ES2371647T3 - ENDOSOMOLYTIC POLYMERS. - Google Patents

Abstract

A nanoparticle for delivering a polynucleotide to a cell comprising: said polynucleotide associated with a polymer with reversibly inhibited membrane activity, wherein said polymer with reversible inhibited membrane activity consists of a plurality of inhibitors with membrane activity, bound together reversible to a polyamine with membrane activity through a pH-labile bond, where said inhibitors are selected from the group consisting of: disubstituted maleic anhydride derivatives, and wherein the nanoparticle additionally comprises a polycation.

Description

Endosomolytic polymers

Background of the invention

The current paradigm of the development of non-viral vectors for DNA delivery is to present a viral unit and a gene transfer by incorporating, in a combinatorial way, functional groups that allow a particular unit and some stages for transfer. Cationic polymers or lipids are used to condense DNA into small virus-like particles. This condensation stage is considered important for several reasons: a) DNA protection against inactivation through blood components, b) DNA protection against degradation with extracellular nucleases, c) particle extravascularization through small holes (fenestrations) in the endothelial barrier (for intravascular administration routes), and d) cell endocytosis. Functional groups are incorporated into synthetic vectors to improve cell target localization, endosomal escape and nuclear DNA target localization, to be delivered. These signals include ligands on the cell surface, designed to direct the vector to a particular type of cell and / or improve endocytic uptake of the particle by adsorption or through a receptor. The vector may also contain molecules designed to improve the release of the phagocytized DNA particle, within the cell cytoplasm. Although the components of a DNA delivery vehicle are known in theory, the creation of an effective non-viral delivery vector has been difficult in practice. Cationic polymers or lipids, which are suitable for the condensation of DNA, tend to be toxic or have a low biodistribution. Similarly, compounds that may possess good endosomal disruption activity are also often toxic.

Although polymers and cationic lipids are essential for condensing DNA into nanoparticles, its cationic nature limits its most extensive utility for in vivo applications, not only because of low gene expression, but also toxicity. The intravascular route of administration, an attractive route for delivery with wide diffusion, is particularly affected by toxicity, as well as biodistribution problems. A decrease in the efficiency of transfection in vivo is due in part to the interaction of polyplejos or lipoplejos (polymer-DNA and liposome-DNA complexes) with blood components, such as serum proteins that inhibit transfection. This effect is generally attributed to the opsonization of DNA complexes by serum components. In addition, cationic DNA complexes injected intravenously also encounter unwanted cell types, such as macrophages, monocytes, neutrophils, platelets and erythrocytes, which are important potential mediators of toxicity. Toxic manifestations of systemically administered cationic DNA complexes can range from a red blood cell agglutination to a strong inflammatory reaction and elevated serum levels of liver enzymes. Several studies have tried to avoid such adverse interactions, by including polyethylene glycol (PEG) or proteins, such as albumin or transferrin, in DNA complexes. Another proposed method to decrease the charge of a condensed DNA particle in a polycation and, therefore, to reduce the interaction with the serum components, is to recharge the DNA / polycation complex by the addition of a polyanion. Alternate complexions of polycations and polyanions form layered structures when absorbed onto macrosurfaces of aqueous solutions. It has been shown that a similar phenomenon occurs on the surface of DNA particles condensed with polycations, when they form additional complexes with a third layer polyanion (US Patent Application Document Serial No. 09/328975, incorporated in this report as a reference).

The liver is one of the most important target tissues for gene therapy, given its fundamental role in metabolism (for example, lipoprotein metabolism in various hypercholesterolemias) and in the secretion of circulating proteins (for example, coagulation factors in the hemophilia). At least one hundred different genetic disorders could be corrected, at least partially, with a gene therapy directed towards the liver. Its cumulative frequency is approximately one percent of all births. In addition, acquired disorders such as chronic hepatitis and cirrhosis are common and could also be treated with polynucleotide-based liver therapies. Gene therapies that involve the expression of heterotopic genes would further expand the number of treatable disorders by gene transfer directed to the liver. For example, diabetes mellitus could be treated by expressing the insulin gene in hepatocytes, whose physiology can allow glucose-regulated insulin secretion. Gene therapy encompasses the deliberate delivery of genetic material to cells in order to treat a disease, as well as for study or biomedical research. The research can be used to study gene function or to facilitate drug discovery or validation.

Although viral vectors are the basis of most preclinical studies and clinical trials in humans, for the delivery of DNA to liver cells, non-viral pathways continue to advance. Poliplejos, lipoplejos and lipopoliplejos have all been proposed as delivery vectors in the liver. Most of the studies of non-viral transfer in the liver have used polyplejos that normally contain poly-L-lysine or PEI and ligands for the receptor of asialoglycoprotein (ASGPr). Liposomes and lipopoliplejos have also been described for gene transfer in the liver. We are oriented to develop DNA nanoparticles that are better to save two decisive stages: pass through the circulatory system to access hepatocytes and release their genetic load from endosomes. The particles may contain ligands to improve target localization in hepatocytes and absorption.

Summary of the Invention

This report describes labile polymers as a function of pH, reversibly modified, particles for the delivery of polynucleotides containing said polymers and methods for the generation of said polymers and particles. The described particles incorporate endosomolytic chemicals and nanotechnologies to assemble nanoparticles capable of delivering polynucleotides to cells from the peripheral circulation, with subsequent release from the endosomes.

In a preferred embodiment, compounds and particles are described which incorporate said compounds to release molecules from an endosome into the cytoplasm of a cell. The compounds comprise reversibly inhibited polymers with membrane activity that undergo selective chemical cleavage events in the acidic medium of the endosome. Before entering the endosome, the activity of the polymer with membrane activity is inhibited by the covalent and reversible fixation of an inhibitor. After exposure to acidic conditions, such as in an endosome or in an acidified lysosome, the bond that binds the inhibitor to the polymer is broken, exposing the membrane's membrane activity.

In a preferred embodiment, nanoparticles for delivering a polynucleotide to a cell are described. The particles comprise a polyanion ionically associated with a binary polynucleotide / polycation complex. Polycation, polyanion, or both, consist of a polymer with membrane activity. A polycation with membrane activity can be reversibly inhibited. The polyanion can be a polyamine with membrane activity, reversibly modified. The polyanion recharges the complex to reduce the positive charge of the binary complex or to increase the negative charge of the nanoparticle. The nanoparticle can be positively charged, negatively charged or neutrally charged. The particle may contain a plurality of polynucleotides, polycations or polyanions. To increase the stability of the particle, polycations and polyanions of molecular weight greater than about 10,000 Daltons are preferred. The stability of the particles can be further increased by the cross-linking of the polyanion with the polycation.

In a preferred embodiment, methods for the formation of nanoparticles competent for transfection are described, comprising: condensation of a polynucleotide with a polycation to form a binary complex and recharge of the binary complex by adding a polyanion to form a complex small ternary (<150 nm), stable, containing a negatively charged polynucleotide. Polycation, polyanion or both consist of polymers with membrane activity. A polycation with membrane activity can be reversibly inhibited. The polyanion can be a polymer with reversibly modified / inhibited membrane activity. The particle may contain a plurality of polynucleotides, polycations or polyanions. To increase the stability of the particle, polycations and polyanions of molecular weight greater than about 10,000 Daltons are preferred. The stability of the particles can be further increased by cross-linking the polyanion with the polycation.

In a preferred embodiment a method is described for the delivery of a molecule in the cytoplasm of the cell comprising: the association of the molecule with a polymer with membrane activity that is reversibly inhibited to form a complex and the delivery of the complex to the cell, where the complex is phagocytic. Before entering the endosome, the membrane's membrane activity is masked by the covalent fixation of an inhibitor. The selective chemical cleavage of the polymer inhibitor (s) with membrane activity occurs in the acidic medium of the endosome, restoring the activity of the polymer. The rupture of the endosomal membrane then facilitates the release of the molecule in the cytoplasm of the cell. Endosomal release is of paramount importance for the supply of a wide variety of molecules that are unable to diffuse through cell membranes.

In a preferred embodiment, large acid-cleavable polyanions are described that have strong endosomolytic activity and that can recharge complexes containing cationic polynucleotides, and methods for the synthesis of large acid-cleavable polyanions. The negatively charged nanoparticles are small and stable in physiological saline and have transfecting activity.

Other objects, features and advantages of the invention will be apparent from the following detailed description, together with the accompanying drawings.

Brief description of the drawings

Fig. 1. Illustration of a reversible chemical modification, labile as a function of pH, of an amine-containing molecule with a maleic anhydride derivative, to form a maleamate followed by excision in the acid medium of the modification. For monosubstituted maleic anhydrides, R1 or R2 is hydrogen and R2 or R1 is linked to the anhydride through a carbon-carbon bond. For disubstituted maleic anhydrides, R1 and R2 are attached to the anhydride through carbon-carbon bonds.

Fig. 2. Illustration of a reversible chemical modification, labile as a function of pH, of an amine-containing molecule, with the derivative of CDM disubstituted maleic anhydride (2-propionic-3-methylmaleic anhydride).

Fig. 3. Illustration of the synthesis of polyvinyl ether amphiphilic polycations.

Fig. 4. Illustration of the reversible masking of the membrane activity of the PBAVE polymer through labile fixation as a function of pH, of inhibitors, to the polymer.

Fig. 5. Illustration of labile cross-linking as a function of pH of two molecules containing amines using CDM-thioester.

Fig. 6. Illustrations of the chemical structures of CDM-PEG, CDM2-PEG and CDM3-PEG.

Detailed description

We have developed a strategy for the endosomal release of membrane impermeable molecules. This strategy involves the reversible inactivation of an agent with membrane or membrane lysis activity. Reversible inactivation of the agent with membrane activity is achieved by attaching an inhibitor or a variety of inhibitors, to the agent with membrane activity through a bond or a variety of bonds that break in the acidic medium of an endosome. The inhibitor prevents the agent from lysing the cytoplasmic membrane and therefore causing cell death. The inhibitor is removed from the agent in the acidic medium of the endosome, by breaking a labile bond, thus allowing the agent with membrane activity to destroy the endosomal membrane so that the release of endosomal contents in the cytoplasm is performed.

A key element to limit the membrane activity in the endosome is the labile bond, which must be stable under extracellular conditions, but very unstable in the endosomal vesicle. In particular, we have focused on identifying links that break in an acidic medium. Acidification is a characteristic of the endosomal environment that is generally exploited by viral and non-viral delivery vehicles. Protonation-dependent agents to become active for the membrane, such as polypropyl acrylic acid and the peptide derivatives of the viral envelope protein, hemagglutinin, have a serious defect. Activation of the agent causes partial destruction of the endosome, thereby destroying the pH gradient and leading to the inactivation of the agent with membrane activity. This cycle may limit the effectiveness of the agent with membrane activity in the delivery of macromolecules in the cell cytoplasm. On the contrary, the invention, as described herein, results in the essentially irreversible reactivation of agents with membrane activity, after exposure to a medium with acidic pH.

An important consideration in the selection of labile bonds for use in cellular delivery systems is the kinetics of the link rupture after exposure of the bond at an acidic pH. The kinetics of endosome acidification and endosome maturation in a lysosome are very fast compared to the cleavage rates for most labile bonds in acid medium, described in the literature. Once endocytosis occurs, the pH drops from the extracellular pH (about 7.4) to a pH of approximately 5, in about 10 min. Endosomal contents are rapidly exposed to active lysosomal enzymes and degradation of the molecule to be delivered can take place. Therefore, bonds that break in a few minutes in the pH range 5-7 are preferred.

A well-studied bond that is unstable as a function of pH is the maleamate bond, which is obtained from the reaction of an amine and a maleic anhydride or a maleic anhydride derivative (Figure 1). The maleamate cleavage rate depends on the structure of the maleic anhydride used to form the maleamate. In general, disubstituted maleamates are more labile than monosubstituted maleamates, which are more labile than unsubstituted maleamates. Monosubstituted maleamates are the most studied members of this family, and have half-lives of hours at a pH <5. According to the bibliography, maleamate disubstitution results in an increase of approximately two orders of magnitude of the cleavage rate. We have found that the disubstituted maleamate bond obtained from dimethylmaleic anhydride (R1 and R2 = CH3 in Fig. 1) has a half-life of approximately 2 minutes at pH 5. This rate is in the same order as endosome maturation. On the contrary, we have observed that the monosubstituted maleamate bonds obtained from methylmaleic anhydride (R1 or 2 = H and R2 or 1 = CH3 in Fig. 1), have a half-life of excision of approximately 300 minutes (5 hours) at pH 5. To increase the loading and solubility, derivatives of dimethylmaleic anhydrides, such as 2-propionic-3-methylmaleic anhydride (Naganawa et al. 1994; Carboxylated DimethylMaleic anhydride or CDM) (Fig. 2) can be used.

The ability of a disubstituted maleic anhydride to reversibly inhibit the membrane activity of the melitin peptide, until reaching the acidic medium of the endosome, has been described by us (Rozema et al. 2003). We have demonstrated the ability of inhibited melitin reversibly to deliver membrane impermeable molecules, polyethylene glycol and an oligonucleotide, in the cytoplasm of the cell. In these delivery examples, the delivery reagent (CDM modified melitin) and the compound were not connected or associated with each other, but were delivered independently to common endocytic compartments in the cell. For the delivery of membrane impermeable molecules, in the cytoplasm of cells in vivo, there must be an association between the molecule and the delivery agent. We now provide agents with membrane activity that may be non-covalently associated with the membrane impermeable molecule or covalently bound thereto, for delivery of the molecule in the cytoplasm of a cell.

DNA particle recharge:

The DNA can be condensed with excess polycation in aqueous solutions, to form nanoparticles with positive surface charge. This phenomenon is fundamental, not only for the chromatin and virus unit, but it is also important in the construction of vehicles for gene delivery. The positive charge surplus contained in a complex of condensed DNA and polycation can be used to deposit a layer of polyanions on the surface of the DNA / polycation complex, which results in negatively charged particles (or complexes) in a process called recharge (US Patent Application Document Serial No. 09 / 328,975). Negatively charged particles can reduce the non-specific interactions that cationic particles have with whey proteins, cell surfaces and extracellular matrix. Recharging is a two stage process. In the first stage, the DNA or other polynucleotide is condensed by the addition of an excess of polycation to form a positively charged polynucleotide nanoparticle. Typical polynucleotide delivery formulations stop at this point and add the nanoparticle to the cell. In the recharge process, a third polyion (a polyanion) is added to the positively charged polycation / polynucleotide particle, to form a ternary complex that has a neutral to negative surface charge. Under suitable formulation conditions, the particles are small (<150 nm), and are called nanoparticles.

The negatively charged complexes must be better able to circulate and target a specific cell in vivo, by reducing non-specific interactions with negatively charged cell surfaces, whey proteins and the extracellular matrix.

Polymers with membrane activity for particle formulation with polynucleotides:

In order for the agent with membrane activity, reversibly masked to facilitate the delivery of polynucleotides or other molecules impervious to the membrane, to the cells, the agent with masked membrane activity must be associated with the molecule. Small agents with low overall charge membrane activity, such as the lytic membrane peptide, melitin, can form particles with polynucleotides. However, these particles are large (> 150 nm) and unstable (that is, they increase in size, in the presence of physiological salt concentrations). Larger polymers with membrane activity can be used to form small, stable particles with polynucleotides. Previously we have synthesized polymers with membrane activity composed of amines and alkyl groups, through the copolymerization of several alkyl vinyl ethers, with a monomer with the protected amine (polyvinyl ether amphiphilic polycations; Fig. 3 and the patent application document U.S. Serial No. 10 / 772,502, incorporated herein by reference). By way of example, a 50:50 mixture of alkyl groups and amines produces polymers containing ethyl (PEAVE), propyl (PPAVE) and butyl (PBAVE) groups, using trifluoride etherate as the initiator. Deprotection of the phthalimide amine protecting groups results in water soluble polymers with a molecular weight of approximately 20,000 Daltons.

The PBAVE polymer containing butyl was found to be approximately 60% as hemolytic as melitin, when the lytic activity of the red blood cells was tested. Reversible inhibition of PBAVE can be achieved by modifying CDM. Incubation of the modified polymer at pH 5, restored the lytic capacity with a half-life of approximately 10 minutes. Therefore, the membrane activity of the PBAVE polymer can be controlled by modifying the polymer with CDM. Under basic conditions, the polymer does not produce lysis of the membrane. After acidification, the CDM inhibitor is cleaved from the polymer and the membrane membrane activity is restored (Fig. 4). The endosomolytic activity of CDM-PBAVE is demonstrated by its ability to deliver a polynucleotide to cells (see example 5 below). CDM and CDM derivatives can be used to modify any polymer with membrane activity that contains amine.

Recharge of nanoparticles with polymers with reversible inhibited membrane activity:

In addition to masking the membrane activity of an amine-containing polymer, the modification of a polymer with the maleic anhydride derivative CDM, additionally reversibly converts the positive charges in the polymer into negatively charged carboxyl groups. Therefore, a polycation can become a polyanion. After the condensation of a polynucleotide with a first polycation to form a binary complex or a smaller particle, a polyanion can then be used to recharge the binary complex and form a complex or ternary particle that has a less positive surface charge or more negative than the binary complex. In the recharge of a particle containing a polynucleotide, with a second polycation modified with CDM, the recharge layer is labile in an acid medium.

Exposure of this charged nanoparticle to acidic conditions results in the cleavage of the CDM groups of the polyanion, with the consequent loss of the negative charge of the recharge polymer. The reversion of polyanion to a polycation with membrane activity (second polycation) can have several effects that include: destabilization of the particle, the release of the agent with membrane activity in the endocytic vesicle and increased interaction of the first polycation with the membrane of the endocytic vesicle. The first polycation can also be a polymer with membrane activity and could be of the same species as the second polycation with membrane activity. Rupture of the endosome by means of the polymer (s) with membrane activity, results in the delivery into the cytoplasm of the polynucleotide or another molecule originally present in the recharged particle.

Transfection with small nanoparticles, negatively charged, unstable against acid:

It has been shown that endosomolysis can be achieved by reversibly modifying a peptide with membrane activity, such as melitin, with maleic anhydride derivatives (Rozema et al. 2003; U.S. Patent Application Document Serial No. 10 / 444.662). The ability of CDM-melitin to deliver polyethylene glycol macromolecules and an oligonucleotide analog without charge has been shown. However, in order to incorporate masked agents with membrane activity into polynucleotide delivery vectors, we synthesize polymers of sufficient size and charge to be formulated in the form of stable nanoparticles containing polynucleotides. As an example, PBAVE polycation was synthesized and it was shown that it had membrane activity and the ability to form small, stable particles with DNA. Masking of the membrane activity of PBAVE by reaction with CDM, resulted in a polyanion that can be used to recharge DNA / polycation particles to produce small, negatively charged and unstable nanoparticles in acidic medium.

The nanoparticles composed of DNA: PBAVE: CDM-PBAVE were formulated in a weight ratio of 10:20:80 and applied to mouse liver cell cultures (Hepa-1c1c7) in tissue culture, in the presence of DMEM and 10 % serum The DNA used in the delivery formulations was pCILuc, which contains a gene encoding luciferase. The transfection capacity of the complexes was determined by measuring the relative luciferase light units, produced by cells that had been treated with nanoparticles containing pCILuc. As a control for the polymer with reversibly inhibited membrane activity (CDM-PBAVE), the particles were also constructed using PBAVE-succinylated (PBAVE-S) and cis-aconitylated PBAVE (PBAVE-A). Cisaconitic anhydride is a monosubstituted derivative of maleic anhydride having a carboxylate substituent (CH2CO2H) in maleic anhydride. Succinylation is irreversible and cis aconitilation is cleaved with a half-life of approximately 300 minutes at pH 5.

There is a dependence on transfection, on the lability of the group used to modify / inhibit the agent with PBAVE membrane activity. Membrane activity polymers, reversibly masked, CDM-PBAVE and PBAVE-A were able to transfect cells, while the irreversibly modified polymer (PBAVE-S) was inactive. In addition, nanoparticles containing CDM-PBAVE (disubstituted maleamate bonds) had 30 times more transfecting activity than nanoparticles formed with PBAVE-A (monosubstituted maleamate bonds). The increase in the transfection capacity of the particles containing CDM-PBAVE is more likely related to the greater lability of the maleic anhydride derivative disubstituted with CDM, with respect to the cis-aconitic monosubstituted maleic anhydride derivative. Similar results are expected for other polymers with membrane activity that contain amine.

Particle stability increased by covalent bonding between polymers:

In addition to the stability of the particles due to electrostatic forces between polycation and polyanion, the stability of the particle can also be improved by the formation of covalent bonds, that is, crosslinking between the polymers. However, irreversible cross-linking of polycation and polyanion results in particles that are ineffective for the delivery of biologically active nucleic acids. In order to provide the particles with crosslinking stability, while still providing the intracellular instability to the particles, the polycation and polyanion of a nanoparticle can be covalently linked through a plurality of unstable maleamate bonds in acidic medium. In order to couple a CDM-based polyanion with a polyamine, it is necessary to use a cross-linking group that can react with amines only after the anhydride has reacted to form the CDM-based maleamate group. This selectivity of the reaction is necessary because both the formation of the maleamate and the cross-linking between polyanion and polycation, involve reactions with amines. As a consequence, to selectively couple a CDM-based polyanion and a polyamine, there must be selectivity in the reactions of the amine. One method of carrying out this selectivity is to provide, on a CDM derivative, a functional cross-linking group that is less reactive than the anhydride group involved in the formation of maleamate. An example of such a functional group is a thioester. A thioester is moderately reactive with an amine in relation to an anhydride. By using a thioester derivative of CDM, it is possible to connect two amines to each other, through an unstable maleamate bond as a function of pH (Fig. 5).

In addition to the maleamate bond, other unstable pH-dependent bonds can be incorporated into cross-linking reagents that include acetals, enol ethers and hydrazones. In particular, acetals obtained from benzaldehyde and benzaldehyde derivatives are very unstable depending on the pH.

Increase in particle stability by covalently fixing polyethylene glycol:

In addition to increasing stability in the presence of a salt, the location of targets with particles in vivo requires the reduction of non-specific interactions, with serum components and with non-target cells. In order to reduce such interactions with delivery vehicles, many researchers have fixed polyethylene glycol (PEG) (Kircheis et al. 2001; Woodle et al. 1992), a water-soluble polymer without charge, to particles containing nucleic acid. However, PEG also decreases the transfection capacity of the particles. In order to obtain the benefits of PEGylation, while maintaining the transfection capacity, a series of PEGylation reagents obtained from dimethylmaleic anhydride have been synthesized. The fixation of a plurality of dimethylmaleic anhydride groups to a single group of PEG allows the formation of a plurality of reversible covalent bonds, thereby increasing the stability of a particle with the particle (Fig. 6). A plurality of PEG groups can be covalently fixed to a particle.

Membrane activity - Polymers or compounds with membrane activity are molecules that are capable of altering the structure of the membrane. This change in structure can be observed with the compound, which induces one or more of the following effects on a membrane: an alteration that allows the permeability of small molecules, the formation of pores in the membrane, a fusion and / or fission of membranes, an alteration that allows the permeability of large molecules, or a dissolution of the membrane. This alteration can be functionally defined by the activity of the compound in at least one of the following tests: red blood cell lysis (hemolysis), liposome leakage, liposome fusion, cell fusion, cell lysis and endosome delivery.

Polymer - A polymer is a molecule formed by the repetitive binding of smaller units called monomers. A polymer can be linear, branched, star, comb or ladder type. A polymer can be a homopolymer in which a single monomer is used or it can be a copolymer in which two or more monomers are used.

The main chain of a polymer is composed of atoms whose bonds are necessary for the propagation of the length of the polymer. For example, in poly-L-lysine, carbonyl carbon, carbon-a and a-amine groups are necessary for the length of the polymer and therefore are atoms of the main chain. The side chain of a polymer is composed of atoms whose bonds are not necessary for the propagation of the length of the polymer. For example, in poly-L-lysine, carbons �, and, 5 and E, and nitrogen E, are not necessary for the propagation of the polymer and, therefore, are side chain atoms.

Polycation - A polycation can be a polymer that has a net positive charge, for example, poly-L-lysine hydrobromide or a histone. The polymeric polycation may contain monomer units that are positively charged, neutrally charged or negatively charged, however, the net polymer charge must be positive. A polycation can also be a non-polymeric molecule that contains two or more positive charges.

Polyanion - A polyanion can be a polymer that contains a net negative charge, for example, poly (glutamic acid). The polymeric polyanion may contain units of monomers that are negatively charged, neutral charged or positively charged, however, the net polymer charge must be negative. A polyanion can also be a non-polymeric molecule that contains two or more negative charges.

Other components of monomers and polymers: polymers can have functional groups that enhance their usefulness. These groups can be incorporated into the monomers before the formation of polymers or they can be fixed to the polymer after their formation. Functional groups can be selected from the list consisting of: target locator groups, interaction modifiers, steric stabilizers and compounds with membrane activity, affinity groups and reactive groups.

Target locator groups - Target locator groups or ligands are used to locate the target with the polymer or with complex polymers in cells, in specific cells, in tissues or in specific places in a cell. Target locator groups improve the association of molecules with a cell. Examples of target locator groups include those that target the asialoglycoprotein receptor target, using asialoglycoproteins or galactose residues. Other proteins, such as insulin, EGF or transferrin can be used for the localization of a target. Other target locator groups include molecules that interact with membranes, such as fatty acids, cholesterol, dansyl compounds and amphotericin derivatives. A variety of ligands have been used to direct drugs and genes to specific cells and cell receptors. The ligand can search for a target within the cell membrane, on the cell membrane or near a cell. Binding of a ligand with a receptor can initiate endocytosis.

Steric Stabilizer - A steric stabilizer is a long chain hydrophilic group that prevents aggregation of the final polymer by means of a particle that sterically prevents electrostatic interactions of particles. Some examples include: alkyl groups, PEG chains, polysaccharides, hydrogen molecules, alkyl amines.

Interaction Modifier - An interaction modifier changes the way a molecule interacts with itself or with other molecules, relative to the molecule that does not contain any interaction modifier. The result of this modification is that self-interaction or interactions with other molecules increase

or decrease. For example, the signals for the localization of cellular targets are modifiers of the interaction with a change in the interaction between a molecule and a cell or a cellular component. Polyethylene glycol is an interaction modifier that decreases interactions between molecules and with themselves and with other molecules.

An unstable joint is a chemical compound that contains an unstable bond and provides a joint or spacer between two other groups. The groups that are attached can be chosen from compounds such as biologically active compounds, compounds with membrane activity, compounds that inhibit membrane activity, reactive functional groups, monomers and localizing signals of a cell target. The spacer group may contain chemical moieties chosen from a group that includes alkanes, alkenes, esters, ethers, glycerol, amide, saccharides, polysaccharides and heteroatoms, such as oxygen, sulfur or nitrogen. The spacer can be electronically neutral, can have a positive or negative charge, or can have positive and negative charges with a neutral, positive or negative total charge.

Labile as a function of pH refers to the selective rupture of a covalent bond under acidic conditions (pH <7). That is, the unstable pH-dependent bond can be broken under acidic conditions in the presence of other covalent bonds that do not break. The labile expression as a function of pH includes both the junctions and the bonds that are unstable as a function of pH, very unstable as a function of pH and extremely unstable as a function of pH.

Target locator groups - Target locator groups or ligands are used to locate the target of the polymer or complex polymers in cells, in specific cells, in tissues or in specific places in a cell. Target locator groups improve the association of molecules with a cell. Examples of target locator groups include those that target the asialoglycoprotein receptor target, using asialoglycoproteins or galactose residues. Other proteins, such as insulin, EGF or transferrin can be used for the localization of a target. Other target locator groups include molecules that interact with membranes, such as fatty acids, cholesterol, dansyl compounds and amphotericin derivatives. A variety of ligands have been used to direct drugs and genes to specific cells and cell receptors. The ligand can search for a target within the cell membrane, on the cell membrane or near a cell. Binding of a ligand with a receptor can initiate endocytosis.

Polynucleotide - The term polynucleotide, or nucleic acid or poly (nucleic acid), is a technical term that refers to a polymer that contains at least two nucleotides. Nucleotides are the monomer units of polynucleotide polymers. Polynucleotides with less than 120 monomer units are often referred to as oligonucleotides. Natural nucleic acids have a main chain of deoxyribose or ribose phosphate. An artificial or synthetic polynucleotide is any polynucleotide that is polymerized in vitro or in a cell-free system and that contains the same or similar bases, but may contain a backbone of a type other than the natural ribose phosphate main backbone. These main chains include: PNAs (peptide nucleic acids), phosphorothioates, phosphorodiamidates, morpholinos and other variants of the phosphate main chain of natural nucleic acids. The bases include purines and pyrimidines, which also include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine and natural analogs. Synthetic derivatives of purines and pyrimidines include modifications, but are not limited thereto, that place new reactive groups, such as, but not limited to, amines, alcohols, thiols, carboxylates and alkyl halides. The term "base" encompasses any of the known analogs of DNA and RNA bases. The term polynucleotide includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and combinations of DNA, RNA and other natural and synthetic nucleotides.

A polynucleotide can be delivered to a cell to express an exogenous nucleotide sequence, to inhibit, eliminate, increase or alter the expression of an endogenous nucleotide sequence, or to affect a specific physiological characteristic that is not naturally associated with the cell.

A polynucleotide-based gene expression inhibitor comprises any polynucleotide that contains a sequence whose presence or expression in a cell causes degradation or inhibits the function, transcription or translation of a gene in a specific sequence form. Polynucleotide-based expression inhibitors can be selected from the group comprising: siRNA, microRNA, interfering RNA or RNAi, RNAs, ribozymes, non-coding polynucleotides and DNA expression cassettes encoding siRNA, microRNA, RNAds, ribozymes or non-coding nucleic acids. The siRNA comprises a double stranded structure that typically contains 15-50 base pairs and, preferably, 19-25 base pairs, and that has a nucleotide sequence identical to or almost identical to that of a gene or a target RNA expressed within the cell. An siRNA can be composed of two reassociated polynucleotides or a single polynucleotide that forms a hairpin structure. MicroRNAs (miRNAs) are small, non-coding polynucleotides, approximately 22 nucleotides in length, that direct destruction or repress the translation of their mRNA targets. Non-coding polynucleotides comprise the sequence that is complementary to a gene or mRNA. Non-coding polynucleotides include, but are not limited to: morpholinos, 2'-O-methyl polynucleotides, DNA, RNA and the like. The polynucleotide-based expression inhibitor can be polymerized in vitro, it can be recombinant, contain chimeric sequences, or it can be a derivative of these groups. The polynucleotide-based expression inhibitor may contain ribonucleotides, deoxyribonucleotides, synthetic nucleotides or any suitable combination so that the RNA and / or the target gene are inhibited.

Transfection - The process of delivering a polynucleotide to a cell has been commonly called transfection.

or the transfection process and has also been called transformation. The term "transfection," as used herein, refers to the introduction into the cells of a polynucleotide or other biologically active compound. The polynucleotide can be used for research purposes or to produce a change in a cell that can be therapeutic. The delivery of a polynucleotide can lead to the modification of the genetic material present in the target cell. A transfection reagent or delivery vehicle is a compound or compounds that binds (n) or forms (n) a complex with oligonucleotides and polynucleotides, and is a mediator of its entry into cells.

Examples

Example 1. Synthesis of 2-propionic-3-methylmaleic anhydride (carboxydimethylmaleic anhydride or CDM).

To a suspension of sodium hydride (0.58 g, 25 mmol) in 50 ml of anhydrous tetrahydrofuran, 2 triethyl phosphonopropionate (7.1 g, 30 mmol) was added. When the evolution of hydrogen gas was stopped, dimethyl 2-oxoglutarate (3.5 g, 20 mmol) in 10 ml of anhydrous tetrahydrofuran was added and stirred for 30 minutes. Water, 10 ml, was then added, and the tetrahydrofuran was removed by rotary evaporation. The resulting mixture of solids and water was extracted with 3 x 50 ml of ethyl ether. The ether extractions were combined, dried with magnesium sulfate, and concentrated until a light yellow oil was obtained. The oil was purified by elution with silica gel chromatography, with 2: 1 ether: hexane, to yield 4 g (82% yield) of pure triester. The 2-propionic-3-methylmaleic anhydride was then formed, dissolving this triester in 50 ml of a 50/50 mixture of water and ethanol, containing 4.5 g (5 equivalents) of potassium hydroxide. This solution was heated at reflux for 1 hour. The ethanol was then removed by rotary evaporation and the solution was acidified to pH 2 with hydrochloric acid. This aqueous solution was then extracted with 200 ml of ethyl acetate, which was isolated, dried over magnesium sulfate, and concentrated to a white solid. This solid was then recrystallized from dichloromethane and hexane to produce 2 g (80%) of 2-propionic-3-methylmaleic anhydride.

Example 2. Synthesis of thioester of CDM.

To a solution of 2-propionic-3-methylmaleic anhydride (30 mg, 0.16 mmol) in 5 ml of methylene chloride, oxalyl chloride (200 mg, 10 eq) and dimethylformamide (1 IL) were added. The reaction was allowed to proceed overnight, in which the excess of oxalyl chloride and methylene chloride was removed by rotary evaporation, to obtain the acid chloride, a clear oil. The acid chloride was dissolved in 1 ml of methylene chloride. To this solution, 2 equivalents of thioglycolic acid and pyridine (20 IL, 1.5 eq) in 10 ml of methylene chloride were added. The solution was then stirred overnight. The solvent was then removed and the resulting solid was dissolved in 5 ml of water and purified by reverse phase HPLC, using a gradient of 0.1% TFA in water / acetonitrile.

Example 3. Synthesis of poly (vinyl ethers).

2-Vinyloxy ethyl phthalimide (1 g, 4.6 mmol) was added to a round base flask dried in an oven, under a blanket of nitrogen in anhydrous dichloromethane, to this solution was added ethyl vinyl ether (0.332 g, 4, 6 mmol), propyl vinyl ether (0.396 g, 4.6 mmol) or butyl vinyl ether (0.460 g, 4.6 mmol). These solutions were then brought to 78 ° C and BF3 • OEt2 (0.065 g, 0.46 mmol) was added and the reaction was allowed to proceed for 2 hours at -78 ° C. The polymerization was then stopped by the addition of a 50/50 mixture. of ammonium hydroxide in methanol. The solvents were then removed by rotary evaporation. The polymer was then dissolved in 30 ml of 1,4-dioxane / methanol (2/1). To this solution was added hydrazine (0.147 g, 46 mmol) and the mixture was heated at reflux for 3 hours. The solvents were then removed by rotary evaporation and the resulting solid was then completed to 20 ml of 0.5 M HCl and refluxed for 15 minutes, diluted with 20 ml of distilled water, and refluxed for An additional hour. This solution was then neutralized with NaOH, cooled to room temperature and transferred to a 3,500 molecular weight cellulose tube and dialyzed for 24 hours (2 x 20 L) against distilled water, and lyophilized.

Example 4. Hemolysis by melitin, PEAVE, PPAVE, PBAVE and PBAVE modified with CDM.

The membrane activity of the amphiphilic cationic polymers was tested according to the published procedure. 108 red blood cells were added to 500 Il phosphate buffer. To this solution, 20 g of melitine, PEAVE, PPAVE, PBAVE or CDM-PBAVE were added, which had been prepared by acylation of PBAVE with 2 eq. of CDM in relation to amines. The samples were incubated for 15 minutes at 37 ° C, then centrifuged for 1 minute at 15,000 RCF. Lysis was determined by measuring the absorbance of the supernatant material at 541 nm. The percentage of hemolysis was calculated assuming that the absorbance of hemoglobin released after the addition of distilled water was 100% lysis. It was determined that all polymers that were hemolytic, with PBAVE and melitin being the most lithic. The PBAVE polymer modified with CDM was not hemolytic until acidification.

Example 5. Luciferase induction after oligonucleotide delivery.

HeLa Luc / 705 cells (Gene Tools, Philomath OR) were cultured under the conditions used for HeLa cells. The cells were spread on 24-well culture plates, at a density of 3 x 10 6 cells / well and incubated for 24 hours. Media was replaced with 1.0 ml DMEM with 10% fetal calf serum and 2.5 nmol PMO (CCT CTT ACC TCA GTT ACA ATT TAT A, SEQ ID 1, Gene Tools, Philomath, OR) with or without 20 Ig of PBAVE modified with CDM. The cells were then incubated for 48 hours in a humidified incubator with 5% CO2 at 37 ° C. Cells were collected and lysates were analyzed to study luciferase expression, using a Lumat LB 9507 luminometer (EG & G Berthold, Bad-Wildbad, Germany). The addition of PBAVE modified with CDM resulted in a 2-3-fold increase in luciferase activity.

Example 6. Transfection with unstable DNA particles in acid medium:

Hepa cells (a mouse hepatocyte cell line) were cultured in 1 ml of Dulbecco-modified Eagle medium containing 10% fetal calf serum, using 12-well plates. PBAVE nanoparticles were formulated in accordance with the aforementioned proportions, with pCIluc plasmid DNA (10 Ig / ml, pCIluc, prepared according to the procedure published in 0.5 mL of 5 mM HEPES pH 7.5). As controls for modification with unstable CDM 5 as a function of pH, polyanions were generated from the polyamines using succinic anhydride that irreversibly modifies the amine, and aconitic anhydride that reversibly modifies the amine, but is much slower to cleave than CDM, to form PBAVE-S and PBAVE-A, respectively. The nanoparticles, 2 Ig of DNA, were then added (200 IL) to the cells. The cells were incubated for 48 h. Cells were collected and luciferase expression was tested, as previously reported. A

10 Lumat LB 9507 luminometer (EG & G Berthold, Bad-Wildbad, Germany). The amount of transfection is the average transfection of two separate cell wells. The amount of luciferase in picograms = 5.1 x 10-5 (URL) +3.683.

Formulation

Relative Light Units

DNA: PBVE: CDM-PBAVE 10:20:80 Ig / ml

1,875,801

DNA: PBVE: PBAVE-S 10:20:80 Ig / ml

195

DNA: PBVE: PBAVE-A 10:20:80 Ig / ml

68,549

Naked DNA

200

Example 7. Transfection with unstable charged particles in acidic medium in vivo.

PBAVE nanoparticles were formulated according to the aforementioned proportions with pCIluc plasmid DNA

15 (30 Ig / ml, pCIluc; prepared according to the published procedure, in 0.5 mL of 5 mM HEPES pH 7.5). The nanoparticles, 9 Ig of DNA, were then injected into the tail vein (300 IL) of the mice. Twenty-four hours after the injection, the mice were sacrificed, their livers were removed and luciferase expression was analyzed, as previously reported. A Lumat LB 9507 luminometer (EG & G Berthold, Bad-Wildbad, Germany) was used. The amount of transfection is the average transfection of a group of three mice. The

20 luciferase amount in picograms = 5.1 x 10-5 (URL) +3.683.

Formulation

Relative Light Units

DNA: PBVE: CDM-PBAVE 30: 60: 240 Ig / ml

30,123

Naked DNA

1,021

Example 8. Particle size in the absence and in the presence of salt and measurement of potential z.

Nanoparticles between DNA and PEAVE and CDM / PEAVE modified with CDM thioester were formulated in 20 mM HEPES buffer pH 7.5, according to the weight ratios presented above, with a DNA concentration of 10 Ig / ml. For CDM / CDM thioester modified polymers, the CDM thioester was mixed with

25 CDM in a 9: 1 weight ratio, before mixing with the polymer. The size of the nanoparticles and the z potential were determined by light scattering at 532 nm, using an I90 ZetaPlus particle meter from Brookhaven Instruments Corporation. The stability of the nanoparticles against a salt was determined by adding sodium chloride up to 150 mM and measuring the size after 10 min.

Prop particles by weight of the polymer (Ig / ml)

Size (nm) in 20 mM HEPES pH 7.5 Size (nm) in 150 mM NaCl

DNA: PEAVE: CDM-PEAVE

 10: 20: 100

90-110 > 1000

5: 10: 100

90-130 > 1000

DNA: PEAVE: CDM / CDMthioester-PEAVE

 10: 20: 100

90-110 114

5: 10: 100

90-130 118

Example 9. Transfection of DNA particles crosslinked with CDM-thioester.

30 Hepa cells (a mouse hepatocyte cell line) were cultured in 1 ml of Dulbecco-modified Eagle medium containing 10% fetal calf serum, using 12-well plates. PBAVE nanoparticles were formulated according to the aforementioned proportions with pCIluc plasmid DNA (10 Ig / ml, pCIluc; prepared according to the procedure published in 0.5 mL of 5 mM HEPES pH 7.5). For the CDM / CDM thioester modified polymers, the CDM thioester was mixed with CDM in a 9: 1 weight ratio, before mixing

35 with the polymer. The nanoparticles, 2 Ig of DNA, were then added (200 IL) to the cells. The cells were incubated for 48 h. Cells were collected and luciferase expression was analyzed, as previously reported. A Lumat LB 9507 luminometer (EG & G Berthold, Bad-Wildbad, Germany) was used. The amount of transfection is the average transfection for two separate cell wells. The amount of luciferase in picograms = 5.1 x 10-5 (URL) + 3.683.

Formulation

Relative Light Units

DNA: PBAVE: CDM-PBAVE 10: 40: 100 Ig / ml

774,432

DNA: PBAVE: CDM-PBAVE 10:20:50 Ig / ml

4,967,879

DNA: PBAVE: CDM / CDM-thioester-PBAVE 10: 40: 100 Ig / ml

1,040,076

DNA: PBAVE: CDM / CDM-thioester-PBAVE 10:20:50 Ig / ml

2,276,733

5 Example 10. Synthesis of monomethyl ethers of amino polyethylene glycol.

To a solution of 10% by weight of PEG monomethyl ether of different molecular weights in methylene chloride, 3 equivalents of mesyl chloride and triethylamine are added. After stirring overnight, the solution is washed with an equal volume of water saturated with NaHCO3. The organic layer is then dried with sodium sulfate and PEG precipitates in the solution, by adding 9 volume equivalents of diethyl ether. Be

10 allows precipitation of PEG mesylate overnight at -78 ° C. PEG mesylate is then dissolved up to 15% by weight in water and 10 equivalents of amine (ethylenediamine or Tris (2-aminoethyl) amine). The reaction is allowed to run for 48 hours and the amine modified PEG is purified using reverse phase HPLC with a gradient of 0.1% TFA in water / acetonitrile.

Example 11. Synthesis of CDM-PEG derivatives.

To a solution of 2-propionic-3-methylmaleic anhydride (30 mg, 0.16 mmol) in 5 ml of methylene chloride, oxalyl chloride (200 mg, 10 eq) and dimethylformamide (1 IL) were added. The reaction was allowed to proceed overnight during which the excess of oxalyl chloride and methylene chloride was removed by rotary evaporation, to obtain the acid chloride, a clear oil. The acid chloride was dissolved in 1 ml of methylene chloride. To this solution 2 equivalents of amino polyethylene glycol monomethyl ether, of different molecular weights were added

20 res, and pyridine (20 IL, 1.5 eq) in 10 ml of methylene chloride. The solution was then stirred overnight. The solvent was then removed and the resulting solid was dissolved in 5 ml of water and purified using reverse phase HPLC with a gradient of 0.1% TFA in water / acetonitrile.

Example 12. Particle size in the absence and in the presence of salt and measurement of potential z.

The nanoparticles between 10 Ig / ml of DNA and 20 Ig / ml of PBAVE were formulated in 20 mM HEPES buffer pH 7.5. TO

25 This solution did not add anything or 100 Ig of CDM-PEG2 (the molecular weight of the PEG was 1100). The size of the nanoparticles was determined by light scattering at 532 nm, using an I90 ZetaPlus particle meter from Brookhaven Instruments Corporation. The stability of the nanoparticles against salt was determined by adding sodium chloride up to 150 mM and measuring the size after 10 min. Without the addition of CDM-PEG2, the DNA / polycation particles grew from 100 to> 1000 nm after the addition of sodium chloride.

30 After a modification with CDM-PEG2, this increase in particle size did not take place in the presence of salt.

Example 13. Condensation and decondensation of DNA after the addition of salt and polyacrylic acid.

The DNA was labeled with the reagent to label DNA with LabelIT tetramethylrodamine (Mirus Corporation) with a 1: 1 weight ratio of DNA: LabelIT, according to the manufacturer's protocol. A solution of 1 Ig / ml of tetramethylrodamine labeled DNA was condensed by the addition of 10 Ig / ml of PBAVE in the presence of TAPS buffer at pH 9. Various amounts of CDM-PEG2 and CDM-PEG3 were added to this solution. Then, NaCl was added to the solution to bring the concentration to 150 mM. Finally poly (acrylic acid) was added up to 100 Ig / ml. After the addition of each reagent, rhodamine fluorescence was measured using a Varian spectrofluorometer by stimulating at 555 nm and measuring the emission at 575 nm. A decrease in fluorescence is

40 an indicator of DNA condensation, while an increase indicates a decondensation of DNA.

Sample

Relative fluorescence

DNA alone + PBAVE + 40 Ig of PEG (1100) -CDM2 + 150 mM NaCl + 100 Ig / ml of PACAC

1.0 0.2 0.3 0.3 0.75

DNA alone + PBAVE

1.0 0.2

Sample

Relative fluorescence

+ 40 Ig of PEG (1100) -CDM3 + 150 mM NaCl + 100 Ig / ml of PACAC

0.3 0.3 0.79

DNA alone + PBAVE + 150 Ig of PEG (1100) -CDM3 + 150 mM NaCl + 100 Ig / ml of PACAC

1.0 0.2 0.3 0.3 0.56

DNA alone + PBAVE + 150 mM NaCl + 100 Ig / ml PACAC

1.0 0.2 0.3 0.95

The foregoing is considered only illustrative of the principles of the invention. In addition, since those skilled in the art will make numerous modifications and changes, it is not desired to limit the invention to the exact construction and operation, shown and described. Therefore, all modifications and suitable equivalents are within the scope of the invention.

Claims (11)

1. A nanoparticle for delivering a polynucleotide to a cell comprising: said polynucleotide associated with a polymer with reversible inhibited membrane activity, wherein said polymer with reversible inhibited membrane activity consists of a plurality of inhibitors with membrane activity, reversibly linked to a polyamine with membrane activity through a pH-labile bond, wherein said inhibitors are selected from the group consisting of: disubstituted maleic anhydride derivatives, and wherein the nanoparticle additionally comprises a polycation. 2. The nanoparticle according to claim 1, wherein said polymer with reversibly inhibited membrane activity is associated with said polynucleotide through an electrostatic interaction. 3. The nanoparticle according to claim 1 or 2, wherein said polycation is a polymer with membrane activity. 4. The nanoparticle according to claim 1 or 2, wherein said polycation is selected from PBAVE, PPAVE or PBAVE. 5. The nanoparticle according to any of claims 1-4, wherein said polycation is crosslinked with said polyamine with reversible inhibited membrane activity, through a labile bond as a function of pH. 6. The nanoparticle according to any of claims 1-5, wherein said disubstituted maleic anhydride derivatives are selected from the groups consisting of CDM, CDM-thioester and CDM-PEG. 7. The nanoparticle according to claim 1, wherein the nanoparticle consists of a stable nanoparticle against a salt. 8. The nanoparticle according to claim 1, wherein said polymer with membrane activity has a molecular weight of at least about 10,000 Daltons. 9. The nanoparticle according to any of claims 1-8, wherein said polymer with reversibly inhibited membrane activity breaks an endocytic membrane, thereby providing delivery of said polynucleotide into the cytoplasm of the cell. 10. A method for in vitro delivery of a nanoparticle according to any of claims 1-9, in the cytoplasm of a cell comprising: culturing mammalian cells in a suitable culture medium, adding said nanoparticle to said cells cultured and incubate the cells for a suitable period of time. 11. Use for in vivo delivery of a nanoparticle according to any of claims 1-9, in the cytoplasm of a cell.