere were grown to confluency and plated at a density of
2.times.10.sup.4 cells per well in a 96-well plate. Firefly
Luciferase (GL2+GL3) siRNA was combined with the formulations at
N:P 10:1. Formulations were allowed to combine with lipid
formulations for 10 m at room temperature prior to dilution with
DMEM. Transfection efficiency was tested in serum-free, 10%, and
20% serum conditions. Culture medium was removed and replaced with
70 .mu.L transfection medium per well (siRNA 100 nM). Cells were
treated for 4 h before washing three times with 1.times.PBS. 48 h
after treatment, luciferase expression (FIG. 9) was analyzed by a
luciferase assay kit. Formulations containing both gramicidin as
well as ASGR showed greater transfection efficiency than either LN
modification alone.
[0139] Uptake of LLNs was analyzed by fluorescent microscopy (FIG.
10) and flow cytometry (FIG. 11). DAPI nuclear was applied to
hepatocellular carcinoma (HCC) cells cy3-labeled oligonucleotides
were added to indicate the cellular uptake. As demonstrated by the
fluorescent images, ASGR targeting is necessary for efficacious
delivery of siRNA and ODN to the cytosol of cells. Flow cytometry
data showed an approximate 3.3 fold difference between targeted
LLNs and non-targeted LLNs, further substantiating the advantages
of targeted delivery. Downregulation of miR-155 by LN-antagomir
formulations was assessed by RT-PCR (FIG. 12). About 60%
downregulation of miR-155 was achieved relative to RNU6B. 100 nM
anti-miR-155 was used.
Example 5
[0140] Lipids (DDAB, CHOL, Tween 80 at a molar ratio of 60:35:5)
were dissolved in 100% ethanol. 100 .mu.L of this solution was
diluted in 900 .mu.L 1.times.PBS. LNs at various w/w ratios were
combined with LOR-1284 (siRNA purchased from Dharmacon) (0.1 .mu.g)
for gel mobility shift analysis (FIG. 13). Retardation occurred at
1:8 (siRNA:LN). LNs were combined with 0.1 .mu.M siRNA for
downregulation studies. Downregulation of RNR R2 by LN-siRNA
LOR-1281 formulations was assessed by RT-PCR using actin as a
control and w/w ratios 1:20 and 1:30 (siRNA:LN). KB cells, grown in
RPMI 1640 medium at 37.degree. C. under 5% CO.sub.2 atmosphere,
were plated 24 h prior to transfection at a density of
3.0.times.10.sup.5 cells per well in a 6-well plate. Cells were
grown to approximately 80% confluency and the serum containing
media was removed. Cells were transfected with 1000 .mu.L
transfection media and treated for 4 h. Transfection occurred in
the presence of 10% serum-containing RPMI 1640 media. Experiments
were performed with 3 replicates. After treatment was completed,
cells were washed with 1.times.PBS and serum-containing RPMI 1640
was restored. 48 h after treatment was completed, cells were
analyzed for RNR R2 expression levels by RT-PCR with actin as a
housekeeping gene. Results are located in FIG. 14. Significant
downregulation occurred for the 1:30 siRNA:LN formulation.
[0141] The LNs (1:30, siRNA:LN) of the previous step were combined
with various amounts of PrK pre-mixed with LOR-1284 for 15 min in
room temperature. KB (mouth carcinoma) cells, grown in RPMI 1640
medium at 37.degree. C. under 5% CO.sub.2 atmosphere, were plated
were plated 24 h prior to transfection at a density of
3.0.times.10.sup.5 cells per well in a 6-well plate. Cells were
grown to approximately 80% confluency and the serum containing
media was removed. Cells were transfected with 1000 .mu.L
transfection media and treated for 4 h. Transfection occurred in
the presence of 0%, 5%, and 10% serum-containing RPMI 1640 media.
Experiments were performed with 3 replicates. After treatment was
completed, cells were washed with 1.times.PBS and serum-containing
RPMI 1640 was restored. 48 h after treatment was completed, cells
were analyzed for RNR R2 expression levels by RT-PCR with actin as
a housekeeping gene. Results are shown in FIG. 15. For
serum-containing media, the 0.3:1:LN-PrK:siRNA LOR-1281 formulation
showed significant downregulation compared to the formulation
without PrK. Cell viability studies were also carried out under the
same transfection parameters with 10% serum-containing media.
Neither the LNs nor the PrK-LNs showed significant toxicity at the
treated levels (FIG. 16). The protective effect of PrK-siRNA
complexes in serum was investigated by incubating LN-siRNA and
PrK-LN-siRNA complexes in fresh mouse plasma (FIG. 17). Inclusion
of PrK in the formulation showed significant protective activity
over the non-protected formulation over a three day period.
[0142] Further study investigated the temperature effect of
PrK-LNs. PrK, LOR-1284, and LNs were combined at a weight ratio of
0.3:1:30 with vortexing and were maintained at temperatures of
4.degree. C., 18.degree. C., 37.degree. C., and 55.degree. C. Zeta
potentials (FIG. 18) of the formed complexes were measured.
Complexes mixed at 18.degree. C. and 37.degree. C. had a much
higher zeta potential than those mixed at 4.degree. C. and
55.degree. C. The complexes formed at the various temperatures were
tested for in vitro activity. KB cells, grown in RPMI 1640 medium
at 37.degree. C. under 5% CO.sub.2 atmosphere, were plated 24 h
prior to transfection at a density of 3.times.10.sup.5 cells per
well in a 96-well plate. Cells were grown to approximately 80%
confluency and the serum containing media was removed. Cells were
transfected with 1000 .mu.L transfection media and treated for 4 h.
Transfection occurred in the presence of 10% serum-containing RPMI
1640 media. Experiments were performed with 2 replicates. After
treatment was completed, cells were washed with 1.times.PBS and
serum-containing RPMI 1640 was restored. 48 h after treatment was
completed, cells were analyzed for RNR R2 expression levels by
RT-PCR with actin as a housekeeping gene. Results are shown in FIG.
19. Formulations complexed at higher temperatures (37.degree. C.
and 55.degree. C.) displayed a small increase in transfection
efficiency relative to formulations formed at lower
temperatures.
Example 6
[0143] SPLN-G20 for anti-miR delivery into MDA-MB-468 cells was
studied. SPLN-G20 were prepared as described above. MDA-MB-468
(triple negative breast cancer) cells were plated 24 h prior to
transfection in a 6-well plate at a density of 2.times.10.sup.4
cells/cm.sup.2 in DMEM/F12 media supplemented with 1%
penicillin/streptomycin and 10% FBS. SPLN-G20 was combined with
anti-miR-221 to gauge its ability to upregulate the downstream
target of miR-221, p27/Kip1, a tumor suppressor. The sequence of
anti-miR-221 was as follows:
5'-g.sub.sa.sub.saacccagcagacaaugu.sub.sa.sub.sg.sub.sc.sub.su-Chol-3'
[SEQ ID NO. 1]. This sequence included 2'-O-Methyl-modified
oligonucleotides (lower case letters) and phosphorothioate linkages
(s subscript) to aid in nuclease stability of antisense
oligonucleotides. Furthermore, the addition of a hydrophobic moiety
(cholesterol (Chol)) to the 3' end was added to better facilitate
association with the lipid nanoparticle formulation. MDA-MB-468
cells were transfected using SPLN-G20 with 50, 100, and 250 nM
anti-miR-221 in the presence of 20% serum. Treatment was allowed to
proceed for 4 h at which time the transfection medium was removed
and replaced with fresh media (supplemented with 10% FBS). Cells
were allowed to proliferate for an additional 44 h before the start
of RT-PCR. RNA from cells was extracted by TRIzol Reagent (Life
Technologies) and cDNA was generated by SuperScript.RTM. III
First-Strang Synthesis System (Life Technologies) per the
manufacturer's instructions. RT-PCR was then performed using SYBR
green (Life Technologies) and primers for p27/kip1 (Alpha DNA)
[0144] forward: 5'CGTGCGAGTGTCTAACGG-3' [SEQ ID NO. 2],
[0145] reverse: 5'-CGGATCAGTCTTTGGGTC-3' [SEQ ID NO. 3]).
[0146] .beta.-actin (forward: 5'-CGTCTTCCCCTCCATCG-3' [SEQ ID NO.
4],
[0147] reverse: 5'-CTCGTTAATGTCAC GCAC-3') [SEQ ID NO. 5] was used
as a control. As seen in FIG. 24, the mRNA was upregulated several
fold in a dose dependent manner through the treatment of
SPLN-G/anti-miR-221.
Example 7
[0148] SPLN-G20 version 1 (SPLN-G20v1) composed of DMHDA, DOTAP,
GRAM, DOPE, TPGS at a molar ratio of 40:5:20:30:5 was prepared as
described previously. A second version of SPLN-G20 (SPLN-G20v2) was
also generated, replacing DMHDA and DOPE with DODAP and Soy PC
(SPC), respectively. DODAP is also a tertiary amine and is better
characterized than DMHDA in transfection. In this embodiment, the
choice to replace the helper lipid with SPC was made because DOPE
based formulations generally show reduced activity in vivo due to
interaction with serum proteins. The final composition was set at a
molar ratio of 40:5:20:30:5 (DODAP:DOTAP:GRAM:SPC:TPGS). All lipids
and peptide were purchased from Avanti Polar Lipids (USA) or
Sigma-Aldrich (USA) and used without further purification. Lipids
and peptides were dissolved in ethanol and combined at the
appropriate ratio. Additional ethanol and citric acid buffer, pH
4.0, was added to reach a final ethanol content of 30%. At this
point, the 2.0 mg/mL lipid nanoparticle solution was combined with
anti-miR at an N:P of 15:1, bath-sonicated for 5 min, and allowed
to form electrostatic complexes at room temperature for 15 min
before further dilution. Particle size of the formulation ranged
between 100-200 nm.
[0149] Anti-miR-221 was combined with SPLN-G20v1 and SPLN-G20v2 at
an N:P of 15:1. BT-549, a triple negative breast cancer cell line,
was tested for upregulation of miR-221's downstream targets
p27/kip1 (a gene involved in apoptosis regulation) and estrogen
receptor alpha (ER.alpha., a gene responsible for expression of the
estrogen receptor and thus sensitization to hormone based therapy).
Cells were plated in 6-well plates 24 h prior to transfection at a
density of 2.times.10.sup.4 cells/cm.sup.2 in DMEM/F12 cell culture
media supplemented with 5% FBS and 1% penicillin/streptomycin (Life
Technologies). At the time of transfection, culture medium was
replaced with 20% serum containing media. 20% serum was used to
simulate the high serum conditions in vivo. Cells were transfected
with appropriate controls or 250 nM anti-miR-221 loaded SPLN-G
based formulations for 4 h at 37.degree. C. At the end of the
treatment period, cells were washed twice with 1.times.PBS. Cells
were allowed to proliferate for 44 h before the start of RT-PCR.
RNA from cells was extracted by TRIzol Reagent (Life Technologies)
and cDNA was generated by SuperScript.RTM. III First-Strand
Synthesis System (Life Technologies) per the manufacturer's
instructions. RT-PCR was then performed using SYBR green (Life
Technologies) and primers for p27/kip1 and estrogen receptor
.alpha.. .beta.-actin was used as a reference gene. As demonstrated
in FIG. 25, free anti-miR-221 displayed minimal activity while
SPLN-G20v1 and SPLN-G20v2 demonstrated .about.1.75 and
.about.2.5-fold upregulation in p27/Kip1 respectively. Similar
results were observed for ER.alpha. expression, as seen in FIG. 26.
SPLN-G20v1 upregulated ER.alpha. by nearly 3-fold while SPLN-G20v2
upregulated ER.alpha. by over 5-fold. These data show that
SPLN-G20v2 is an especially useful nanocarrier for in vivo anti-miR
delivery.
Example 8
[0150] A lipophilic asialoglycoprotein receptor (ASGR) targeting
ligand composed of lactobionic acid (LA), bearing a galactose
moiety, and linked to a phospholipid, was synthesized and
incorporated into a LN for liver-specific delivery of anti-miR-155.
Gramicidin A was also incorporated into the LN to facilitate
endosomal release of the anti-miR. This formulation is referred to
herein as lactosylated gramicidin-based LN (Lac-GLN). The
hepatocyte targeting was evaluated in HepG2 cells and in mice. The
physiochemical properties, cellular uptake, in vitro and in vivo
delivery efficacy were investigated.
[0151] 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), and
L-.alpha.-dioleoyl phosphatidylethanolamine (DOPE) were purchased
from Avanti Polar Lipids (Alabaster, Ala.); 1,
2-dimyristoyl-sn-glycerol and methoxypolyethylene glycol (DMG-PEG)
were purchased from NOF America Corporation (Elysian, Minn.);
1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) were from Thermo Scientific
(Rockford, Ill.). Monomethoxy polyethylene glycol 2000-distearoyl
phosphatidylethanolamine (mPEG-DSPE) was obtained from Genzyme
Pharmaceuticals (Cambridge, Mass.). Cholesterol, lactobionic acid,
gramicidin A and all other reagents were purchased from
Sigma-Aldrich (St. Louis, Mo.) without further purification.
Firefly Luciferase (GL2+GL3) siRNA (Luci-siRNA) (AM 4629), negative
scrambled control (AM 17010), and Lipofectamine 2000 were purchased
from Invitrogen (Grand Island, N.Y.).
[0152] Anti-miR-155 (sequence: 5'-A*C*CCCUAUCACGAUUAGCAUU*A*A-3'
(SEQ ID NO. 6), containing phosphorothioate linkages (*) and
2'-O-Methyl), Cy3 labeled anti-miR-155 (Cy3-anti-miR-155), and
Cy5.5 labeled anti-miR-155 (Cy5.5-anti-miR-155) were synthesized by
Alpha DNA (Montreal, Canada). The Taqman kits for real-time RT-PCR
assay of miR-155 (002623) and RNU6B (001093) were purchased from
Applied Biosystems (Carlsbad, Calif.).
[0153] Lactobionic acid was activated by EDC and converted to its
NHS ester, which was then reacted with DOPE to yield
n-lactobionyl-DOPE (Lac-DOPE). The product was characterized by
Fourier transform infrared (FTIR) spectrometry on a Nexus 470 FTIR
Spectrometer (Thermo Scientific, Rockford, Ill.). Lac-GLNs were
prepared by the ethanol injection method. The lipid mixture,
composed of DODAP, Lac-DOPE, DOPE, DMG-PEG and gramicidin A at a
molar ratio of 50:10:28:2:10, was dissolved in ethanol, and rapidly
injected into RNAse- and DNAse-free HEPES buffered solution (20 mM,
pH 7.4). The resulting lipid nanoparticles were sonicated for 2 min
by a bath sonicator and dialyzed against RNAse- and DNAse-free
water for 4 hr at room temperature to remove ethanol using a
molecular weight cut-off (MWCO) 10,000 Dalton Float-A-Lyzer
(Spectrum Laboratories Inc., Ranco Dominguz, Calif.).
[0154] The anti-miR-155 containing Lac-GLN was prepared by adding
an equal volume of anti-miR-155 dissolved in RNAse- and DNAse-free
HEPES buffer to Lac-GLN, followed by brief vortexing for 10 sec and
incubation at room temperature for 10 min. The weight ratio of
lipids:anti-miR was fixed at 10:1, and the concentration of
anti-miR-155 was 1 mg/kg. The resulting nanoparticles were
sterilized using 0.22 .mu.m filters (Fisher Scientific, Pittsburgh,
Pa.). Control formulations were prepared by the same method.
[0155] The particle size of anti-miR-155 containing Lac-GLN was
determined by dynamic light scattering on a Model 370 NICOMP
Submicron Particle Sizer (NICOMP, Santa Barbara, Calif.) in the
volume-weighted distribution mode. Particles were dispersed in cell
culture medium. The morphology of Lac-GLN was examined by a FEI
Tecnai G2 Bio TWIN transmission electron microscope (FEI Company,
OR, USA). Samples were prepared as described above, and a drop of
the sample was negatively stained with uranyl acetate for 1 min on
a perforated carbon grid for analysis. Images were recorded using a
Gatan 791 MultiScan CCS camera and processed by the Digital
Micrograph 3.1 software package.
[0156] The surface charge of anti-miR-155 containing Lac-GLN was
examined in 20 mM HEPES buffer using ZetaPALS zeta potential
analyzer (Brookhaven Instruments Corp., Holtsville, N.Y.).
Encapsulation efficiency of Lac-GLN was determined by Quant-iT.TM.
RiboGreen RNA Kit (Invitrogen, Grand Island, N.Y.) following the
manufacturer's protocol, and the fluorescence intensity (H) was
determined using a luminescence spectrometer (KS 54B, Perkin Elmer,
UK) at an excitation of 480 nm and an emission of 520 nm. The
encapsulation efficiency was calculated.
[0157] The colloidal stability of anti-miR-155 containing Lac-GLN
was determined by monitoring changes in its particle size over a
30-day period during storage at 4.degree. C. or 25.degree. C. The
serum stability test was used to investigate the ability of Lac-GLN
to protect anti-miR from serum nuclease degradation.
Anti-miR-155-lac-GLN and free anti-miR-155 were exposed to 50%
fetal bovine serum (FBS) and incubated at 37.degree. C. for various
time periods. Aliquots of each sample were then loaded onto a 1.5%
(w/v) agarose gel containing ethidium bromide.
[0158] Human HCC cell lines SK-Hep-1 and HepG2 cells were cultured
in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100
U/ml penicillin and 100 .mu.g/ml streptomycin at 37.degree. C. and
5% CO.sub.2.
[0159] For Luci-siRNA transfection, 2.times.10.sup.4 SK-Hep-1 cells
stably expressing luciferase, were seeded per well in 800 .mu.l
culture medium in 48-well plates and allowed to grow overnight at
37.degree. C. under 5% CO.sub.2 atmosphere. Next day, the culture
medium was replaced with medium containing 0%, 10% and 20% FBS, and
cells were transfected with Luci-siRNA containing Lac-GLN and
various control formulations at 100 nM for 4 hr. After
transfection, the medium was replaced with fresh medium containing
10% FBS and 48 hrs post transfection cells were washed with PBS and
luciferase activity in cell lysates was determined using Luciferase
Assay Kit (Promega, Madison, Wis.) following manufacturer's
instruction. Briefly, the total amount of protein of each well was
determined using BCA Assay Kit (Pierce, Rockford, Ill.), and
luciferase activity was determined by normalization to the total
amount of protein. The luciferase down-regulation was then
calculated as the relative value compared to the untreated negative
control.
[0160] For anti-miR-155 transfection, HepG2 cells were plated at
2.times.10.sup.5 cells per well in 6-well plates with 2 ml cultured
medium, and incubated overnight at 37.degree. C. under 5% CO.sub.2
atmosphere. Culture medium was then replaced with fresh medium, and
cells were transfected with 100 nM anti-miR-155 using Lipofectamine
2000, Lac-GLN, and control formulations and after 4 hr incubation,
the medium was replaced with fresh medium. Cells were incubated for
an additional 48 hr at 37.degree. C., then miR-155 and its target
gene expression level was determined by real time RT-PCR analysis.
As a positive control, cells transfected with Luc-siRNA and
anti-miR-155 using Lipofectamine 2000 were performed following
manufacturer's protocol. Untreated cells and empty Lac-GLN were
used as negative controls.
[0161] The cytotoxicity of Lac-GLN was evaluated by MTS assay
(Promega, Madison, Wis.). HepG2 cells were seeded in 96-well plates
at a density of 1.times.10.sup.4 cells per well. After overnight
incubation, cells were treated with empty Lac-GLN, negative control
RNA alone, anti-miR-155 alone, negative control RNA containing
Lac-GLN, and anti-miR-155 containing Lac-GLN at RNA concentration
of 100 nM for 24 hr. MTS reagent (20 .mu.L was then added to each
well, and cells were incubated for another 2 hr. The optical
density (OD) at 490 nm of each well was measured using a Multiskan
Ascent automatic plate reader. Untreated cells were used as control
and defined as 100% viability. Cell viability was calculated as a
percentage of the untreated cells.
[0162] Analysis of the cellular uptake of Lac-GLN was performed by
transfecting fluorescent Cy3-anti-miR-155 in HepG2 cells, and
evaluated by confocal microscopy and flow cytometry. For confocal
microscopy, 2.times.10.sup.5 HepG2 cells per well were seeded in
6-well plates containing a sterile glass coverslip at the bottom of
each well (Fisher Scientific, 12-545-82, Pittsburgh, Pa.) and
allowed to grow overnight. Cells were then treated with 100 nM
Cy3-anti-miR-155 containing GLN, Lac-GLN, and Lac-GLN with 20 mM
lactose and 1% BSA for 1 hr at 37.degree. C., followed by a wash
step with PBS five times. Cells were fixed with 4% paraformaldehyde
for 15 min, and stained with Hoechst 33342 (Invitrogen, Grand
Island, N.Y.) and Alexa-488 phalloidin (Invitrogen, Grand Island,
N.Y.) for 10 min each at room temperature. The glass coverslip with
the cells was then detached from the plates and covered with a
regular glass slide. Confocal analysis was performed on an Olympus
FV1000 Filter Confocal Microscope (Olympus Optical Co., Tokyo,
Japan).
[0163] For the flow cytometric analysis, 2.times.10.sup.5 HepG2
cells were treated with 100 nM Cy3-anti-miR-155 containing GLN,
Lac-GLN, GLN with 20 mM lactose and 1% BSA, and Lac-GLN with 20 mM
lactose and 1% BSA for 1 hr at 37.degree. C. Cells were suspended
using 0.25% trypsin, washed with PBS five times, and fixed with 4%
paraformaldehyde. The fluorescence intensity was measured on a
Becton Dickinson FACScalibur Flow Cytometer (Franklin Lakes, N.J.),
and a total of 10,000 events were collected for each sample.
[0164] Total RNA from transfected cells or tissue extracts was
isolated by TriZol reagent (Invitrogen, Grand Island, N.Y.) and
purified by following the standard protocol. The miR-155 cDNA was
synthesized using TaqMan MicroRNA reverse transcription Kit
(Applied Biosystems, Carlsbad, Calif.), and the cDNA was amplified
and quantified using the TaqMan MicroRNA Kit (Applied Biosystems,
Carlsbad, Calif.). The cDNA of C/EBP.beta. and FOXP3 was
synthesized using the first-strand cDNA synthesis kit (Invitrogen,
Grand Island, N.Y.) and resulting cDNA was amplified and quantified
using SYBR Green method (Applied Biosystems, Carlsbad, Calif.).
[0165] Primers were designed by the Primer Express Program (Applied
Biosystems):
TABLE-US-00001 C/EBP.beta.: [SEQ ID NO. 7] forward:
5'-AGAAGACCGTGGACAAGCACAG-3', [SEQ ID NO. 8] reverse:
5'-TTGAACAAGTTCCGCAG GGTGC-3'; FOXP3 [SEQ ID NO. 9] forward:
5'-AATGGCACTGACCAAGGCTTC-3', [SEQ ID NO. 10] reverse: 5'-TGTG
GAGGAACTCTGGGAATGTG-3'; and GAPDH: [SEQ ID NO. 11] forward:
5'-CCCCTGGCCAAGGTCATC CATGACAACTTT-3, [SEQ ID NO. 12] reverse:
5'-GGCCATGAGGTCCACCACCCTGTTGCTGTA-3').
[0166] miR-155 level was normalized to that of RUN6B, while
C/EBP.beta. and FOXP3 levels were normalized to that of GAPDH.
Their expressions were calculated using the 2.sup.-.DELTA.CT
approach.
[0167] Fluorescent Cy3-anti-miR-155 containing GLN and Lac-GLN were
used for confocal microscopy analysis. Male C57BL/6 mice were given
Cy3-anti-miR-155 (50 .mu.g) containing GLN and Lac-GLN
intravenously with a total injection volume of 200 .mu.l. After 4
hr, mice were sacrificed and tissues were collected. Harvested
tissues were fixed in 4% paraformaldehyde for 6 hr and soaked in
30% sucrose overnight at 4.degree. C. Tissues were then transferred
to block holders, embedded with O.C.T. freezing medium (Fisher
Scientific, Pittsburgh, Pa.), and frozen in liquid nitrogen. Tissue
samples were processed for tissue sectioning, and stained with
Hoechst 33342 (Invitrogen, Grand Island, N.Y.) and Alexa-488
phalloidin (Invitrogen, Grand Island, N.Y.) for 10 min each at room
temperature. The Fluorescent images were captured using an Olympus
FV1000 Filter Confocal Microscope (Olympus Optical Co., Tokyo,
Japan).
[0168] Fluorescent Cy5-anti-miR-155 containing GLN and Lac-GLN were
used for measuring in vivo uptake in different tissues by IVIS
imaging. The same treatment as described above was applied for this
experiment. Whole tissues were harvested and fixed in 4%
paraformaldehyde for 6 hr and immersed in 30% sucrose for 12 hr at
4.degree. C. Whole tissue Cy5 fluorescent signals were measured
using Xenogen IVIS-200 Optical In Vivo Imaging system (Caliper Life
Sciences, Hopkinton, Mass.).
[0169] Negative control RNA or anti-miR-155 containing Lac-GLN and
other controls were administered to male C57BL/6 mice by
intravenous injection at a dose of 1.5 mg/kg. 48 hr post
administration, mice were anesthetized, and liver tissues were
harvested and immediately frozen in liquid nitrogen. RNA extraction
and RT-PCR were performed as described in the previous section.
[0170] Results were reported as mean.+-.standard deviation, and a
minimum of triplicates were performed for each experiment.
Comparisons between the groups were analyzed by Student's t test
for two groups or ANOVA for multiple groups. Results were
considered as statistically significant when p values were
<0.05. All the statistical analysis was performed by Microsoft
Excel 2003 software.
[0171] FTIR was used to confirm the formation of the conjugate.
FIG. 27 is an FTIR spectrum of Lac-DOPE, DOPE, and lactobionic
acid. The absorption peaks of lac-DOPE are in blue at 1660
cm.sup.-1 and 1540 cm.sup.-1, indicating amide bond formation.
[0172] The particle size and zeta potential of GLN with various
molar percentages of Lac-DOPE were evaluated and this
characterization is shown in FIGS. 28A-B and 29A-B. 10% Lac-DOPE in
the formulation was the optimal composition with an average
diameter of 72.66 nm and a zeta potential of 3.49 mV. This
composition was selected as the delivery vehicle for the following
experiments and termed Lac-GLN. The size and morphology of Lac-GLN
was further examined by TEM. The image in FIG. 28B shows the
spherical shape and a uniform size distribution of Lac-GLN with
less than 100 mm diameter, which was in accordance with data
obtained by DLS.
[0173] The encapsulation efficiency was calculated from the
particle's ability to condense oligonucleotides. As shown in FIG.
30, the encapsulation efficiency of Lac-GLN was >85%. The
colloidal stability was determined by monitoring the change of
particle size over time. As shown in FIG. 29A, the average diameter
of Lac-GLN remained unchanged over a 30 day period at 4.degree. C.,
but a significant increase in the average diameter was observed
under storage at 25.degree. C.
[0174] The ability of Lac-GLN to protect anti-miR was evaluated by
a serum stability test. In this test, free anti-miR and
anti-miR-155-Lac-GLN were mixed with FBS and culture at 37.degree.
C. for different time periods. As shown in FIG. 31, Lac-GLN was
able to protect anti-miR-155 from nuclease degradation for up to 12
hrs, while free anti-miR-155 was completely digested within 4 hr
serum incubation. This result demonstrated good serum stability for
Lac-GLN.
[0175] The delivery efficiency was first examined by comparing the
performances of GLN and Lac-GLN in HCC cell-specific uptake. HepG2
cells with a high expression of ASGR on the surface were treated
with Cy3-anti-miR-155 containing non-targeted GLN and ASGR-targeted
Lac-GLN. The pre-incubation with 20 mM lactose and 1% BSA was
applied to block ASGR-mediated and non-specific uptake,
respectively. Cells were evaluated by confocal microscopy. As shown
in FIG. 29, cells treated with Lac-GLN showed a significantly
stronger fluorescence signal than those treated with non-targeted
GLN. This uptake enhancement was reduced in cells pretreated with
blocking agents, which demonstrated that the cellular uptake of
Lac-GLN was ASGR-specific. This result indicated a successful ASGR
targeting of Lac-GLN.
[0176] Cellular uptake of GLN and Lac-GLN was further quantified by
flow cytometry. As shown in FIG. 30A, the uptake of Lac-GLN was
about 3.58-fold higher than that of non-targeted GLN in HepG2
cells. The fluorescence signal did not reduce significantly in the
GLN pretreated cells with 20 mM lactose and 1% BSA, indicating
non-specific uptake of GLN by HCC cells (FIG. 30C). However, the
uptake of Lac-GLN was reduced by 3.51-fold in cells pre-incubated
with blocking agents while in absence of blocking agents the uptake
between GLN and Lac-GLN treated cells was comparable (FIG. 30B).
This result further confirmed that ASGR-targeted delivery improved
cellular uptake in HCC cells.
[0177] HCC SK-Hep1 cells, stably expressing firefly luciferase
mRNA, were used to determine the transfection efficiency of
different vehicles and the effect of several factors on
transfection efficiency including targeting ligand, gramicidin A,
and serum, by analyzing the silencing ability of siRNAs targeting
luciferase gene. FIG. 32A shows a significantly lesser expression
of luciferase in Lac-LN treated group (78.95%) compared to that in
the LN treated group (96.35%) in FBS-free medium, which confirmed
the advantage of ASGR-targeted strategy. However, media containing
20% FBS affected this transfection efficiency by only 6%. Treatment
with the commercial Lipofectamine 2000 caused 7.84% reduction in
luciferase expression, close to the LN treated group, and this
transfection was strongly inhibited by serum at high concentration.
Moreover, the effect of increasing concentration of gramicidin A as
a competitor was analyzed. Surprisingly, in the Lac-GLN treated
groups, neither 5% nor 10% gramicidin A was affected by the
presence of FBS during transfection. This finding was in contrast
to the previously reported studies, where transfection activities
were sensitive to the presence of serum. Similar results were
obtained in HepG2 cells (data not shown). Thus, this Lac-GLN
formulation was advantageous since serum was the main barrier for
in vivo delivery in a clinical setting.
[0178] To assess the application of this vehicle in further in
vitro and in vivo delivery, its cytotoxicity was first investigated
on HCC cells. HepG2 cells were treated with equal amount of empty
Lac-GLN, negative control RNA alone, anti-miR-155 alone, negative
control RNA-Lac-GLN, and anti-miR-155-Lac-GLN. As shown in FIG.
33B, no significant change in cell viability was observed between
treated cells and untreated cells. This result revealed a low
cytotoxicity of Lac-GLN in HepG2 cells.
[0179] Next, the effects of Lac-GLN containing anti-miR-155 on
miR-155 and its downstream targets expression were evaluated in
HepG2 cells. Cells were treated with anti-miR-155 containing
Lac-GLN and other control formulations for 4 hr, and the miR-155
and its targeting gene expression was measured 48 hr after
transfection by real time RT-PCR. FIG. 33C shows the miR-155
expression level from different treatment groups relative to the
untreated group. The positive control, treated with Lipofectamine
2000, had 92.4% miR-155 expression of the untreated. In addition,
LN, GLN, Lac-LN and Lac-GLN treated groups exhibited a similar
miR-155 expression level to that of Lipofectamine 2000 treated
group, and the differences among these groups were small. Based on
the minor difference in miR-155 expression between the positive
control and Lac-GLN treatment group, a doubled anti-miR-155
concentration was applied to examine whether the down-regulation of
miR-155 was anti-miR-155 concentration-dependent. As shown in FIG.
34A, the miR-155 expression in the Lipofectamine 2000 treated group
changed from 92.4% to 89.5% when the concentration of anti-miR-155
was doubled from 100 nm to 200 nM. This difference between the two
treatments was still not statistically significant. In the Lac-GLN
treated group, a similar trend was observed, where the expression
of miR-155 was 87.2% and 82.9% in 100 nM and 200 nM anti-miR-155
treatments, respectively. These indicated that the miR-155
expression did not depend on anti-miR-155 concentration and
anti-miR-155 delivery did not lead to miR-155 degradation.
[0180] To further examine the delivery efficiency, the expression
of miR-155 targeting genes, C/EBP.beta. and FOXP3, were evaluated.
The results are summarized in FIG. 34B. In contrast to the steady
expression of miR-155 (FIG. 32A), there were a 16.1- and 4.1-fold
increase in C/EBP.beta. and FOXP3 expression, respectively, in the
Lac-GLN 100 nM anti-miR-155 treatment group. Only a 1.4-, 1.9-fold
increase of C/EBP.beta. and FOXP3 expression was observed in cells
transfected with Lipofectamine 2000, respectively. Furthermore,
doubling the anti-miR-155 concentration resulted in an improved
up-regulation of C/EBP.beta. and FOXP3 expression, clearly
demonstrating that the miR-155 targeting gene expression was
dependent on anti-miR-155 concentration. Thus, the delivery of
anti-miR-155 most likely resulted in functional inhibition of
miR-155 rather than its degradation. In sum, these results show
Lac-GLN's superiority over the commercial available agent in
anti-miR delivery.
[0181] In order to assess the in vivo delivery efficiency and
tissue specificity of Lac-GLN, tissue distribution study was
performed in C57BL/6 mice that were administrated Cy5-anti-miR-155
containing GLN and Lac-GLN intravenously at a dose of 1.5 mg/kg.
After 4 hr, organs were harvested and fluorescence signals were
compared. As shown in FIG. 35, lung, spleen and liver were the
major organs exhibiting fluorescence signals when mice were
injected with non-targeted GLN. In contrast, maximal fluorescence
signals accumulated in the liver when mice were treated with
Lac-GLN with very weak signals in the spleen and kidney and no
detectable signal in lung. These results show that the delivery of
Cy5-anti-miR-155 by Lac-GLN was liver-specific and that Lac-GLN was
able to minimize off-target uptake, thus improving the overall
delivery efficiency.
[0182] Confocal microscopy was performed on the liver and other
organs to further evaluate the delivery efficiency between GLN and
Lac-GLN. Besides hepatocytes, the liver also contains a large
population of Kupffer cells, known as residential macrophage. As
shown in FIG. 36A, a larger proportion of fluorescence signals was
taken up by hepatocytes than by Kupffer cells in liver when mice
were treated with Lac-GLN, while the uptake was predominantly by
Kupffer cells in the non-targeted GLN-Cy5-anti-miR-155 treated
liver.
[0183] The distribution of fluorescence signals in lung and spleen
were also examined to evaluate Lac-GLN delivery. As shown in FIG.
36B, fluorescence signals accumulating in lung and spleen in the
Lac-GLN treated mice were less than those in the GLN treated mice,
indicating high specificity of Lac-GLN for delivery to the
liver.
[0184] Next, the delivery efficiency of Lac-GLN-anti-miR-155 in
C57BL/6 mouse liver was studied. For this purpose, mice were
injected a single dose of 1.5 mg/kg anti-miR-155 formulated in
Lipofectamine 2000, GLN, Lac-LN or Lac-GLN through tail vein.
Injections of empty Lac-GLN, negative control RNA containing
Lac-GLN or free anti-miR-155 were used as negative controls. 48 hr
post administration, mice were sacrificed and livers were
harvested. The expression of miR-155 and its target, C/EBP.beta.,
was evaluated by real time RT-PCR. FIG. 37A illustrates the
expression of miR-155. As noted, miR-155 level was not altered in
the negative control groups. A slight decrease in miR-155
expression by 13% and 20% was observed when anti-miR-155 was
delivered using Lipofecamine 2000 and Lac-GLN, respectively,
compared to the untransfected control. Moreover, the differences
among GLN, Lac-LN and Lac-GLN were not significant. On the
contrary, the delivery efficiency reflected by C/EBP.beta.
expression varied considerably among these groups as demonstrated
by a 2.8-, 3.7- and 6.9-fold increase in its expression in GLN,
Lac-LN and Lac-GLN treated groups, respectively, compared to the
untreated group (FIG. 37B). No significant changes were observed in
the negative control groups, and the Lipofectamine 2000 treated
group only exhibited a 1.4-fold up-regulation of C/EBP.beta.. In
addition, another miR-155 target gene, FOXP3 expression, was
increased by 1.1-, 1.2-, and 2.1-fold in GLN, Lac-LN and Lac-GLN
treated groups, respectively (FIG. 37C). These data demonstrate the
improvement of delivery efficiency by Lac-GLN and agree with the
results of in vitro experiments (FIG. 34).
Example 9
[0185] cRGD-PEG-DSPE conjugates were synthesized. cRGDfC and
PEG-DSPE-maleimide was conjugated via -SH and -maleimide reaction
resulting in a thioether linkage. The cRGDfC and PEG-PSPE-maleimide
molar ratio used during the reaction was 1.5:1. cRGDfC and
PEG-DSPE-maleimide was each dissolved in PBS buffer containing 5 mM
EDTA (pH=7.0). The cRGDfC and PEG-DSPE solutions were combined and
reacted at room temperature for 6 h with stirring. The product was
purified by gel filtration on a PD-10 column to remove
unreacted/excess cRGDfC from the product. For scaled-up reactions,
the gel filtration can be replaced with GPC, dialysis using MWCO
2000 membrane, or tangential flow diafiltration. The product can be
frozen or lyophilized for long-term stability. The product purity
was confirmed by HPLC and by LC-MS. Minimum cRGDfC conjugation
level (e.g., 80%) and free peptide content (e.g., <1%) can be
established as specifications. The cRGDfC content in the product
can be determined by BCA protein assay.
[0186] Certain embodiments of the formulations and methods
disclosed herein are defined in the above examples. It should be
understood that these examples, while indicating particular
embodiments of the invention, are given by way of illustration
only. From the above discussion and these examples, one skilled in
the art can ascertain the essential characteristics of this
disclosure, and without departing from the spirit and scope
thereof, can make various changes and modifications to adapt the
compositions and methods described herein to various usages and
conditions. Various changes may be made and equivalents may be
substituted for elements thereof without departing from the
essential scope of the disclosure. In addition, many modifications
may be made to adapt a particular situation or material to the
teachings of the disclosure without departing from the essential
scope thereof.
Sequence CWU 1
1
17123DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1ccctgtggat gactgagtac ctg 23219DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2ccagcctccg ttatcctgg 19320DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 3ccggcacctg cacacctgga
20423RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotidemisc_feature(1)..(3)Phosphorothioate
linkagemisc_feature(1)..(23)2'-O-methyl-modified
nucleotidemisc_feature(19)..(23)Phosphorothioate
linkagemisc_feature(23)..(23)U-Chol 4gaaacccagc agacaaugua gcu
23518DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 5cgtgcgagtg tctaacgg 18618DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
6cggatcagtc tttgggtc 18717DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 7cgtcttcccc tccatcg
17818DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 8ctcgttaatg tcacgcac 18921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
9cggagcacgg ggacgggtat c 211024DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 10aagacgaagg ggaagacgca catc
241122DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 11agaagaccgt ggacaagcac ag 221222DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
12ttgaacaagt tccgcagggt gc 221321DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 13aatggcactg accaaggctt c
211423DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 14tgtggaggaa ctctgggaat gtg 231530DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
15cccctggcca aggtcatcca tgacaacttt 301630DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
16ggccatgagg tccaccaccc tgttgctgta 301723RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotidemisc_feature(1)..(3)Phosphorothioate
linkagemisc_feature(21)..(23)Phosphorothioate linkage 17accccuauca
cgauuagcau uaa 23
* * * * *