DOUBLE-DERIVED CHITOSAN NANOPARTICLES AND METHODS FOR THEIR MANUFACTURE AND USE FOR IN VIVO GENE TRANSFER

Abstract

A chitosan-derived nanoparticle comprising chitosan functionalized with a cationic amino acid and a hydrophilic polyol is provided herein, along with methods for preparing and using it, for example, for in vivo gene delivery.

Description

The present invention relates generally to nanoparticles comprising double-derivative chitosan and methods for manufacturing and using them to deliver nucleic acids, e.g., gene transfer, in vivo. BACKGROUND OF THE INVENTION Chitosan is a non-toxic cationic copolymer of N-acetyl-D-glucosamine and D-glucosamine. Chitosan can form a complex with nucleic acid and, as a biocompatible and non-toxic polysaccharide, has been used as a vehicle for delivering DNA to transfected cells. There has been considerable interest in using chitosan for non-viral nucleic acid delivery due to the complexities and potential toxicity of viral vectors. A number of chitosan / DNA complexes, including complexes between chitosan and modified nucleic acids, have been examined in an attempt to identify suitable compositions for gene transfection. See, for example, WO2010 / 088565; WO2008 / 082282. The complexes have been found to vary in, among other properties, solubility, aggregation propensity, complex stability, particle size, DNA release capacity, and transfection efficiency. Therefore, there is a need for new compositions and methods for in vivo gene transfer with improved transfection efficiency. The compositions and methods described herein help to meet these and other needs. SUMMARY OF THE INVENTION The discovery herein is provided that chitosan, functionalized with (1) arginine (Arg) and (2) a hydrophilic polyol (HP) at a particular Arg:HP ratio, exhibits a dramatic improvement in transfection efficiency. Arginine and gluconic acid have been shown to act synergistically as functional groups to enhance the transfection efficiency of chitosan nanoparticles. See, for example, PCT / CA2013 / 050218. The discovery herein is described that gluconic acid can be substituted with other molecules as chitosan derivatives and still exhibit this synergistic effect. Furthermore, optimized functionalization ratios of the two substituents are provided, within which the derived chitosan exhibits maximum transfection efficiency.Therefore, novel compositions are provided herein to facilitate the delivery of nucleic acids to cells, tissues, and organs, for example, in vivo. Specifically, double-derivative chitosan-based nanoparticles are provided, wherein the chitosan is functionalized with the cationic amino acid arginine (Arg) and a hydrophilic polyol (HP) in final degree of functionalization ratios of Arg:final degree of functionalization of HP (i.e., Arg:HP). In a preferred embodiment, the nanoparticles comprise chitosan coupled to both multiple arginines and multiple hydrophilic polyols, see, for example, Formula I ινΐΛ / a / zuzz / uuuo iz (i) ινΐΛ / a / zuzz / uuuo iz where n is an integer from 1 to 650, α is the degree of arginine functionalization, β is the degree of hydrophilic polyol functionalization and each R1 is independently selected from hydrogen, acetyl, arginine and a hydrophilic polyol. In a preferred embodiment, the nanoparticles comprise chitosan coupled to arginine. See Formula II. In a preferred embodiment, the arginine contributes to increased solubility of the chitosan and / or enables the derived chitosan to bind to nucleic acids at a higher pH. (ii) In another embodiment, the nanoparticles comprise chitosan coupled to a hydrophilic polyol, preferably having a carboxyl or aldehyde group for coupling to the chitosan. In one embodiment, the hydrophilic polyol is selected from the group consisting of a molecule having a carboxyl or aldehyde group for coupling to the chitosan, and a saccharide, wherein said hydrophilic polyol is not gluconic acid. In one embodiment, the hydrophilic polyol has a carboxyl group. In one embodiment, the hydrophilic polyol having a carboxyl group for coupling to the chitosan is selected from the group consisting of gluconic acid and threonic acid. In one embodiment, the hydrophilic polyol is gluconic acid; see, for example, Formula III. In another embodiment, the hydrophilic polyol is threonic acid; see, for example, Formula IV. In another form, the hydrophilic polyol is a saccharide, which can be natural or synthetic, or the acidic form of a saccharide.Non-exhaustive examples include glyceraldehyde, threose, erythrose, ribose, arabinose, xylose, lyxose, allose, glucose, altrose, mannose, gulose, idose, galactose, and talose. In one embodiment, the hydrophilic polyol is glucose; see, for example, Formula V. In another embodiment, the hydrophilic polyol is threose; see, for example, Formula VI. HO HO iviA / a / zuzz / uuuo i ¿ (III) (IV) HO HO (V) (VI) The double-derivative chitosan, as described herein, comprises arginine and a hydrophilic polyol at an optimized final degree of functionalization ratio of Arg:HP. In a preferred embodiment, the double-derivative chitosan has a molar or final degree of functionalization ratio of Arg:HP of between about 1:1 and about 10:1. In another embodiment, the final degree of functionalization ratio of Arg:HP is between about 3:1 and about 7:1. In yet another embodiment, the final degree of functionalization ratio of Arg:HP is about 5:1. In particular, chitosan-nucleic acid polyplexes formed with such double-derivative chitosan (“DD chitosan”) exhibit greater transfection efficiency than nucleic acid polyplexes formed with non-functionalized chitosan, single-derivative chitosan, or double-derivative chitosan with a final Arg:HP functionalization degree ratio between 1:1 and 10:1. Other desirable properties conferred by the use of double-functionalized chitosan in the polyplexes described herein include improved mucosal barrier penetration, improved polyplex stability at pH above 6.5, reduced accumulation at higher polyplex concentrations, reduced cellular toxicity, and increased intracellular nucleic acid release. Furthermore, in some preferred formulations, the DD chitosan polyplex compositions described herein can be administered at physiological pH (e.g., systemic administration). Accordingly, in one aspect, the invention provides chitosan DD and nucleic acid polyplexes. The chitosan DD and nucleic acid polyplexes comprise chitosan having a double derivation with arginine and a hydrophilic polyol. In one form, the nucleic acid of the chitosan DD polyplex and nucleic acid is DNA. In one modality, the nucleic acid of the chitosan DD polyplex and nucleic acid is RNA. invention to a patient, where the nucleic acid encodes an immunomodulatory cytokine. BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the in vivo transfection efficiency (ng of SEAP / mg of protein; y-axis) of 5 kDa functionalized chitosan (x-axis) with gluconic acid alone to a final degree of functionalization of 3% or 10%, with arginine alone to a final degree of functionalization of 10%, or with both gluconic acid and arginine to a final degree of functionalization ratio of arginine to gluconic acid of about 1:1 or about 3.3:1. Figure 2 shows the transfection efficiency (ng of SEAP / mg of protein; y-axis) of 5 kDa functionalized chitosan (x-axis) with gluconic acid alone to a final degree of functionalization of 3% to 10%, with arginine alone to a final degree of functionalization of 52%, or with both gluconic acid and arginine to a final degree of functionalization ratio of arginine to gluconic acid of about 6:1 or about 17:1. Figure 3 shows the transfection efficiency (ng of SEAP / mg of protein; y-axis) of 5 kDa functionalized chitosan (x-axis) with gluconic acid only to a final degree of functionalization of 3% to 10%, with arginine only to a final degree of functionalization of 26%, or with both gluconic acid and arginine to a final degree of functionalization ratio of arginine to gluconic acid of about 4:1. Figure 4 shows the transfection efficiency (ng of SEAP / mg of protein; y-axis) of 5 kDa functionalized chitosan (x-axis) with only threonic acid to a final degree of functionalization of 3%, or with only arginine to a final degree of functionalization of 29%, or with both threonic acid and arginine to a final degree of functionalization ratio of arginine to threonic acid of about 10:1. DETAILED DESCRIPTION Chitosan is the deacetylated form of chitin, a polymer of N-acetylglucosamine that is the main component of the exoskeletons of crustaceans (e.g., shrimp, crab, lobster). Chitosan is formed from chitin by deacetylation, and as such, it is not a single polymer molecule, but rather a class of molecules with varying molecular weights and degrees of deacetylation. The percentage of deacetylation in commercial chitosans is typically 50–100%. The chitosan derivatives described herein are generated by functionalizing the resulting free amino groups with positively charged and / or hydrophilic residues, as described herein. The chitosan derivatives described herein possess a number of properties that are favorable for a nucleic acid delivery vehicle, including: efficient binding and complexation with negatively charged nucleic acids, the ability to form nanoparticles of a controllable size, cell uptake, and the ability to release nucleic acids at the appropriate time within cells. The present invention uses chitosans with any degree of deacetylation (DDA) greater than 50%, with a functionalization between 1% and 50% (the percentage of functionalization being determined with respect to the amount of free amino residues in the chitosan polymer). The degrees of deacetylation and functionalization impart a specific charge density to the functionalized chitosan derivative. The resulting charge density affects the solubility and binding. ML / a / ZUZZ / UUUó I z of nucleic acid and subsequent release and interaction with mammalian cell membranes. Therefore, according to the present invention, these properties must be optimized to achieve optimal efficiency. Examples of chitosan derivatives are described in Baker et al.; 11 / 657,382 filed on January 24, 2007, which is incorporated herein by reference. In one embodiment, the double-derivative chitosan described herein comprises chitosan with a degree of deacetylation of at least 50%. In another embodiment, the degree of deacetylation is at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and even more preferably at least 95%. In a preferred embodiment, the double-derivative chitosan described herein comprises chitosan with a degree of deacetylation of at least 98%. The chitosan derivatives described herein have a range of average molecular weights that are soluble at neutral and physiological pH and, for the purposes of this invention, include molecular weights ranging from 3 to 110 kDa. The embodiments described herein feature lower average molecular weight chitosan derivatives (<25 kDa, for example, from approximately 5 kDa to approximately 25 kDa), which may have desirable delivery and transfection properties, are small in size, and have favorable solubility. A chitosan derivative with a lower average molecular weight is generally more soluble than one with a higher molecular weight; therefore, the former produces a nucleic acid / chitosan complex that will release the nucleic acid more readily and provide greater cell transfection. Much literature has been devoted to optimizing all these parameters of chitosan-based delivery systems. A person skilled in the art will recognize that chitosan refers to multiple molecules having a structure of Formula I, where n is any integer, and each R1 is independently selected from either acetyl or hydrogen, with the degree of hydrogen selection in R1 ranging from 50% to 100%. Furthermore, when chitosan is referred to as having an average molecular weight, for example, from 3 kDa to 110 kDa, it generally refers to multiple chitosan molecules with an average weight molecular weight of, for example, 3 kDa to 110 kDa, respectively, where each of the chitosan molecules can have different chain lengths (n+2). It is also recognized that when chitosan is referred to as “n-mer chitosan,” it does not necessarily comprise chitosan molecules of Formula I, where each chitosan molecule has a chain length of n+2.Conversely, as used herein, “n-mer chitosan” refers to multiple chitosan molecules, each of which may have different chain lengths, where the plurality has an average molecular weight substantially similar to or equal to a chitosan molecule with a chain length of n. For example, 24-mer chitosan may comprise multiple chitosan molecules, each with different chain lengths ranging from, for example, 7–50, but having an average molecular weight substantially similar to or equivalent to a chitosan molecule with a chain length of 24. The functionalized chitosan derivatives described herein are doubly derived chitosan compounds, for example, chitosan-Arg-HP compounds. In general, chitosan-Arg-HP compounds have the following structure of Formula I ινΐΛ / a / zuzz / uuuó iz iviA / a / zuzz / uuuo 1 ¿ (i) where n is an integer from 1 to 650, a is the final degree of functionalization of Arg, β is the final degree of functionalization of HP, and each R1 is selected independently from hydrogen, acetyl, an Arg and an HP. A double-derivative chitosan of the invention can be functionalized with the cationic amino acid, arginine. A double-derivative chitosan of the invention can also be functionalized with a hydrophilic polyol, which can help increase the hydrophilicity of the chitosan (including Arg-chitosan) and / or can donate a hydroxyl group. Chitosan-derived nanoparticles comprising arginine-functionalized chitosan (Arg) and a hydrophilic polyol (HP) of Formula Vil are provided: EITHER X R3 VII where: R2 is selected from: H and hydroxyl; R3 is selected from: H and hydroxyl; and X is selected from: C2-C6 alkylene optionally substituted with one or more hydroxyl substituents. In some embodiments, the chitosan-derived nanoparticle comprises a hydrophilic polyol of Formula V: X R3 where: R2 is selected from: H and hydroxyl; R3 is selected from: H and hydroxyl; and VII X is selected from: C2-C6 alkylene© optionally substituted with one or more hydroxyl substituents; provided that said hydrophilic polyol is not gluconic acid. As used herein, the term “C2-C6 alkylene” refers to a linear or branched divalent hydrocarbon radical that optionally contains one or more carbon-carbon multiple bonds. For the avoidance of doubt, as used herein, the term “C2-C6 alkylene” includes divalent radicals of alkanes, alkenes, and alkynes. A hydrophilic polyol according to the present invention may have a backbone of 3, 4, 5, 6, or 7 carbons. In one embodiment, a hydrophilic polyol according to the present invention having 3 to 7 carbons may have 2, 3, 4, 5, or 6 hydroxyl groups. In another embodiment, a hydrophilic polyol according to the present invention having 3 to 7 carbons may have one or more carbon-carbon multiple bonds. In a preferred embodiment, a hydrophilic polyol according to the present invention comprises a carboxyl group. In another preferred embodiment, a hydrophilic polyol according to the present invention comprises an aldehyde group. A person skilled in the art will recognize that when a hydrophilic polyol according to the present invention comprises an aldehyde group, such hydrophilic polyol encompasses both open-chain (aldehyde) and cyclic (hemiacetal) formations. Non-exhaustive examples of hydrophilic polyols include gluconic acid, threonic acid, glucose, and threose; see, for example, Formulas III–VI. Examples of other hydrophilic polyols, which may have a carboxyl and / or aldehyde group, or may be a saccharide or an acid form thereof, are included in Tables 1–3 below. A person skilled in the art will recognize that Tables 1–3 provide non-exhaustive examples of hydrophilic polyols and, furthermore, that the hydrophilic polyols shown are not limited to the stereochemistry described. iviA / a / zuzz / uuuo 1 ¿ Table 1: Chemical structures of certain aldehydes and carboxylic acids of hydrophilic polyols D-glyceric acid 0 HO^y^OH OH L-glyceric acid 0 ΗΟ^γ^ΟΗ OH D-threonic acid OH 0 HO- Λ. A OH OH L-threonic acid OH O HO^ A ^^< OH OH D-erythronic acid OH O <YCh OH L-erythronic acid OH 0 OH D-ribonic acid OH O HO^Y^Y^OH OH OH L-ribonic acid OH O HO^γ^γ^OH OH OH D-arabonic acid OH O HO^γ^γ^OH OH OH L-arabonic acid OH O HO^γ^-^OH OH OH D-xylonic acid OH 0 HO^^Y^Y^OH OH OH L-xylonic acid OH 0 HO^γ^γ^OH OH OH D-lyxonic acid OH 0 ho^^^Y^oh OH OH L-lyxonic acid OH 0 ho^^y^y^oh OH OH D-alonic acid OH OH 0 OH OH L-alonic acid OH OH O HO^ OH OH OH D-gluconic acid H0\ OH OH O OH OH OH L-gluconic acid HO\ OH OH O OH OH OH D-altronic acid HO^ OH OH O OH OH OH L-altronic acid OH OH O H0\ OH OH OH D-manonic acid OH OH O HO\ 1 - OH OH OH L-manonic acid OH OH O HO^ OH OH OH D-gulonic acid OH OH O OH OH OH L-gulonic acid OH OH O HO^ - ~ ; I ΌΗ ÓH OH D-idonic acid HO^ OH OH 0 OH OH OH L-Idonic acid OH OH O HO. A^ A ^Υ Υ ΟΗ OH OH D-galactonic acid OH OH O OH OH L-galactonic acid OH OH O 0' ¡ O(. OH OH D-talonic acid OH OH 0 HO, A. A OH OH OH L-talonic acid OH OH 0 OH OH D-fuconic acid OH OH 0 ^Y^Y^OH OH OH L-fuconic acid OH OH O ΧΧ^γ^ΟΗ OH OH L-glycero-D-mannoheptonic acid OH OH 0 HO^Y^Y^OH OH OH OH ÓH L-glycero-D-mannoheptonic acid OH OH 0 HO^^^^^^Y^OH ÓH ÓH OH D-glyceraldehyde 0 HO^Y^H ÓH L-glyceraldehyde O HO^ OH D-treose OH 0 HO^ ^Y^H OH L-treose OH 0 HO^ OH D-erythrose OH 0 H0\ ^^V^H OH L-erythrose OH 0 HO\ ^Y^H OH D-ribose OH 0 HO^ H OH OH L-ribose HO^ OH 0 H OH OH D-arabose OH 0 HO^ H OH OH L-arabose OH 0 HO^ H OH OH D-xylose OH 0 HO^ H ÓH ÓH ML / a / ZUZZ / UUU 01 of L-xylose OH 0 HO^ OH OH D-lyxose OH 0 HO^ OH OH L-lyxose OH 0 HO^ γΥ Ή OH OH D-alose OH OH O HO^ Ή OH OH L-alose OH OH 0 ΗΗ OH Hχ D OH OH OH OH OH OH OH OH L-glucose OH OH 0 HO^ H OH OH D-altrosa OH OH 0 HO^ Ή OH OH L-altrosa OH OH 0 HO^ Ή OH OH D-mannose OH OH 0 HO^ Ή OH OH L-mannosa OH OH O OH OH D-glucose OH OH 0 OH OH L-glucose OH OH 0 H0 ¡ ' H OH OH D-idosa OH OH O OH L-idosa OH OH O ho^ / AUh OH OH0 D-galactose OH OH OH 0 OH-OH-Hgala D-talosa OH OH 0 OH OH L-talosa OH OH 0 HoAAAh OH OH D-fucosa OH OH 0 ^Y^Y^H OH OH ΜΛ / a / ZUZZ / UUU OI z L-fucose OH OH 0 OH OH L-glycero-D-mannoheptose HO' OH 0 OH OH OH D-glycero-L-manoheptose HO' OH 0 ^Υ^Υ^γ^Η OH OH OH iviA / a / zuzz / uuuo and ¿ Table 2: Chemical Names of Certain Carboxylic Acids of Hydrophilic Polyols Number of carbons Number of hydroxyls IUPAC name 7 carbons 6 hydroxyls 2,3,4,5,6,7-hexahydroxyheptanoic acid 5 hydroxyls 2,3,4,5,7-pentahydroxyheptanoic acid 2,3,4,6,7-pentahydroxyheptanoic acid 2,3,5,6,7-pentahydroxyheptanoic acid 2,4,5,6,7-pentahydroxyheptanoic acid 3,4,5,6,7-pentahydroxyheptanoic acid 4 hydroxyls 2,3,4,5-tetrahydroxyheptanoic acid 2,3,4,6-tetrahydroxyheptanoic acid 2,3,4,7-tetrahydroxyheptanoic acid 2,3,5,6-tetrahydroxyheptanoic acid 2,3,5,7-tetrahydroxylheptanoic acid 2,3,6,7-tetrahydroxylheptanoic acid 2,3,6,7-tetrahydroxylheptanoic acid 16 2,4,5,6-tetrahydroxyleptanoic acid 2,4,5,7-tetrahydroxyleptanoic acid 2,4,6,7-tetrahydroxyleptanoic acid 2,5,6,7-tetrahydroxyleptanoic acid 2,5,6,7-tetrahydroxyleptanoic acid 3,4,5,6-tetrahydroxyleptanoic acid 3,4,5,7-tetrahydroxyleptanoic acid 3,4,6,7-tetrahydroxyleptanoic acid 3,5,6,7-tetrahydroxyleptanoic acid 4,5,6,7-tetrahydroxyleptanoic acid 3 hydroxyls 2,3,4-trihydroxyleptanoic acid 2,3,5-trihydroxyleptanoic acid 2,3,6-trihydroxyleptanoic acid 2,3,7-trihydroxyheptanoic acid 2,4,5-trihydroxyheptanoic acid 2,4,6-trihydroxyheptanoic acid 2,5,6-trihydroxyheptanoic acid 2,5,7-trihydroxyheptanoic acid 2,6,7-trihydroxyheptanoic acid 3,4,5-trihydroxyheptanoic acid 3,4,6-trihydroxyheptanoic acid 3,5,6-trihydroxyheptanoic acid 4,5,6-trihydroxyheptanoic acid 4,5,7-trihydroxyheptanoic acid 5,6,7-trihydroxyheptanoic acid 2 hydroxyls 2,3-dihydroxyheptanoic acid 2,4-dihydroxyheptanoic acid 2,5-dihydroxylheptanoic acid, 2,6-Dihydroxyheptanoic acid 2,7-Dihydroxyheptanoic acid 3,4-Dihydroxyheptanoic acid 3,5-Dihydroxyheptanoic acid 3,6-Dihydroxyheptanoic acid 3,7-Dihydroxyheptanoic acid 4,5-Dihydroxyheptanoic acid 4,6-Dihydroxyheptanoic acid 4,7-Dihydroxyheptanoic acid 5,6-Dihydroxyheptanoic acid 5,7-Dihydroxyheptanoic acid 6,7-Dihydroxyheptanoic acid 6 carbons 5 hydroxyls 2,3,4,5,6-Pentahydroxyheptanoic acid 4 hydroxyls 2,3,4,5-Tetrahydroxyheptanoic acid 2,3,4,6-Tetrahydroxyheptanoic acid 2,3,5,6-tetrahydroxyhexanoic acid 2,4,5,6-tetrahydroxyhexanoic acid 3,4,5,6-tetrahydroxyhexanoic acid 3 hydroxyls 4,5,6-trihydroxyhexanoic acid 3,4,5-trihydroxyhexanoic acid 2,3,4-trihydroxyhexanoic acid 3,4,6-trihydroxyhexanoic acid 2,3,6-trihydroxyhexanoic acid 1,3,6-trihydroxyhexanoic acid 1,5,6-trihydroxyhexanoic acid 2,5,6-trihydroxyhexanoic acid 2,3,5-trihydroxyhexanoic acid 2,4,5-trihydroxyhexanoic acid 2 hydroxyls 5,6-dihydroxylhexanoic acid 4,6-dihydroxylhexanoic acid 3,6-dihydroxylhexanolco acid 2,6-dihydroxylhexanoic acid 4,5-dihydroxylhexanoic acid 3,5-dihydroxylhexanoic acid 2,5-dihydroxylhexanoic acid 3,4-dihydroxylhexanoic acid 2,4-Dihydroxylhexanoic acid 2,3-dihydroxylhexanoic acid 5 carbons 4 hydroxyls 2,3,4,5-tetrahydroxylpentanoic acid 3 hydroxyls 3,4,5-trihydroxylpentanoic acid 2,3,5-trihydroxylpentanoic acid 1,4,5-trihydroxyl pentanoic acid 2,4,5-trihydroxylpentanoic acid 2,3,5-Trihydroxypentanoic acid 2,3,4-Trihydroxypentanoic acid 2 hydroxyls 4,5-Dihydroxypentanoic acid 3,5-Dihydroxypentanoic acid 2,5-Dihydroxypentanoic acid 3,4-Dihydroxypentanoic acid 2,4-Dihydroxypentanoic acid 2,3-Dihydroxypentanoic acid 4 carbons 3 hydroxyls 2,3,4-Trihydroxybutanoic acid 2 hydroxyls 3,4-Dihydroxybutanoic acid 2,4-Dihydroxybutanoic acid 2,3-Dihydroxybutanoic acid 3 carbons 2 hydroxyls 2,3-dihydroxylpropanoic acid, ΜΛ / a / ZUZZ / UUU OI z Table 3: Chemical names of certain aldehydes of hydrophilic polyoles ΜΛ / a / ZUZZ / UUUO 1 z Cantidad de carbonos Cantidad de polyoles Nombre IUPAC 7 carbonos 6 hydroxylos 2,3,4,5,6,7-hexahydroxylheptanal 5 hydroxy los 2,3,4,5,7-pentahydroxylheptanal 2,3,4,6,7-pentahydroxylheptanal 2,3,5,6,7-pentahydroxylheptanal 2,4,5,6,7-pentahydroxylheptanal 3,4,5,6,7-pentahydroxylheptanal 4-hydroxyl 2,3,4,5-tetrahydroxylheptanal 2,3,4,6-tetrahydroxylheptanal 2,3,4,7-tetrahydroxylheptanal 2,3,5,6-tetrahydroxylheptanal 2,3,5,7-tetrahydroxylheptanal 2,3,6,7-tetrahydroxylheptanal 2,3,6,7-tetrahydroxyheptanal 2,4,5,6-tetrahydroxyheptanal 2,4,5,7-tetrahydroxylheptanal 2,4,6,7-tetrahydroxylheptanal 2,5,6,7-tetrahydroxylheptanal 2,5,6,7-tetrahydroxylheptanal 2,5,6,7-tetrahydroxylheptanal 3,4,5,6-tetrahydroxylheptanal 3,4,5,7-tetrahydroxylheptanal 3,4,6,7-tetrahydroxylheptanal 3,5,6,7-tetrahydroxylheptanal 4,5,6,7-tetrahydroxylheptanal 3 hydroxyl 2,3,4-trihydroxylheptanal 20 2,3,5-trihydroxylheptanal 2,3,6-trihydroxylheptanal 2,3,7-trihydroxylheptanal 2,4,5-trihydroxylheptanal 2,4,6-trihydroxylheptanal 2,5,6-trihydroxylheptanal 2,5,7-trihydroxylheptanal 2,6,7-trihydroxylheptanal 3,4,5-trihydroxylheptanal 3,4,6-trihydroxylheptanal 3,5,6-trihydroxylheptanal 4,5,6-trihydroxylheptanal 4,5,7-trihydroxylheptanal 5,6,7-trihydroxylheptanal 2 hydroxyl 2,3-dihydroxylheptanal 2,4-dihydroxylheptanal 2,5-dihydroxylheptanal 2,6-dihydroxylheptanal 2,7-dihydroxylheptanal 3,4-dihydroxylheptanal 3,5-dihydroxylheptanal 3,6-dihydroxylheptanal 3,7-dihydroxylheptanal 4,5-dihydroxylheptanal 4,6-dihydroxylheptanal 4,7-dihydroxylheptanal 5,6-dihydroxylheptanal 5,7-dihydroxylheptanal 6,7-dihydroxylheptanal 6 carbons 5 hydroxyls 2,3,4,5,6-pentahydroxylhexanal 4 hydroxyls 2,3,4,5-tetrahydroxylhexanal 2,3,4,6-tetrahydroxylhexanal 2,3,5,6-tetrahydroxylhexanal 2,4,5,6-tetrahydroxylhexanal 3,4,5,6-tetrahydroxylhexanal 3 hydroxyls 4,5,6-trihydroxylhexanal 3,4,5-trihydroxylhexanal 2,3,4-trihydroxylhexanal 3,4,6-trihydroxylhexanal 2,3,6-trihydroxylhexanal 1,3,6-trihydroxylhexanal 1,5,6-trihydroxylhexanal 2,5,6-trihydroxylhexanal 2,3,5-trihydroxylhexanal 2,4,5-trihydroxylhexanal 2 hydroxyls 5,6-dihydroxylhexanal 4,6-dihydroxylhexanal 3,6-dihydroxylhexanal 2,6-dihydroxylhexanal 4,5-dihydroxylhexanal 3,5-dihydroxylhexanal 2,5-dihydroxylhexanal 3,4-dihydroxylhexanal 2,4-dihydroxylhexanal 2,3-dihydroxylhexanal 5 carbon 4 hydroxyl 2,3,4,5-tetrahydroxylpentanal 3 hydroxyl 3,4,5-trihydroxylpentanal ΜΛ / a / ZUZZ / UUU 01 z 2,3,5-trihydroxylpentanal 1,4,5-trihydroxylpentanal 2,4,5-trihydroxylpentanal 2,3,5-trihydroxylpentanal 2,3,4-trihydroxylpentanal 2-hydroxyls 4,5-dihydroxylpentanal 3,5-dihydroxylpentanal 2,5-dihydroxylpentanal 3,4-dihydroxylpentanal 2,4-dihydroxylpentanal 2,3-dihydroxylpentanal 4-carbon 3-hydroxyl 2,3,4-trihydroxylbutanal 2-hydroxyl 3,4-dihydroxylbutanal 2,4-dihydroxylbutanal 2,3-dihydroxylbutanal 3-carbon 2-hydroxyl 2,3-dihydroxylpropanal iviA / a / zuzz / uuuo i ¿ In a preferred embodiment, HP can be selected from the group consisting of 2,3-dihydroxypropanoic acid, 2,3,4,5,6,7-hexahydroxyheptanal, 2,3,4,5,6-pentahydroxyhexanal, 2,3,4,5-tetrahydroxyhexanal and 2,3-dihydroxypropanal. In a preferred embodiment, HP can be selected from the group consisting of D-glyceric acid, L-glyceric acid, L-glycero-D-mannoheptose, D-glycero-L-mannoheptose, D-glucose, L-glucose, D-fucose, L-fucose, D-glyceraldehyde, and L-glyceraldehyde. In a preferred embodiment, the α:β ratio is from about 1:1 to about 10:1. In a preferred embodiment, the ratio is from about 3:1 to about 7:1. In a most preferred embodiment, the ratio is about 5:1. A non-exhaustive method for conjugating chitosan with arginine or a hydrophilic polyol in an aqueous medium is described herein, according to the present invention. The method uses water-soluble 1-ethyl-3-(3-dimethylaminopropyl)-carboamide (EDC) and N-hydroxysuccinimide (NHS) to catalyze amide formation between an amine of the chitosan backbone and a carboxylic acid in an Arg protected with Boc or a hydrophilic polyol having a carboxyl group. Generally, chitosan in dilute HCl solution with a pH adjusted to a target coupling pH of, for example, 6.0 ± 0.5 and more preferably 6.0 ± 0.2, is first coupled to an argon (e.g., Boc-Arg) or a hydrophilic polyol with a carboxyl group, purified, and then coupled to the second functional group. For example, if chitosan is first coupled to an argon, the argon-coupled chitosan (Arg-chitosan) can be purified and then coupled to the hydrophilic polyol. Conversely, if chitosan is first coupled to the hydrophilic polyol, the chitosan coupled to the hydrophilic polyol can be purified and then coupled to an argon. Regardless of the order of coupling, the argon and the hydrophilic polyol can be coupled to chitosan using known methods. For example, an Arg can be coupled to chitosan or to polyol-functionalized chitosan (polyol-chitosan) by adding a mixture of Boc-Arg and pH-adjusted aqueous NHS solution to chitosan in dilute HCl, followed by the addition of EDC water to initiate the coupling at room temperature for 24 hours. The chitosan-amine concentration, reaction pH, and molar ratios of Arg-COOH to chitosan-amine and EDC:NHS:Arg-COOH can be pre-calculated and ensured to achieve a reproducible degree of Arg functionalization. Boc-Arg-chitosan can be purified before the De-Boc reaction. De-Boc can proceed in HCl medium with controlled reaction time and HCl concentration.Any depolymerization of chitosan during de-Boc can be monitored by measuring the change in viscosity of the reaction solution, which was shown to be negligible, and the effectiveness of de-Boc can be determined by nuclear magnetic resonance (NMR) methods on de-Boc-Arg-chitosan and Boc-Arg-chitosan. The degree of functionalization can be determined from elemental C, N analysis of the purified de-Boc-Arg-chitosan. A hydrophilic polyol having a carboxyl group can be coupled to chitosan or to Arg-coupled chitosan (Arg-chitosan) at a reaction pH of 6.0 ± 0.3. At this pH, the carboxylic acid group of the hydrophilic polyol can be attacked by uncoupled amines on the chitosan backbone according to a nucleophilic substitution reaction mechanism. A person skilled in the art will recognize that, upon coupling such a hydrophilic polyol to Arg-chitosan, it is also possible for a small amount of the hydrophilic polyol to form a covalent bond with an amine group of the Arg via the same mechanism, although the nucleophilic substitution reaction is likely to occur predominantly with the amine group of the chitosan backbone. A hydrophilic polyol that is a natural saccharide can be coupled to chitosan or Arg-coupled chitosan (Arg chitosan) by using reductive amination followed by reduction with NaCNBHs or NaBH4. Boc-Arg-chitosan, de-Boc-Arg-chitosan, polyol-chitosan and / or double-bypass chitosan can be purified by precipitation or column treatment, or regular dialysis, or reverse flow dialysis against water using suitable molecular weight cutoff (MWCO) dialysis tubes, or through tangential flow filtration (TFF) and diafiltration cartridges. Therefore, “double-derivative chitosan” or “DD chitosan” also refers to chitosan that has been functionalized twice (“double-functionalized chitosan” or “DF chitosan”), for example, coupled with both an Arg and a hydrophilic polyol, where both are covalently linked to the chitosan. The Arg can be covalently linked to chitosan as a single amino acid or as a polypeptide. As used herein, unless otherwise stated, the terms “peptide” and “polypeptide” are used interchangeably. The term “polypeptide” is used in its broadest sense to refer to conventional polypeptides (i.e., short polypeptides containing L or D amino acids), as well as peptide equivalents, peptide analogs, and peptidomimetics that retain the desired functional activity. Peptide equivalents may differ from conventional peptides by replacing one or more amino acids with related organic acids, amino acids, or similar compounds, or by substituting or modifying side chains or functional groups. As is known in the art, an alternative bond can replace one or more peptide bonds in peptidomimetics. Parts or all of the peptide backbone can also be replaced with cyclic alkyl or aryl substituents that have conformational restrictions to limit the mobility of functional amino acid side chains, as is known in the art. The polypeptides of the present invention can be produced by known methods, such as recombinant and synthetic methods known in the art. Techniques for peptide synthesis are known and include those described in Merrifield, J. Amer. Chem. Soc. 85:2149-2456 (1963), Atherton, et al., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press (1989), and Merrifield, Science 232:341-347 (1986). As used herein, “linear polypeptide” refers to a polypeptide that does not have branching groups covalently attached to its constituent amino acid side chains. As used herein, “branched polypeptide” refers to a polypeptide comprising branching groups covalently attached to its constituent amino acid side chains. As mentioned above, the final degree of functionalization of the Arg or the final degree of functionalization of the polyol of a DD chitosan of the invention can be determined by elemental analysis. An example of calculating the final degree of functionalization of, for example, arginine and gluconic acid attached to the chitosan backbone is described below. For clarity, the following example uses a 20-mer chitosan molecule of 100% DDA. DDA 100% chitosan is a homopolymer of D-glucosamine; this repeating unit has 6 C atoms and 1 N atom, so the molar ratio of C / N is 6 / 1. Due to the depolymerization process, the end unit at one end of the polymer is a 2,5-anhydro-Dmannose. This unit has a C / N ratio of 6 / 0. Therefore, in the 20-mer chitosan example, there are 19 units of D-glucosamine and 1 unit of 2,5-anhydro-D-mannose. Thus, the overall C / N ratio is: C _ (19 x 6) + (1 x 6) _ 120 Ñ “ (19 x 1) + (1 x 0) ΤΓ This ratio is normalized to 6 carbons (which is the average number of C atoms per unit) over a known whole number of nitrogen atoms on average: 120 _ 6 “ 0^95 ινΐΛ / a / zuzz / uuuó iz In the coupling, for example, of arginine to chitosan, the arginine moiety has 6 C and 4 N. For an arginine-modified chitosan of 20 repeating units with an average degree of arginine functionalization of R%, the following relationship describes the C and N contribution of chitosan and arginine: C 6 + 6R% Ñ “ 0.95 + 4R% The C / N molar ratio is calculated from the experimentally derived C / N mass ratio. Therefore, once the C / N molar ratio is known, the degree of functionalization R% can be derived; that is, the percentage of amino groups in the chitosan backbone that are coupled with an arginine group. Gluconic acid coupling to chitosan: The gluconic acid residue has 6 C and 0 N. For a gluconic acid-modified chitosan of 20 repeating units with a degree of functionalization G%, the following relationship describes the contribution of C and N from quitO^^nn v áririn nhinónir.n· C _ 6 + 6G% Ñ “ 0.95 The C / N molar ratio is calculated from the experimentally derived C / N mass ratio. Therefore, once the C / N molar ratio is known, the degree of functionalization of G% can be derived; that is, the percentage of amine groups in the chitosan backbone that are coupled to a gluconic acid moiety. Arginine coupling to chitosan, followed by gluconic acid coupling: By coupling gluconic acid to 20-repeating R-chitosan with a previously determined final degree of arginine functionalization (R%), the following relationship describes the contribution of C and N from chitosan, arginine, and gluconic acid: C _ 6 + 6R% + 6G% Ñ “ 0.95 + 47?% The C / N molar ratio is calculated from the experimentally derived C / N mass ratio. Therefore, since R% was determined previously, the degree of functionalization G% can be derived once the C / N molar ratio is known. As described above, and as used herein, the “final degree of functionalization” of Arg or HP refers to the percentage of amino groups on the chitosan backbone functionalized with Arg or HP, respectively. Accordingly, “α:β ratio,” “final degree of functionalization ratio” (e.g., final degree of functionalization of Arg:final degree of functionalization ratio of HP), and similar terms may be used interchangeably with the expression “molar ratio” or “numerical ratio.” In one embodiment, the doubly derived chitosan is not a chitosan-derived nanoparticle or chitosan-derived nanoparticle polyplex as shown in PCT application no. PCT / CA2013 / 050218, particularly in the Examples in PCT / CA2013 / 050218. In one embodiment, the chitosan-derived nanoparticle is not one of the following nanoparticles: a. a chitosan-derived nanoparticle, wherein the nanoparticle comprises 24-mer chitosan coupled with arginine to a final degree of functionalization of 52% and gluconic acid to a final degree of functionalization of 8%; b. a chitosan-derived nanoparticle having an amine / phosphate (N / P) ratio of 20, wherein the nanoparticle comprises 24-mer chitosan coupled with arginine to a final degree of functionalization of 26% and gluconic acid to a final degree of functionalization of 3%; c. a chitosan-derived nanoparticle having an amine / phosphate (N / P) ratio of 20, wherein the nanoparticle comprises 24-mer chitosan coupled with arginine to a final degree of functionalization of 26% and gluconic acid to a final degree of functionalization of 5%; d. a chitosan-derived nanoparticle having an amine / phosphate (N / P) ratio of 20, wherein the nanoparticle comprises 24-mer chitosan coupled with arginine to a final degree of functionalization of 26% and gluconic acid to a final degree of functionalization of 6%; e. a chitosan-derived nanoparticle having an amine / phosphate (N / P) ratio of 20, wherein the nanoparticle comprises 24-mer chitosan coupled with arginine to a final degree of functionalization of 26% and gluconic acid to a final degree of functionalization of 9%; f. a chitosan-derived nanoparticle having an amine / phosphate (N / P) ratio of 40, wherein the nanoparticle comprises 24-mer chitosan coupled with arginine to a final degree of functionalization of 26% and gluconic acid to a final degree of functionalization of 5%; g. a chitosan-derived nanoparticle having an amine / phosphate (N / P) ratio of 10, wherein the nanoparticle comprises 24-mer chitosan coupled with arginine to a final degree of functionalization of 26% and gluconic acid to a final degree of functionalization of 5%; h. a chitosan-derived nanoparticle, wherein the nanoparticle comprises 24-mer chitosan coupled with arginine to a final degree of functionalization of 26% and gluconic acid to a final degree of functionalization of 5%; i. a chitosan-derived nanoparticle, wherein the nanoparticle comprises 24-mer chitosan coupled with arginine to a final degree of functionalization of 26% and gluconic acid to a final degree of functionalization of 6%; or j. a chitosan-derived nanoparticle, wherein the nanoparticle comprises 24-mer chitosan coupled with arginine to a final degree of functionalization of 52% and gluconic acid to a final degree of functionalization of 8%. In one embodiment, the chitosan-derived nanoparticle comprises chitosan coupled with gluconic acid to a final degree of functionalization of 1%, 2%, 4%, 7%, 8%, 10%, 15%, 20%, 25%, 30%, or higher. In one embodiment, the chitosan-derived nanoparticle comprises chitosan coupled with arginine to a final degree of functionalization of about 1% to about 25%. In another embodiment, the chitosan-derived nanoparticle comprises chitosan coupled with arginine to a final degree of functionalization of about 27% to about 51%. In one embodiment, the chitosan-derived nanoparticle comprises chitosan coupled with arginine to a final degree of functionalization of about 53% to about 70% arginine. In another embodiment, the chitosan-derived nanoparticle comprises chitosan coupled with arginine to a final degree of functionalization that is neither 26% nor 52%. In one embodiment, the chitosan-derived nanoparticle comprises chitosan coupled with arginine to a final degree of functionalization of 26% and coupled with gluconic acid to a final degree of functionalization that is not 3%. In one embodiment, the chitosan-derived nanoparticle comprises chitosan coupled with arginine to a final degree of functionalization of 26% and coupled with gluconic acid to a final degree of functionalization that is not 5%. In one embodiment, the chitosan-derived nanoparticle comprises chitosan coupled with arginine to a final degree of functionalization of 26% and coupled with gluconic acid to a final degree of functionalization that is not 6%. In one embodiment, the chitosan-derived nanoparticle comprises chitosan coupled with arginine to a final degree of functionalization of 26% and coupled with gluconic acid to a final degree of functionalization that is not 9%. In one embodiment, the chitosan-derived nanoparticle comprises chitosan coupled with arginine to a final degree of functionalization of 26% and coupled with gluconic acid to a final degree of functionalization that is not 3%, 5%, 6%, or 9%.In one embodiment, the chitosan-derived nanoparticle comprises chitosan coupled with arginine to a final degree of functionalization of 52% and coupled with gluconic acid to a final degree of functionalization other than 8%. In another embodiment, the chitosan-derived nanoparticle comprises chitosan coupled with gluconic acid to a final degree of functionalization selected from 3%, 5%, 6%, and 9% and coupled with arginine to a final degree of functionalization other than 26%. In yet another embodiment, the chitosan-derived nanoparticle comprises chitosan coupled with gluconic acid to a final degree of functionalization of 8% and coupled with arginine to a final degree of functionalization other than 52%. In some forms, where applicable, chitosan DD includes chitosan DD derivatives, for example, chitosan DD incorporating additional functionalization, such as chitosan DD with an attached ligand. “Derivatives” shall be understood to include the broad category of chitosan-based polymers comprising covalently modified N-acetyl-D-glucosamine and / or D-glucosamine units, as well as chitosan-based polymers incorporating other units or linked to other residues. Derivatives are frequently based on a modification of the hydroxyl or amine group of glucosamine, as is the case with arginine-functionalized chitosan. Examples of chitosan derivatives include, but are not limited to, trimethylated chitosan, PEGylated chitosan, thiolated chitosan, galactosylated chitosan, alkylated chitosan, PEI-incorporated chitosan, uranic acid-modified chitosan, glycol chitosan, and the like.For further information on chitosan derivatives see, for example, pp.63-74 of “Non-viral Gene Therapy”, K. Taira, K. Kataoka, T. Nildome (eds.), Springer-Verlag Tokyo, 2005, ISBN 4-431-25122-7; Zhu et al., Chinese Science Bulletin, December 2007, vol. 52 (23), pp. 3207-3215; and Varma et al., Carbohydrate Polymers 55 (2004) 77-93. ινΐΛ / a / zuzz / uuuo i ¿ Dispersed systems consist of particulate matter, known as the dispersed phase, distributed throughout a continuous medium. A “dispersion” of chitosan DD polyplexes and nucleic acid is a composition comprising hydrated chitosan DD polyplexes and nucleic acid, where the polyplexes are distributed throughout the medium. As used herein, a “preconcentrated” dispersion is one that has not been subjected to the concentration process to form a concentrated dispersion. As used herein, “substantially without” polyplex precipitate means that the composition is essentially devoid of particles that can be observed by visual inspection. As used herein, physiological pH refers to a pH between 6 and 8. “Doubly derived chitosan polyplex and nucleic acid” or its grammatical equivalents refer to a complex comprising multiple molecules of doubly derived chitosan and multiple molecules of nucleic acid. In a preferred embodiment, the doubly derived chitosan forms complexes with said nucleic acid. Chitosan DD and nucleic acid polyplexes comprise a nucleic acid component and a chitosan DD component. Chitosan and chitosan DD and nucleic acid polyplexes can be prepared by any method known in the art. For example, the concentrations of nucleotide and functionalized chitosan raw materials can be adjusted to suit various amine-to-phosphate (N / P) ratios, mixing ratios, and target nucleotide concentrations. In some embodiments, particularly for small batches (e.g., batches less than 2 mL), the nucleotide and functionalized chitosan raw materials can be mixed by slowly dripping the nucleotide raw material into the functionalized chitosan raw material while vortexing the container. In other embodiments, the functionalized chitosan and nucleotide raw materials can be mixed by in-line mixing of the two fluid streams.In other embodiments, the resulting polyplex dispersion can be concentrated by TFF. A preferred method for polyplex formation is described in WO 2009 / 039657, which is expressly incorporated herein in its entirety by this reference. A nucleic acid of the present invention generally contains phosphodiester linkages, although in some cases nucleic acid analogs are included that may have alternative backbones or other modifications or incorporated residues for any of a variety of purposes, for example, stability and protection. Other contemplated nucleic acid analogs include those having backbones that are not ribose. In addition, mixtures of naturally occurring nucleic acids, analogs, and both are possible. The nucleic acids may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences.Nucleic acids include, but are not limited to, DNA, RNA, and hybrids where the nucleic acid contains any combination of deoxyribonucleotides and ribonucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthan, hypoxanthan, isocytosine, isoguanine, etc. Nucleic acids include DNA in any form, RNA in any form, including triple-stranded, double-stranded, single-stranded, antisense, siRNA, riboximes, deoxyribozymes, polynucleotides, oligonucleotides, chimeras, microRNAs, and derivatives thereof. Nucleic acids include artificial nucleic acids, which include, but are not limited to, peptide nucleic acid (PNA), morpholinophosphodiamidate oligo (PMO), locked nucleic acid (LNA), glycolic nucleic acid (GNA), and threose nucleic acid (TNA). In one embodiment, the nucleic acid component comprises a therapeutic nucleic acid. The chitosan DD and nucleic acid polyplexes hereof are suitable for use with any therapeutic nucleic acid known in the art. Therapeutic nucleic acids include therapeutic RNAs, which are RNA molecules capable of eliciting a therapeutic effect in a mammalian cell. Therapeutic RNAs include, but are not limited to, antisense RNA, siRNA, short hairpin RNA, microRNA, and enzyme RNAs. Therapeutic nucleic acids also include, but are not limited to, nucleic acids desired to form triplet molecules, protein-binding nucleic acids, ribozymes, deoxyribozymes, and small nucleotide molecules. Many types of therapeutic RNAs are known in this technique. For example, see Grimm et al., Therapeutic application of RNAi is mRNA targeting finally ready for prime time? J. Clin. Invest., 117:3633-3641, 2007; Aagaard et al., RNAi therapeutics: Principles, prospects and challenges, Adv. Drug Deliv. Rev., 59:75-86, 2007; Dorsett et al., siRNAs: Applications in functional genomics and potential as therapeutics, Nat. Rev. Drug Discov., 3:318-329, 2004. These include small double-stranded interfering RNA (sRNA). Therapeutic nucleic acids also include nucleic acids that encode therapeutic proteins, including cytotoxic proteins and prodrugs. In a preferred embodiment, the nucleic acid component comprises a therapeutic nucleic acid construct. A therapeutic nucleic acid construct is a nucleic acid construct capable of producing a therapeutic effect. Therapeutic nucleic acid constructs may comprise nucleic acids that encode therapeutic proteins, as well as nucleic acids that produce transcripts that are therapeutic RNAs. A therapeutic nucleic acid can be used to effect gene therapy by serving as a replacement for or enhancement of a defective gene or to compensate for the lack of a particular gene product by encoding a therapeutic product. A therapeutic nucleic acid can also inhibit the expression of an endogenous gene.A therapeutic nucleic acid may encode all or part of a translation product and may function by recombining with DNA already present in a cell, thereby replacing a defective portion of a gene. It may also encode part of a protein and produce its effect by simultaneously suppressing a gene product. In a preferred embodiment, the therapeutic nucleic acid is selected from those described in USSN 11 / 694,852, which is expressly incorporated herein by reference. In a preferred modality, the therapeutic nucleic acid encodes a therapeutic protein selected from the group consisting of hormones, enzymes, cytokines, chemokines, antibodies, mitogenic factors, growth factors, differentiation factors, factors influencing angiogenesis, factors influencing blood clot formation, factors influencing blood glucose levels, factors influencing glucose metabolism, factors influencing lipid metabolism, factors influencing blood cholesterol levels, factors influencing LDL or HDL levels in the blood, factors influencing cell apoptosis, factors influencing food intake, factors influencing energy expenditure, factors influencing appetite, factors influencing nutrient absorption, factors influencing inflammation, and factors influencing the ML / a / ZUZZ / UUUó I z bone formation. Therapeutic nucleic acids encoding insulin, leptin, glucagon antagonist, GLP-1, GLP-2, ghrelin, cholecystokinin, growth hormone, coagulation factors, PYY, erythropoietin, inflammation inhibitors, IL-10, IL-17 antagonists, TNFα antagonists, growth hormone-releasing hormone, or parathyroid hormone are particularly preferred. Expression control regions In a preferred embodiment, a polyplex of the invention comprises a therapeutic nucleic acid, which is a therapeutic construct, comprising an expression control region operatively linked to a coding region. The therapeutic construct produces therapeutic nucleic acid, which may be therapeutic in itself or may encode a therapeutic protein. In some embodiments, the expression control region of a therapeutic construct possesses constitutive activity. In a number of preferred embodiments, the expression control region of a therapeutic construct does not have constitutive activity. This facilitates the dynamic expression of a therapeutic nucleic acid. “Dynamic” expression means expression that changes over time. Dynamic expression may include several periods of low or absent expression separated by periods of detectable expression. In a number of preferred embodiments, the therapeutic nucleic acid is operatively linked to a tunable promoter. This facilitates the tunable expression of therapeutic nucleic acids. Expression control regions comprise regulatory polynucleotides (sometimes referred to herein as elements), such as promoters and enhancers, which influence the expression of an operationally bound therapeutic nucleic acid. The expression control elements included herein may be from bacteria, yeast, plants, or animals (mammalian or non-mammalian). Expression control regions include full-length promoter sequences, such as natural promoter and enhancer elements, as well as polynucleotide subsequences or variants that retain all or part of the full-length or non-variant function (e.g., retain a certain amount of nutrient regulation or cell / tissue-specific expression). As used herein, the term "functional" and its grammatical variants, when used in reference to a nucleic acid sequence, subsequence, or fragment, means that the sequence has one or more natural nucleic acid sequence functions (e.g., non-variant or unmodified sequence).As used herein, the term variant means a sequence substitution, deletion or addition, or other modification (e.g., chemical derivatives such as nuclease-resistant modified forms). As used herein, the term operational link refers to a physical juxtaposition of the components so described, enabling them to function in their intended manner. In the example of an expression control element operationally linked to a nucleic acid, the relationship is such that the control element modulates the expression of the nucleic acid. Typically, an expression control region that modulates transcription is juxtaposed near the 5' end of the transcribed nucleic acid (i.e., upstream). Expression control regions may also be located at the 3' end of the transcribed sequence (i.e., downstream) or within the transcript (e.g., in a (ML / a / ZUZZ / UUUó I z intron). Expression control elements can be located at a distance from the transcribed sequence (e.g., 100 to 500, 500 to 1000, 2000 to 5000) or more nucleotides of the nucleic acid. A specific example of an expression control element is a promoter, which is usually located at the 5' position of the transcribed sequence. Another example of an expression control element is an enhancer, which can be located at the 5' or 3' position of the transcribed sequence or within the transcribed sequence. Some expression control regions confer adjustable expression to an operatively bound therapeutic nucleic acid. A signal (sometimes called a stimulus) can increase or decrease the expression of a therapeutic nucleic acid operatively bound to such an expression control region. These expression control regions that increase expression in response to a signal are frequently referred to as inducible. These expression control regions that decrease expression in response to a signal are frequently referred to as repressible. Typically, the amount of increase or decrease conferred by such elements is proportional to the amount of signal present; the greater the amount of signal, the greater the increase or decrease in expression. Several tunable promoters are known in the art. Preferred inducible expression control regions include those comprising an inducible promoter that is stimulated by a small-molecule chemical compound. In one embodiment, an expression control region responds to a chemical that can be administered orally but is not normally found in food. Particular examples can be found, for instance, in U.S. Patent Nos. 5,989,910; 5,935,934; 6,015,709; and 6,004,941. In one embodiment, the therapeutic construct also comprises an integration sequence. In another embodiment, the therapeutic construct comprises a single integration sequence. In yet another embodiment, the therapeutic construct comprises first and second integration sequences for integrating the therapeutic nucleic acid, or a portion thereof, into the genome of a target cell. In a preferred embodiment, the integration sequence(s) are functional in combination with an integration medium selected from the group consisting of Mariner, Sleeping Beauty, FLP, Cre, ΦO31, R, lambda, and integration media for integrating viruses such as AAV, retroviruses, and lentiviruses. In one embodiment, the composition hereof also comprises a non-therapeutic construct in addition to a therapeutic construct, wherein the non-therapeutic construct comprises a nucleic acid sequence encoding an integration medium operatively linked to a second expression control region. This second expression control region and the expression control region operatively linked to the therapeutic nucleic acid may be the same or different. The encoded integration medium is preferably selected from the group consisting of Mariner, Sleeping Beauty, FLP, Cre, ΦO31, R, lambda, and integration media for integrating viruses such as AAV, retroviruses, and lentiviruses. For other indications, see WO2008020318, which is expressly incorporated herein in its entirety by this reference. In one embodiment, the nucleic acid of the chitosan DD polyplex and nucleic acid is an artificial nucleic acid. iviA / a / zuzz / uuuo i ¿ Preferred artificial nucleic acids include, but are not limited to, peptide nucleic acid (PNA), morpholino phosphorodiamidate oligo (PMO), locked nucleic acid (LNA), glycolic nucleic acid (GNA), and threose nucleic acid (TNA). In one modality, the nucleic acid of the chitosan DD polyplex and nucleic acid is a therapeutic nucleic acid. In another modality, the therapeutic nucleic acid is a therapeutic RNA. Preferred therapeutic RNAs include, but are not limited to, antisense RNA, siRNA, short hairpin RNA, microRNA, and enzyme RNA. In one modality, the therapeutic nucleic acid is DNA. In one modality, the therapeutic nucleic acid comprises a nucleic acid sequence that encodes a therapeutic protein. In preferred embodiments, the double-derivative (DD) nucleic acid-chitosan polyplex is not a polyplex comprising a nucleic acid encoding secreted alkaline phosphatase (SEAP) or luciferase that forms complexes with one of the following chitosan-derived nanoparticles: a. a chitosan-derived nanoparticle, wherein the nanoparticle comprises 24-mer chitosan coupled with arginine to a final degree of functionalization of 52% and gluconic acid to a final degree of functionalization of 8%; b. a chitosan-derived nanoparticle having an amine / phosphate (N / P) ratio of 20, wherein the nanoparticle comprises 24-mer chitosan coupled with arginine to a final degree of functionalization of 26% and gluconic acid to a final degree of functionalization of 3%; c. a chitosan-derived nanoparticle having an amine / phosphate (N / P) ratio of 20, wherein the nanoparticle comprises 24-mer chitosan coupled with arginine to a final degree of functionalization of 26% and gluconic acid to a final degree of functionalization of 5%; d. a chitosan-derived nanoparticle having an amine / phosphate (N / P) ratio of 20, wherein the nanoparticle comprises 24-mer chitosan coupled with arginine to a final degree of functionalization of 26% and gluconic acid to a final degree of functionalization of 6%; e. a chitosan-derived nanoparticle having an amine / phosphate (N / P) ratio of 20, wherein the nanoparticle comprises 24-mer chitosan coupled with arginine to a final degree of functionalization of 26% and gluconic acid to a final degree of functionalization of 9%; f. a chitosan-derived nanoparticle having an amine / phosphate (N / P) ratio of 40, wherein the nanoparticle comprises 24-mer chitosan coupled with arginine to a final degree of functionalization of 26% and gluconic acid to a final degree of functionalization of 5%; g. a chitosan-derived nanoparticle having an amine / phosphate (N / P) ratio of 10, wherein the nanoparticle comprises 24-mer chitosan coupled with arginine to a final degree of functionalization of 26% and gluconic acid to a final degree of functionalization of 5%; h. a chitosan-derived nanoparticle, wherein the nanoparticle comprises 24-mer chitosan coupled with arginine to a final degree of functionalization of 26% and gluconic acid to a final degree of functionalization of 5%; i. a chitosan-derived nanoparticle, wherein the nanoparticle comprises 24-mer chitosan coupled with arginine to a final degree of functionalization of 26% and gluconic acid to a final degree of functionalization of 6%; or j. a chitosan-derived nanoparticle, wherein the nanoparticle comprises 24-mer chitosan coupled with arginine to a final degree of functionalization of 52% and gluconic acid to a final degree of functionalization of 8%. Poly feet In a preferred embodiment, the polyplexes of the compositions comprise chitosan molecules with an average molecular weight less than 110 kDa, more preferably less than 65 kDa, more preferably less than 50 kDa, more preferably less than 40 kDa, and more preferably less than 30 kDa before functionalization. In some embodiments, the polyplexes of the compositions comprise chitosan with an average molecular weight less than 15 kDa, less than 10 kDa, less than 7 kDa, or less than 5 kDa before functionalization. In a preferred embodiment, the polyplexes comprise chitosan molecules having on average fewer than 680 glucosamine monomer units, more preferably fewer than 400 glucosamine monomer units, more preferably fewer than 310 glucosamine monomer units, more preferably fewer than 250 glucosamine monomer units, and even more preferably fewer than 190 glucosamine monomer units. In some embodiments, the polyplexes comprise chitosan molecules having on average fewer than 95 glucosamine monomer units, fewer than 65 glucosamine monomer units, fewer than 45 glucosamine monomer units, or fewer than 35 glucosamine monomer units. In a preferred embodiment, the polyplexes hereof have an amine-to-phosphate (N / P) ratio of 2 to 100, for example, 2 to 50, for example, 2 to 40, for example, 2 to 30, for example, 2 to 20, for example, 2 to 5. Preferably, the N / P ratio is inversely proportional to the molecular weight of the chitosan, i.e., a lower molecular weight DD chitosan requires a higher N / P ratio and vice versa. In a preferred embodiment, the present polyplexes have an average hydrodynamic diameter less than 1000 nm, more preferably less than 500 nm, and even more preferably less than 200 nm. In one modality, chitosan DD and nucleic acid polyplexes have an average zeta potential of at least 0 mV at an acidic pH, for example, a pH less than 7, more preferably a pH of around 4 to 6. In one modality, chitosan DD and nucleic acid polyplexes have an average zeta potential between +1 and +60 mV, more preferably +1 to +40 mV, more preferably +1 to +30 mV at an acidic pH. In a preferred modality, the polyplexes have a low net positive, neutral, or net negative charge at physiological pH and a pKa less than 6. These chitosan DD and nucleic acid polyplexes exhibit lower cellular toxicity and better intracellular nucleic acid release. The chitosan DD and nucleic acid polyplexes of the composition are preferably homogeneous with respect to polyplex size. Accordingly, in a preferred embodiment, the composition has a low average polydispersity index (“PDI”). In a particularly preferred embodiment, the dispersion of the chitosan DD and nucleic acid polyplex has a PDI less than 0.5, more preferably less than 0.4, more preferably less than 0.3, and even more preferably less than 0.25. The polyplexes of the compositions hereof are preferably of substantially stable size within the composition. In a preferred embodiment, a composition of the invention comprises polyplexes whose average diameter increases by less than 100%, more preferably less than 50%, and even more preferably less than 25%, at room temperature for 6 hours, more preferably 12 hours, more preferably 24 hours, and even more preferably 48 hours. In a particularly preferred embodiment, a composition of the invention comprises polyplexes whose average diameter increases by less than 25% at room temperature for at least 24 hours or at least 48 hours. The polyplexes of the compositions herein are preferably of substantially stable size under cold conditions. In a preferred embodiment, a composition of the invention comprises polyplexes whose average diameter increases by less than 100%, more preferably less than 50%, and even more preferably less than 25%, at 2-8 degrees Celsius for 6 hours, more preferably 12 hours, more preferably 24 hours, and even more preferably 48 hours. The polyplexes of the compositions herein preferably have a substantially stable size under freeze-thaw conditions. In a preferred embodiment, a composition of the invention comprises polyplexes whose average diameter increases by less than 100%, more preferably less than 50%, and even more preferably less than 25%, at room temperature for 6 hours, more preferably 12 hours, more preferably 24 hours, and even more preferably 48 hours after thawing from freezing at -20 to 80 degrees Celsius. In a preferred embodiment, the composition has a nucleic acid concentration greater than 0.5 mg / ml and is substantially free of precipitated polyplexes. More preferably, the composition has a nucleic acid concentration of at least 0.6 mg / ml, more preferably at least 0.75 mg / ml, more preferably at least 1.0 mg / ml, more preferably at least 1.2 mg / ml, and even more preferably at least 1.5 mg / ml, and is substantially free of precipitated polyplexes. The compositions are hydrated. In a preferred embodiment, the composition is substantially free of nucleic acid that did not form complexes. In a preferred embodiment, the chitosan DD polyplex and nucleic acid composition is isotonic. Achieving isotonicity while maintaining polyplex stability is highly desirable in pharmaceutical formulations, and these preferred compositions are suitable for pharmaceutical formulation and therapeutic applications. Generally, compositions comprising chitosan DD polyplexes and nucleic acid are used to bring a target cell into contact with the nucleic acid. This contact typically results in the delivery of the nucleic acid for expression by the target cell. Suitable chitosan DD polyplex and nucleic acid compositions described herein are known in the art and are generally described below. In a preferred embodiment, the polyplexes of the compositions comprise chitosan molecules having an average molecular weight of less than 50 kDa before functionalization, an average of less than 310 glucosamine monomeric units, an N / P ratio of 2 to 20, an average hydrodynamic diameter of less than 500 nm, an average zeta potential of at least 0 mV at acidic pH, a PDI of less than 0.5, a nucleic acid concentration greater than 0.5 mg / ml, are substantially free of precipitated polyplexes, and the polyplexes are size stable, in that their average diameter increases by less than 100% at room temperature for at least 6 hours, at least 12 hours, at least 24 hours, or at least 48 hours. In a preferred embodiment, the chitosan DD polyplex composition and nucleic acid is isotonic and has a substantially stable size under freeze-thaw conditions. In a preferred embodiment, the polyplexes of the compositions comprise chitosan molecules having an average molecular weight of less than 65 kDa before functionalization, an average of fewer than 400 glucosamine monomeric units, an N / P ratio of 2 to 20, an average hydrodynamic diameter of less than 500 nm, an average zeta potential of +1 to 40 mV at acidic pH, a PDI of less than 0.4, a nucleic acid concentration greater than 0.5 mg / ml, are substantially free of precipitated polyplexes, and the polyplexes are size-stable, in that their average diameter increases by less than 50% at room temperature for at least 6 hours, at least 12 hours, at least 24 hours, or at least 48 hours. In a preferred embodiment, the chitosan DD and nucleic acid polyplex composition is isotonic and substantially size-stable under freeze-thaw conditions. In a preferred embodiment, the polyplexes of the compositions comprise chitosan molecules having an average molecular weight of less than 40 kDa before functionalization, an average of fewer than 250 glucosamine monomeric units, an N / P ratio of 2 to 20, an average hydrodynamic diameter of less than 500 nm, an average zeta potential of +1 to +30 mV at acidic pH, a PDI of less than 0.3, a nucleic acid concentration greater than 0.5 mg / ml, are substantially free of precipitated polyplexes, and the polyplexes are size-stable, in that their average diameter increases by less than 50% at room temperature for at least 6 hours, at least 12 hours, at least 24 hours, or at least 48 hours. In a preferred embodiment, the chitosan DD and nucleic acid polyplex composition is isotonic and substantially size-stable under freeze-thaw conditions. In a preferred embodiment, the polyplexes of the compositions comprise chitosan molecules having an average molecular weight of less than 30 kDa before functionalization, an average of fewer than 190 glucosamine monomeric units, an N / P ratio of 2 to 5, an average hydrodynamic diameter of less than 500 nm, an average zeta potential of +1 to +30 mV at acidic pH, a PDI of less than 0.25, a nucleic acid concentration greater than 0.5 mg / ml, are substantially free of precipitated polyplexes, and the polyplexes are size-stable, in that their average diameter increases by less than 25% at room temperature for at least 6 hours, at least 12 hours, at least 24 hours, or at least 48 hours. In a preferred embodiment, the chitosan DD and nucleic acid polyplex composition is isotonic and substantially size-stable under freeze-thaw conditions. In a preferred embodiment, the polyplexes of the compositions comprise chitosan molecules having an average molecular weight of less than 30 kDa before functionalization, an average of less than 190 glucosamine monomeric units, an N / P ratio of 2 to 5, an average hydrodynamic diameter of less than 500 nm, an average zeta potential of +1 to +30 mV at acidic pH, a PDI of less than 0.25, a nucleic acid concentration greater than 0.75 mg / ml, substantially lacking precipitated polyplexes, and the polyplexes are size stable, in that their average diameter increases by less than 25% at room temperature for at least 6 hours, at least 12 hours, at least 24 hours, or at least 48 hours. In a preferred embodiment, the chitosan DD polyplex composition and nucleic acid is isotonic and has a substantially stable size under freeze-thaw conditions. In a preferred embodiment, the polyplexes of the compositions comprise chitosan molecules having an average molecular weight of less than 15 kDa before functionalization, an average of fewer than 95 glucosamine monomeric units, an N / P ratio of 2 to 5, an average hydrodynamic diameter of less than 200 nm, an average zeta potential of +1 to +30 mV at acidic pH, a PDI of less than 0.25, a nucleic acid concentration greater than 1.0 mg / ml, are substantially free of precipitated polyplexes, and the polyplexes are size-stable, in that their average diameter increases by less than 50% at room temperature for at least 6 hours, at least 12 hours, at least 24 hours, or at least 48 hours. In a preferred embodiment, the chitosan DD and nucleic acid polyplex composition is isotonic and substantially size-stable under freeze-thaw conditions. Powder formulations The chitosan DD polyplex and nucleic acid compositions of the invention include powders. In a preferred embodiment, the invention provides a dry powder chitosan DD polyplex and nucleic acid composition. In a preferred embodiment, the dry powder chitosan DD polyplex and nucleic acid composition is produced by dehydrating a dispersion of the chitosan DD polyplex and nucleic acid of the invention. Pharmaceutical formulations The present invention also provides “pharmaceutically acceptable” or “physiologically acceptable” formulations comprising chitosan DD polycomplex compositions and nucleic acid of the invention. These formulations can be administered in vivo to a subject to implement the treatment methods. As used herein, the terms pharmaceutically acceptable and physiologically acceptable refer to carriers, diluents, excipients, and the like that can be administered to a subject, preferably without producing excessive adverse side effects (e.g., nausea, abdominal pain, headache, etc.). These preparations for administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Pharmaceutical formulations may be manufactured from carriers, diluents, excipients, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption retardants, and similar components compatible with administration to a subject. These formulations may be contained in a tablet (coated or uncoated), capsule (hard or soft), microbead, emulsion, powder, granule, crystal, suspension, syrup, or elixir. Additional active compounds and preservatives, among other additives, may also be present, for example, antimicrobials, antioxidants, chelating agents, and inert gases. The excipients may include a salt, an isotonic agent, a serum protein, a buffer or other pH control agent, an antioxidant, a thickener, an uncharged polymer, a preservative, or a cryoprotectant. The excipients used in the compositions of the invention may also include an isotonic agent and a buffer or other pH control agent. These excipients may be added to achieve preferred pH ranges (around 6.0–8.0) and osmolarity ranges (around 50–400 mmol / L). Examples of suitable buffers are acetate, borate, carbonate, citrate, phosphate, and sulfonated organic molecule buffer. These buffers may be present in a composition at concentrations of 0.01 to 1.0% (w / v). An isotonic agent may be selected from any known in the art, for example, mannitol, dextrose, glucose, and sodium chloride or other electrolytes. Preferably, the isotonic agent is glucose or sodium chloride.Isotonic agents may be used in quantities that give the composition the same or a similar osmotic pressure as the biological environment into which it is introduced. The concentration of isotonic agent in the composition will depend on the nature of the particular isotonic agent used and may be in the range of approximately 0.1 to 10%. When glucose is used, it is preferably used at a concentration of 1 to 5% w / v, more particularly 5% w / v. When the isotonic agent is sodium chloride, it is preferably used in quantities up to 1% w / v, particularly 0.9% w / v. The compositions of the invention may also contain a preservative. Examples of preservatives include polyhexamethylene biguanidine, benzalkonium chloride, stabilized oxychlor complexes (such as those known as Purite®), phenylmercuric acetate, chlorobutanol, sorbic acid, chlorhexidine, benzyl alcohol, parabens, and thimerosal.Typically, these preservatives are present at concentrations of approximately 0.001 to 1.0%. In addition, the compositions of the invention may also contain a cryopreservative. Preferred cryopreservatives include glucose, sucrose, mannitol, lactose, trehalose, sorbitol, colloidal silicon dioxide, dextran (preferably with a molecular weight less than 100,000 g / mol), glycerol, and polyethylene glycols (with molecular weights less than 100,000 g / mol), or mixtures thereof. The most preferred are glucose, trehalose, and polyethylene glycol. Typically, these cryopreservatives are present at concentrations of approximately 0.01 to 10%. A pharmaceutical formulation can be formulated to be compatible with its intended route of administration. For example, for oral administration, a composition can be combined with excipients and used in the form of tablets, lozenges, capsules (e.g., gelatin capsules), or coatings (e.g., enteric coatings such as Eudragit® or Sureteric®). Pharmaceutically compatible binding agents and / or adjuvant materials can be included in oral formulations.Tablets, pills, capsules, pastilles and the like may contain any of the following ingredients or compounds of a similar nature: a binder, such as microcrystalline cellulose, tragacanth gum or gelatin; an excipient, such as starch or lactose, a disintegrating agent, such as alginic acid, Primogel or corn starch; a lubricant, such as magnesium stearate or other stearates; a gluing agent, such as colloidal silicon dioxide; a sweetening agent, such as sucrose or saccharin; or an agent. ML / a / ZUZZ / UUUó I z flavoring, such as mint, methyl salicylate or flavoring. Formulations may also include carriers to protect the composition from rapid degradation or elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. For example, a time-delay material such as glyceryl monostearate or glyceryl stearate may be used alone or in combination with a wax. Suppositories and other formulations that can be administered rectally (e.g., those that can be administered by enema) are also considered. Regarding rectal administration, see, for example, Song et al., Mucosal drug delivery: membranes, methodologies, and applications, Crit. Rev. Ther. Drug. Carrier Syst., 21:195-256, 2004; Wearley, Recent progress in protein and peptide delivery by noninvasive routes, Crit. Rev. Ther. Drug. Carrier Syst., 8:331-394, 1991. Other pharmaceutical formulations suitable for administration are known in the art and can be applied in the methods and compositions of the invention (see, for example, Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, NJ; and Pharmaceutical Principles of Solid Dosage Forms, Technonic Publishing Co., Inc., Lancaster, Pa., (1993)). Administration In one formulation, the use of chitosan DD in chitosan DD and nucleic acid polyplexes provides prolonged polyplex stability at physiological pH. This allows for efficient systemic administration, as well as other delivery methods. A number of administration routes are possible, and the choice of a particular route will depend in part on the target tissue. Syringes, endoscopes, cannulas, intubation tubes, catheters, and other devices can be used for administration. The effective doses or quantities for treating a subject are preferably sufficient to improve one, several, or all of the symptoms of the condition, in a measurable or detectable way, although preventing or inhibiting the progression or deterioration of the disorder or condition, or a symptom, is a satisfactory outcome. Therefore, in the case of a condition or disorder that can be treated by expressing a therapeutic nucleic acid in target tissue, the amount of therapeutic RNA or protein produced to improve a condition treatable by a method of the invention will depend on the condition and the desired outcome, and can be readily determined by someone skilled in the art. The appropriate quantities will depend on the condition being treated, the desired therapeutic effect, and the individual subject (e.g., bioavailability within the subject, sex, age, etc.).The effective amount can be determined by measuring the relevant physiological effects. Veterinary applications are also contemplated in the present invention. Accordingly, in one embodiment, the invention provides methods for treating non-human mammals, involving the administration of a chitosan-based nanoparticle of the invention to a non-human mammal in need of treatment. Parenteral administration iviA / a / zuzz / uuuo i ¿ The compounds of the invention can be administered directly into the bloodstream, muscle, or an internal organ. Suitable means for parenteral administration include intravenous, intra-arterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, and subcutaneous. Suitable devices for parenteral administration include needle injectors (including microneedles), needle-free injectors, and infusion techniques. Parenteral formulations are typically aqueous solutions that may contain excipients such as salts, carbohydrates, and buffering agents, but, for some applications, they may be more appropriately formulated as a sterile non-aqueous solution or in dry form for use with a suitable vehicle such as sterile, pyrogen-free water. The preparation of parenteral formulations under sterile conditions, for example by lyophilization, can be easily achieved by using standard pharmaceutical techniques known to experts in the art. The solubility of compounds used in the preparation of parenteral solutions can be increased by using appropriate formulation techniques, such as incorporating solubility-enhancing agents. Parenteral formulations can be formulated for immediate and / or modified release. Modified-release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release. Therefore, the compounds of the invention can be formulated as a thixotropic solid, semisolid, or liquid for administration as an implanted depot that provides modified release of the active compound. Oral administration The compositions herein can be administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract. The compositions of the invention can also be administered directly into the gastrointestinal tract. Suitable formulations for oral administration include solid formulations such as tablets, capsules, coated capsules containing particles or coated particles, liquids, or powders, dragees (including liquid-filled), chewable tablets, multiparticles and nanoparticles, gels, films, ovules, and sprays. Liquid formulations include suspensions, solutions, syrups, and elixirs. Liquid formulations can be prepared by reconstituting a solid. Tablet dosage forms generally contain a disintegrant. Examples of disintegrants include sodium starch glycolate, sodium carboxymethylcellulose, calcium carboxymethylcellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methylcellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropylcellulose, starch, pregelatinized starch, and sodium alginate. The disintegrant typically comprises 1% to 25% by weight, preferably 5% to 20% by weight, of the dosage form. Binders are generally used to provide cohesive qualities to a tablet formulation. Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinized starch, hydroxypropylcellulose, and hydroxypropylmethylcellulose. Tablets may also contain diluents, such as lactose (monohydrate, dry monohydrate powder, anhydrous, and the like), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch, and dibasic calcium phosphate dihydrate. The tablets may also optionally contain surfactants, such as sodium lauryl sulfate and polysorbate 80, and gluants, such as silicon dioxide and talc. When present, the surfactants may comprise from 0.2% to 5% by weight of the tablet, and the gluants may comprise from 0.2% to 1% by weight of the tablet. The tablets generally also contain lubricants such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl sulfate. The lubricants generally comprise from 0.25% to 10% by weight, preferably from 0.5% to 3% by weight of the tablet. Other possible ingredients include antioxidants, colorings, flavoring agents, preservatives, and flavor masking agents. Tablet mixtures may be compressed directly or by roller to form tablets. Tablet mixtures or portions thereof may be alternatively wet, dry, or granulated and melted, frozen and melted, or extruded prior to tablet formation. The final formulation may comprise one or more layers and may be coated or uncoated; it may even be encapsulated. The formulation of the tablets is described in Pharmaceutical Dosage Forms: Tablets, Vol. 1, by H. Lieberman and L. Lachman (Marcel Dekker, New York, 1980). Consumable oral films for human and veterinary use are typically water-soluble, flexible or thin, water-swellable dosage forms that can be rapidly dissolved or mucoadhesive and typically comprise a film-forming polymer, a binder, a solvent, a wetting agent, a plasticizer, a stabilizer or emulsifier, a viscosity modifier, and a solvent. Some formulation components may perform more than one function. The invention also includes multiparticle beads comprising a composition of the invention. Other possible ingredients include antioxidants, colorings, flavorings and flavor enhancers, preservatives, saliva-stimulating agents, cooling agents, cosolvents (including oils), emollients, bulking agents, antifoaming agents, surfactants, and flavor-masking agents. The films according to the invention are typically prepared by evaporative drying of thin aqueous films coated onto a peelable backing paper or carrier. This can be achieved in a drying oven or furnace, typically a combination coating dryer, or by freeze-drying or vacuum drying. Solid oral formulations can be formulated for immediate and / or modified release. Modified-release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release. Other suitable delivery technologies are known, such as high-energy dispersions and osmotic particles, and ML / a / ZUZZ / UUUó I z coated. Topical administration The compounds of the invention can also be administered topically to the skin or mucous membranes, that is, dermal or transdermal. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, absorbent powders, bandages, foams, films, dermal patches, wafers, implants, sponges, fibers, dressings, and microemulsions. Other means for topical administration include administration by electroporation, iontophoresis, phonophoresis, sonophoresis, and microneedle or needle-free injection (e.g., Powderject™, Bioject™, etc.). Topical formulations can be formulated for immediate and / or modified release. Modified-release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release. Inhaled / intranasal administration The compounds of the invention can also be administered intranasally or by inhalation, typically in the form of a dry powder (either alone, as a mixture, for example, in a dry mixture with lactose, or as a particle of mixed components) from a dry powder inhaler or as an aerosol from a pressurized container, pump, aerosol, spray, or nebulizer, with or without the use of a suitable propellant. Capsules, ampoules, and cartridges can be formulated for use in an inhaler or insufflator to contain a powder mixture of the compound of the invention, a suitable powder base such as lactose or starch, and a performance modifier such as l-leucine, mannitol, or magnesium stearate. Inhaled / intranasal formulations can be formulated for immediate and / or modified release. Modified-release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release. Straight administration l / i ntra vag i nal The compounds of the invention can be administered rectally or vaginally, for example, in the form of a suppository, enema, or other oral form. Cocoa butter is a traditional base for suppositories, but various alternatives may be used as appropriate. Formulations for rectal / vaginal administration can be formulated for immediate and / or modified release. Modified-release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release. Eye / ear administration The compounds of the invention can also be administered directly to the eye or ear, typically in the form of drops. Other formulations suitable for administration to the eye and ear include ointments, biodegradable (e.g., absorbent gel sponges, collagen) and non-biodegradable (e.g., silicone) implants, wafers, lenses, and particulate systems. The formulations can also be administered by iontophoresis. Eye / ear formulations can be formulated to be immediate and / or modified release. iviA / a / zuzz / uuuo i ¿ Modified-release formulations include delayed, sustained, pulsed, controlled, targeted, or programmed release. Methods of use In one embodiment, the nucleic acid and chitosan DD polyplex compositions of the invention can be used for therapeutic treatment. Such compositions are sometimes referred to herein as therapeutic compositions. The therapeutic proteins of the invention, as described below, are produced by polyplexes of the invention comprising therapeutic nucleic acids. The use of the proteins herein as described below refers to the use of the polyplexes herein to affect such protein use. The therapeutic proteins contemplated for use in the invention have a wide variety of activities and can be used in the treatment of a wide variety of disorders. The following description of the therapeutic protein activities and treatable indications of the invention is an example and is not intended to be exhaustive. The term "subject" refers to an animal; mammals are preferred, and humans are especially preferred. Table 4 shows a partial list of therapeutic proteins and target diseases. Table 4. iviA / a / zuzz / uuuo i ¿ MAIN COMPOUNDS TARGET DISEASE FUNCTION THERAPEUTIC EFFECT Insulin Diabetes Insulin replacement Improved glucose tolerance. Delay / Prevention of diabetes. Glucagon antagonists Diabetes Reduction of endogenous glucose production Improved glucose tolerance GLP-1 Diabetes Obesity Stimulation of β-lymphocyte growth, improved insulin sensitivity, appetite suppression Improved glucose tolerance. Induction of weight loss Leptin Obesity Diabetes Appetite suppression and improved insulin sensitivity Induction of weight loss. Improved glucose tolerance CCK Obesity Appetite suppression Induction of weight loss Growth hormone (GH) GH deficiency, wasting and anti-aging GH replacement Growth enhancement Coagulation factors Hemophilia Coagulation factor replacement Improved clotting time Therapeutic antibodies and antibody parts / fragments Infections Cancer Pathogen neutralization or immune modulation Prevention of infections or transplant rejection iviA / a / zuzz / uuuo i ¿ Inflammation inhibitors, e.g., IL-10, TGF-β, TNFα antagonists, IL-17 antagonists. Inflammation of gastrointestinal organs, e.g., inflammatory bowel disease (IBD). Immune modulation. Prevention of inflammation in gastrointestinal organs. In another embodiment, the therapeutic compositions of the invention comprise therapeutic nucleic acids that do not encode therapeutic proteins, for example, therapeutic RNAs. For instance, by selecting therapeutic RNAs that target genes involved in disease mechanisms and / or unwanted cellular or physiological conditions, the compositions herein can be used in the treatment of a wide range of diseases and conditions. The compositions herein are such that the therapeutic RNAs used are not limited with respect to the scope of the target selection. Accordingly, the compositions herein can be used in any disease or condition involving a suitable target. Preferred tissues, diseases, and conditions include the following, which are examples and not exhaustive: Target Organ Target Disease Gastrointestinal Organs (Gl) Diabetes Obesity Inflammatory Bowel Disease Irritable Bowel Syndrome Infection Gl Peptic Ulcers Gastroesophageal Reflux Gastroparesis Hemorrhoids Nutrient Malabsorption Cancers Gl (colorectal, pancreatic, stomach, esophageal, bile duct, gallbladder cancer) Pancreatitis Hemochromatosis Celiac Disease Food Allergies Induction of Immune Tolerance Hormone Deficiency Eye Macular degeneration Age-related macular degeneration Uveitis Retinitis pigmentosa Iritis Sclerosis Glaucoma Keratitis Retinopathy Eye infection (e.g., keratomycosis) Cancer of the uterus, vagina, ovary, and cervix Cancers Infections Endometriosis Cervicitis Urologic pain Polyps Fibroids Endometrial hyperplasia Bladder and urinary tract Urinary incontinence Bladder and urinary tract infection Overactive bladder Erectile dysfunction Diabetic neuropathy ML / a / ZUZZ / UUU 01 z Kidney Diabetic nephropathy Membranous nephropathy Hypertension Kidney cancer Hypertension Polycystic kidney disease Glomerulonephritis Liver Dyslipidemia / hypercholesterolemia Diabetes Metabolic syndrome Hepatoma Hepatitis A / B / C Hemochromatosis Cirrhosis Steatohepatitis Glycogen storage diseases Skin Psoriasis Acne Rosacea Granulomatous dermatitis Anti-wrinkle Depigmentation ML / a / ZUZZ / UUU 01 z Lung / Respiratory Organs Lung Cancer Chronic Obstructive Pulmonary Disease Respiratory Tract Infection Cystic Fibrosis Pulmonary Vascular Diseases Myasthenia Gravis Fibrosis Asthma Brain Huntington's Disease Alzheimer's Disease Parkinson's Disease Brain Cancer Obesity Neurological Disorders Blood Cells Cancers Infectious Disease Autoimmune Disease Muscle Metabolic Syndrome Atherosclerosis Diabetes Sarcoma Inflammation (e.g., polymyositis) Glycogen Storage Diseases Myopathy Heart Myocardial infarction Atherosclerosis Angina Cardiomyopathy Ischemia Hypertensive heart disease Thrombosis Aneurysm Adipose tissue Diabetes Obesity Metabolic syndrome Atherosclerosis Dyslipidemia iviA / a / zuzz / uuuo i ¿ Hyperglycemia and body mass Therapeutic proteins include insulin and insulin analogues. Diabetes mellitus is a debilitating metabolic disease caused by the absent (type 1) or insufficient (type 2) production of insulin by pancreatic beta cells (Unger, RH et al., Williams Textbook of Endocrinology Saunders, Philadelphia (1998)). Beta cells are specialized endocrine cells that create and store insulin for release after a meal (Rhodes, et al., J. Cell Biol. 105:145 (1987)), and insulin is a hormone that facilitates the transfer of glucose from the blood into the tissues where it is needed. Patients with diabetes must frequently monitor their blood glucose levels, and many require multiple daily insulin injections to survive. However, such patients rarely achieve ideal glucose levels through insulin injection alone (Turner, RC et al., JAMA 281:2005 (1999)).Furthermore, prolonged elevation of insulin levels can produce harmful side effects such as hypoglycemic shock and desensitization of the body's response to insulin. As a consequence, diabetic patients developed similar long-term complications, such as cardiovascular disease, kidney disease, blindness, nerve damage, and impaired wound healing (UK Prospective Diabetes Study (UKPDS) Group, Lancet 352, 837 (1998)). The disorders treatable by a method of the invention include a hyperglycemic condition, such as insulin-dependent (type 1) or insulin-independent (type 2) diabetes, as well as physiological conditions or disorders associated with and arising from the hyperglycemic condition. Therefore, hyperglycemic conditions treatable by a method of the invention also include a histopathological change associated with chronic or acute hyperglycemia (e.g., diabetes). Particular examples include degeneration of the pancreas (β-cell destruction), calcification of the renal tubules, liver degeneration, eye damage (diabetic retinopathy), diabetic foot, mucosal ulcers, such as those in the mouth and gums, excessive bleeding, delayed blood clotting or wound healing, and an increased risk of coronary heart disease, stroke, peripheral vascular disease, dyslipidemia, hypertension, and obesity. The compositions herein are useful for lowering glucose, improving glucose tolerance, treating a hyperglycemic condition (e.g., diabetes), or treating physiological disorders associated with or resulting from a hyperglycemic condition. Such disorders include, for example, diabetic (autonomic) neuropathy, nephropathy (kidney damage), skin infections and other skin disorders, slow or delayed healing of wounds or injuries (e.g., leading to diabetic carbuncles), eye damage (retinopathy, cataracts) which can lead to blindness, diabetic foot, and accelerated periodontitis. Such disorders also include an increased risk of developing coronary heart disease, stroke, peripheral vascular disease, dyslipidemia, hypertension, and obesity. As used herein, the term hyperglycemic or hyperglycemia, when used in reference to a condition in a person, refers to an abnormally high, transient or chronic, level of glucose present in the person's blood. The condition may result from a delay in glucose metabolism or absorption, such that the person exhibits glucose intolerance or a state of elevated glucose not normally found in healthy individuals (e.g., in subdiabetic individuals with glucose intolerance at risk of developing diabetes, or in diabetic individuals). Fasting plasma glucose (FPG) levels for normoglycemia are less than approximately 110 mg / dL, for impaired glucose metabolism, approximately 110 to 126 mg / dL, and for diabetics, greater than approximately 126 mg / dL. Disorders that can be treated by producing a protein in digestive mucosal tissue also include obesity or unwanted body mass. Leptin, cholecystokinin, PYY, and GLP-1 decrease hunger, increase energy expenditure, induce weight loss, or provide normal glucose homeostasis. Therefore, in several embodiments, a method of the invention for treating obesity or unwanted body mass, or hyperglycemia, involves the use of a leptin encoding a therapeutic nucleic acid, cholecystokinin, PYY, or GLP-1. In another embodiment, a ghrelin targeting therapeutic RNA is used. Ghrelin increases appetite and hunger. Therefore, in several embodiments, a method of the invention for treating obesity or unwanted body mass, or hyperglycemia, involves the use of a ghrelin targeting therapeutic RNA to reduce its expression.Disorders that can be treated also include those typically associated with obesity, e.g., abnormally elevated plasma / serum LDL, VLDL, triglycerides, cholesterol, plaque formation leading to narrowing or blockage of blood vessels, increased risk of hypertension / stroke, coronary heart disease, etc. As used herein, the term obese or obesity refers to an individual who has at least a 30% increase in body mass compared to a normal-weight individual of the same age and sex. “Unwanted body mass” refers to individuals who have more than 1%–29% more body mass than a normal-weight individual, as well as individuals who have a normal body mass but wish to reduce or prevent further increase in their body mass. In one embodiment, a therapeutic protein of the invention is a glucagon antagonist. Glucagon is a peptide hormone produced by β cells in pancreatic islets and is a key regulator of glucose metabolism (Unger RH & Orel LN Eng. J. Med. 304:1518(1981); Unger RH Diabetes 25:136 (1976)). As with insulin, the The concentration of blood glucose mediates glucagon secretion. However, unlike insulin, glucagon is secreted in response to a decrease in blood glucose. Therefore, circulating concentrations of glucagon are higher during periods of fasting and lower during a meal. Glucagon levels increase to prevent insulin from promoting glucose storage and to stimulate the release of glucose from the liver into the bloodstream. A specific example of a glucagon antagonist is [des-His1, des-Phe6, Glu9]glucagon-NH2. In streptozotocin-diabetic rats, blood glucose levels are reduced by 37% within 15 minutes of an intravenous bolus (0.75 pg / g body weight) of this glucagon antagonist (Van Tiñe BA et al. Endocrinology 137:3316 (1996)).In another embodiment, the invention provides a method for treating diabetes or hyperglycemia, comprising the use of a therapeutic RNA to reduce glucagon production levels from the pancreas. In another embodiment, a therapeutic protein of the invention useful for treating a hyperglycemic condition or unwanted body mass (e.g., obesity) is glucagon-like peptide-1 (GLP-1). GLP-1 is a hormone released from L cells in the intestine during a meal while stimulating pancreatic β cells to increase insulin secretion. GLP-1 has additional activities that make it an attractive therapeutic agent for the treatment of obesity and diabetes. For example, GLP-1 reduces gastric emptying, suppresses appetite, reduces glucagon concentration, increases β-cell mass, stimulates glucose-dependent insulin biosynthesis and secretion, and possibly increases tissue sensitivity to insulin (Kieffer TJ, Habener JF Endocrin. Rev. 20:876 (2000)).Therefore, the regulated release of GLP-1 in the intestine to coincide with a meal may provide a therapeutic benefit for a hyperglycemic condition or unwanted body mass. GLP-1 analogs that are resistant to dipeptidyl peptidase IV (DPP-IV) provide a longer duration of action and better therapeutic value. Therefore, GLP-1 analogs are preferred therapeutic polypeptides. In another embodiment, the invention provides a method for treating diabetes or hyperglycemia, comprising the use of a therapeutic RNA to reduce DPP-IV levels. In another embodiment, a therapeutic protein of the invention useful for treating a hyperglycemic condition is an antagonist of the hormone resistin. Resistin is an adipocyte-derived factor whose expression is elevated in genetic and diet-induced forms of obesity. Neutralizing circulating resistin improves blood glucose and insulin action in obese mice. Conversely, administering resistin to normal mice impairs glucose tolerance and insulin action (Steppan CM et al. Nature 409:307 (2001)). The production of a protein that antagonizes the biological effects of resistin in the gut may therefore provide an effective therapy for insulin resistance associated with obesity and hyperglycemic conditions.In another embodiment, the invention provides a method for treating diabetes or hyperglycemia, comprising the use of a therapeutic RNA to reduce resistin expression levels in adipose tissue. In another embodiment, a therapeutic polypeptide of the invention useful for treating a hyperglycemic condition or unwanted body mass (e.g., obesity) is leptin. Although primarily produced by fat cells, leptin is also produced in smaller amounts in the stomach in a food-dependent manner. Leptin transmits information about fat cell metabolism and body weight to the appetite centers in the brain, where it signals reduced food intake (promoting satiety) and increased energy expenditure. In another embodiment, a therapeutic polypeptide of the invention useful for treating a hyperglycemic condition or unwanted body mass (e.g., obesity) is the C-end globular head domain of the adipocyte complement-related protein (Acrp30). Acrp30 is a protein produced by differentiated adipocytes. Administration of a proteolytic cleavage product of Acrp30 consisting of the globular head domain to mice results in substantial weight loss (Fruebis J. et al., Proc. Nati Acad. Sci USA 98:2005 (2001)). In another embodiment, a therapeutic polypeptide of the invention useful for treating a hyperglycemic condition or unwanted body mass (e.g., obesity) is cholecystokinin (CCK). CCK is a gastrointestinal peptide secreted by the intestine in response to particular nutrients in the gut. CCK release is proportional to the amount of food consumed and is believed to signal the brain to end a meal (Schwartz MW et al., Nature 404:661-71 (2000)). Consequently, elevated CCK may reduce meal size and promote weight loss or weight stabilization (i.e., prevent or inhibit weight gain). Regarding PYY, see for example le Roux et al., Proc Nutr Soc. May 2005; 64(2):213-6. Immunological disorders In one embodiment, a therapeutic composition of the invention possesses immunomodulatory activity. For example, a therapeutic polypeptide of the present invention may be useful in the treatment of deficiencies or disorders of the immune system by activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells. Immune cells develop through the process of hematopoiesis, producing myeloid cells (platelets, red blood cells, neutrophils, and macrophages) and lymphoid cells (B and T lymphocytes) from pluripotent stem cells. The etiology of these immune deficiencies or disorders may be genetic, somatic, such as cancer or some acquired autoimmune disorders (e.g., due to chemotherapy or toxins), or infectious. A therapeutic composition of the present invention may be useful in the treatment of hematopoietic cell deficiencies or disorders. For example, a therapeutic polypeptide of the present invention may be used to increase the differentiation or proliferation of hematopoietic cells, including pluripotent stem cells, in an effort to treat disorders associated with a reduction in certain (or many) types of hematopoietic cells. Examples of immunodeficiency syndromes include, but are not limited to: blood protein disorders (e.g., agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia, common variable immunodeficiency, DiGeorge syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome, lymphopenia, bactericidal phagocyte dysfunction, severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, anemia, thrombocytopenia, or hemoglobinuria. A therapeutic composition of the present invention may also be useful in the treatment of autoimmune disorders. Many autoimmune disorders occur when immune cells inappropriately recognize foreign material. This inappropriate recognition results in an immune response that leads to the destruction of host tissue. Therefore, the administration of a therapeutic composition of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T cells, may be an effective therapy in the prevention of autoimmune disorders. Examples of autoimmune disorders that can be treated by the present invention include, but are not limited to: Addison's disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture syndrome, Graves' disease, multiple sclerosis, myasthenia gravis, neuritis, ophthalmia, vesicular pemphigus, pemphigus, polyendocrinopathies, purpura, Reiter's disease, stiff-person syndrome, autoimmune thyroiditis, systemic lupus erythematosus, autoimmune pulmonary inflammation, Guillain-Barré syndrome, insulin-dependent diabetes mellitus, Crohn's disease, ulcerative colitis, and autoimmune inflammatory eye disease. Similarly, allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems, can also be treated using a therapeutic composition of the present invention. Furthermore, these molecules can be used to treat anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility. A therapeutic composition of the present invention can also be used to treat and / or prevent organ rejection or graft-versus-host disease (GVHD). Organ rejection occurs through the destruction of host immune cells in the transplanted tissue via an immune response. Similarly, an immune response is also involved in GVHD, but in this case, foreign transplanted immune cells destroy the host tissues. Administration of a therapeutic composition of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T lymphocytes, can be an effective therapy in preventing organ rejection or GVHD. Similarly, a therapeutic composition of the present invention may also be useful for modulating inflammation. For example, the therapeutic polypeptide may inhibit the proliferation and differentiation of cells involved in an inflammatory response. These molecules can be used to treat inflammatory conditions, both chronic and acute, including inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, pancreatitis, complement-mediated hyperacute rejection, nephritis, cytokine- or chemokine-induced lung injury, inflammatory bowel disease (IBD), Crohn's disease, or those resulting from the overproduction of cytokines (e.g., TNF or IL-1). In one embodiment, a therapeutic RNA targeting TNFα is used in the compositions hereof to treat inflammation.In another preferred embodiment, a therapeutic RNA directed against IL-1 is used in the compositions hereof to treat inflammation. SiRNA therapeutic RNAs are particularly preferred. Inflammatory disorders of interest for treatment in the present invention include, but are not limited to, chronic obstructive pulmonary disease (COPD), interstitial cystitis, and inflammatory bowel disease. Coagulation disorders. In some embodiments, a therapeutic composition of the present invention can also be used to modulate hemostatic (bleed-stopping) or thrombolytic (clot-forming) activity. For example, by increasing hemostatic or thrombolytic activity, a therapeutic composition of the present invention can be used to treat blood clotting disorders (e.g., afibrinogenemia, factor deficiency), platelet disorders (e.g., thrombocytopenia), or wounds resulting from trauma, surgery, or other causes. Alternatively, a therapeutic composition of the present invention that can reduce hemostatic or thrombolytic activity could be used to inhibit or dissolve clots. Such therapeutic compositions may be important in the treatment of heart attacks (myocardial infarctions), strokes, or wound healing.In one embodiment, a therapeutic polypeptide of the Invention is a clotting factor, useful for the treatment of hemophilia or another clotting disorder (e.g., Factor VIII, IX or X). Hyperproliferative disorders In one embodiment, a therapeutic composition of the invention is capable of modulating cell proliferation. This therapeutic polypeptide can be used to treat hyperproliferative disorders, including malignant neoplasms. Examples of hyperproliferative disorders that can be treated with a therapeutic composition of the present invention include, but are not limited to, malignant neoplasms located in: abdomen, bones, breasts, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testes, ovaries, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic and urogenital. Similarly, other hyperproliferative disorders can also be treated with a therapeutic composition of the present invention. Examples of such hyperproliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sézary syndrome, Waldenström macroglobulinemia, Gaucher disease, histiocytosis, and any other hyperproliferative disease, as well as malignant neoplasms, located in an organ system listed above. Administration to the circulatory system facilitates access of therapeutic proteins to a wide variety of tissues. Alternatively, a therapeutic composition of the present invention may stimulate the proliferation of other cells that can inhibit the hyperproliferative disorder. For example, hyperproliferative disorders can be treated by enhancing the immune response, particularly by increasing the antigenic qualities of the hyperproliferative disorder or by proliferating, differentiating, or mobilizing T cells. This immune response can be enhanced either by boosting an existing immune response or by initiating a new one. Alternatively, suppressing the immune response can also be a method for treating hyperproliferative disorders, such as with a chemotherapeutic agent. Infectious disease In one embodiment, a therapeutic composition of the present invention can be used to treat an infectious disease. For example, by enhancing the immune response, particularly by increasing the proliferation and differentiation of B and / or T cells, infectious diseases can be treated. The immune response can be enhanced either by potentiating an existing immune response or by initiating a new immune response. Alternatively, the therapeutic composition of the present invention can also directly inhibit the agent. ML / a / ZUZZ / UUUó I z infectious, without necessarily provoking an immune response. Viruses are an example of an infectious agent that can cause a disease or symptoms treatable with a therapeutic composition of the present invention. Examples of viruses include, but are not limited to, the following DNA and RNA viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza), Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae (such as smallpox or vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g., Rubivirus).The viruses included in these families can cause a variety of diseases or symptoms, including but not limited to: arthritis, bronchiolitis, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, chronic active, delta), meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt lymphoma, chickenpox, hemorrhagic fever, mumps, measles, parainfluenza, rabies, influenza, polio, leukemia, rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's warts), and viremia. A therapeutic composition of the present invention can be used to treat any of these symptoms or diseases. Similarly, the bacterial or fungal agents that can cause the disease or symptoms and that can be treated with a therapeutic composition of the present invention include, but are not limited to, the following families and Gram-negative and Gram-positive bacteria and fungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia), Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia, Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, Enterobacteriaceae (Klebsiella, Salmonella, Serratia, Yersinia), Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Meningococcal), Pasteurellacea infections (e.g., Actinobacillus, Heamophilus, Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, and Staphylococcal.These bacterial or fungal families can cause the following diseases or symptoms, including, but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (e.g., AIDS-related infections), paronychia, denture-related infections, Reiter's syndrome, respiratory tract infections such as whooping cough or empyema, sepsis, Lyme disease, cat scratch disease, dysentery, paratyphoid fever, food poisoning, typhoid, pneumonia, gonorrhea, meningitis, chlamydia, syphilis, diphtheria, leprosy, paratuberculosis, tuberculosis, lupus, botulism, gangrene, tetanus, impetigo, rheumatic fever, scarlet fever, sexually transmitted diseases, skin diseases (e.g., cellulitis, dermatomycosis), toxemia, urinary tract infections, and wound infections.A therapeutic composition of the present invention can be used to treat any of these symptoms or diseases. Furthermore, the parasitic agents that cause the disease or symptoms and that can be treated with a therapeutic composition of the present invention include, but are not limited to, the following families: Amebiasis, Babesiosis, ινΐΛ / a / zuzz / uuuó i ¿ Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic infections, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas. These parasites can cause a variety of diseases or symptoms, including but not limited to: scabies, thrombocytopenia, eye infections, intestinal disease (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS-related), malaria, pregnancy complications, and toxoplasmosis. A therapeutic composition of the present invention can be used to treat any of these symptoms or diseases. Regeneration A therapeutic composition of the present invention can be used to differentiate, proliferate, and attract cells, thereby promoting tissue regeneration. (See Science 276:59-87 (1997).) Tissue regeneration could be used to repair, replace, or protect tissues damaged by congenital defects, trauma (wounds, burns, incisions, or ulcers), age, disease (e.g., osteoporosis, osteoarthritis, periodontal disease, liver dysfunction), surgery, including cosmetic plastic surgery, fibrosis, reperfusion injury, or systemic cytokine damage. The therapeutic compositions of the invention can promote the regeneration of a variety of tissues, including but not limited to organs (e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscles (soft, skeletal, or cardiac), vascular tissues (including vascular endothelium), nervous tissue, hematopoietic tissue, and skeletal tissue (bone, cartilage, tendon, and ligament). Preferably, the regeneration results in a small amount of scarring or occurs without scarring. The regeneration may also include angiogenesis. Furthermore, a therapeutic composition of the present invention can enhance the regeneration of tissues that are difficult to heal. For example, increased regeneration of tendons / ligaments would accelerate recovery time after injury. A therapeutic composition of the present invention could also be used prophylactically in an effort to prevent injury. Specific conditions that could be treated include tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. Additional examples of tissue regeneration in non-healing wounds include pressure ulcers, ulcers associated with vascular insufficiency, and surgical and traumatic wounds. Similarly, nerve and brain tissue could also be regenerated using a therapeutic composition of the present invention to promote the proliferation and differentiation of nerve cells. Diseases that could be treated using this method include diseases of the central and peripheral nervous system, neuropathies, and mechanical and traumatic disorders (e.g., bone marrow disorders, head trauma, cerebrovascular disease, and stroke). Specifically, diseases associated with peripheral nerve injury, peripheral neuropathy (e.g., resulting from chemotherapy or other medical therapies), localized neuropathies, and diseases of the central nervous system (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome) could be treated using therapeutic compositions of the present invention.Regarding CNS disorders, several techniques are known to facilitate therapeutic access to brain tissue, including methods to alter the blood-brain barrier and methods to attach therapeutic agents to molecules that provide transport to the CNS. In one approach, a therapeutic nucleic acid is designed to encode a fusion protein, which comprises one of these factors (e.g., orthoclone OKT-e (anti-CD3), monoclonal antibody GPIIb / 11). Therapeutic RNAs targeting nucleic acids that encode such factors are also being considered. Vaccine In one embodiment, the invention provides methods for vaccinating a patient. The methods comprise administering a composition of the invention capable of producing the desired epitope. In a preferred embodiment, the composition comprises a therapeutic nucleic acid construct capable of expressing a protein comprising the epitope. Cosmetic applications In one embodiment, the invention provides chitosan DD and nucleic acid polyplexes for cosmetic use. The subjects hereof comprise chitosan DD-nucleic acid polyplexes in a formulation suitable for cosmetic use. EXAMPLES Example 1: Formation of double-derivative chitosan and formation of DNA polyplexes Chitosan was dual-derived (DD chitosan) with arginine and gluconic acid, or with arginine and threonic acid, according to known methods. DD chitosan formed a polyplex with the DNA vector encoding either secreted alkaline phosphatase (SEAP) or luciferase siRNA. Example 2: In vitro transfection with DNA polyplex In general, in vitro transfection of 293T cells with DD chitosan polyplex formulations and nucleic acid was performed in two steps: cell preparation followed by transfection. Example 3: Cell line maintenance The 293T cell line was courtesy of Dr. Timothy Kieffer's laboratory at the University of British Columbia (Vancouver, Canada) and was prepared as follows. Human kidney cells were transformed with the SV40 T antigen; cultured in modified high-glucose Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and penicillin / streptomycin; and maintained below 80% confluency. Example 4: Preparing cells for transfection Cells were prepared for transfection as follows. The day before transfection, 293T cells were added to 6-well tissue culture plates (4.5 x 10⁵ cells / well) in 3 mL of complete medium (DMEM with elevated glucose + 10% FBS + penicillin / streptomycin). On the day of transfection, cell counts were determined for two selected wells by washing the cells 1X with phosphate-buffered saline (PBS), treating the cells with 0.5 mL of 0.05% trypsin, adding 0.5 mL of complete medium, and counting 10 pL using a hemocytometer. If the cells were 50% confluent (~7 x 10⁵ cells / well), then transfection occurred (if the cells were either too dispersed or too confluent, then transfection did not occur). Example 5: Cell transfection. The transfection was performed as follows. First, the medium was removed from each well, followed by the addition of 1 mL of Opti-mem (pH 7.4) to each well. The medium was gently swirled and then removed (six wells were washed at the same time to prevent cell detachment). Then, an additional 1 mL of Opti-mem (pH 7.4) was carefully added. ML / a / ZUZZ / UUUó I z 7.4) to each well in a way that did not dislodge the cells. Then, the polyplex samples (target of 2 pg of DNA) were added to each well, shaken, and incubated at 37 °C for 2 hours. After incubation, the medium was discarded and replaced with 2 mL of complete medium and re-incubated at 37 °C. At the required time points, the supernatant was discarded and stored at -20 °C for the subsequent SEAP assay. Example 6: SEAP Protein Assay The SEAP assay was performed using the SEAP chemiluminescent assay kit. All reagents for the assay were equilibrated at 25 °C for 30 minutes before use. Assay standards were prepared by dissolving placental alkaline phosphatase at 1 mg / mL in 1X dilution buffer from the kit enriched with 0.1% bovine serum albumin and 50% glycerol, and then serially diluting 10-fold with DMEM to 0.01 pg / µL. Standards and frozen samples were then diluted 1:4 with dilution buffer, heat-inactivated at 65 °C for 30 minutes, incubated on ice for 2 minutes, centrifuged (16,100 x rcf for 2 minutes at room temperature), and the supernatants were transferred to new tubes. After equilibrating at 25 °C for 5 min, 50 uL of the samples and standards were added to each well of a Microlite-1 plate in duplicate.The inactivation buffer (50 µL) was then added to each well and gently pipetted up and down to mix, avoiding the creation of bubbles, and incubated for 5 minutes. The substrate / enhancer reagent was prepared during the 5-minute incubation at a 1:19 substrate-to-enhancer ratio. The substrate / enhancer was then added to each well, incubated for 20 minutes, and the plate was read on a luminometer (Lmax11384, Molecular Devices) with an integration time of 1 second. Example 7: Results Figures 1-3 show the transfection efficiencies of chitosan derived with varying percentages of arginine or gluconic acid, and of chitosan doubly derived with both gluconic acid and arginine. Figures 1-3 show a synergistic effect when chitosan is doubly derived with both arginine and gluconic acid. This synergistic effect can be observed when chitosan is doubly functionalized with arginine to a final functionalization degree of 10% and gluconic acid to final functionalization degrees of 3% to 10%, although the greatest effect was observed at a final gluconic acid functionalization degree of approximately 3% (Figure 1).The synergistic effect can also be observed when chitosan is doubly functionalized with arginine to a final functionalization degree of 52% and gluconic acid to final functionalization degrees ranging from 3% to 8%, although the greatest effect was observed at a final gluconic acid functionalization degree of around 8% (Figure 2). Furthermore, chitosan doubly functionalized with arginine and gluconic acid to final functionalization degrees of 26% and 6%, respectively, demonstrated a greater synergistic effect on transfection efficiency (Figure 3). Finally, the synergistic effect can also be observed when chitosan is doubly derived with arginine and an alternative moiety, such as threonic acid instead of gluconic acid. The synergistic effect is observed when chitosan is doubly functionalized with arginine to a final functionalization degree of 29% and threonic acid to a final functionalization degree within the 3% range (Figure 4). Example 8: ινΐΛ / a / zuzz / uuuo i ¿ Chitosan has a double derivation (DD chitosan) with arginine and a hydrocarbon selected from the group consisting of 2,3-dihydroxypropanoic acid; 2,3,4,5,6,7-hexahydroxyheptanal, 2,3,4,5,6-pentahydroxyhexanal, 2,3,4,5-tetrahydroxyhexanal and 2,3-dihydroxypropanal, and analyzed according to Examples 1-7. DD chitosan forms a polyplex with a DNA vector encoding either secreted alkaline phosphatase (SEAP) or luciferase siRNA. All references are expressly incorporated in their entirety by this reference. All patents and patent publications mentioned herein are incorporated herein by reference. Skilled practitioners may think of certain modifications and improvements after reading the preceding description. It should be understood that all such modifications and improvements have been removed from the description for the sake of readability and conciseness, but are adequately included within the scope of the following claims.

Claims

1. A chitosan-derived nanoparticle comprising chitosan functionalized with arginine (Arg) iviA / a / zuzz / uuuo 1 and a hydrophilic polyol (HP) of Formula Vil: wherein: R2 is selected from: H and hydroxyl; R3 is selected from: H and hydroxyl; and X is selected from: C2-C6 alkylene optionally substituted with one or more hydroxyl substituents, provided that said hydrophilic polyol is not gluconic acid.

2. The chitosan-derived nanoparticle of claim 1, wherein the hydrophilic polyol has a carboxyl group.

3. The chitosan-derived nanoparticle of claim 2, wherein the hydrophilic polyol is threonic acid.

4. The chitosan-derived nanoparticle of claim 1, wherein the hydrophilic polyol is a saccharide selected from the group consisting of glyceraldehyde, threose, erythrose, ribose, arabinose, xylose, lyxose, allose, glucose, altrose, mannose, gulose, idose, galactose, and talose.

5. The chitosan-derived nanoparticle of claim 1, wherein the hydrophilic polyol is selected from the group consisting of 2,3-dihydroxypropanoic acid, 2,3,4,5,6,7-hexahydroxyheptanal; 2,3,4,5,6-pentahydroxyhexanal, 2,3,4,5-tetrahydroxyhexanal and 2,3-dihydroxypropanal.

6. The chitosan-derived nanoparticle of claim 4, wherein the hydrophilic polyol is glucose.

7. The chitosan-derived nanoparticle of claim 1, wherein the nanoparticle has a final degree of functionalization ratio of Arg:final degree of functionalization ratio of HP of 1:1 to 10:

1.

8. The chitosan-derived nanoparticle of claim 7, wherein the final degree of functionalization ratio of Arg:final degree of functionalization ratio of HP is from 3:1 to 7:

1.

9. The chitosan-derived nanoparticle of claim 8, wherein the final degree of functionalization ratio of Arg:final degree of functionalization ratio of HP is 5:

1.

10. A composition comprising the nanoparticle according to any of claims 1-6 and 7-9, wherein said chitosan is complexed with a nucleic acid to form a dually derivatized (DD) chitosan nucleic acid polyplex.

11. The composition according to claim 10, wherein said nucleic acid is DNA or RNA.

12. The composition according to claim 11, wherein the amine to phosphate ratio of said chitosan DD nucleic acid polyplex is between 2 and 100.

13. The composition according to claim 12, chitosan DD nucleic acid polyplex is between 2 and 50.

14. The composition according to claim 13, chitosan DD nucleic acid polyplex is between 2 and 30.

15. The composition according to claim 14, wherein the amine to phosphate ratio of said chitosan nucleic acid polyplex DD is between 2 and 15.

16. A composition according to claim 11 for use in the delivery of a nucleic acid molecule to a cell.

17. The composition according to claim 10, wherein said nucleic acid is an artificial nucleic acid.

18. The composition according to claim 17, wherein said artificial nucleic acid is selected from the group consisting of locked nucleic acid (LNA), glycolic nucleic acid (GNA), and threose nucleic acid (TNA).

19. The composition according to claim 10, wherein said nucleic acid is a therapeutic nucleic acid.

20. The composition according to claim 19, wherein said therapeutic nucleic acid is a therapeutic RNA.

21. The composition according to claim 19, wherein said therapeutic RNA is an antisense RNA, siRNA, short hairpin RNA, mRNA, microRNA or enzyme RNA.

22. The composition according to claim 10, wherein said polyplex comprises chitosan molecules having an average molecular weight of less than 65kDa before functionalization.

23. The composition according to claim 22, wherein said polyplexes comprise chitosan molecules having an average molecular weight of less than 30kDa before functionalization.

24. The composition according to claim 10, wherein said composition has a pH between 4.0-7.

0.

25. The composition according to claim 24, wherein said composition has a pH between 4.5-6.

5.

26. The composition according to claim 10, wherein said chitosan DD nucleic acid polyplex has an average polydispersity index (PDI) of less than 0.

4.

27. The composition according to claim 26, wherein said chitosan DD nucleic acid polyplex has an average polydispersity index (PDI) of less than 0.

25.

28. The composition according to claim 10, wherein said chitosan nucleic acid polyplexes comprise chitosan molecules with an average molecular weight of less than 50 kDa before functionalization, an average of less than 310 glucosamine monomeric units, an N / P ratio of 2 to 20, an average hydrodynamic diameter of less than 500 nm, an average zeta potential of at least 0 mV at acidic pH, a polydispersity index (PDI) of less than 0.5, and a nucleic acid concentration greater than 0.5 mg / ml, wherein said chitosan nucleic acid polyplexes are free from precipitated polyplexes and have a stable size, in that their average diameter increases by less than 100% at room temperature for at least 6 hours, at least 12 hours, at least 24 hours, or at least 48 hours.

29. The composition according to claim 10, wherein said chitosan nucleic acid polyplexes comprise chitosan molecules having an average molecular weight of less than 65 kDa before functionalization, an average of less than 400 glucosamine monomeric units, an N / P ratio of 2 to 20, an average hydrodynamic diameter of less than 500 nm, an average zeta potential of at least +1 to +40 mV at acidic pH, a polydispersity index (PDI) of less than 0.4, and a nucleic acid concentration greater than 0.5 mg / ml, wherein said chitosan nucleic acid polyplexes are free from precipitated polyplexes and have a stable size, in that their average diameter increases by less than 50% at room temperature for at least 6 hours, at least 12 hours, at least 24 hours, or at least 48 hours.

30. The composition according to claim 10, wherein said chitosan nucleic acid polyplexes comprise chitosan molecules having an average molecular weight of less than 40 kDa before functionalization, an average of less than 250 glucosamine monomeric units, an N / P ratio of 2 to 20, an average hydrodynamic diameter of less than 500 nm, an average zeta potential of at least +1 to +30 mV at acidic pH, a polydispersity index (PDI) of less than 0.3, and a nucleic acid concentration greater than 0.5 mg / ml, wherein said chitosan nucleic acid polyplexes are free from precipitated polyplexes and have a stable size, in that their average diameter increases by less than 50% at room temperature for at least 6 hours, at least 12 hours, at least 24 hours, or at least 48 hours.

31. The composition according to claim 10, wherein said chitosan nucleic acid polyplexes comprise chitosan molecules having an average molecular weight of less than 30 kDa before functionalization, an average of less than 190 glucosamine monomeric units, an N / P ratio of 2 to 5, an average hydrodynamic diameter of less than 500 nm, an average zeta potential of at least +1 to +30 mV at acidic pH, a polydispersity index (PDI) of less than 0.25, and a nucleic acid concentration greater than 0.5 mg / ml or 0.75 mg / ml, wherein said chitosan nucleic acid polyplexes are free from precipitated polyplexes and have a stable size, in that their average diameter increases by less than 25% at room temperature for at least 6 hours, at least 12 hours, at least 24 hours, or at least 48 hours.

32. The composition according to claim 10, wherein said chitosan nucleic acid polyplexes comprise chitosan molecules having an average molecular weight of less than 15 kDa before functionalization, an average of less than 95 glucosamine monomeric units, an N / P ratio of 2 to 5, an average hydrodynamic diameter of less than 200 nm, an average zeta potential of at least +1 to +30 mV at acidic pH, a polydispersity index (PDI) of less than 0.25, and a nucleic acid concentration greater than 1.0 mg / ml, wherein said chitosan nucleic acid polyplexes are free from precipitated polyplexes and have a stable size, in that their average diameter increases by less than 50% at room temperature for at least 6 hours, at least 12 hours, at least 24 hours, or at least 48 hours.

33. The chitosan-derived nanoparticle of claim 1, wherein the hydrophilic polyol has an aldehyde group. ML / a / ZUZZ / UUUó I z 34. The composition according to claim 19, wherein the therapeutic nucleic acid is therapeutic DNA.

35. The composition according to claim 34, wherein said DNA encodes insulin, a glucagon antagonist, GLP-1 or leptin.

36. The composition according to claim 34, wherein said DNA encodes IL-10 or a TNFα angatonist.

37. The composition according to claim 34, wherein said DNA encodes leptin, cholecystokinin, PYYoGLP-1.

38. The composition according to claim 34, wherein said DNA encodes a cytokine.

39. The composition according to claim 38, wherein said cytokine is an interleukin.

40. A dually derivatized (DD) chitosan nucleic acid polyplex for use in administering a therapeutic acid to a subject in need thereof, wherein said DD chitosan nucleic acid polyplex comprises a chitosan-derived nanoparticle and a therapeutic nucleic acid, wherein said chitosan-derived nanoparticle comprises chitosan functionalized with arginine (Arg) and a hydrophilic polyol (HP) of Formula V1: R3 iviA / a / zuzz / uuuo 1 where: R2 is selected from: H and hydroxyl; R3 is selected from: H and hydroxyl; and X is selected from: C2-C6 alkylene optionally substituted with one or more hydroxyl substituents, provided that said hydrophilic polyol is not gluconic acid.

41. The dually derivatized (DD) chitosan nucleic acid polyplex for use according to claim 40, wherein the nanoparticle has a final degree of functionalization ratio of Arg:final degree of functionalization ratio of HP of 1:1 to 10:1, of 3:1 to 7:1, or is 5:

1.

42. The dually derivatized (DD) chitosan nucleic acid polyplex for use according to claim 40, wherein the amine-to-phosphate ratio of said DD chitosan nucleic acid polyplex is between 2 to 100, between 2 to 50, between 2 to 30, or between 2 to 15.

43. The dually derivatized (DD) chitosan nucleic acid polyplex for use according to claim 40, wherein said polyplexes comprise chitosan molecules having an average molecular weight of less than 65 kDa or less than 30 kDa before functionalization.

44. The dually derivatized (DD) chitosan nucleic acid polyplex for use according to claim 40, wherein said DD chitosan nucleic acid polyplex has a pH between 4.0-7.0 or between 4.5-6.

5.

45. The dually derivatized (DD) chitosan nucleic acid polyplex for use according to claim 40, wherein said DD chitosan nucleic acid polyplex has an average polydispersity index (PDI) of less than 0.4 or less than 0.

25.

46. ​​The dually derivatized (DD) chitosan nucleic acid polyplex for use according to claim 40, wherein said therapeutic nucleic acid is either DNA or RNA.

47. The use of claim 46, wherein said therapeutic nucleic acid is an RNA selected from the group consisting of antisense RNA, siRNA, short hairpin RNA, mRNA, microRNA or enzyme RNA.

48. The use of claim 47, wherein said DNA encodes insulin, a glucagon antagonist, GLP-1 or leptin.

49. The use of claim 46, wherein said DNA encodes IL-10 or a TNFα angatonist.

50. The use of claim 46, wherein said DNA encodes leptin, cholecystokinin, PYY or GLP-1.

51. The use of claim 46, wherein said DNA encodes a cytokine.

52. The use of claim 51, wherein said cytokine is an interleukin.

53. The dually derivatized (DD) chitosan nucleic acid polyplex for use according to claim 40, wherein said hydrophilic polyol has an aldehyde group.

54. A composition comprising a dually derivatized (DD) chitosan nucleic acid polyplex for use in the treatment of diabetes in a patient in need thereof, said DD chitosan nucleic acid polyplex comprising a chitosan-derived nanoparticle and a therapeutic nucleic acid, wherein said chitosan-derived nanoparticle comprises chitosan functionalized with arginine (Arg) and a hydrophilic polyol (HP) of Formula V1: ML / a / ZUZZ / UUU or 1z wherein: R2 is selected from: H and hydroxyl; R3 is selected from: H and hydroxyl; and X is selected from: Cs-Cs alkylene optionally substituted with one or more hydroxyl substituents, provided that said hydrophilic polyol is not gluconic acid, and wherein said therapeutic nucleic acid encodes insulin, a glucagon antagonist, GLP-1, or leptin.

55. A composition comprising a dually derivatized (DD) chitosan nucleic acid polyplex for use in the treatment of inflammatory bowel disease in a patient in need thereof, said DD chitosan nucleic acid polyplex comprising a chitosan-derived nanoparticle and a therapeutic nucleic acid, wherein said chitosan-derived nanoparticle comprises chitosan functionalized with arginine (Arg) and a hydrophilic polyol (HP) of Formula VII: wherein: R2 is selected from: H and hydroxyl; R3 is selected from: H and hydroxyl; and X is selected from: C2-C6 alkylene optionally substituted with one or more hydroxyl substituents, provided that said hydrophilic polyol is not gluconic acid, and wherein said therapeutic nucleic acid encodes IL-10, a TNFα antagonist, or an IL-17 antagonist.

56. A composition comprising a dually derivatized (DD) chitosan nucleic acid polyplex for use in the treatment of obesity in a patient in need thereof, said DD chitosan nucleic acid polyplex comprising a chitosan-derived nanoparticle and a therapeutic nucleic acid, wherein said chitosan-derived nanoparticle comprises chitosan functionalized with arginine (Arg) and a hydrophilic polyol (HP) of Formula Vil: iviA / a / zuzz / uuuo 1 where: R2 is selected from: H and hydroxyl; R3 is selected from: H and hydroxyl; and X is selected from: C2-C6 alkylene optionally substituted with one or more hydroxyl substituents, provided that said hydrophilic polyol is not gluconic acid, and wherein said therapeutic nucleic acid encodes leptin, cholestokinin, PYY, or GLP-1.

57. The composition of claims 54, 55, or 56, wherein the hydrophilic polyol has a carboxyl group.

58. The composition of claims 54, 55, or 56 wherein the hydrophilic polyol has an aldehyde group.

59. The composition of claims 54, 55, or 56, wherein the hydrophilic polyol is a saccharide selected from the group consisting of glyceraldehyde, threose, erythrose, ribose, arabinose, xylose, lyxose, allose, glucose, altrose, mannose, gulose, idose, galactose, and talose.

60. The composition of claims 54, 55, or 56, wherein the nanoparticle has a final degree of functionalization ratio of Arg:final degree of functionalization ratio of HP of 1:1 to 10:

1.

61. The composition of claims 54, 55, or 56, wherein said polyplexes comprise chitosan molecules having an average molecular weight of less than 110kDa before functionalization.

62. The composition of claims 54, 55, or 56, wherein the amine-to-phosphate ratio of said chitosan DD nucleic acid polyplex is between 2 and 100.

63. The composition of claim 62, wherein the nanoparticle has a final degree of functionalization ratio of Arg:final degree of functionalization ratio of HP of 3:1 to 7:

1.

64. The composition of claim 62, wherein the amine to phosphate ratio of said chitosan DD nucleic acid polyplex is between 2 to 50, 2 to 30 or 2 to 15.