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Charge reversal of polyion complexes

Charge reversal of polyion complexes description/claims

This application is continuation of Application No. 09/328,975, filed Jun. 9, 1999, which claims the benefit of U.S. Provisional Application No. 60/093,153, filed Jul. 17, 1998.

The invention relates to compounds and methods for use in biologic systems. More particularly, polyions are utilized for reversing the charge (“recharging”) particles, such as molecules, polymers, nucleic acids and genes for delivery to cells.

Background Polymers are used for drug delivery for a variety of therapeutic purposes. Polymers have also been used in research for the delivery of nucleic acids (polynucleotides and oligonucleotides) to cells with an eventual goal of providing therapeutic processes. Such processes have been termed gene therapy or anti-sense therapy. One of the several methods of nucleic acid delivery to the cells is the use of DNA-polycation complexes. It has been shown that cationic proteins like histones and protamines or synthetic polymers like polylysine, polyarginine, polyornithine, DEAE dextran, polybrene, and polyethylenimine may be effective intracellular delivery agents while small polycations like spermine are ineffective. The following are some principles involving the mechanism by which polycations facilitate uptake of DNA:

Polycations provide attachment of DNA to the target cell surface. The polymer forms a cross-bridge between the polyanionic nucleic acids and the polyanionic surfaces of the cells. Polycations protect DNA in complexes against nuclease degradation. Polycations can also facilitate DNA condensation. The volume which one DNA molecule occupies in a complex with polycations is drastically lower than the volume of a free DNA molecule. The size of a DNA/polymer complex is important for gene delivery in vivo.

In terms of intravenous injection, DNA must cross the endothelial barrier and reach the parenchymal cells of interest. The largest endothelia fenestrae (holes in the endothelial barrier) occur in the liver and have an average diameter of 100 nm. The trans-epithelial pores in other organs are much smaller, for example, muscle endothelium can be described as a structure which has a large number of small pores with a radius of 4 nm, and a very low number of large pores with a radius of 20-30 nm. The size of the DNA complexes is also important for the cellular uptake process. After binding to the target cells the DNA-polycation complex should be taken up by endocytosis. Since the endocytic vesicles have a homogenous internal diameter of about 100 nm in hepatocytes and are of similar size in other cell types, DNA complexes smaller than 100 nm are preferred.

A significant number of multivalent cations with widely different molecular structures have been shown to induce condensation of DNA.

Two approaches for compacting (used herein as an equivalent to the term condensing) DNA:

1. Multivalent cations with a charge of three or higher have been shown to condense DNA. These include spermidine, spermine, Co(NH3)63+,Fe3+, and natural or synthetic polymers such as histone Hi, protamine, polylysine, and polyethylenimine. Analysis has shown DNA condensation to be favored when 90% or more of the charges along the sugar-phosphate backbone are neutralized. 2. Polymers (neutral or anionic) which can increase repulsion between DNA and its surroundings have been shown to compact DNA. Most significantly, spontaneous DNA self-assembly and aggregation process have been shown to result from the confinement of large amounts of DNA, due to excluded volume effect.

Depending upon the concentration of DNA, condensation leads to three main types of structures:

1) In extremely dilute solution (about 1 μg/mL or below), long DNA molecules can undergo a monomolecular collapse and form structures described as toroid. 2) In very dilute solution (about 10 μg/mL) microaggregates form with short or long molecules and remain in suspension. Toroids, rods and small aggregates can be seen in such solution. 3) In dilute solution (about 1 mg/mL) large aggregates are formed that sediment readily.

Toroids have been considered an attractive form for gene delivery because they have the smallest size. While the size of DNA toroids produced within single preparations has been shown to vary considerably, toroid size is unaffected by the length of DNA being condensed. DNA molecules from 400 bp to genomic length produce toroids similar in size. Therefore one toroid can include from one to several DNA molecules. The kinetics of DNA collapse by polycations that resulted in toroids is very slow. For example, DNA condensation by Co(NH3)6Cl3 needs 2 hours at room temperature.

The mechanism of DNA condensation is not clear. The electrostatic force between unperturbed helices arises primarily from a counterion fluctuation mechanism requiring multivalent cations and plays a major role in DNA condensation. The hydration forces predominate over electrostatic forces when the DNA helices approach closer then a few water diameters. In a case of DNA—polymeric polycation interactions, DNA condensation is a more complicated process than the case of low molecular weight polycations. Different polycationic proteins can generate toroid and rod formation with different size DNA at a ratio of positive to negative charge of 0.4. T4 DNA complexes with polyarginine or histone can form two types of structures; an elongated structure with a long axis length of about 350 nm (like free DNA) and dense spherical particles. Both forms exist simultaneously in the same solution. The reason for the co-existence of the two forms can be explained as an uneven distribution of the polycation chains among the DNA molecules. The uneven distribution generates two thermodynamically favorable conformations.

The electrophoretic mobility of DNA -polycation complexes can change from negative to positive in excess of polycation. It is likely that large polycations don't completely align along DNA but form polymer loops that interact with other DNA molecules. The rapid aggregation and strong intermolecular forces between different DNA molecules may prevent the slow adjustment between helices needed to form tightly packed orderly particles.

Cationic molecules with charge greater than +2 are able to condense DNA into compact structures (Bloomfield V. A., DNA condensation, (1996) Curr, Opinion in Struct. Biol., 6:334-341). This phenomenon plays a role in chromatin and viral assembly and is of particular importance in the construction of artificial gene delivery vectors. Morphologies of condensed DNA during titration of DNA with polycations are now well documented. When DNA is in excess (DNA/polycation charge ratio >1), complexes assemble into “daisy-shaped” particles that stabilized with loops of uncondensed DNA (Hansma, G. H., Golan, R., Hsieh, W., Lollo, C. P., Mullen-Ley, P. and Kwoh. D. (1998) DNA condensation for gene therapy as monitored by atomic force microscopy, Nucleic Acids Res. 26:2481-2487). When polycation is in excess (DNA/polycation ratio <1), DNA condenses completely within particles that adopt customarily toroid morphology (Tang, M. X., and Szoka, F. C., Jr. 1997, The influence of polymer structure on the interactions of cationic polymers with DNA and morphology of the resulting complexes, Gene Ther. 4:823-832). In low salt aqueous solutions the excess of polycation stabilizes these highly condensed structures and maintains them in soluble state (Kabanov A V, Kabanov V A., Interpolyelectrolyte and block ionomer complexes for gene delivery: physico-chemical aspects, Adv. Drug Delivery Rev. 30:49-60 (1998)).

Several methods can be used to determine the condensation state of DNA. They include the prevention of fluorescent molecules such as ethidium bromide from intercalating into the DNA. The condensation state of DNA was monitored as previously described (Dash, RR, Toncheva V, Schacht E, Seymour L W J. Controlled Release 48:269-276). Alternatively the condensation of fluorescein-labeled DNA (or any fluorescent group) causes self-quenching by bringing the fluorescent groups on the DNA closer together (Trubetskoy, V S, Budker, V G, Slattum, P M, Hagstrom, J E and Wolff, J A. Analytical Biochemistry 267:309-313, 1999).

As previously stated, preparation of polycation-condensed DNA particles is of particular importance for gene therapy, more specifically, particle delivery such as the design of non-viral gene transfer vectors. Optimal transfection activity in vitro and in vivo can require an excess of polycation molecules. However, the presence of a large excess of polycations may be toxic to cells and tissues. Moreover, the non-specific binding of cationic particles to all cells forestalls cellular targeting. Positive charge also has an adverse influence on biodistribution of the complexes in vivo.

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