We describe a process for generating multilayer particles comprising condensing a polymer with an oppositely charged polymer to form a particle and sequentially adding oppositely charged polymers to the particle forming at least three layers of polymers. The process is used to form a composition for delivering a biologically active compound to a cell.

SUMMARY OF THE INVENTION

This invention pertains to the formation of multilayered polyelectrolyte complexes containing various macromolecules or compounds (i.e. polymers, nucleic acids, proteins, drugs, etc.). The formation of multilayered complexes provides a method for increasing the overall amount of polymer in each individual complex. When the polymer provides a specific functionality to the complex, the increasing amount of polymer present in the successive layers allows for increased overall functionality of the complex. The inclusion of more layers (i.e. multilayering) allows for increased amounts of polymers containing functionalities comprising: ligands, membrane active compounds, endosomal lytic activity, hydrophilicity, hydrophobicity, biologically active molecules, cell targeting signals, protonateable groups, etc.

In a preferred embodiment, we describe a process for fabrication in solution of nanometer-scale multilayer complexes wherein alternating layers are composed of polyanions and polycations comprising: condensing a polyanion with a polycation in a solution to form a core complex, sequentially adding alternating solutions of polycations and polyanions, and forming the multilayer complex. Any polyion in the core complex or subsequent layers may be or may include as a component a biologically active compound or functional group. A preferred core polyanion is nucleic acid.

In a preferred embodiment, we describe a process for fabrication in solution of nanometer-scale multilayer complexes wherein alternating layers are composed of polyanions and polycations comprising: condensing a polyanion with a polycation in a solution to form a core complex, sedimenting by centrifugation the core complex through an appropriate density gradient consisting of alternating layers of polyanions, polycations and buffer in increasing concentrations of density gradient-forming solute, and forming the multilayer complex. Any polyion in the core complex or subsequent layers may be or may include as a component a biologically active compound or functional group. A preferred core polyanion is nucleic acid. A preferred density gradient is a step gradient consisting of increasing concentrations of sucrose (Example 1, FIG. 1), but other gradients that do not cause aggregation, dissociation or decondensation of the polymers or complexes is acceptable. The gradient and polyions are chosen such that the complexes of interest sediment through the gradient when centrifuged at an appropriate speed more readily than free polyions or blank particles. Blank complexes are those complexes that do not contain the core complex.

In a preferred embodiment, the process can be used to form nanometer-scale complexes that contain more polycation than is present in the core complex. In another preferred embodiment, the complex may contain more polycation than is present in the core complex but has a surface charge that is negative, i.e. a negative ζ-potential. Such particles are recharged by the addition of a polyanion as the final layer.

In a preferred embodiment, we describe a composition for delivering a biologically active compound to a cell comprising: a multilayer complex consisting of alternating layers of polycation and polycation wherein one or more of the layers is or includes as a component a biologically active compound. A preferred biologically active compound is a nucleic acid, such as DNA or RNA. In one aspect of the invention, the multilayered polyelectrolyte complexes can be used to deliver biologically active compounds to cells in vitro, in vivo, in situ or ex vivo.

In a preferred embodiment, we describe a process for delivering a biologically active polyanion to a cell comprising: condensing the biologically active polyanion with a polycation in a solution to form a core complex, sedimenting by centrifugation the core complex through a density gradient consisting of alternating layers of polyanion, polycation and buffer in increasing concentrations of solute, forming a multilayer complex, collecting the multilayer complex, associating the multilayer complex with a cell, and delivering the biologically active polyanion to the cell. A preferred biologically active polyanion is nucleic acid. The multilayer complex may have a positive or negative ζ-potential or surface charge. The multilayered polyelectrolyte complexes can be used to deliver biologically active compounds to cells in vitro, in vivo, in situ or ex vivo.

In a preferred embodiment, centrifugation of condensed DNA complexes through a sucrose step gradient consisting of alternating polycation-containing and polyanion-containing layers results in fabrication of multilayered electrostatic assemblies with condensed DNA at the core and does not contain the admixture of non-DNA-containing blank complexes. Other density gradients and density gradient-forming solutes may also be used.

In a preferred embodiment, a process is described for delivering a complex to a mammalian cell, comprising: forming a complex having multiple alternating layers of polyanion and polycation and inserting the complex into a mammal.

In a preferred embodiment, a polyion may be modified with neutral hydrophilic polymers for steric stabilization of the whole complex.

In another preferred embodiment, a polyion layer in a complex may be covalently or noncovalently attached to the same or a different polyion layer in the complex in order to crosslink the layers. Bifunctional molecules may be added that crosslink the complex. A polyanion may contain groups that covalently or noncovalently attach to a polycation. Alternatively, a polycation may contain groups that covalently or noncovalently attach to a polyanion. Crosslinking can occur during formation of the multilayer complex or after formation of the multilayer complex.

DETAILED DESCRIPTION OF THE INVENTION

We describe methods for the fabrication of multilayer polyelectrolyte complexes (particles). In one method, a core complex is first generated by condensation of a polyion with a polyion of opposite charge in a low salt buffer such as water. The amounts of polyions are chosen so that small, <100 nm, particles are formed. The precise amounts are determined empirically for different polyions but are typically related to charge density of the polyions (charge per molecular weight). Successive layers of alternating polyanion and polycation are then added sequentially to the complex. The amounts of polyion are chosen such that upon addition of each layer the particles remain small, <100 nm, and the surface of the complex is recharged without causing aggregation (see example 5). Polyanions of lower charge density, such as succinylated PLL and poly(glutamic acid), do not decondense DNA in DNA/polycation complexes, even when added in 20-fold charge excess to polycation. Further studies have found that displacement effects are salt-dependent. In the absence of salt such the complexes may exist indefinitely. Measurement of the ζ-potential of DNA/PLL particles during titration with SPLL revealed the change of particle surface charge at approximately the charge equivalency point. Using this method, the surface charge of a polyelectrolyte complex can be reversed and thus the complex can be "recharged".

In another method a core complex is first fabricated as described above, for example with DNA and lPEI. This core complex is then applied to the top of a density gradient wherein the gradient contains alternating layers of polyanions and polycations in increasing concentrations of a density gradient-forming solute, such as sucrose (FIGS. 1 and 2). The solute is chosen such that its presence does not cause aggregation or dissociation of the complexes. This method takes advantage of the higher density of condensed nucleic acid-containing particles compared with polyanion/polycation complexes that do not contain nucleic acid. The DNA-containing particles sediment through the gradient during centrifugation at appropriate rpm while particles that do not contain DNA, blank particles, float in the gradient. The bottom layer of the gradient may be a cushion, such a 40% metrizamide, such that the DNA-containing particles float on the surface of the metrizamide and are not pelleted in the bottom of the tube. This method allows the fabrication of nanometer-scale multilayer complexes that contain the core complex, without contamination of excess blank particles.

By forming multilayer particles, more of a given polyion—or functional molecule attached to a polyion—is incorporated into the complex. This quality can improve delivery of a biologically active compound to a cell. For instance, multilayer DNA/lPEI particles—which contain more lPEI within the complex—are more efficient transfection vectors than compositions that contain similar amounts of lPEI, but with less lPEI incorporated into DNA-containing complexes and more excess lPEI free in solution.

Multilayer particles with a negative surface charge (i.e. complexes in which the outermost layer is composed of polyanions), may be used to enhance delivery. Negative surface charge on the particles may reduce non-specific interactions with cells and serum proteins [Wolfert et al. Hum. Gene Therapy 7:2123-2133 (1996); Dash et al., Gene Therapy 6:643-650 (1999); Plank et al., Hum. Gene Ther. 7:1437-1446 (1996); Ogris et al., Gene Therapy 6:595-605 (1999); Schacht et al. Brit. Patent Application 9623051.1 (1996)]

A wide a variety of polyanions can be used to recharge the DNA/polycation particles comprising: succinylated PLL, succinylated PEI (branched), polyglutamic acid, polyaspartic acid, polyacrylic acid, polymethacrylic acid, polyethylacrylic acid, polypropylacrylic acid, polybutylacrylic acid, polymaleic acid, dextran sulfate, heparin, hyaluronic acid, polysulfates, polysulfonates, polyvinyl phosphoric acid, polyvinyl phosphonic acid, copolymers of polymaleic acid, polyhydroxybutyric acid, acidic polycarbohydrates, DNA, RNA, negatively charged proteins, pegylated derivatives of above polyanions, pegylated derivatives carrying specific ligands, block and graft copolymers of polyanions, any hydrophilic polymers (PEG, poly(vinylpyrrolidone), poly(acrylamide), etc), and other water-soluble polyanions

Any polyion in the multilayer complex may be cleavable or labile. Cleavable means that a chemical bond between atoms is broken. Labile also means that a chemical bond between atoms is breakable.

Polyions within the complex may also be crosslinked to enhance stability of the complex or to enable attachment of a ligand or signal or other functional group to be attached. Crosslinking refers to the chemical attachment of two or more molecules with a bifunctional reagent. A bifunctional reagent is a molecule with two reactive ends. The reactive ends can be identical as in a homobifunctional molecule, or different as in a heterobifucnctional molecule. A polyion in a complex can be covalently or noncovalently attached to another polyion in the complex using a variety of chemical reactions.

The multilayer particles may be used to delivery a biologically active compound to a cell that is in vitro or in vivo. The biologically active compound is delivered to a cell if the biologically active compound becomes associated with the cell. The biologically active compound may can be on the membrane of the cell or inside the cytoplasm, nucleus, or other organelle of the cell.

For delivery in vivo, biologically active compound-containing multilayer particles may be delivered intravasculary, intrarterially, intravenously, orally, intraduodenaly, via the jejunum (or ileum or colon), rectally, transdermally, subcutaneously, intramuscularly, intraperitoneally, intraparenterally, via direct injections into tissues such as the liver, lung, heart, muscle, spleen, pancreas, brain (including intraventricular), spinal cord, ganglion, lymph nodes, lymphatic system, adipose tissues, thyroid tissue, adrenal glands, kidneys, prostate, blood cells, bone marrow cells, cancer cells, tumors, eye retina, via the bile duct, or via mucosal membranes such as in the mouth, nose, throat, vagina or rectum or into ducts of the salivary or other exocrine glands. Intravascular herein means within a tubular structure called a vessel that is connected to a tissue or organ within the body. Within the cavity of the tubular structure, a bodily fluid flows to or from the body part. Examples of bodily fluid include blood, lymphatic fluid, or bile. Examples of vessels include arteries, arterioles, capillaries, venules, sinusoids, veins, lymphatics, and bile ducts. The intravascular route includes delivery through the blood vessels such as an artery or a vein. An administration route involving the mucosal membranes is meant to include nasal, bronchial, inhalation into the lungs, or via the eyes.
 

Claim 1 of 18 Claims

1. A process for delivering a polynucleotide to a cell comprising:

a) condensing a first polyanion with a polycation to form a particle;

b) adding a second polyanion to the particle to form a complex wherein at least one of the polyanions is the polynucleotide;

c) crosslinking the second polyanion to the polycation; and,

d) contacting the cell with the complex.
 

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