Porous nanoparticle-supported lipid bilayers (protocells) for targeted delivery and methods of using same

Patent metadata

Field	Value
Publication number	US-9579283-B2
Application number	US-201214113371-A
Country	US
Kind code	B2
Filing date	Apr 27, 2012
Priority date	Apr 28, 2011
Publication date	Feb 28, 2017
Grant date	Feb 28, 2017

Abstract

The present invention is directed to protocells for specific targeting of hepatocellular and other cancer cells which comprise a nanoporous silica core with a supported lipid bilayer; at least one agent which facilitates cancer cell death (such as a traditional small molecule, a macromolecular cargo (e.g. siRNA or a protein toxin such as ricin toxin A-chain or diphtheria toxin A-chain) and/or a histone-packaged plasmid DNA disposed within the nanoporous silica core (preferably supercoiled in order to more efficiently package the DNA into protocells) which is optionally modified with a nuclear localization sequence to assist in localizing protocells within the nucleus of the cancer cell and the ability to express peptides involved in therapy (apoptosis/cell death) of the cancer cell or as a reporter, a targeting peptide which targets cancer cells in tissue to be treated such that binding of the protocell to the targeted cells is specific and enhanced and a fusogenic peptide that promotes endosomal escape of protocells and encapsulated DNA. Protocells according to the present invention may be used to treat cancer, especially including hepatocellular (liver) cancer using novel binding peptides (c-MET peptides) which selectively bind to hepatocellular tissue or to function in diagnosis of cancer, including cancer treatment and drug discovery.

First claim

The invention claimed is:

1. A cell-targeting porous protocell composition comprising a plurality of protocells, the protocells comprising:
a nanoporous silica or metal oxide core with a supported lipid bilayer;
a Met binding peptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, wherein the Met binding peptide is attached to the supported lipid bilayer;
a fusogenic peptide that promotes endosomal escape, wherein the fusogenic peptide is attached to the supported lipid bilayer; and
a cargo loaded into the protocell, wherein the cargo is selected from the group consisting of:
double stranded linear DNA;
plasmid DNA;
a drug;
an imaging agent;
small interfering RNA;
small hairpin RNA; and
microRNA;

wherein the cargo is optionally conjugated to a nuclear localization sequence.

2. The protocell composition according to claim 1 wherein said silica cores of the protocells are spherical and range in diameter from about 10 nm to about 250 nm.

3. The protocell composition according to claim 2 wherein said silica cores of the protocells have a mean diameter of about 150 nm.

4. The protocell composition according to any one of claims 1 - 3 wherein said protocells are monodisperse.

5. The protocell composition according to any one of claims 1 - 3 wherein said protocells are polydisperse.

6. The protocell composition according to claim 1 wherein said lipid bilayer is comprised of lipids selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyi-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-[phosphor-L-serine] (DOPS), 1,2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (18:1 PEG-2000 PE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (16:0 PEG-2000 PE), 1-Oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl]-sn-Glycero-3-Phosphocholine (18:1-12:0 NBD PC), 1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl}-sn-glycero-3-phosphocholine (16:0-12:0 NBD PC), cholesterol and mixtures thereof.

7. The protocell composition according to claim 6 wherein said lipid bilayer comprises DOPC in combination with DOPE.

8. The protocell composition according to claim 6 wherein said lipid bilayer comprises DOTAP, DOPG, DOPC or mixtures thereof.

9. The protocell composition according to claim 6 wherein said lipid bilayer comprises DOPG and DOPC.

10. The protocell composition according to claim 6 wherein said lipid bilayer comprises cholesterol.

11. The protocell composition according to claim 1 wherein said lipid bilayer comprises DOPC in combination with about 5 wt % DOPE, about 30 wt % cholesterol, and about 10 wt % PEG-2000 PE (18:1).

12. The protocell composition according to claim 1 wherein lipid bilayer comprises about 5% by weight DOPE, about 30% by weight cholesterol, and about 60% by weight DOPC and/or DPPC.

13. The protocell composition according to claim 1 wherein the lipid bilayer comprises PEG-conjugated to DOPE.

14. The protocell composition according to claim 1 wherein said protocells further comprise an additional targeting peptide.

15. The protocell composition according to claim 14 wherein said targeting peptide is a SP94 peptide.

16. The protocell composition according to claim 14 wherein said targeting peptide is SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.

17. The protocell composition according to claim 1 wherein said fusogenic peptide is H5WYG peptide (SEQ ID NO: 13) or an eight mer of polyarginine (SEQ ID NO: 14).

18. The protocell composition according to claim 17 wherein said fusogenic peptide is SEQ ID NO: 13.

19. The protocell composition according to claim 1 wherein the cargo comprises plasmid DNA.

20. The protocell composition according to claim 19 wherein said plasmid DNA is supercoiled or packaged plasmid DNA.

21. The protocell composition according to claim 20 wherein said DNA is both supercoiled and packaged plasmid DNA.

22. The protocell according to claim 19 wherein said plasmid DNA is conjugated to a nuclear localization sequence.

23. The protocell composition according to claim 19 wherein said plasmid DNA is histone-packaged supercoiled plasmid DNA and comprises a mixture of human histone proteins.

24. The protocell composition according to any of claim 1 wherein said plasmid DNA encodes a polypeptide toxin, a small hairpin RNA (shRNA) or a small interfering RNA (siRNA).

25. The protocell composition according to claim 24 wherein said polypeptide toxin is selected from the group consisting of ricin toxin chain-A and diphtheria toxin chain-A.

26. The protocell composition according to claim 24 wherein said shRNA or said siRNA induces apoptosis of a cell.

27. The protocell composition according to claim 1 wherein said DNA encodes a reporter protein.

28. The protocell composition according to claim 27 wherein said reporter protein is green fluorescent protein or red fluorescent protein.

29. The protocell composition according to claim 27 wherein said MET binding peptide is a peptide according to SEQ ID NO: 1.

30. The protocell composition according to claim 1 wherein said nuclear localization sequence is a peptide according to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.

31. The protocell composition according to claim 1 wherein said cargo comprises said drug, and said drug is an anticancer agent.

32. The protocell composition according to claim 31 wherein said anticancer agent is everolimus, trabectedin, abraxane, TLK 286, AV-299, DN-101, pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, a FLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an aurora kinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDAC inhibitor, a c-MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFR TK inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a PI3 kinase inhibitors, an AKT inhibitor, a JAK/STAT inhibitor, a checkpoint-1 or 2 inhibitor, a focal adhesion kinase inhibitor, a Map kinase kinase (mek) inhibitor, a VEGF trap antibody, pemetrexed, erlotinib, dasatanib, nilotinib, decatanib, panitumumab, amrubicin, oregovomab, Lep-etu, nolatrexed, azd2171, batabulin, ofatumumab, zanolimumab, edotecarin, tetrandrine, rubitecan, tesmilifene, oblimersen, ticilimumab, ipilimumab, gossypol, Bio 111 , 131-I-TM-601, ALT-110, BIO 140, CC 8490, cilengitide, gimatecan, IL13-PE38QQR, INO 1001, IPdR 1 KRX-0402, lucanthone, LY 317615, neuradiab, vitespan, Rta 744, Sdx 102, talampanel, atrasentan, Xr 311, romidepsin, ADS-100380, sunitinib, 5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin, 5′-deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709, seliciclib; PD0325901, AZD-6244, capecitabine, L-Glutamic acid, N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-, disod

Assignees

• Brinker C Jeffrey
• Carnes Eric C
• Ashley Carlee Erin
• Willman Cheryl L
• Stc Unm
• Sandia Corp

Inventors

• Brinker C Jeffrey
• Carnes Eric C
• Ashley Carlee Erin
• Willman Cheryl L

Classifications

• A61K9/1271 Primary

  Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers (liposomes as conjugates {A61K47/6911}) · CPC title

• A61K48/0016

  wherein the nucleic acid is delivered as a 'naked' nucleic acid, i.e. not combined with an entity such as a cationic lipid · CPC title

• C12N2310/14

  interfering nucleic acids [NA] · CPC title

• A61K47/6901

  Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors · CPC title

• C12N15/88

  using microencapsulation, e.g. using {amphiphile} liposome vesicle · CPC title