United States Patent:  6,110,490

Assignee:  The United States of America as represented by the Department of Health (Washington, DC)

Filed:  January 29, 1996

The present invention is directed to a liposomal preparation which is based on specific lipid components. The liposomal compounds are also combined with a biologically active agent, forming liposomal compounds. These compounds are useful in drug delivery, where specific therapeutic compounds are provided in the liposomes. The specific lipid components of the present invention provide a highly efficient and stable delivery system for nucleic acids. Consequently, one embodiment of the invention provide the liposomal preparations which are suitable for use in gene therapy.

SUMMARY OF THE INVENTION

The present invention relates to liposome compositions and a method of preparing such liposomes. In addition, the present invention relates to the administration of the biologically active agent-liposome preparations to cells and further to the administration of the liposome preparations to patients as a therapeutic agent.

The liposome compositions of the present invention provide highly efficient delivery of biologically active agents to cells. Liposome vesicles are prepared from a mixture of a cationic lipopolyamine and a neutral lipid and form a bi- or multilamellar membrane structure (referred to herein as "DLS-liposomes") A preferred embodiment of the present invention uses a spermine-5-carboxy-glycinedioctadecylamide (referred to herein as "DOGS") as the cationic lipopolyamine and dioleylphosphatidyl ethanolamine (referred to herein as "DOPE") as the neutral lipid.

The liposomes of the present invention efficaciously deliver biologically active agents into the cytoplasmic compartment of human cells. Use of such liposomal vehicles make possible high transfection efficiency of biologically active materials into cells.

The present invention also encompasses a method of preparing such a liposome composition. The presence of at least one neutral lipid in combination with at least one cationic lipopolyamine makes possible the formation of liposomes after hydration. According to the method of the present invention, liposomes are prepared by mixing together each of a cationic lipopolyamine and a neutral lipid in a molar ratio ranging from a ratio of 0.02:1 to a ratio of 2:1; evaporating the mixture to dryness; and rehydrating. In order to introduce a biologically active agent into the liposomes, such agent can be added prior to or after rehydration of the dried film.

In one aspect of the invention, nucleic acids may be associated with the liposomes. This association may be accomplished in at least in two ways: (1) complex formation between the cationic liposome vesicle and negatively charged polyaminon, such as nucleic acid or (2) encapsulation in the cationic liposome vesicle.

The present invention is further directed to a method of treating a subject with a suitable pharmaceutical formulation of nucleic acid-liposomes in order to deliver specific nucleic acids to target cells of the subject. Such a method of treating subjects provides effective delivery of oligonucleotides or gene-expressing nucleic acid vectors (e.g. plasmids or viral vectors) into cells. Therefore, such a method of drug delivery is useful for the transport of nucleic acid based therapeutics.

Another embodiment of the present invention is directed to a combination of a DOGS/DOPE liposome preparation externally anchored through hydrophobic interactions with an adenovirus particle. Since adenoviruses enter cells via receptor-mediated endocytosis, the combination of adenovirus particles and the DLS-liposomes produces an enhanced transduction efficiency.

Adenovirus particles may also serve as a nucleic acid which is carried in the liposome internally.

The present invention provides a therapeutic method of treating ailments and conditions based upon a liposome-facilitated transfer of biologically active agents. For example, the present invention provides a pharmaceutical liposomal formulation for the delivery of nucleic acids using systemic administration to provide long-term expression of a given gene. One such method provides direct systematic nucleic acid transfer combining the DLS-liposomes with episomally replicative DNA vectors carrying the nucleic acid of interest. Alternatively in vitro cell transfection followed by tissue transplantation such that the transfected cells are incorporated in transplanted tissue. This method is referred to as in vitro/ex vivo transfer. Other biologically active agents may be encapsulated in the liposomes of the present invention and delivered to cells using systemic or in vitro/ex vivo transfer methods.

Claim 1 of 21 Claims

1. A composition comprising a bi- or multi-layer membrane surrounding an internal aqueous liposome comprising at least one cationic lipopolyamine and at least one neutral lipid provided in a molar ratio range said ratio from about 0.02:1 to about 2.0:1.

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