Title:  Sub-100nm biodegradable polymer spheres capable of transporting and releasing nucleic acids

United States Patent:  6,254,890

Assignee:  Massachusetts Institute of Technology (Cambridge, MA)

Filed:  December 10, 1998

The present invention provides biodegradable polymer nanospheres capable of transporting and releasing therapeutic agents, specifically nucleic acids. In preferred embodiments, a sub-150 nm nanosphere is formed containing nucleic acids. Thereafter, the agent is released from the nanosphere. In one embodiment a biodegradable polymer nanosphere surface has attached to it a targeting moiety. In another embodiment, the biodegradable polymer nanosphere surface has attached to it a masking moiety. In yet another embodiment both targeting and masking moieties are attached to the nanosphere surface.

DETAILED DESCRIPTION OF THE INVENTION

Recognizing the importance of the development of systems that are effective not only at encapsulating biological agents such as nucleic acids, but that are also able to release these biological agents controllably, the present invention provides a system for the delivery of nucleic acids comprising: 1) encapsulating nucleic acids in biodegradable polymers and 2) releasing these encapsulated nucleic acids from the biodegradable polymers. The present invention also provides a method for the delivery of nucleic acids to cells comprising 1) formation of a nanosphere encapsulating nucleic acids and 2) contacting the nanospheres encapsulating the nucleic acids with cells, whereby the encapsulated nucleic acids are released from the nanospheres.

In general, the delivery agent is comprised of a biodegradable polymer, a condensation agent, and a desired therapeutic agent, most preferably nucleic acids, that are formulated as a nucleic acid encapsulating nanosphere. In preferred embodiments, these nanospheres are formulated as sub-150 nm spheres of which at least 50% of the size distribution of nanospheres is sub-100 nm. In particularly preferred embodiments, the nanospheres are all sub-100 nm. In particularly preferred embodiments, the nanosphere compositions utilized in the inventive method have attached to the surface a masking moiety, a targeting moiety, or alternatively a masking moiety and a targeting moiety.

Various characteristics of the inventive compositions utilized in preferred embodiments of the present invention are discussed in more detail below; certain examples of inventive compositions for use in the method of the present invention are also presented.

Delivery and Release Compositions and Systems

As mentioned above, the method for delivery and release of a therapeutic agent of the present invention comprises 1) the formation of a nucleic acid encapsulating nanosphere, comprised of a biodegradable polymer, an encapsulating component and a desired therapeutic agent, and 2) controlled release of the therapeutic agent from the inventive composition. In general, formation of the nucleic acid encapsulating nanospheres comprises dissolving a biodegradable polymer in a solvent with a condensing agent and a therapeutic agent, and subsequent precipitation. The removal of the solvent then yields the nucleic acid encapsulating nanospheres. The precipitation/solvent evaporation method is preferred for the formulation of the nanosphere composition because this method allows for the encapsulation of drug molecules, especially shear-sensitive molecules such as DNA, without exposing the molecules to undue mechanical stress or harsh chemical processing. One of ordinary skill in the art will appreciate that a variety of polymers, condensing agents, and therapeutic agents can be used in the formation of the inventive composition. For example, polymers that can be utilized include, but are not limited to, poly (e-caprolactone) and poly (hydroxybutyrate) and poly (orthoesters). Condensing agents include, but are not limited to, poly(l-lysine), poly (d-lysine), spermine, spermidine, poly (lactic acid-co-lysine), dendrimers, 1,2-diacyl-3-trimethylammonium-propane (TAP) and 1,2-diacyl-3-dimethylammonium-propane (DAP), dimethyldioctadecylammonium bromide (DDAB), and other cationic lipids. In particularly preferred embodiments, the therapeutic to be delivered is a nucleic acid, however, one of ordinary skill in the art will realize that a variety of therapeutic agents can be utilized, including but not limited to, lipophilic drugs, biomolecules, and small organic molecules.

In particularly preferred embodiments, the therapeutic agent utilized in the formation of the composition is a nucleic acid, and the polymer spheres formulated are sub-150 nm, of which 50-100% of the distribution is sub-100 nm. In most preferred embodiments, all of the polymer spheres formulated are sub-100 nm.

In but one example, the method of encapsulation includes dissolving a polymer such as poly(oactic-co-glycolic acid) in an appropriate solvent such as 2,2,2-trifluoroethanol (TFE) in the presence of a condensing agent, such as a cationic lipid, and a therapeutic agent. It will be appreciated that different polymer concentrations, preferably 2.5 to 7.5 mg/ml, more preferably lower concentrations and most preferably about 2.5 mg/ml, can be used. Additionally, it will be appreciated that a variety of compatible solvents can be utilized as will be readily determined by one of ordinary skill in the art. Furthermore, a range of 0 to 100% ethanol, or other similar non-solvents, or precipitation agents, as will be readily discernable to one of ordinary skill in the art, can be used in the process as a precipitation agent. The solvent:non-solvent ratio can be varied from 1.about.2.5. DNA concentrations are preferably in the range of 0.1 to 1 mg/ml, more preferably from 0.25 to 0.68, and most preferably about 0.68 mg/ml. Finally, the nanospheres can be formed under conditions where positive:negative charge ratios of condensing agent:nucleic acid were varied preferably from 1 to 1000, more preferably from 25 to 100 and most preferably about 25. This system affords a method for preparing nanospheres of different sizes, including those less than 100 nm.

In a preferred embodiment of the present invention, targeting moieties are attached to the surface of the nanospheres. As discussed previously, such small nanospheres have the potential to be targeted to specific cells in vivo and to be taken up by receptor-mediated endocytosis. The targeting moieties can be selected by one of ordinary skill in the art keeping in mind the specific cell surface to be targeted. For example, if one wishes to target the asialoglycoprotein receptor on the hepatocytes in the liver, an appropriate targeting moiety would be clustered trigalactosamine. Once a specific targeting moiety has been selected for a particular cell to target, the different targeting moieties can be attached either by covalent linkage directly onto the particle surface, or by indirect linkage via, for example, a biotin-avidin bridge. More specifically, in one embodiment, avidin is attached covalently to the polymer and a biotinylated ligand attaches non-covalently to the avidin. In another embodiment, biotin is covalently attached to the polymer, and then avidin is used as a bridge between the biotinylated polymer and the biotinylated ligand. Preferred targeting agents are biocompounds, or portions thereof, that interact specifically with individual cells, small groups of cells, or large categories of cells. Examples of useful targeting agents include, but are in no way limited to, low-density lipoproteins (LDS's), transferrin, asiaglycoproteins, gp120 envelope protein of the human immunodeficiency virus (HIV), and diptheria toxin, antibodies, and carbohydrates. A variety of agents that direct compositions to particular cells are known in the art (see, for example, Cotten et al., Methods Enzym, 1993, 217, 618).

In another embodiment of the presently claimed invention, masking moieties are attached to the surface of the nanospheres. These masking moieties prevent the recognition by a specific cell surface and instead allows for intravenous administration applications. For example, the surface masking characteristics are provided by poly(ethylene glycol) (PEG) by using various PEG-PLA and PLGA mixtures in the initial polymer solution.  It will be appreciated by those skilled in the art that other masking moieties can also be employed for use in the presently claimed invention. As mentioned earlier, both targeting and surface masking (for example oligosaccharides and other surfactants) moieties may be attached or adsorbed to the surface of the nanospheres. When both targeting and masking moieties are utilized, some optimization must be done in order to obtain enough of a specificity for a specific target cell but to retain enough of the masking capacity to avoid non-specific uptake in vivo. Other means can be co-employed to achieve cell specificity. Such means can be directly programmed into the nucleic acids themselves, such as using the albumin promoter for targeting hepatocytes.

Once the desired compositions have been prepared, the particle size and polydispersity of the nanospheres can be measured using quasi-elastic light scattering, and the particle size confirmed by transmission electron microscopy. The loading efficiency of the nucleic acid into these nanospheres has been shown to be 30-60% and can be determined using radiolabeled plasmid nanospheres.

As discussed previously, the system of the present invention ultimately provides for the release of the therapeutic agent, preferably nucleic acids, from these nanospheres. In an exemplary embodiment, the release of the plasmid DNA occurs preferably over 200 hours or alternatively so that there is not a burst that would release DNA prior to the nanosphere's arrival at a particular target, such as a cell.  These spheres release at a constant rate for a long time despite the initial expectation of instant release of therapeutic reagents from such small spheres with such a high surface area: volume ratio. These spheres can potentially be made to release at different rates as desired. The inset autoradiograph shows a band of original linear DNA on the left lane with a sample at greater than 600 hours. The higher molecular weight bands are the DNA complexed to the spheres, unable to penetrate the agarose gel.

Uses

Those of ordinary skill in the art will immediately appreciate that the present invention can be utilized in a wide variety of applications to deliver agents into cells. A few particularly preferred applications are discussed in more detail here in order to highlight some of the characteristics and advantages of the inventive systems.

As discussed at length above, the present invention is particularly well adapted for delivery of nucleic acids into cells. As such, the inventive compositions are useful for various applications including gene therapy and antisense regulation. To give but a few examples of particular embodiments of nucleic acid delivery applications of the present invention, inventive compositions can be employed to introduce a gene into specific cells or tissue that will express the protein encoded by that gene and thereby correct a defect caused by a deficiency in that gene in the cells or tissue. Alternatively, inventive compositions can also be used to turn off the function of a specific gene, for example an oncogene in a tumor cell, by delivering antisense messenger RNA into a cell that will bind with the sense messenger RNA so that translation of the message and therefore expression of the protein encoded by that message will not occur.

Inventive compositions can be used in therapeutic gene delivery applications, for example to introduce "suicide genes" into cancer cells that will turn on the cell death pathway. Drug sensitivity genes can also be introduced into tumor cells. For example, cells can be genetically engineered to express prodrug activating enzyme, such as herpes simplex virus thymidine kinase, which phosphorylates ganciclovir creating toxic metabolites that kill tumor cells upon exposure to prodrug.

In the arena of immunotherapy, inventive compositions can be employed in "adoptive immunotherapy" preparations, in which genetically engineered tumor-infiltrating lymphocytes are prepared that express tumor necrosis factor and can be used to treat patients with melanoma. Immunomodulation of tumor cells to invoke an immune response directed toward specific target cell population is yet another area to which this invention can be applied.

Claim 1 of 41 Claims

What we claim is:

1. A method for the delivery of nucleic acids comprising:

forming nucleic acid containing nanospheres, wherein said nanospheres are sub-150 nm polymer spheres of which at least 50% of the size distribution of nanospheres is sub-100 nm; and

releasing said nucleic acids from said nanospheres over a period of time.

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