Methods of forming targeted liposomes loaded with a therapeutic agent

Abstract

The present invention provides for liposomes loaded with a therapeutic agent, which liposomes are targeted to a cell of interest by incubation with a targeting protein protein conjugated to a linker molecule comprising a hydrophobic domain, a hydrophilic polymer chain terminally attached to the hydrophobic domain, and a chemical group reactive to one or more functional groups on a protein molecule and attached to the hydrophilic polymer chain at a terminus contralateral to the hydrophobic domain, for a time sufficient to permit the hydrophobic domain to become stably associated with the liposome.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. application Ser. No. 10 / 847,982, filed May 17, 2004, which is a continuation of U.S. application Ser. No. 10 / 177,939, filed Jun. 21, 2002, now U.S. Pat. No. 6,803,053, which is a continuation of U.S. application Ser. No. 09 / 765,107, filed Jan. 16, 2001, now U.S. Pat. No. 6,528,087, which is a continuation of U.S. application Ser. No. 09 / 076,618, filed May 12, 1998, now U.S. Pat. No. 6,210,707, which was a continuation-in-part of U.S. patent application Ser. No. 08 / 967,791, filed Nov. 10, 1997, now U.S. Pat. No. 6,071,533 which claims the benefit of U.S. Provisional Patent Application No. 60 / 030,578, filed Nov. 12, 1996. The contents of all of these applications are hereby incorporated by referenceFEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] Not applicable. FIELD OF THE INVENTION [0003] The present invention relates to the field of cationic lipid:DNA complexes (“CLDC”). In particular, the ...

AI Technical Summary

Benefits of technology

The present invention provides a novel method for preparing cationic lipid:nucleic acid complexes with increased shelf life. These complexes can be prepared by contacting a nucleic acid with an organic polycation, such as spermidine or spermine, and an amphiphilic cationic lipid plus a helper lipid, such as cholesterol. Optionally, a hydrophilic polymer can also be added. The resulting complex has an increased shelf life compared to a similar complex without the hydrophilic polymer. The nucleic acid can be any nucleic acid, such as a DNA, and can be combined with a liposome before or after formation of the complex. The lipid:nucleic acid complex can also be targeted with a targeting moiety, such as an antibody, for specific cell types. The invention also provides a method for transfecting a nucleic acid into a mammalian cell.

Problems solved by technology

Moreover, they cannot replicate or recombine to form an infectious agent and have low integration frequency.

However, the technical problems for preparation of complexes that have stable shelf-lives have not been addressed.

It is therefore difficult to obtain homogeneous lipid:nucleic acid complexes with a size distribution suitable for systemic injection.

The structural instability along with the loss of transfection activity of lipid:nucleic acid complex with time have been challenges for the future development of lipid-mediated gene therapy.

The disadvantages of methods of this type are: often uncontrollable, incomplete reaction of the protein with the linker; the presence of excess linker on the resulting conjugate, potentially adverse effect of the linker on the stability of the particle, and the inability to incorporate components reactive with the linker into the composition of the particle.

These methods have a number of disadvantages, including the imposition of severe limitations on the range of methods by which the particle can be formed, (e.g. the detergent removal technique is required) and by which the drug or other agent can be loaded into the microparticle.

These methods are therefore unable to attach a protein to a premade particle without first destroying it.

The presence of detergent in these methods is unavoidable because without a detergent the hydrophobically modified protein is insoluble in aqueous medium

These studies therefore provide no guidance for inserting into liposomes or other lipidic microparticles proteins, such as antibodies, or fragments thereof, conjugated to linkers significantly smaller than the protein.

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