Title:  Non-ligand polypeptide and liposome complexes as intracellular delivery vehicles

United States Patent:  6,245,427

Appl. No.:  111265

The present invention discloses compositions and methods of using intracellular delivery vehicles for delivery and transfection of DNA, RNA, polypeptides, genes, proteins, drugs and biologically active agents into cells in vitro and in vivo. The vehicle comprises a mixture of a liposome and a polypeptide lacking specificity for cellular receptors. In another embodiment, a method for intracellular delivery of biologically active agents comprising combining a non-receptor-binding protein and a liposome, incubating the mixture for a period of time, adding the biologically active agent, incubating again, and finally, introducing the resulting mixture to the cell. Preferably, the liposome is a cationic liposome. The charge ratio of cationic liposome to DNA can effectively be varied from 2:1 to 1:2. Preferably, the non-receptor-binding protein is the serum albumin of the animal source of the cell to be transfected. This inention is an improvement over, and offers several advantages compared to, previously disclosed cationic liposomal delivery vehicles which utilize receptor ligands.

SUMMARY OF THE INVENTION

This invention describes unique liposomal complexes for the intracellular delivery of particular agents or molecules such as DNA, RNA or proteins. This invention also describes methods for the use of these complexes for intracellular delivery. A unique feature of this invention is that it utilizes polypeptides that lack cell-receptor specificity (non-ligands, also referred to as non-receptor-binding polypeptides) combined with cationic liposomes as a delivery vehicle. Molecules desirable for intracellular delivery include, but are not limited to DNA, polydeoxyribonucleotides, RNA, polyribonucleotides, proteins, peptides, polypeptides and the like.

It is an object of this invention to provide carrier compositions for intracellular delivery of a biologic molecule or agent.

It is an object of this invention to provide carriers that do not include viral components.

It is an object of this invention to provide carriers that do not utilize receptor-ligands or receptor-mediated transport molecules.

It is an object of this invention to provide carriers that comprise liposomes and serum proteins that bind to cells non-specifically.

It is an object of this invention to utilize vehicle components that are abundant, inexpensive and easy to isolate in large quantity.

It is an object of this invention to provide a vehicle whose components are not likely to cause an undesirable biological reaction.

It is an object of this invention to provide a vehicle that is effective in the presence of high concentrations of blood serum.

It is an object of this invention to provide methods of using carrier cdmpositions to effect intracellular delivery of particular molecules.

In accordance with the above objects and other described herein, the carrier composition for intracellular delivery of a biologically active molecule of the present invention comprises a mixture of a cationic lipid and a non-receptor-binding protein.

In another embodiment of the invention the cationic lipid is combined with a neutral lipid forming a cationic liposome thereby.

In another embodiment, the carrier composition comprises a cationic lipid and serum albumin of human or non-human origin.

In another embodiment, the carrier composition comprises a cationic lipid, a protein that lacks receptor specificity (a non-receptor-binding protein) and a biologically active molecule.

In further accordance with the above objects and others described herein, the method for intracellular delivery of a biologically active agent to a target cell comprises:

a) combining a non-receptor-binding polypeptide and a cationic lipid to form a first mixture such that said polypeptide and cationic lipid become associated;

b) adding to said first mixture said biologically active agent to form a second mixture such that said agent becomes associated with said cationic lipid; and,

c) introducing said second mixture to said cell.

In another embodiment of the method, the non-receptor-binding polypeptide is a protein. In a preferred embodiment, the protein is a serum albumin, most preferably, the serum albumin of the animal source of the target cell to be inoculated.

In another embodiment of the method, the cationic lipid is combined with a neutral lipid forming a cationic liposome thereby.

Claim 1 of 41 Claims

What is claimed is:

1. A method for intracellular delivery of a biologically active agent to a target cell comprising:

a) combining a non-receptor-binding polypeptide and a cationic lipid to form a first mixture such that said polypeptide and lipid become associated;

b) adding to said first mixture said biologically active agent to form a second negatively charged complex mixture such that said agent becomes associated with said lipid; and,

c) introducing said second mixture to said cell.

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