US 2003O23.5916A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2003/0235916A1 Monahan et al. (43) Pub. Date: Dec. 25, 2003

(54) NOVEL METHODS FOR THE DELIVERY OF Related U.S. Application Data POLYNUCLEOTIDES TO CELLS (60) Provisional application No. 60/388,685, filed on Jun. (76) Inventors: Sean D. Monahan, Madison, WI (US); 14, 2002. Lisa Nader, Madison, WI (US); Jon A. Wolff, Madison, WI (US); Vladimir G. Publication Classification Budker, Middleton, WI (US); James E. Hagstrom, Middleton, WI (US) (51) Int. Cl." ...... A61K 48/00; C12N 15/85 (52) U.S. Cl...... 435/455; 514/44 Correspondence Address: Mark K. Johnson (57) ABSTRACT Mirus Corporation Process are described for the delivery of a polynucleotide to 505 S. Rosa Rd. a cell. The proceSS comprises forming a Salt Stable complex Madison, WI 53719 (US) between the polynucleotide and a cationic Surfactant. Ter nary complexes are also made by associating an amphipathic (21) Appl. No.: 10/462,138 compound with the binary complex. The resultant com plexes are suitable for delivery of the polynucleotide to cells (22) Filed: Jun. 16, 2003 in vitro and in vivo. Patent Application Publication Dec. 25, 2003 Sheet 1 of 7 US 2003/023.5916A1

Rhodamine Fluorescence (Ex: 555 nm and Em: 585 nm)

000 -0- Rh-DNA

800 -- Rh-DNA-NC12-PyrDet 600 -A-Rh-DNA/FITC-Chit-O 400 -X-Pyret 200 - A - Rh-DNA-NC 2 () PyrDet/FITC-Chit-Ol O 2 4 6 8 10 12 14 -e- FITC-Chit-Ol Fraction

------FITC Fluorescence (Ex: 495 nm and Em: 530 nm)

--Rh-DNA -- Rh-DNA-NC12-PyrDet

-A-Rh-DNA/FITC-Chit-Ol

-be-PyrDet

-A-Rh-DNA-NC2 PyrDet/FITC-Chit-Ol -e-FITC-Chit-Ol

Pyrene Fluorescence (Ex: 340 nm and Em: 377 nm)

-- Rh-DNA

-- Rh-DNA-NC 12-PyrDet

-A-Rh-DNA/FITC-Chit-Ol

-be-PyrDet

-A-Rh-DNA-NC 2 PyrDet/FITC-Chit-Ol 0 2 4 6 8 10 12 14 -e-FITC-Chit-Ol Fraction

FIG. 1 Patent Application Publication Dec. 25, 2003 Sheet 2 of 7 US 2003/023.5916 A1

1 2 3 4 5 6 7 8 9 10 11 12 13 14

FIG. 2

Patent Application Publication Dec. 25, 2003 Sheet 4 of 7 US 2003/023.5916A1

RhDNA + NC2 titrated with NaC (RT 20hr)

s an 3. -0-0.01ug/uL- i --0. lug/ul 0 250 500 750 000 - A 0.2ugful mM NaCl

Rh)NA + NC12 titrated with NaCl (37C 20hr)

3. ot, s:3 -0-0.01ug/ul------H 0.1ugful 0 250 500 750 1000 0.2ugul M. NaCl

RhDNA + NC2 titrated with NaCl (50C 20hr)

g 2 - 1000 3g5 500 -0-0.01ugful i 0 --0. lug/ul 0 250 500 750 1000 - A 0.2ugful mM NaC

l - --

RhDNA - NC12 titrated with NaC - (70C 20hr)

2w s uu ------mu E -0-0.01ug/ul : -- 0.1ugful - A - 0.2ugful

E 0 250 500 750 1000

RhDNA-NC12 titrated with NaC

2 --DNA-NC2 DMF -HDNA-NC2 f - BZOH

mM NaCl

FIG. 4 Patent Application Publication Dec. 25, 2003 Sheet 5 of 7 US 2003/023.5916A1

RhDNA-NC12 Stability at 37C 1000 ------rr------

2 800 5 600 3. 400 e 2 200

O - RhDNA-NC12 RhDNA + NC12 RhDNA + p LL RhDNA (1:2) (1:2) (1:3)

-- Initial E4hr 37C 24hr 37C

RhDNA-NC12 Stability in 150mM NaCl at 37C s 1000 - s 800 E 600 --

sE 400 2. s 200 E O RhDNA-NC12 RhDNA + NC12 RhDNA + p L RhDNA (1:2) (1:2) (1:3) 3 Initial 4hr 37C 24hr 37C

FIG. 5 Patent Application Publication Dec. 25, 2003 Sheet 6 of 7 US 2003/023.5916A1

-- . . .-----, mn- ---- Stability of RhDNA-NC12 in 10% Serum

1000 -

800

600 -

400 -

200 -

0% Serum 10% Serum 10min 20min 4hr 24hr RhDNA-NC 2 (1:2) RhDNA + NC 2 (1:2) RhDNA + pLL (1:3)

FIG. 6 Patent Application Publication Dec. 25, 2003. Sheet 7 of 7 US 2003/023.5916A1

CD: DNA/Cationic Surfactant Complexes

8.0 - 6.0 4.0 - DNA E2. 2.0' DNA + NC 12 0.0 -2.0 DNA - NC 1237C -4.0 DNA-NC2 -6.0 - -8.0 20 225 240 255 27O 285 300 35 330 Wavelength (nm)

FIG. 7 US 2003/023.5916 A1 Dec. 25, 2003

NOVEL METHODS FOR THE DELIVERY OF liver, lung, and brain. Other methods are most effective POLYNUCLEOTIDES TO CELLS when applied to cells or tissues that have been removed from the body and the genetically-modified cells are then trans CROSS-REFERENCE TO RELATED planted back into the body. Indirect approaches in conjunc APPLICATIONS tion with retroviral vectors are being developed to transfer genes into bone marrow cells, lymphocytes, hepatocytes, 0001. This application is related to prior provisional myoblasts and skin cells. application U.S. Serial No. 60/388,685 filed Jun. 14, 2002. 0007 Gene Therapy And Nucleic Acid-Based Thera FIELD OF THE INVENTION pies-Gene therapy promises to be a revolutionary advance ment in the treatment of disease. With gene therapy, a 0002 The technical field of the present invention is a disease State can be directly treated by inserting a corrective composition comprising polynucleotide, amphipathic com polynucleotide into cells. In contrast, traditional drug based pounds and polymers, and the use of Such compositions for approaches act downstream on the products of the genes delivering the polynucleotide to an animal cell. (proteins, enzymes, enzyme Substrates and enzyme prod ucts). Although, the initial motivation for gene therapy was BACKGROUND the treatment of genetic disorders, it is becoming increas 0.003 Gene And Nucleic Acid-Based Delivery-Gene or ingly apparent that gene therapy will be useful for the polynucleotide transfer to cells is an important technique for treatment of a broad range of acquired diseaseS Such as biological and medical research as well as potentially thera cancer, infectious diseases, heart disease, arthritis, and neu peutic applications. The polynucleotide needs to be trans rodegenerative disorders (such as Parkinson's and Alzhe ferred across the cell membrane and into the cell. For imer's). polynucleotides encoded expressible genes, the polynucle 0008. In addition to providing an exogenous gene, gene otide must be delivered to the cell nucleus where the gene therapy also has the potential to inhibit endogenous genes. can be transcribed. Gene transfer methods currently being Several mechanisms exist for Specifically inhibiting expres explored include Viral vectors and non-viral methods. Sion of an endogenous gene. These include antisense nucleic acid, ribozymes, and small inhibitory RNA (siRNA) medi 0004 Viruses have evolved over millions of year to ated RNA interference (RNAi). Antisense inhibition transfer their genes into mammalian cells. Viruses can be involves Single Stranded polynucleotide that is complemen modified to carry a desired gene and become a “vector' for tary to the target mRNA. Ribozymes are catalytic RNAS gene delivery. Using recombinant techniques, harmful or capable of specifically cleaving a target mRNA. SiRNAS are Superfluous viral genes can be removed and replaced with a short double stranded RNAS that are identical in sequence to desired gene. This technique was first accomplished with a Segment of the expressed target gene and, in conjunction mouse retroviruses. The development of retroviral vectors is with cellular proteins, cause the degradation of the target being pursued for gene therapy applications. However, these RNA. Specific inhibition of endogenous gene expression has vectors cannot infect all cell types efficiently, especially in great potential in the treatment of dominant genetic disor Vivo. Therefore, Several viral vectors, including Herpes ders, Viral infections, and cancer Virus, Adenovirus, Adeno-associated virus and others are being developed to enable more efficient gene transfer 0009 Gene transfer can also be used as a vaccination different cell types, including brain and lung. against infectious diseases and cancer. When a foreign gene is transferred to a cell and expressed, the resultant protein is 0005 Non-viral vectors are also being developed to presented to the immune System. This presentation differs transfer polynucleotides into mammalian cells. For these from the antigen presentation resulting from Simply inject non-viral vectors, an expressible gene is typically cloned ing the protein into the body and is more likely to cause a into a plasmid that is capable of replicating in bacteria, cell-mediated immune response. Expression of the viral usually E. coli. The desired gene is recombinantly inserted gene within a cell Simulates a viral infection without the into the bacterial plasmid along with a mammalian pro danger of an actual viral infection and induces a more moter, enhancer, or other Sequences that enable the gene to effective immune response. This approach may be more be expressed in mammalian cells. Milligrams of the plasmid effective in for fighting latent Viral infections Such as human DNA can then be prepared and purified from bacterial immunodeficiency virus, Herpes and cytomegalovirus. cultures. Alternatively, polynucleotides for delivery to cells can by made enzymatically such as by PCR, or they can be 0010 Polymers for Nucleic Acid Delivery-Polymers Synthesized chemically. The polynucleotides can be incor have been used in research for the delivery of polynucle porated into lipid vesicles (liposomes including cationic otides to cells. One of the several methods of polynucleotide lipids Such as Lipofectin) which transfer the polynucleotide delivery to cells is the use of polynucleotides/polycation into the target mammalian cell. Polynucleotides can also be complexes. It has been shown that cationic proteins, like complexed with polymerS Such as polylysine, polyethylen histones and protamines, or Synthetic polymers, like polyl imine, and proteins. Other methods of polynucleotide deliv ySine, polyarginine, polyornithine, DEAE dextran, poly ery to cells include electroporation and "gene gun' tech brene, and polyethylenimine may be effective intracellular nologies. polynucleotide delivery agents. The following are Some important principles involving the mechanism by which 0006 Gene delivery approaches can be classified into polycations facilitate uptake of polynucleotides: direct and indirect methods. Some of these gene transfer methods are most effective when directly injected into a 0011 Polycations facilitate nucleic acid condensation. tissue Space. Direct methods using many of the above gene The Volume which one polynucleotide molecule occupies in transfer techniques are being used to target tumors, muscle, a complex with polycations is drastically lower than the US 2003/023.5916 A1 Dec. 25, 2003

volume of the free polynucleotide molecule. The size of a nate over electrostatic forces when the nucleic acid helices polynucleotides/polymer complex is probably critical for approach closer then a few water diameters. In a case of gene delivery in vivo and possible for in vitro as well. For DNA/polymeric polycation interactions, DNA condensation intravascular delivery, the polynucleotide needs to croSS the is a more complicated process than the case of low molecu endothelial barrier in order to reach the parenchymal cells of lar weight polycations. Different polycationic proteins can interest. The largest endothelial fenestrae (holes in the generate toroid and rod formation with different size DNA at endothelial barrier) occur in the liver and have an average a ratio of positive to negative charge of two to five. DNA diameter of 100 nm. The trans-epithelial pores in other complexes with polyarginine or histone can form two types organs are much Smaller. For example, muscle endothelium of Structures, an elongated Structure with a long axis length can be described as a structure which has a large number of of about 350 nm (like free DNA) and dense spherical Small pores with a radius of 4 nm, and a very low number particles. Both forms exist Simultaneously in the same of large pores with a radius of 20-30 nm. The size of the Solution. The reason for the co-existence of the two forms polynucleotide complexes is also important for the cellular can be explained as an uneven distribution of the polycation uptake process. After binding to the cells the polynucleotide/ chains among the DNA molecules. The uneven distribution polycation complex is likely taken up by endocytosis. Since generates two thermodynamically favorable conformations. endocytic vesicles have a typical internal diameter of about 100 nm, polynucleotide complexes smaller than 100 nm are 0016. The electrophoretic mobility of polynucleotide/ preferred. polycation complexes can change from negative to positive in exceSS polycation. It is likely that large polycations do not 0012 Polycations may provide attachment of polynucle completely align along the polynucleotide but form polymer otides to the cell Surface. The polymer forms a cross-bridge loops which interact with other polynucleotide molecules. between the polyanionic polynucleotide and the polyanionic The rapid aggregation and Strong intermolecular forces Surface of a cell. As a result, the mechanism of polynucle between different nucleic acid molecules may prevent the otide translocation to the intracellular space might be non Slow adjustment between helices needed to form tightly Specific adsorptive endocytosis. This proceSS may be more packed orderly particles. effective than either fluid phase endocytosis or receptor mediated endocytosis. Furthermore, polycations provide a 0017 Surface charging-AS discussed previously, poly convenient linker for attaching Specific ligands to the com cations can help adhere polynucleotide complexes to a cell plex. The polynucleotide/polycation complexes could then Surface. However, negative Surface charge would be more be targeted to Specific cell types. desirable for many practical applications, i.e. in Vivo deliv ery. The phenomenon of Surface recharging is well known in 0013 The polynucleotides in polycation complexes are colloid chemistry and is described in great detail for lyo protected against nuclease degradation. This protection is phobic/lyophilic Systems (i.e., Silver halide hydrosols). important for both extra- and intracellular preservation of Addition of polyion to a Suspension of lateX particles with an polynucleotide Since nucleases are present in Serum and oppositely-charged Surface leads to the permanent absorp endoSomeS/lySOSomes. Protection from degradation in endo tion of the polyion onto the Surface. Upon reaching appro SomeS/lySOSomes is enhanced by preventing organelle acidi priate Stoichiometry, the Surface is changed to the opposite fication. One method of preventing acidification is the use of charge. NHCl or chloroquine. Other polymers, such as polyethyl enimine, probably disrupt endoSomal/lySOSomal function 0018. The Use of pH-Sensitive Lipids, Aniphipathic without additional treatments. Disruption of endoSomal/ Conipounds, and Liposomes for Nucleic Acid Delivery lySOSomal function has also been accomplished by linking Cationic liposomes may deliver DNA either directly across endoSomal or membrane disruptive agents Such as fusion the plasma membrane or via the endoSome compartment. peptides or adenoviruses to the polycation or complex. Regardless of its exact entry point, much of the DNA within cationic liposomes accumulates in the endoSome compart 0.014 Condensation of nucleic acid-A significant num ment. Several approaches have been investigated to prevent ber of multivalent cations with widely different molecular loss of the foreign DNA in the endosomal compartment by Structures have been shown to induce(condensation of protecting it from hydrolytic digestion or enabling its escape nucleic acid. Multivalent cations with a charge of three or into the cytoplasm. These approaches include the use of higher have been shown to condense DNA. These include acidotropic (lysomotrophic) weak amines, such as chloro Spermidine, Spennine, Co(NH3) "...Fe", and natural or Syn quine, which presumably prevent DNA degradation by thetic polymerS Such as histone H1, protamine, polylysine, inhibiting endoSomal acidification Legendre et al. 1992). and polyethylenimine. Analysis has shown DNA condensa Alternatively viruses and Viral fusion peptides have been tion to be favored when 90% or more of the charges along included to disrupt endoSomes or promote fusion of lipo the Sugar-phosphate backbone are neutralized. Neutral and Somes with endoSomes and facilitate release of DNA into the anionic polymers can increase repulsion between DNA and cytoplasm Kamata et al. 1994; Wagner et al. 1994). its Surroundings, therefore compacting the DNA. Most Sig nificantly, Spontaneous DNA Self-assembly and aggregation 0019 Knowledge of lipid phases and membrane fusion processes have been shown to result from the confinement of has been used to design potentially more versatile liposomes large amounts of DNA due to excluded volume effect. which exploit endoSomal acidification to promote fusion with endoSomal membranes. Such an approach is best 0.015 The mechanism of polynucleotide condensation is exemplified by anionic, pH-sensitive liposomes that have not clear. The electrostatic force between unperturbed heli been designed to destabilize or fuse with the endoSome ces arises primarily from a counter-ion fluctuation mecha membrane at acidic pH Duzgunes et al. 1991). All of the nism requiring multivalent cations and plays a major role in anionic, pH-sensitive liposomes have utilized phosphati polynucleotide condensation. The hydration forces predomi dylethanolamine (PE) bilayers that are stabilized at non US 2003/023.5916 A1 Dec. 25, 2003 acidic pH by the addition of lipids that contain a carboxylic cationic Surfactant in an aqueous Solution and incubating the acid group. Liposomes containing only PE are prone to the Solution at elevated temperature. The incubation may be inverted hexagonal phase (H). In pH-sensitive, anionic from Several minutes to hours, depending on the tempera liposomes, the carboxylic acid's negative charge increases ture. The elevated temperature may be 30° C. to 100° C. or the size of the lipid head group at pH greater than the higher. More preferably, the elevated temperature is 35 C. carboxylic acid's PK, and thereby stabilizes the phosphati to 50 C. A stable particle comprises condensed polynucle dylethanolamine bilayer. At acidic pH conditions found otide wherein the size of the complex does not rapidly within endoSomes, the uncharged or reduced charge Species increase nor does the polynucleotide rapidly decondense if is unable to Stabilize the phosphatidylethanolamine-rich the complex is exposed to physiological Salt concentrations. bilayer. Anionic, pH-sensitive liposomes have delivered a 0023. In a preferred embodiment, we describe a process variety of membrane-impermeable compounds including for delivering a polynucleotide to a cell comprising: asso DNA. However, the negative charge of these pH-sensitive ciating a polynucleotide with a cationic Surfactant to form a liposomes prevents them from efficiently taking up DNA binary complex, associating the complex with an amphip and interacting with cells; thus decreasing their utility for athic compound to form a ternary complex, and associating transfection. We have described the use of cationic, pH the complex with a cell. The amphipathic compound may be sensitive liposomes to mediate the efficient transfer of DNA Selected from the list comprising: polymers containing into a variety of cells in culture U.S. Ser. No. 08/530,598, hydrophobic moieties, peptides containing hydrophobic and U.S. Ser. No. 09/020,566. moieties, targeting groups containing hydrophobic moieties, 0020. The Use of pH-Sensitive Polymers for Nucleic Steric Stabilizers containing hydrophobic moieties, Surfac Acid Delivery-Polymers that are pH-sensitive have found tants and lipids. The amphipathic compound may be cat broad application in the area of drug delivery because of ionic, anionic, neutral, or Zwitterionic. The resultant ternary their ability to exploit various physiological and intracellular complex can have a net Surface charge that is positive, pH gradients for the purpose of controlled release of drugs. negative or neutral. The amphipathic compound may also be pH sensitivity can be broadly defined as any change in modified to contain one or more functional groups that polymer's physico-chemical properties over a range of pH. increase transfection efficiency. The amphipathic compound Narrower definitions demand Significant changes in the may be modified prior to ternary complex formation of after polymer's ability to retain or release a bioactive Substance in ternary complex formation. The binary complex may be a physiologically tolerated pH range (typically pH 5.5-8). Stabilized prior to formation of the ternary complex. All polyions can be divided into three categories based on their ability to donate or accept protons in aqueous Solutions: 0024 Stabilizing the binary complex comprises: incubat polyacids, polybases and polyampholytes. Use of pH-sen ing the complex in aqueous Solution at elevated temperature. The incubation may be from Several minutes to hours, Sitive polyacids in drug delivery applications usually relies depending on the temperature. Alternatively, Stabilizing the on their ability to a) become soluble with a pH increase complex comprises: drying the complex, dissolving the (acid/salt conversion), b) form a complex with other poly complex in an appropriate organic Solvent, and diluting with mers over a change of pH, or c) undergo significant change an appropriate aqueous Solution. A stable particle comprises in hydrophobicity/hydrophilicity balance. Combinations of condensed polynucleotide wherein the size of the complex all three above factors are also possible. does not rapidly increase nor does the polynucleotide rapidly decondense if the complex is exposed to Salt at physiological SUMMARY OF THE INVENTION concentrations. The ternary complex may be delivered to a 0021. In a preferred embodiment we describe an in vivo cell in Vivo or in vitro. Delivering the ternary complex to a proceSS for delivering a polynucleotide to a cell comprising: cell in Vivo may comprise: directly injecting the complex in asSociating the polynucleotide with a cationic Surfactant in an aqueous Solution into a tissue or inserting the complex in an aqueous Solution to form a complex, Stabilizing the an aqueous Solution into a vessel in a mammal for delivery complex, and bringing the complex into contact with the to cell in a tissue to which the vessel either Supplies or drains cell. Stabilizing the complex comprises: incubating the a bodily fluid. complex in the aqueous Solution at elevated temperature. 0025. In a preferred embodiment, we describe a process The incubation may be from Several minutes to hours, for forming Small, less than 50 nm diameter, polynucleotide depending on the temperature. Alternatively, Stabilizing the containing complexes comprising: associating a polynucle complex comprises: drying the complex, dissolving the otide with a cationic Surfactant to form a binary complex, complex in an appropriate organic Solvent, and diluting with Stabilizing the binary complex, and asSociating the binary an appropriate aqueous Solution. A stable particle comprises complex with an amphipathic compound to form a ternary condensed polynucleotide wherein the size of the complex complex. Stabilizing the binary complex comprises: incu does not rapidly increase nor does the polynucleotide rapidly bating the complex in aqueous Solution at elevated tempera decondense if the complex is exposed to Salt at physiological ture. The incubation may be from Several minutes to hours, concentrations. Bringing the complex into contact with the depending on the temperature. Alternatively, Stabilizing the cell may comprise: directly injecting the complex in an complex comprises: drying the complex, dissolving the aqueous Solution into a tissue or inserting the complex in an complex in an appropriate organic Solvent, and diluting with aqueous Solution into a vessel-in a mammal for delivery to an appropriate aqueous Solution. A stable particle comprises cell in a tissue to which the vessel either Supplies or drains condensed polynucleotide wherein the size of the complex a bodily fluid. does not rapidly increase nor does the polynucleotide rapidly 0022. In a preferred embodiment, we describe a process decondense if the complex is exposed to Salt at physiological for forming a stable polynucleotide/cationic Surfactant com concentrations. The amphipathic compound may be Selected pleX comprising: associating the polynucleotide with the from the list comprising: polymers containing hydrophobic US 2003/023.5916 A1 Dec. 25, 2003 moieties, peptides containing hydrophobic moieties, target exhibit condensed DNATrubetskoy et al. 1999 and similar ing groups containing hydrophobic moieties, Steric Stabiliz particle sizes. However, the complexes show vastly different erS containing hydrophobic moieties, Surfactants and lipids, Stability, based on particle sizing and fluorescence decon The amphipathic compound may be cationic, anionic, neu densation assays, in the presence of physiological Salt con tral, or Zwitterionic. The resultant ternary complex can have centrations. The complex that is dried and taken up in a net Surface charge that is positive, negative or neutral. The organic solutions, shows stability to 150 mM NaCl without amphipathic compound may also be modified to contain one an increase in particle size. However, the non-precipitated or more functional groups that increase transfection effi complex is not stable in 150 mM NaCl, showing increasing ciency. The amphipathic compound may be modified prior particle size and decondensation. The present invention to ternary complex formation of after ternary complex provides for the process by which Stable complexes can be formation. formed (indicated by salt stability assays, DNA condensa 0.026 Further objects, features, and advantages of the tion, and particle size stability) from non-precipitative con invention will be apparent from the following detailed centrations of DNA and cationic Surfactants. We show that description when taken in conjunction with the accompa DNA/cationic Surfactant complexes can be formed in aque nying drawings. ous Solutions, and heated at various temperatures for Several minutes to hours, resulting in the formation of a complex that is salt stable. We further show that DNA/cationic BRIEF DESCRIPTION OF THE FIGURES Surfactant complexes can be formed in aqueous Solutions, 0027 FIG. 1. Fluorescence of DNA/cationic surfactant lyophilized to dryness, dissolved in an appropriate organic complexes after ultracentrifugation. Solvent, and diluted with appropriate aqueous Solutions resulting in the formation of a complex that is Salt Stable. 0028 FIG. 2. DNA/Cationic Surfactant Complex Deg radation with DNAse I. 0036) The present invention provides for the transfer of polynucleotides to cells in Vivo and in vitro. Processes are 0029 FIG. 3. RhDNA Condensation with Dodecylamine described for the preparation and delivery of DNA/cationic Hydrochloride. Surfactant complexes in Vivo. Additionally, the present 0030 FIG. 4. RhDNA/Cationic Surfactant Complex Sta invention provides for the preparation and delivery of DNA/ bility with NaCl. cationic Surfactant/third component complexes in Vivo and in vitro. 0031 FIG.5. RhDNA-NC12 Stability at 37° C. or in 150 mM NaCl. 0037 Although the literature indicates that DNA/cationic Surfactant complexes do not transfect cells in Vitro, we have 0032 FIG. 6. RhDNA-NC12 Stability with 10% Serum. found that the complexes are capable of DNA delivery in 0033 FIG. 7. Circular Dichroism of DNA/Cationic Sur Vivo. IntrVascular injection can be accomplished by dissolv factant Complexes. ing the complex in an organic Solvent and diluting the Sample with an appropriate aqueous buffer prior to injection. DETAILED DESCRIPTION OF THE Studies indicate that the complex remains a particle in the INVENTION Solution and that the particle is able to transfect cells. Mouse tail vein injection can also be accomplished by forming the 0034 Several reports have been presented in the litera DNA/cationic Surfactant complex in an aqueous buffer, ture describing the formation of a complex between DNA heating the Sample for an amount of time, and diluting the and cationic surfactants Melnikov et al. 1995a; Sergeyev et Sample with an appropriate aqueous buffer prior to injection. al. 1999a; Sergeyev et al. 1999b; Sukhorukov et al. 2000; Studies indicate that the complex remains a particle in the Tanaka et al. 1996; Ijiro et al. 1992; Melnikoz et al. 1995b). Solution and that the particle is able to transfect cells. For example, Melnikov et al. showed a phase transition between random coil and compact globule states for DNA 0038. Additionally, the DNA/cationic surfactant complex upon the addition of cetyltrimethylammonium bromide can be mixed with polymers containing hydrophobic moi (CTAB). This complex formation was further shown to be eties to form a new complex. The new ternary complexes reversible upon the addition of a competing polyanion increase transfections in vivo relative to the DNA/cationic (polyacrylic acid). The complexes can be prepared in Such a Surfactant complex, and are also functional for the transfec way as to precipitate the complex. Upon thorough drying, tion of the DNA to cells in vitro. Complex formation with the complexes can be Solubilized in organic Solvents Such as the hydrophobically modified polymers can occur below the ethanol, chloroform, THF, and cyclohexane. Although pro critical micelle concentration (cmc) of the polymer. The posed in Several of the reports, gene transfer has not been polymer can be either a polycation or a polyanion and can demonstrated with the DNA/cationic surfactant complexes. contain targeting groups. Additional components can be In fact, Clamme et all Clamme et al. 2000 demonstrated added to the ternary complex, Such as but not limited to, that DNA/CTAB complexes were trapped onto the external additional polymers (including charge modified polymers), face of the plasma membrane, thereby constituting a major peptides, hydrophobically modified peptides, charge modi limitation in efficient transfection. fied peptides, amphipathic compounds, anionic Surfactants, 0.035 Depending on the concentrations used, a precipi membrane active compounds, and Salts. tate may or may not form. The literature describes isolating 0039. Additionally, the DNA/cationic surfactant complex the Solid precipitate and dissolving the sample in organic can be mixed with peptides containing hydrophobic moieties solutions. We have found that the precipitated complex to form a new complex. The new ternary complexes can be (DNA/cationic Surfactant) and non-precipitated complex used for cell transfections in vivo and in vitro. Complex form inherently different complexes. Both complexes formation with the hydrophobically modified peptides can US 2003/023.5916 A1 Dec. 25, 2003 occur below the critical micelle concentration (cmc) of the noncovalent interactions Such as electrostatic interactions, hydrophobically modified peptide. The peptide can be net hydrogen bonding interactions, or hydrophobic interactions. cationic, net anionic, or net neutral charge. Additional com 0046 Binary complex-A binary complex is meant to ponents can be added to the ternary complex, Such as but not include the complex formed between a polynucleotide and a limited to, polymers (including charge modified polymers), cationic Surfactant. The cationic Surfactant can be a single peptides, hydrophobically modified peptides, charge modi Surfactant, a mixture of Surfactants (positive, negative, and fied peptides, amphipathic compounds, anionic Surfactants, neutral) with a net Overall charge of positive, or one or more membrane active compounds, and Salts. Surfactants with one or more other components, Such as a 0040 Additionally, the DNA/cationic surfactant complex Salt. can be mixed with targeting groups containing hydrophobic 0047 Ternary complex-A ternary complex is the com moieties to form a new complex. The new ternary com plex formed when one or more components is added to a plexes can be used for cell transfections in Vivo and in vitro. binary complex. Complex formation with the hydrophobically modified tar geting group can occur below the critical micelle concen 0048 Elevated Temperature-The term elevated tem tration (cmc) of the hydrophobically modified targeting perature in ment to mean temperatures greater than 25 C. group. The hydrophobically modified targeting group can be and less than 100° C. Elevated temperature also means the net cationic, net anionic, or net neutral charge. Additional combination of temperature and time that affords Salt Sta components can be added to the ternary complex, Such as bility to a binary complex. but not limited to, polymers (including charge modified 0049 Polynucleotide- The term polynucleotide, or polymers), peptides, hydrophobically modified peptides, nucleic acid or polynucleic acid, is a term of art that refers charge modified peptides, amphipathic compounds, anionic to a polymer containing at least two nucleotides. Nucle Surfactants, membrane active compounds, and Salts. otides are the monomeric units of polynucleotide polymers. 0041 Additionally, the DNA/cationic surfactant complex Polynucleotides with less than 120 monomeric units are can be mixed with Steric Stabilizers containing hydrophobic often called oligonucleotides. Natural nucleic acids have a moieties to form a new complex. The new ternary com deoxyribose- or ribose-phosphate backbone. An artificial or plexes can be used for cell transfections in Vivo and in vitro. Synthetic polynucleotide is any polynucleotide that is poly Complex formation with the hydrophobically modified merized in vitro or in a cell free System and contains the Steric Stabilizer can occur below the critical micelle concen Same or Similar bases but may contain a backbone of a type tration (cmc) of the hydrophobically modified steric stabi other than the natural ribose-phosphate backbone. These lizer. The hydrophobically modified targeting group can be backbones include: PNAS (peptide nucleic acids), phospho net cationic, net anionic, or net neutral charge. Additional rothioates, phosphorodiamidates, morpholinos, and other components can be added to the ternary complex, Such as variants of the phosphate backbone of native nucleic acids. but not limited to, polymers (including charge modified BaseS include purines and pyrimidines, which further polymers), peptides, hydrophobically modified peptides, include the natural compounds adenine, thymine, guanine, charge modified peptides, amphipathic compounds, anionic cytosine, uracil, inoSine, and natural analogs. Synthetic Surfactants, membrane active compounds, and Salts. derivatives of purines and pyrimidines include, but are not limited to, modifications which place new reactive groups 0.042 Additionally, the DNA/cationic surfactant complex Such as, but not limited to, amines, alcohols, thiols, car can be mixed with anionic Surfactants to form a new boxylates, and alkylhalides. The term base encompasses any complex. The new ternary complexes can be used for cell of the known base analogs of DNA and RNA including, but transfections in vivo and in vitro. Complex formation with not limited to, 4-acetylcytosine, 8-hydroxy-N6-methylad the anionic Surfactants can occur below the critical micelle enosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxy concentration (cmc) of the anionic Surfactants. Additional hydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-car components can be added to the ternary complex, Such as boxymethylaminomethyl-2-thiouracil, 5-carboxymethyl but not limited to, polymers (including charge modified aminomethyluracil, dihydrouracil, inosine, polymers), peptides, hydrophobically modified peptides, N6-isopentenyladenine, 1 methyladenine, 1 methylpseudo charge modified peptides, amphipathic compounds, mem uracil, 1 methylguanine, 1 methylinosine, 2,2-dimethyl-gua brane active compounds, and Salts. nine, 2-methyladenine, 2-methylguanine, 3-methyl-cy 0043. In another preferred embodiment, a process is tosine, 5-methylcytosine, N6-methyladenine, described in that the DNA/cationic Surfactant complexes can 7-methylguanine, 5-methylaminomethyluracil, 5-methoxy combined with amphipathic compounds, evaporated into a amino-methyl-2-thiouracil, beta-D-mannosylqueosine, film, hydrated to from complex containing liposomes. The 5'-methoxycarbonylmethyluracil, 5 methoxyuracil, 2-meth complexes can contain additional components including but ylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid not limited to polymers (including charge modified poly methylester, uracil-5-oxyacetic acid, oxybutoxosine, mers, hydrophobically modified polymers), peptides, hydro pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiou phobically modified peptides, charge modified peptides, racil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5- amphipathic compounds, anionic Surfactants, membrane oxyacetic acid methylester, uracil-5-oxyacetic acid, active compounds, and Salts. pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopu rine. The term polynucleotide includes deoxyribonucleic 0044) Definitions: acid (DNA) and ribonucleic acid (RNA) and combinations 0.045 Complex-Two molecules are combined to form a on DNA, RNA and other natural and synthetic nucleotides. complex through a process called complexation or complex 0050 DNA may be in form of cDNA, in vitro polymer formation if the are in contact with one another through ized DNA, plasmid DNA, parts of a plasmid DNA, genetic US 2003/023.5916 A1 Dec. 25, 2003 material derived from a virus, linear DNA, vectors (PI, PAC, include, but are not limited to, Sequences required for BAC, YAC, artificial chromosomes), expression cassettes, replication or Selection of the polynucleotide in a host chimeric Sequences, recombinant DNA, chromosomal organism. DNA, an oligonucleotide, anti-sense DNA, or derivatives of 0054) A polynucleotide can be used to modify the these groups. RNA may be in the form of oligonucleotide genomic or extrachromosomal DNA sequences. This can be RNA, tRNA (transfer RNA), snRNA (small nuclear RNA), achieved by delivering a polynucleotide that is expressed. rRNA (ribosomal RNA), mRNA (messenger RNA), in vitro Alternatively, the polynucleotide can effect a change in the polymerized RNA, recombinant RNA, chimeric Sequences, DNA or RNA sequence of the target cell. This can be anti-sense RNA, siRNA (small interfering RNA), achieved by hybridization, multistrand polynucleotide for ribozymes, or derivatives of these groups. An anti-Sense mation, homologous recombination, gene conversion, or polynucleotide is a polynucleotide that interferes with the other yet to be described mechanisms. function of DNA and/or RNA. Antisense polynucleotides 0055. The term gene generally refers to a polynucleotide include, but are not limited to: morpholinos, 2'-O-methyl Sequence that comprises coding Sequences necessary for the polynucleotides, DNA, RNA and the like. SiRNA comprises production of a therapeutic polynucleotide (e.g., ribozyme) a double Stranded Structure typically containing 15-50 base or a polypeptide or precursor. The polypeptide can be pairs and preferably 21-25 base pairs and having a nucle encoded by a full length coding Sequence or by any portion otide Sequence identical or nearly identical to an expressed of the coding Sequence So long as the desired activity or target gene or RNA within the cell. Interference may result functional properties (e.g., enzymatic activity, ligand bind in Suppression of expression. The polynucleotide can be a ing, Signal transduction) of the full-length polypeptide or Sequence whose presence or expression in a cell alters the fragment are retained. The term also encompasses the cod expression or function of cellular genes or RNA. In addition, ing region of a gene and the including Sequences located DNA and RNA may be single, double, triple, or quadruple adjacent to the coding region on both the 5' and 3' ends for Stranded. Double, triple, and quadruple Stranded polynucle a distance of about 1 kb or more on either end Such that the otide may contain both RNA and DNA or other combina gene corresponds to the length of the full-length mRNA. The tions of natural and/or Synthetic nucleic acids. Sequences that are located 5' of the coding region and which are present on the mRNA are referred to as 5' untranslated 0051. A delivered polynucleotide can stay within the Sequences. The Sequences that are located 3' or downstream cytoplasm or nucleus apart from the endogenous genetic of the coding region and which are present on the mRNA are material. Alternatively, DNA can recombine with (become a referred to as 3' untranslated Sequences. The term gene part of) the endogenous genetic material. Recombination encompasses both cDNA and genomic forms of a gene. A can cause DNA to be inserted into chromosomal DNA by genomic form or clone of a gene contains the coding region either homologous or non-homologous recombination. interrupted with non-coding Sequences termed introns, inter Vening regions or intervening Sequences. Introns are Seg 0.052 A polynucleotide can be delivered to a cell to ments of a gene which are transcribed into nuclear RNA. express an exogenous nucleotide Sequence, to inhibit, elimi Introns may contain regulatory elements Such as enhancers. nate, augment, or alter expression of an endogenous nucle Introns are removed or spliced out from the nuclear or otide Sequence, or to affect a specific physiological charac primary transcript, introns therefore are absent in the mes teristic not naturally associated with the cell. senger RNA (mRNA) transcript. The mRNA functions dur Polynucleotides may contain an expression cassette coded to ing translation to specify the Sequence or order of amino express a whole or partial protein, or RNA. An expression acids in a nascent polypeptide. The term non-coding cassette refers to a natural or recombinantly produced poly Sequences also refers to other regions of a genomic form of nucleotide that is capable of expressing a gene(s). The term a gene including, but not limited to, promoters, enhancers, recombinant as used herein refers to a polynucleotide mol transcription factor binding Sites, polyadenylation Signals, ecule that is comprised of Segments of polynucleotide joined internal ribosome entry Sites, Silencers, insulating together by means of molecular biological techniques. The Sequences, matrix attachment regions. These Sequences may cassette contains the coding region of the gene of interest be present close to the coding region of the gene (within along with any other Sequences that affect expression of the 10,000 nucleotide) or at distant sites (more than 10,000 gene. A DNA expression cassette typically includes a pro nucleotides). These non-coding Sequences influence the moter (allowing transcription initiation), and a sequence level or rate of transcription and translation of the gene. encoding one or more proteins. Optionally, the expression Covalent modification of a gene may influence the rate of cassette may include, but is not limited to, transcriptional transcription (e.g., methylation of genomic DNA), the sta enhancers, non-coding Sequences, Splicing Signals, tran bility of mRNA (e.g., length of the 3' polyadenosine tail), Scription termination Signals, and polyadenylation signals. rate of translation (e.g., 5' cap), nucleic acid repair, and An RNA expression cassette typically includes a translation immunogenicity. One example of covalent modification of initiation codon (allowing translation initiation), and a nucleic acid involves the action of LabellTreagents (Mirus Sequence encoding one or more proteins. Optionally, the Corporation, Madison, Wis.). expression cassette may include, but is not limited to, translation termination Signals, a polyadenosine Sequence, 0056. As used herein, the term gene expression refers to internal ribosome entry sites (IRES), and non-coding the process of converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) Sequences. through transcription of a deoxyribonucleic gene (e.g., via 0053. The polynucleotide may contain sequences that do the enzymatic action of an RNA polymerase), and for not serve a Specific function in the target cell but are used in protein encoding genes, into protein through translation of the generation of the polynucleotide. Such Sequences mRNA. Gene expression can be regulated at many Stages in US 2003/023.5916 A1 Dec. 25, 2003 the process. Up-regulation or activation refers to regulation has taken up a polynucleotide but has not integrated the that increases the production of gene expression products polynucleotide into its genomic DNA. (i.e., RNA or protein), while down-regulation or repression 0059 Intravascular and vessel-The term intravascular refers to regulation that decrease production. Molecules refers to an intravascular route of administration that enables (e.g., transcription factors) that are involved in up-regulation a polymer, oligonucleotide, or polynucleotide to be deliv or down-regulation are often called activators and repres ered to cells more evenly distributed than direct injections. Sors, respectively. Intravascular herein means within an internal tubular struc ture called a vessel that is connected to a tissue or organ 0057. An RNA function inhibitor comprises any poly within the body of an animal, including mammals. Vessels nucleotide or nucleic acid analog containing a sequence comprise internal hollow tubular structures connected to a whose presence or expression in a cell causes the degrada tissue or organ within the body. Bodily fluid flows to or from tion of or inhibits the function or translation of a specific the body part within the cavity of the tubular structure. cellular RNA, usually an mRNA, in a sequence-specific Examples of bodily fluid include blood, lymphatic fluid, or manner. Inhibition of RNA can thus effectively inhibit bile. Examples of vessels include arteries, arterioles, capil expression of a gene from which the RNA is transcribed. laries, Venules, Sinusoids, veins, lymphatics, and bile ducts. RNA function inhibitors are selected from the group com Afferent blood vessels of organs are defined as vessels which prising: siRNA, interfering RNA or RNAi, dsRNA, RNA are directed towards the organ or tissue and in which blood Polymerase III transcribed DNAS encoding siRNA or anti flows towards the organ or tissue under normal physiologi Sense genes, ribozymes, and antisense nucleic acid, which cal conditions. Conversely, efferent blood vessels of organs may be RNA, DNA, or artificial nucleic acid. SiRNA are defined as vessels which are directed away from the comprises a double Stranded Structure typically containing organ or tissue and in which blood flows away from the 15-50 base pairs and preferably 21-25 base pairs and having organ or tissue under normal physiological conditions. In the a nucleotide Sequence identical or nearly identical to an liver, the hepatic vein is an efferent blood vessel Since it expressed target gene or RNA within the cell. AntiSense normally carries blood away from the liver into the inferior polynucleotides include, but are not limited to: morpholinos, Vena cava. Also in the liver, the portal vein and hepatic 2'-O-methyl polynucleotides, DNA, RNA and the like. RNA arteries are afferent blood vessels in relation to the liver polymerase III transcribed DNAS contain promoters, Such as since they normally carry blood towards the liver. Insertion the U6 promoter. These DNAS can be transcribed to produce of the inhibitor or inhibitor complex into a vessel enables the small hairpin RNAS in the cell that can function as siRNA inhibitor to be delivered to parenchymal cells more effi or linear RNAS that can function as antisense RNA. The ciently and in a more even distribution compared with direct RNA function inhibitor may be polymerized in vitro, recom parenchymal injections. binant RNA, contain chimeric Sequences, or derivatives of these groups. The RNA function inhibitor may contain 0060 Modification-A molecule is modified, to form a ribonucleotides, deoxyribonucleotides, Synthetic nucle modification through a process called modification, by a otides, or any Suitable combination Such that the target RNA Second molecule if the two become bonded through a and/or gene is inhibited. In addition, these forms of nucleic covalent bond. That is, the two molecules form a covalent acid may be single, double, triple, or quadruple Stranded. bond between an atom form one molecule and an atom from the Second molecule resulting in the formation of a new 0.058 Transfection- The process of delivering a poly Single molecule. A chemical covalent bond is an interaction, nucleotide to a cell has been commonly termed transfection or bond, between two atoms in which there is a sharing of or the process of transfecting and also it has been termed electron density. Modification also means an interaction transformation. The term transfecting as used herein refers between two molecules through a noncovalent bond. For to the introduction of a polynucleotide or other biologically example crown ethers can form noncovalent bonds with active compound into cells. The polynucleotide may be used certain amine groups. for research purposes or to produce a change in a cell that 0061 Salt-A Salt is any compound containing ionic can be therapeutic. The delivery of a polynucleotide for bonds, i.e., bonds in which one or more electrons are therapeutic purposes is commonly called gene therapy. The transferred completely from one atom to another. Salts are delivery of a polynucleotide can lead to modification of the ionic compounds that dissociate into cations and anions genetic material present in the target cell. The term stable when dissolved in Solution and thus increase the ionic transfection or stably transfected generally refers to the introduction and integration of an exogenous polynucleotide Strength of a Solution. into the genome of the transfected cell. The term stable 0062 Pharmaceutically Acceptable Salt-Pharmaceuti transfectant refers to a cell which has stably integrated the cally acceptable Salt means both acid and base addition Salts. polynucleotide into the genomic DNA. Stable transfection 0063 Pharmaceutically Acceptable Acid Addition can also be obtained by using episomal vectors that are Salt-A pharmaceutically acceptable acid addition Salt is a replicated during the eukaryotic cell division (e.g., plasmid Salt that retains the biological effectiveness and properties of DNA vectors containing a papilloma virus origin of repli the free base, is not biologically or otherwise undesirable, cation, artificial chromosomes). The term transient transfec and is formed with inorganic acids Such as hydrochloric tion or transiently transfected refers to the introduction of a acid, hydrobromic acid, Sulfuric acid, nitric acid, phosphoric polynucleotide into a cell where the polynucleotide does not acid and the like, and organic acids Such as acetic acid, integrate into the genome of the transfected cell. If the propionic acid, pyruvic acid, maleic acid, malonic acid, polynucleotide contains an expressible gene, then the Succinic acid, fumaric acid, tartaric acid, citric acid, benzoic expression cassette is Subject to the regulatory controls that acid, mandelic acid, methaneSulfonic acid, ethaneSulfonic govern the expression of endogenous genes in the chromo acid, p-toluenesulfonic acid, Salicylic acid, trifluoroacetic Somes. The term transient transfectant refers to a cell which acid, and the like. US 2003/023.5916 A1 Dec. 25, 2003

0.064 Pharmaceutically Acceptable Base Addition cal groups that react with thiol, Sulfhydryl, or disulfide Salt-A pharmaceutically acceptable base addition Salt is a groups on cells can also be used to target many types of Salts that retains the biological effectiveness and properties cells. Folate and other Vitamins can also be used for target of the free acid, is not biologically or otherwise undesirable, ing. Other targeting groups include molecules that interact and is prepared from the addition of an inorganic organic with membranes Such as lipids, fatty acids, cholesterol, base to the free acid. Salts derived from inorganic bases dansyl compounds, and amphotericin derivatives. In addi include, but are not limited to, Sodium, potassium, calcium, tion viral proteins could be used to bind cells. lithium, ammonium, magnesium, Zinc, and aluminum Salts 0071. After interaction of a compound or complex with and the like. Salts derived from organic bases include, but the cell, other targeting groups can be used to increase the are not limited to, Salts of primary Secondary, and tertiary delivery of the biologically active compound to certain parts amines, Such as methylamine, triethylamine, and the like. of the cell. 0065 Drying Drying means removing the solvent from 0072) Nuclear localization signals-Nuclear localizing a Sample, for example, removing the Solvent from a complex Signals enhance the targeting of the pharmaceutical into under reduced pressure. Drying also means dehydrating a proximity of the nucleus and/or its entry into the nucleus Sample, or lyopholization of a Sample. during interphase of the cell cycle. Such nuclear transport 0.066 Salt Stabilized Comtiplex-A Salt stabilized com Signals can be a protein or a peptide Such as the SV40 large plex is a complex that shows stability when exposed to 150 T antigen NLS or the nucleoplasmin NLS. These nuclear mM NaCl Solution. Stability in this case is indicated by a localizing Signals interact with a variety of nuclear transport Stable particle size reading (less than a 20% change over 60 factors such as the NLS receptor (karyopherin alpha) which min)for the complex in 150 mM NaCl solution. Stability in then interacts with karyopherin beta. The nuclear transport this case is also indicated by no decondensation of the DNA proteins themselves could also function as NLS's since they (less than a 20% change over 60 min) within the complex are targeted to the nuclear pore and nucleus. For example, for the complex in 150 mM NaCl solution. karyopherin beta itself could target the DNA to the nuclear pore complex. Several peptides have been derived from the 0067 Interpolyelectrolyte Complexes-An interpoly SV40 T antigen. Other NLS peptides have been derived electrolyte complex is a noncovalent interaction between from the hnRNP A1 protein, nucleoplasmin, c-myc, etc. polyelectrolytes of opposite charge. These include a short NLS (H-CGYGPKKKRKVGG-OH, 0068 Charge, Polarity, and Sign-The charge, polarity, SEQ ID 1) or long NLS's (H-CKKKSSSDDEATAD or sign of a compound refers to whether or not a compound SOHST-PPKKKRKVEDPKDFPSELLS-OH, SEQID 2 and has lost one or more electrons (positive charge, polarity, or H-CKKKWDDEATADSOHSTPPKKK-RKVEDPKDF sign) or gained one or more electrons (negative charge, PSELLS-OH, SEQ ID 3). Other NLS peptides have been polarity, or sign). derived from M9 protein (CYNDFGNYNNQSSNFGP MKQGNFGGRSSGPY, SEQ ID 4), EIA (H-CKRGP 0069. Functional group-Functional groups include cell KRPRP-OH, SEQ ID 5), nucleoplasmin (H-CK targeting signals, nuclear localization signals, compounds KAVKRPAATKKAGQAKKKKL-OH, SEQ ID 6),and that enhance release of contents from endoSomes or other c-myc (H-CKKKGPAAKRVKLD-OH, SEQ ID 7). intracellular vesicles (releasing signals), and other com 0073 Membrane active compounds-Many biologically pounds that alter the behavior or interactions of the com active compounds, in particular large and/or charged com pound or complex to which they are attached. pounds, are incapable of crossing biological membranes. In 0070 Cell targeting signals-Cell targeting signals are order for these compounds to enter cells, the cells must any signals that enhance the association of the biologically either take them up by endocytosis, i.e., into endoSomes, or active compound with a cell. These signals can modify a there must be a disruption of the cellular membrane to allow biologically active compound Such as drug or nucleic acid the compound to croSS. In the case of endoSomal entry, the and can direct it to a cell location (such as tissue) or location endoSomal membrane must be disrupted to allow for move in a cell (Such as the nucleus) either in culture or in a whole ment out of the endoSome and into the cytoplasm. Either organism. The Signal may increase binding of the compound entry pathway into the cell requires a disruption or alteration to the cell Surface and/or its association with an intracellular of the cellular membrane. Compounds that disrupt mem compartment. By modifying the cellular or tissue location of branes or promote membrane fusion are called membrane the foreign gene, the function of the biologically active active compounds. These membrane active compounds, or compound can be enhanced. The cell targeting Signal can be, releasing Signals, enhance release of endocytosed material but is not limited to, a protein, peptide, lipid, Steroid, Sugar, from intracellular compartments such as endoSomes (early carbohydrate, (non-expressing) polynucleic acid or Syn and late), lysosomes, phagosomes, vesicle, endoplasmic thetic compound. Cell targeting Signals Such as ligands reticulum, golgi apparatus, trans golgi network (TGN), and enhance cellular binding to receptors. A variety of ligands Sarcoplasmic reticulum. Release includes movement out of have been used to target drugs and genes to cells and to an intracellular compartment into the cytoplasm or into an Specific cellular receptors. The ligand may seek a target organelle Such as the nucleus. Releasing Signals include within the cell membrane, on the cell membrane or near a chemicals. Such as chloroquine, bafilomycin or Brefeldin A1 cell. Binding of ligands to receptors typically initiates and the ER-retaining signal (KDEL sequence), Viral com endocytosis. Ligands include agents that target to the asia ponents Such as influenza virus hemagglutinin Subunit HA-2 loglycoprotein receptor by using asiologlycoproteins or peptides and other types of amphipathic peptides. The galactose residues. Other proteins Such as insulin, EGF, or control of when and where the membrane active compound transferrin can be used for targeting. Peptides that include is active is crucial to effective transport. If the membrane the RGD sequence can be used to target many cells. Chemi active agent is operative in a certain time and place it would US 2003/023.5916 A1 Dec. 25, 2003

facilitate the transport of the biologically active compound without the breakage of the other covalent bonds. For acroSS the biological membrane. If the membrane active example, a disulfide bond is capable of being broken in the compound is too active or active at the wrong time, then no presence of thiols without cleavage of other bonds, Such as transport occurs or transport is associated with cell rupture carbon-carbon, carbon-oxygen, carbon-Sulfur, carbon-nitro and cell death. Nature has evolved various Strategies to gen bonds, which may also be present in the molecule. allow for membrane transport of biologically active com Labile also means cleavable. pounds including membrane fusion and the use of mem 0079 Labile Linkage-A labile linkage is a chemical brane active compounds whose activity is modulated Such compound that contains a labile bond and provides a link or that activity assists transport without toxicity. Many lipid Spacer between two other groups. The groups that are linked based transport formulations rely on membrane fusion and may be chosen from compounds Such as biologically active Some membrane active peptides activities are modulated by compounds, membrane active compounds, compounds that pH. In particular, Viral coat proteins are often pH-sensitive, inhibit membrane activity, functional reactive groups, inactive at neutral or basic pH and active under the acidic monomers, and cell targeting Signals. The Spacer group may conditions found in the endoSome. contain chemical moieties chosen from a group that includes 0.074 Cell penetrating compounds-Cell penetrating alkanes, alkenes, esters, ethers, glycerol, amide, Saccharides, compounds, which include cationic import peptides (also polysaccharides, and heteroatoms Such as oxygen, Sulfur, or called peptide translocation domains, membrane transloca nitrogen. The Spacer may be electronically neutral, may bear tion peptides, arginine-rich motifs, cell-penetrating peptides, a positive or negative charge, or may bear both positive and and peptoid molecular transporters) are typically rich in negative charges with an overall charge of neutral, positive arginine and lysine residues and are capable of crossing or negative. biological membranes. In addition, they are capable of transporting molecules to which they are attached acroSS 0080 pH-Labile Linkages and Bonds pH-labile refers membranes. Examples include TAT (GRKKRRQRRR, SEQ to the Selective breakage of a covalent bond under acidic ID 9), VP22 peptide, and an ANTp peptide (RQIKIWFON conditions (pH~7). That is, the pH-labile bond may be RRMKWKK, SEQ ID 10). Cell penetrating compounds are broken under acidic conditions in the presence of other not strictly peptides. Short, non-peptide polymers that are covalent bonds that are not broken. rich in amines or guanidinium groups are also capable of 0081 Amphiphilic and Aniphipathic Compounds-Am carrying molecules crossing biological membranes. Like phipathic, or amphiphilic, compounds have both hydrophilic membrane active peptides, cationic import peptides are (water-soluble) and hydrophobic (water-insoluble) parts. defined by their activity rather than by strict amino acid 0082 Polymers-A polymer is a molecule built up by Sequence requirements. repetitive bonding together of Smaller units called mono 0075 Interaction Modifiers-An interaction modifier mers. In this application the term polymer includes both changes the way that a molecule interacts with itself or other oligomers which have two to about 80 monomers and molecules relative to molecule containing no interaction polymers having more than 80 monomers. The polymer can modifier. The result of this modification is that self-interac be linear, branched network, Star, comb, or ladder types of tions or interactions with other molecules are either polymer. The polymer can be a homopolymer in which a increased or decreased. For example cell targeting Signals Single monomer is used or can be copolymer in which two are interaction modifiers which change the interaction or more monomers are used. Types of copolymers include between a molecule and a cell or cellular component. alternating, random, block and graft. Polyethylene glycol is an interaction modifier that decreases 0083. The main chain of a polymer is composed of the interactions between molecules and themselves and with atoms whose bonds are required for propagation of polymer other molecules. length. The Side chain of a polymer is composed of the 0.076 Linkages-An attachment that provides a covalent atoms whose bonds are not required for propagation of bond or spacer between two other groups (chemical moi polymer length. eties). The linkage may be electronically neutral, or may 0084. To those skilled in the art of polymerization, there bear a positive or negative charge. The chemical moieties are Several categories of polymerization processes that can can be hydrophilic or hydrophobic. Preferred spacer groups be utilized in the described process. The polymerization can include, but are not limited to C1-C12 alkyl, C1-C12 alk be chain or Step. This classification description is more often enyl, C1-C12 alkynyl, C6-C18 aralkyl, C6-C18 aralkenyl, used that the previous terminology of addition and conden C6-C18 aralkynyl, ester, ether, ketone, alcohol, polyol, sation polymer. “Most step-reaction polymerizations are amide, amine, polyglycol, polyether, polyamine, thiol, thio condensation processes and most chain-reaction polymer ether, thioester, phosphorous containing, and heterocyclic. izations are addition processes” (M. P. Stevens Polymer The linkage may or may not contain one or more labile Chemistry: An Introduction New York Oxford University bonds. Press 1990). Template polymerization can be used to form 0.077 Bifunctional-Bifunctional molecules, commonly polymers from daughter polymers. referred to as crosslinkers, are used to connect two mol ecules together, i.e. form a linkage between two molecules. 0085 Step Polymerization-In step polymerization, the Bifunctional molecules can contain homo or heterobifunc polymerization occurs in a stepwise fashion. Polymer tionality. growth occurs by reaction between monomers, oligomers and polymers. No initiator is needed since there is the same 0078 Labile Bond-Alabile bond is a covalent bond that reaction throughout and there is no termination Step So that is capable of being selectively broken. That is, the labile the end groups are still reactive. The polymerization rate bond may be broken in the presence of other covalent bonds decreases as the functional groups are consumed. US 2003/023.5916 A1 Dec. 25, 2003

0.086 Typically, step polymerization is done either of two bonds, ester bonds, Sulfone bonds, acetals, ketals, enol different ways. One way, the monomer has both reactive ethers, enol esters, enamines and imines. functional groups (A and B) in the same molecule So that 0097 Polyelectrolyte-A polyelectrolyte, or polyion, is a 0087 A-B yields-A-B- polymer possessing more than one charge, i.e. the polymer contains groups that have either gained or lost one or more 0088 Or the other approach is to have two difunctional electrons. A polycation is a polyelectrolyte possessing net OOCS. positive charge, for example poly-L-lysine hydrobromide. 0089) A-A+B-B yields-A-A-B-B- The polycation can contain monomer units that are charge positive, charge neutral, or charge negative, however, the net 0090. Yet another approach is to have one difunctional charge of the polymer must be positive. A polycation also monomer So that can mean a non-polymeric molecule that contains two or more positive charges. A polyanion is a polyelectrolyte 0091 A-A plus another agent yields-A-A-. containing a net negative charge. The polyanion can contain 0092 Chain Polymerization-In chain-reaction polymer monomer units that are charge negative, charge neutral, or ization growth of the polymer occurs by Successive addition charge positive, however, the net charge on the polymer of monomer units to limited number of growing chains. The must be negative. A polyanion can also mean a non-poly initiation and propagation mechanisms are different and meric molecule that contains two or more negative charges. there is usually a chain-terminating Step. The polymerization The term polyelectrolyte includes polycation, polyanion, rate remains constant until the monomer is depleted. Zwitterionic polymers, and neutral polymers. The term ZWit 0093. Other Components of the Monomers and Poly terionic refers to the product (salt) of the reaction between merS-The polymers have other groups that increase their an acidic group and a basic group that are part of the same utility. These groups can be incorporated into monomers molecule. prior to polymer formation or attached to the polymer after 0098 Steric Stabilizer-Asteric stabilizer is a long chain its formation. These groups include: Targeting GroupS hydrophilic group that prevents aggregation by Sterically Such groups are used for targeting the polymer-nucleic acid hindering particle to particle or polymer to polymer elec complexes to Specific cells or tissues. Examples of Such troStatic interactions. Examples include: alkyl groups, PEG targeting agents include agents that target to the asialogly chains, polysaccharides, alkyl amines. ElectroStatic interac coprotein receptor by using asialoglycoproteins or galactose tions are the non-covalent association of two or more residues. Other proteins Such as insulin, EGF, or transferrin Substances due to attractive forces between positive and can be used for targeting. Protein refers to a molecule made negative charges. up of 2 or more amino acid residues connected one to another as in a polypeptide. The amino acids may be 0099 Buffers-Buffers are made from a weak acid or naturally occurring or Synthetic. Peptides that include the weak base and their Salts. Buffer Solutions resist changes in RGD sequence can be used to target many cells. Chemical pH when additional acid or base is added to the solution. groups that react with thiol, Sulfhydryl, or disulfide groups 0100 Biological, Chemical, or Biochemical reactions on cells can also be used to target many types of cells. Folate Biological, chemical, or biochemical reactions involve the and other Vitamins can also be used for targeting. Other formation or cleavage of ionic and/or covalent bonds. targeting groups include molecules that interact with mem 0101 Reactive-A compound is reactive if it is capable branes Such as fatty acids, cholesterol, dansyl compounds, of forming either an ionic or a covalent bond with another and amphotericin derivatives. compound. The portions of reactive compounds that are 0094. After interaction of the Supramolecular complexes capable of forming covalent bonds are referred to as reactive with the cell, other targeting groups can be used to increase functional groups or reactive groups. the delivery of the drug or nucleic acid to certain parts of the 0102) Steroid-A steroid derivative means a sterol, a cell. For example, agents can be used to disrupt endoSomes sterol in which the hydroxyl moiety has been modified (for and a nuclear localizing Signal (NLS) can be used to target example, acylated), a steroid hormone, or an analog thereof. the nucleus. The modification can include Spacer groups, linkers, or 0.095) A variety of ligands have been used to target drugs reactive groups. and genes to cells and to specific cellular receptors. The 0.103 Sterics-Steric hindrance, or sterics, is the preven ligand may seek a target within the cell membrane, on the tion or retardation of a chemical reaction because of neigh cell membrane or near a cell. Binding of ligands to receptors boring groups on the same molecule. typically initiates endocytosis. Ligands could also be used for DNA delivery that bind to receptors that are not endocy 0104 Lipid-Any of a diverse group of organic com tosed. For example peptides containing RGD peptide pounds that are insoluble in water, but Soluble in organic Sequence that bind integrin receptor could be used. In Solvents Such as chloroform and benzene. Lipids contain addition viral proteins could be used to bind the complex to both hydrophobic and hydrophilic sections. The term lipids cells. Lipids and Steroids could be used to directly insert a is meant to include complex lipids, simple lipids, and complex into cellular membranes. Synthetic lipids. 0096. The polymers can also contain cleavable groups 0105 Complex Lipids-Complex lipids are the esters of within themselves. When attached to the targeting group, fatty acids and include glycerides (fats and oils), glycolipids, cleavage leads to reduce interaction between the complex phospholipids, and waxes. and the receptor for the targeting group. Cleavable groups 0106 Simple Lipids-Simple lipids include steroids and include but are not restricted to disulfide bonds, diols, diazo terpeneS. US 2003/023.5916 A1 Dec. 25, 2003

0107 Synthetic Lipids-Synthetic lipids includes amides monolayer of Surfactant molecules. The Spontaneous curva prepared from fatty acids wherein the carboxylic acid has ture, HO of the surfactant monolayer at the oil/water inter been converted to the amide, Synthetic variants of complex face dictates the phase behavior and microstructure of the lipids in which one or more oxygen atoms has been Substi vesicle. Hydrophilic Surfactants produce oil in water (O/W) tuted by another heteroatom (such as Nitrogen or Sulfur), microemulsions (H0>0), whereas lipophilic Surfactants pro and derivatives of simple lipids in which additional hydro duce water in oil (W/O) microemulsions. philic groups have been chemically attached. Synthetic 0119) Hydrophobic Groups-Hydrophobic groups indi lipids may contain one or more labile groups. cate in qualitative terms that the chemical moiety is water 0108 Fats-Fats are glycerol esters of long-chain car avoiding. Typically, Such chemical groups are not water boxylic acids. Hydrolysis of fats yields glycerol and a Soluble, and tend not to form hydrogen bonds. carboxylic acid-a fatty acid. Fatty acids may be Saturated 0120 Hydrophilic Groups-Hydrophilic groups indicate or unsaturated (contain one or more double bonds). in qualitative terms that the chemical moiety is water 0109 Oils-Oils are esters of carboxylic acids or are preferring. Typically, Such chemical groups are water glycerides of fatty acids. Soluble, and are hydrogen bond donors or acceptors with 0110 Glycolipids-Glycolipids are sugar containing lip Water. ids. The Sugars are typically galactose, glucose or inositol. EXAMPLES 0111 Phospholipids-Phospholipids are lipids having both a phosphate group and one or more fatty acids (as esters Example 1 of the fatty acid). The phosphate group may be bound to one 0121 Preparation of Dodecylamine Hydrochloride: or more additional organic groups. 0122) Dodecylamine (113 mg, 0.610 mmol, Aldrich 0112 Wax Waxes are any of various solid or semisolid Chemical Company) was converted to its hydrochloride salt Substances generally being esters of fatty acids. by dissolving in methanol (500 ul), and adding hydrogen 0113 Fatty Acids-Fatty acids are considered the chloride (7 mL, 1 M in diethyl ether, Aldrich). The resulting hydrolysis product of lipids (fats, waxes, and phosphoglyc precipitate was washed with diethyl ether and dried under erides). vacuum to afford dodecylamine hydrochloride (115 mg, 85% yield) as a white solid. 0114 Surfactant-A Surfactant is a Surface active agent, Such as a detergent or a lipid, which is added to a liquid to Example 2 increase its spreading or wetting properties by reducing its Surface tension. A Surfactant refers to a compound that 0123 Preparation of a Complex with pl)NA and Dode contains a polar group (hydrophilic) and a non-polar (hydro cylamine Hydrochloride: phobic) group on the same molecule. A cleavable Surfactant 0124) To water (50 ul) was added a solution of pDNA is a Surfactant in which the polar group may be separated (50 uL of 2 mg/mL solution in water, 100 ug, 0.30 umol in from the nonpolar group by the breakage or cleavage of a phosphate) with mixing. A Solution of dodecylamine hydro chemical bond located between the two groups, or to a chloride in water (13.4 uL, 134 ug, 0.60 umol) was added Surfactant in which the polar or non-polar group or both may with mixing. After 10 min, the precipitate was spun down be chemically modified Such that the detergent properties of with centrifugation, and the pellet was washed with water the Surfactant are destroyed. (2x100 uL). The resulting pellet was dried on a lyophilizer 0115 Detergent-Detergents are compounds that are over POs for 3 days. Soluble in water and cause nonpolar Substances to go into solution in water. Detergents have both hydrophobic and Example 3 hydrophilic groups 0.125 Preparation of a Complex with pl)NA and Cetyl 0116 Micelle-Micelles are microscopic vesicles that trimethylammonium Bromide (CTAB): contain amphipathic molecules but do not contain an aque 0126 To water (50 ul) was added a solution of pDNA ous Volume that is entirely enclosed by a membrane. In (50 uL of 2 mg/mL solution in water, 100 ug, 0.30 umol in micelles the hydrophilic part of the amphipathic compound phosphate) with mixing. A Solution of dodecylamine hydro is on the outside (on the Surface of the vesicle). In inverse chloride in water (13.4 uL, 134 ug, 0.60 umol) was added micelles the hydrophobic part of the amphipathic compound with mixing. After 10 min, the precipitate was spun down is on the outside. The inverse micelles thus contain a polar with centrifugation, and the pellet was washed with water core that can Solubilize both water and macromolecules (2x100 uL). The resulting pellet lyophilized over POs for 3 within the inverse micelle. days. 0117 Liposome-Liposomes are microscopic vesicles that contain amphipathic molecules and contain an aqueous Example 4 Volume that is entirely enclosed by a membrane. 0127 Preparation of Cy3-DNA-NC: 0118 Microemulsions-Microemulsions are isotropic, 0128. To HO (15uL) was added Cy3-DNA (100 ug, 85 thermodynamically stable Solutions in which Substantial AiL of 1.17 ug/ull H2O, 0.30 umol phosphate) and gently amounts of two immiscible liquids (water and oil) are mixed. NC (134 ug, 13.4 ul of 10 ug?ul HO, 0.61 umol) brought into a Single phase due to a Surfactant or mixture of was added and gently mixed. A pink precipitate formed. The Surfactants. The Spontaneously formed colloidal particles reaction sat at RT for 10 min and was spun down by are globular droplets of the minor Solvent, Surrounded by a centrifugation to form a pellet. The Supernatant was US 2003/023.5916 A1 Dec. 25, 2003

decanted and the pellet was washed 2x with water. The pellet Example 8 was lyophilized over POs for several days. 0135 Preparation of Cholic Acid Modified Chitosan: Example 5 0136 Cholic acid (CalBiochem) was dissolved in water to a final concentration of 100 mg/mL. To a solution of the 0129. Preparation of Oleic Acid Modified Chitosan: cholic acid in water (11 u , 2.7 umol) was added DMF (407 uL), followed by sulfo-N-hydroxysuccinimide (57 uL of a 0130 Oleic acid (4.15 uL, 13.2 mmol, Aldrich) was taken 10 mg/mL solution in water, 2.7 umol, Pierce Chemical up in DMF (342 uL). To the resulting solution was added Company), and 1-(3-dimethylaminopropyl)-3-ethylcarbo sulfo-N-hydroxysuccinimide (29 uL of a 100 mg/mL solu duimide hydrochloride (25 uL of a 20 mg/mL solution in tion in water, 13.2 umol, Pierce Chemical Company), and DMF, Aldrich) and the resulting solution was stirred at RT. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro After 1 h, chitosan (reagent dissolved in 1% HCl in water to chloride (125ul of a 20 mg/mL solution in DMF, 13.2 umol, a final concentration of 10 mg/mL, 1 mL of concentrate was Aldrich) and the resulting Solution was stirred at room dissolved in 5 mL water and adjusted to pH 6.5 with 3 N temperature. After 1 h, the Solution was added to chitosan NaOH, 10 mg polymer (85% deacylated), 53 umol in amine, (reagent dissolved in 1% HCl in water to a final concentra Fluka Chemical Company) was added and the resulting tion of 10 mg/mL, 500 lull of concentrate was dissolved in solution was stirred for 16 hat room temperature to afford 500 ul water and adjusted to pH 6.5 with 1 N. NaOH, 5.0 mg the cholic acid modified chitosan. polymer (85% deacylated), 31 umol in amine, Fluka Chemi Example 9 cal Company) in DMF (3.5 mL). The resulting solution was 0.137 Preparation of 2-Propionic-3-methylmaleic anhy stirred at room temperature for 16 h to afford oleic acid dride (carboxydimethylmaleic anhydride or CDM): modified chitosan as a 1 mg/ml Solution in 80% DMF/water. 0138 To a suspension of sodium hydride (0.58 g, 25 Example 6 mmol) in 50 mL anhydrous tetrahydrofuran was added triethyl-2-phosphonopropionate (7.1 g, 30 mmol). After 0131 Preparation of Oleic Acid and Lactobionic Acid bubbling of hydrogen gas Stopped, dimethyl-2-oxoglutarate Modified Chitosan: (3.5g, 20 mmol) in 10 mL anhydrous tetrahydrofuran was added and stirred for 30 minutes. Water, 10 mL, was then 0132 A stock solution of lactobionic acid in DMF was added and the tetrahydrofuran was removed by rotary prepared to a final concentration of 100 mg/mL. To 9.45 ul evaporation. The resulting Solid and water mixture was of the lactobionic acid stock Solution (945 ug, 2.64 umol, extracted with 3x50 mL ethyl ether. The ether extractions Aldrich) was added DMF (437 uL), sulfo-N-hydroxysuc were combined, dried with magnesium Sulfate, and concen cinimide (28.7 uL of a 20 mg/mL solution in water, 573 ug, trated to a light yellow oil. The oil was purified by silica gel 2.64 umol, Pierce Chemical Company), and 1-(3-dimethyl chromatography elution with 2:1 ether:hexane to yield 4 gm aminopropyl)-3-ethylcarboduimide hydrochloride (25.3 uL (82% yield) of pure triester. The 2-propionic-3-methylma of a 20 mg/mL solution in DMF, 505 ug, 2.64 umol, leic anhydride then formed by dissolving of this triester into Aldrich). The resulting solution was stirred for 1 h at 50 mL of a 50/50 mixture of water and ethanol containing ambient temperature and added to a Solution of oleic acid 4.5 g (5 equivalents) of potassium hydroxide. This Solution modified chitosan (1 mL of a 1 mg/ml solution in 80% was heated to reflux for 1 hour. The ethanol was then DMF/water). The resulting solution was stirred for 16 hat removed by rotary evaporation and the Solution was acidi ambient temperature to afford oleic acid and lactobionic acid fied to pH 2 with hydrochloric acid. This acqueous solution was then extracted with 200 mL ethyl acetate, which was modified chitosan as a 0.667 mg/ml solution. isolated, dried with magnesium Sulfate, and concentrated to a white solid. This solid was then recrystallized from dichlo Example 7 romethane and hexane to yield 2 g (80% yield) of 2-propi 0133) Preparation of Deoxycholic Acid Modified Chito onic-3-methylmaleic anhydride. S. Example 10 0134) Deoxycholic acid (CalBiochem) was dissolved in 0139 Preparation of 2-(Dodecylpropionamide)-3-meth water to a final concentration of 100 mg/mL. To a solution ylmaleic anhydride (CDMNC): of the deoxycholic acid in water (11 u , 2.7 umol) was 0140) 2-Propionic-3-methylmaleic anhydride (100 mg, added DMF (407 uL), followed by Sulfo-N-hydroxysuccin 0.543 mmol) was dissolved in dichloromethane (3 mL). The imide (57 uL of a 10 mg/mL solution in water, 2.7 umol, resulting Solution was cooled to 0 C. in an ice bath, and Pierce Chemical Company), and 1-(3-dimethylaminopro oxalyl chloride (49.7 ul, 0.57 mmol, Aldrich) was added pyl)-3-ethylcarbodiimide hydrochloride (25 uL of a 20 dropwise by Syringe. The resulting Solution was allowed to mg/mL solution in DMF, Aldrich) and the resulting solution warm to room temperature, and dodecylamine (206 mg, 1.11 was stirred at RT. After 1 h, chitosan (reagent dissolved in mmol, Aldrich) was added, followed by diisopropylethy 1% HCl in water to a final concentration of 10 mg/mL, 1 mL lamine (94.6 uL, 0.543 mmol, Aldrich). After 16 h, the of concentrate was dissolved in 5 mL water and adjusted to Solution was concentrated under reduced pressure (aspira pH 6.5 with 3 N NaOH, 10 mg polymer (85% deacylated), tor), and partitioned in ethyl acetate and water. The organic 53 umol in amine, Fluka Chemical Company) was added layer was washed with IN HCl (3x), brine, dried (NaSO), and the resulting Solution was stirred for 16 h at room filtered, and concentrated to afford 179 mg (94%) of temperature to afford the deoxycholic acid modified chito 2-(dodecylpropionamide)-3-methylmaleic anhydride S. (CDMNC). M+1=352.2 US 2003/023.5916 A1 Dec. 25, 2003

Example 11 Example 16 0141 Preparation of Chitosan-CDMC (Chit-CDMC): 0151. Preparation of MC791-CDMC12-CDM: 0152 MC 791 is a polymer prepared from the polymer 0142 To Chitosan (200 ug, 20 uL of 10 uguL 1% HCl, ization of vinyl ethers. The polymerization feed ratios 1.06 timol amine, Fluka Chemical Company) was added (molar ratios) for MC791 are: 47% ethyl vinyl ether, 3% EtOH (20 uD) and several mg's of diisopropylaminomethyl octadecyl vinyl ether, and 50% 1-aminoethyl vinyl ether polystyrene Solid Support base (Fluka Chemical Company). (protected during the polymerization reaction as the phthal CDMC (185ug, 1.85 uL of 100 ug?u. DMF, 0.53 umol) imide derivative. To MC791 (200 ug, 20 u, of 10 uguL was added and the reaction was stirred at RT for 30 min, and HO, 3.2 umol amine) was added EtOH (80 uD) and a few the Solid Support base was removed by centrifugation. mg's of diisopropylaminomethyl-polystyrene Solid Support base (Fluka Chemical Company). The solution was mixed Example 12 well. CDMC12 (200 ug, 2.0 ul of 100 ug?u. DMF, 0.57 0143 Preparation of Chitosan-CDMC12-CDM (Chit umol) was added and the reaction was stirred at RT for 30 min. CDM (200 ug, 2.0 uL of 100 ug?uL DMF, 1.1 umol) CDMC12-CDM): was added and the reaction was stirred at RT for an addi 0144) To Chit-CDMC12 (200 ug, 100 ul of 2 ug?u L tional 30 min. The solid support base was removed by EtOH, 0.53 umol amine) was added several mg's of diiso centrifugation. propylaminomethyl-polystyrene Solid Support base (Fluka Chemical Company). 2-Propionic-3-methylmaleic anhy Example 17 dride (CDM) (97 ug, 10 ul of 10 ug?u L DMF, 0.53 umol) was added and the reaction was stirred at RT for 30 min, and 0153. Preparation of MC787-CDM: the Solid Support base was removed by centrifugation. 0154 MC 787 is a polymer prepared from the polymer ization of vinyl ethers. The polymerization feed ratios Example 13 (molar ratios) for MC791 are: 47% in-propyl vinyl ether, 3% octadecyl vinyl ether, and 50% 1-aminoethyl vinyl ether 0145 Preparation of Chitosan-Oleic-CDM (Chit-Ol (protected during the polymerization reaction as the phthal CDM): imide derivative. To MC787 (200 ug, 20 u, of 10 uguL 0146) To Chit-O1 (500 lug, 100 uL of 5ug/ull 80% DMF, HO, 3.2 umol amine) was added EtOH (80 uD) and a few 1.3 umol) was added several mg's of diisopropyl-aminom mg's of diisopropylaminomethyl-polystyrene Solid Support ethyl-polystyrene Solid Support base (Fluka Chemical Com base (Fluka Chemical Company). The solution was mixed pany) and mixed well. CDM (244 ug, 2.4 ul of 100 ug?uL well. CDM (200ug, 2.0 ul of 100 ugul DMF, 1.1 umol) DMF, 1.3 umol) was added, the reaction was stirred at RT for was added and the reaction was stirred at RT for 30 min. The 30 min, and the Solid Support base was removed by cen Solid Support base was removed by centrifugation. trifugation. Example 18 Example 14 O155 Preparation of MC787-CDMC12-CDM: 0147 Preparation of Chitosan-Oleic-Succinic Anhydride 0156 MC 787 is a polymer prepared from the polymer ization of vinyl ethers. The polymerization feed ratios (Chit-Ol-SA): (molar ratios) for MC791 are: 47% in-propyl vinyl ether, 3% 0148. To Chit-O1 (500 lug, 100 uL of 5ug/ull 80% DMF, octadecyl vinyl ether, and 50% 1-aminoethyl vinyl ether 1.3 limol) was added several mg's of dilsopropylaminom (protected during the polymerization reaction as the phthal ethyl-polystyrene Solid Support base (Fluka Chemical Com imide derivative. To MC787 (200 ug, 20 u, of 10 uguL pany) and mixed well. Succinic anhydride (132 ug, 1.3 ul. HO, 3.2 umol amine) was added EtOH (80 uD) and a few of 100 ugul DMF, 1.3 umol) was added, the reaction was mg's of diisopropylaminomethyl-polystyrene Solid Support stirred at RT for 30 min, and the solid support base was base (Fluka Chemical Company). The solution was mixed removed by centrifugation. well. CDMC12 (200 ug, 2.0 ul of 100 ug?u. DMF, 0.57 umol) was added and the reaction was stirred at RT for 30 Example 15 min. CDM (200 ug, 2.0 uL of 100 ug?uL DMF, 1.1 umol) was added and the reaction was stirred at RT for an addi 0149 Preparation of MC791-CDM: tional 30 min. The solid support base was removed by 0150 MC 791 is a polymer prepared from the polymer centrifugation. ization of vinyl ethers. The polymerization feed ratios Example 19 (molar ratios) for MC791 are: 47% ethyl vinyl ether, 3% octadecyl vinyl ether, and 50% 1-aminoethyl vinyl ether O157 Condensation of pDNA with Cationic Surfactants. (protected during the polymerization reaction as the phthal imide derivative. To MC791 (200 ug, 20 uL of 10 ug?uL 0158 Part A: Determination of Rhodamine Labeled DNA HO, 3.2 umol amine) was added EtOH (80 uD) and a few Condensation with Cetyltrimethyl-ammonium Bromide mg's of diisopropylaminomethyl-polystyrene Solid Support (CTAB): base (Fluka Chemical Company). The solution was mixed 0159 PCILuc DNA (pDNA) Zhang et al. 1997) was well. CDM (200ug, 2.0 uL of 100 uguL DMF, 1.1 umol) modified to a level of 1 rhodamine per 100 bases using was added and the reaction was stirred at RT for 30 min. The Mirus Label ITOR Rhodamine kit (Rhodamine Containing Solid Support base was removed by centrifugation. DNA Labeling Reagent, Mirus Corporation). The modified US 2003/023.5916 A1 Dec. 25, 2003 pDNA (10 ug) was mixed with plDNA that was not labeled 0163 The results indicate the pdNA is condensed (90 ug) in water and diluted to a final concentration of 1 between 0.036 and 0.054 mM dodecylamine hydrochloride ug?ul with water. The modified plDNA/pDNA mixture (5 concentration. The literature cmc for dodecylamine hydro ug) was diluted with water to a final volume of 500 uL. chloride is 2.5 mM. Different amounts of CTAB were added to the Solution and Example 20 the fluorescence intensity of the Solution was measured using a Cary Eclipse Fluorescence Spectrophotometer (ex= 0164) Mouse Tail Vein Injections of pDNA-NC12 (pCI 555, em=585, Varian, Inc.). Trubetskoy etal have shown that Luc) with Modified Chitosan Polymers: rhodamine labeled pdNA undergoes fluorescence quench 0.165 Fifteen complexes were prepared as follows: ing as the pDNA is condensed Trubetskoy, 1999). 0166 Complex I: plDNA(pCI Luc, 40 ug) in HO (2 mL). Ringers (10 mL) was added prior to injection. 0167 Complex II: pDNA-NC (pCI Luc, 40 ug, 40 N/P Aug CTAB mM CTAB Fluor Int uL of 1 tug/ul EtOH) was added to HO (2 mL) and O O O.OOO 230.896 Vortexed. Ringers (10 mL) was added prior to injec 0.4 2 O.O11 2O1540 tion and mixed well. O.7 4 O.O22 159.277 1.1 6 O.O33 85.253 1.4 8 O.O44 81.625 0168 Complex III: pDNA-NC (pCI Luc, 40 ug, 1.8 1O 0.055 78.471 40 uL of 1 tug/uL EtOH) was added to a solution of 2.2 12 O.O66 74.722 Chit-Ol (84 ug, 0.22 umol) in HO (2 mL) and 2.5 14 0.077 67.267 Vortexed. Ringers (10 mL) was added prior to injec 2.9 16 O.088 62.799 tion and mixed well. 0169 Complex IV: pDNA-NC12 (pCI Luc, 40 ug, 0160 The results indicate that CTAB condenses the 40 uL of 1 tug/uL EtOH) was added to a solution of pDNA at a CTAB concentration of 0.33 mM, which is below Chit-Ol-LBA (84 ug, 0.22 umol) in HO (2 mL) and the literature cmc of CTAB (1 mM)Calbiochem, 2000 Vortexed. Ringers (10 mL) was added prior to injec 2001). tion and mixed well. 0170 Complex V: pDNA-NC (pCI Luc, 40 ug, 40 0161 Part B: Determination of Rhodamine Labeled DNA uL of 1 lugfulL EtOH) was added to a solution of Condensation with Dodecylamine Hydrochloride: Chit-CDMC12 (84 ug, 0.22 umol) in HO (2 mL) 0162 PCILuc DNA (pDNA) Zhang et al. 1997) was and Vortexed. Ringers (10 mL) was added prior to modified to a level of 1 rhodamine per 100 bases using injection and mixed well. Mirus Label ItE Rhodamine kit (Rhodamine Containing 0171 Complex VI: pDNA-NC (pCI Luc, 40 ug, DNA Labeling Reagent, Mirus Corporation). The modified 40 uL of 1 tug/uL EtOH) was added to a solution of pDNA (10 ug) was mixed with plDNA that was not labeled Chit-Ol-SA (84 ug, 0.22 umol) in H2O (2 mL) and (90 ug) in water and diluted to a final concentration of 1 Vortexed. Ringers (10 mL) was added prior to injec ug?ul with water. The modified plDNA/pDNA mixture (5 tion and mixed well. ug) was diluted with water to a final volume of 500 uL. 0172) Complex VII: pDNA-NC (pCI Luc, 40 ug, Different amounts of dodecylamine hydrochloride were 40 uL of 1 tug/uL EtOH) was added to a solution of added to the Solution and the fluorescence intensity of the Chit-Ol-CDM (84 ug, 0.22 umol) in HO (2 mL) and Solution was measured using a Cary Eclipse Fluorescence Vortexed. Ringers (10 mL) was added prior to injec Spectrophotometer (ex=555, em=585, Varian, Inc.). Tru tion and mixed well. betskoy et all have shown that rhodamine labeled pl)NA undergoes fluorescence quenching as the pDNA is con 0173 Complex VIII:pDNA-NC (pCI Luc, 40 ug, densed Trubetskoy, 1999). 40 uL of 1 tug/uL EtOH) was added to a solution of Chit-CDM (84 ug, 0.22 umol) in HO (2 mL) and Vortexed. Ringers (10 mL) was added prior to injec tion and mixed well. N/P Aug NC12 nM NC12 Fluor Int 0174 Complex IX: pdNA-NC (pCI Luc, 40 ug, O O O.OOO 264.039 40 uL of 1 tug/uL EtOH) was added to a solution of O.6 2 O.O18 183.656 1.2 4 O.O36 106.095 Chit-CDM12-CDM (84 ug, 0.22 umol) in HO (2 1.8 6 O.O54 95.586 mL) and vortexed. Ringers (10 mL) was added prior 2.4 8 0.072 86.119 to injection and mixed well. 3.0 1O O.O90 77.670 3.6 12 O.108 68.208 0175 Complex X: pDNA-NC (pCI Luc, 40 ug, 40 4.2 14 O.126 62.115 4.8 16 0.144 60.158 uL of 1 tug/ul EtOH) was added to a solution of 5.4 18 O.162 53.467 Chit-LBA-CDM12 (84 ug, 0.22 umol) in HO (2 6.O 2O O.18O 46.790 mL) and vortexed. Ringers (10 mL) was added prior 6.5 22 O.198 43.375 to injection and mixed well. 7.1 24 O.216 45.OOO 0176) Complex XI: plDNA-NC (pCI Luc, 40 ug, 40 uL of 1 tug/uL EtOH) was added to a solution of US 2003/023.5916 A1 Dec. 25, 2003 15

Chit-Chol (84 ug, 0.22 umol) in HO (2 mL) and 0188 Complex III: pDNA-NC (pCI Luc, 30 ug, Vortexed. Ringers (10 mL) was added prior to injec 30 uL of 1 tug/uL EtOH) was added to a solution of tion and mixed well. Chit-Ol (63 ug, 0.17 umol) in HO (750 uD) and 0177) Complex XII: pDNA-NC (pCI Luc, 40 ug, vortexed. 40 uL of 1 tug/uL EtOH) was added to a solution of 0189 Complex IV: plDNA-NC (pCI Luc, 30 ug, 30 Chit-Chol-CDM12 (84 ug, 0.22 umol) in HO (2 uL of 1 tug/ul EtOH) was added to a solution of mL) and vortexed. Ringers (10 mL) was added prior Chit-CDM12 (63 ug, 0.17 umol) in HO (750 uL) to injection and mixed well. and Vortexed. 0.178 Complex XIII: pDNA-NC (pCI Luc, 40 ug, 0.190 Complex V: pDNA-NC (pCI Luc, 30 ug, 30 40 uL of 1 tug/uL EtOH) was added to a solution of uL of 1 tug/ul EtOH) was added to a solution of Chit-Chol-CDM12-CDM (84 ug, 0.22 umol) in HO Chit-Ol-LBA (63 ug, 0.17 umol) in HO (750 uL) (2 mL) and vortexed. Ringers (10 mL) was added and Vortexed. prior to injection and mixed well. 0191) Complex VI: pDNA-NC (pCI Luc, 30 ug, 0179 Complex XIV: pDNA-NC (pCI Luc, 40 ug, 30 uL of 1 tug/uL EtOH) was added to a solution of 40 uL of 1 tug/uL EtOH) was added to a solution of Chit (63 ug, 0.17 umol) in HO (750 uL) and Chit (84 ug, 0.22umol) in HO (2 mL) and vortexed. vortexed. Ringers (10 mL) was added prior to injection and 0.192 Complex VII: plDNA (pCI Luc, 30 lug) was mixed well. added to a solution of Chit (63 ug, 0.17umol) in H2O 0180 Complex XV: pDNA(pCI Luc, 40 ug) was (750 uL) and vortexed. added to a solution of Chit (84 ug, 0.22umol) in HO 0193 250 uL tail vein injections of 250 uL of the com (2 mL) and vortexed. Ringers (10 mL) was added plex were preformed on ICR mice (n=2) using a 30 gauge, prior to injection and mixed well. 0.5 inch needle, with the total solution injected by hand 0181 Tail vein injections of 1.0 mL per 10 g body weight within 10 Seconds. One day after injection, the animal was (~2.5 ml) were preformed on ICR mice (n=2) using a 30 Sacrificed, and a luciferase assay was conducted on the liver, gauge, 0.5 inch needle. Injections were done manually with Spleen, lung, heart, and kidney. Luciferase expression was injection times of 4-5 sec Zhang et al. 1999; Liu et al. determined as previously reported (Wolff et al. 1990.). A 1999). One day after injection, the livers were harvested and Lumat LB 9507 (EG&G Berthold, Bad-Wildbad, Germany) homogenized in lysis buffer (0.1% Triton X-100, 0.1 M luminometer was used. K-phosphate, 1 mM DTT, pH 7.8). Insoluble material was 0194 Results: 250 uL Injections cleared by centrifugation and 10 ul of the cellular extract or extract diluted 10x was analyzed for luciferase activity as previously reported Wolfetal 1990). Complex I: 266 Complex V: 1,094 0182 Results: 2.5 mL Injections Complex II: 75 Complex VI: 154 Complex III: 5,439 Complex VII: 2O7 Complex IV: 194 Complex I: 11,348,425 Complex IX: 206,866 Complex II: 10,106,551 Complex X: 48,860 Complex III: 17,994,604 Complex XI: 84,423 0.195 Results indicate an increased level of pCI Luc Complex IV: 13,645,295 Complex XII: 61,295 DNA expression in plDNA-NC/modified Chitosan com Complex V: 6,880,568 Complex XIII: 451,827 plexes over pCI Luc DNA/Chitosan complexes. These Complex VI: 87,746 Complex XIV: 268,552 results also indicate that the pdNA is being released from Complex VII: 301,174 Complex XV: 984,729 pDNA-NC2/modified Chitosan complexes, and is acces Complex VIII: 98.434 Sible for transcription. 0183 Results indicate an increased level of pCI Luc Example 22 DNA expression in pl)NA-NC2/modified Chitosan com 0196) Mouse Tail Vein Injections of Cy3-plDNA-NC plexes over pCI Luc DNA/Chitosan complexes. These (pCI Luc) with Modified Chitosan Polymers, Histological results also indicate that the pdNA is being released from Examination: pDNA-NC2/modified Chitosan complexes, and is acces Sible for transcription. 0197) Three complexes were prepared as follows: 0198 Complex I: Cy3-plDNA-NC (pCI Luc, 40 Example 21 ug, 40 uL of 1 tug/uL EtOH) was added to HO (500 0184 Mouse Tail Vein Injections of pDNA-NC12 (pCI AiL) and Vortexed. Luc) with Modified Chitosan Polymers: 0199 Complex II: Cy3-pDNA-NC (pCI Luc, 40 pig, 40 u, of 1 ug?u. EtOH) was added to a Solution 0185. Seven complexesp were preparedprep as follows: of Chit-Ol (84 ug, 0.22 umol) in HO (500 uD) and 0186 Complexp I: pplDNA (pCIp Luc, 30 lug)9. in HO2 vortexed. (750 uL). 0200 Complex III: Cy3-plDNA-NC (pCI Luc, 40 0187 Complex II: plDNA-NC (pCI Luc, 30 ug, 30 pig, 40 u, of 1 ug?u. EtOH) was added to a Solution uL of 1 lug/ul EtOH) was added to HO (750 uL) and of Chit-Ol-LBA (84 ug, 0.22 umol) in HO (500 uL) vortexed. and Vortexed. US 2003/023.5916 A1 Dec. 25, 2003

0201 250 till tail vein injections of 250 till of the com Example 25 plex were preformed on ICR mice (n=2) using a 30 gauge, 0208 Synthesis of 3-Dimethylaminopropyl-1-dimethy 0.5 inch needle, with the total solution injected by hand loctadecyl silyl ether. within 10 Seconds. One day after injection, the animal was Sacrificed, and a luciferase assay was conducted on the liver, 0209 To a solution of 3-dimethylamino-1 propanol (90.0 Spleen, lung, heart, and kidney. Luciferase expression was mg, 0.873 mmol, Aldrich Chemical Company) in 2 mL determined as previously reported (Wolff et al. 1990). A chloroform was added dimethyloctadecyl chlorosilane (378 Lumat LB 9507 (EG&G Berthold, Bad-Wildbad, Germany) mg, 1.09 mmol, Aldrich Chemical Company) and imidazole luminometer was used. (74.2 mg, 1.09 mmol, Aldrich Chemical Company). After 16 hrs at ambient temperature, the Solution was partitioned in Example 23 EtOAc/H2O with 10% sodium bicarbinate. The organic layer was washed with water, and brine. The solvent was 0202 Particle Size of Complexes without pCILuc DNA removed (aspirator) to afford 328 mg (91%) of 3-dimethy NC: laminopropyl-1-dimethyloctadecyl silyl ether as a cream colored Solid. 0203) To a solution of HO (500 uL) was added modified polymer (amount indicated below) and vortexed. The size of Example 26 the complexes was measured on a Zeta Plus Particle Sizer 0210 Synthesis of 3-(dimethylaminopropyl)-1,2-dim (Brookhaven Instrument Corporation). ethyloctadecyl silyl ether. 0211) To a solution of 3-(dimethylamino)-1,2-pro panediol (50.0 mg, 0.419 mmol, Aldrich Chemical Com Polymer Amount (ug) Size (nm) Counts (kcps) pany) in 2 mL chloroform was added dimethyloctadecyl Chit-CDM12 10.5 160 312 chlorosilane (328 mg, 0.944 mmol, Aldrich Chemical Com Chit-CDM12-CDM 10.5 137 538 Chit-O1 10.5 offscale 41 pany) and imidazole (68.1 mg, 0.944 mmol, Aldrich Chemi Chit-O1-SA 10.5 141 143 cal Company). After 16 hrs at ambient temperature, the Chit-O1-CDM 10.5 offscale 109 solution was partitioned in EtOAc/H2O with 10% sodium Chit-LBA-CDM12 1O.O 2865 417 bicarbinate. The organic layer was washed with water, and MC791-CDM12 1O.O 359 42 MC791-CDM12-CDM 1O.O 231 58 brine. The solvent was removed (aspirator) to afford 266 mg MC787-CDM12 1O.O 87 221 (86%) of 3-(dimethylaminopropyl)-1,2-dimethyloctadecyl MC787-CDM12-CDM 1O.O 13686 55 silyl ether as a white solid. Example 27 0204. The particle size data indicates that particles are 0212 Synthesis of 1-(3-lauroylaminopropyl), 4-(3-ole present in Some of the polymer formulations at this concen oylaminopropyl)piperazine (MC763), 1,4-bis(3-lauroylami tration, as polymeric micelles. nopropyl)piperazine (MC762), and 1,4-bis(3-oleoylamino propyl)piperazine (MC798). Example 24 0213 To a 25 mL flame dried flask was added oleoyl 0205 Particle Size of Complexes with pCILuc DNA chloride (freshly distilled, 1.0 ml, 3.0 mmol, Aldrich Chemi NC: cal Company) and lauroyl chloride (0.70 mL, 3.0 mmol, Aldrich Chemical Company) in 15 mL dichloromethane 0206 To a solution of HO (500 uL) was added modified under N. The resulting solution was cooled to 0 C. in an polymer (amount indicated below) and vortexed. DNA ice bath. N,N-Diisopropylethylamine (1.1 ml, 6.1 mmol, NC (5ug, 5uL of 1 u/ug EtOH, 0.015umol phosphate) Aldrich Chemical Company) was added followed by 1,4- was added to the Solution, Vortexed and the size of the bis(3-aminopropyl)piperazine (0.50 ml, 2.4 mmol, Aldrich complexes was measured on a Zeta Plus Particle Sizer Chemical Company). The ice bath was removed and the (Brookhaven Instrument Corporation). solution stirred at ambient temperature for 15 hr. The solution was washed twice with 1N NaOH (10 ml), twice with water (10 ml), and concentrated under reduced pres Polymer Amount (ug) Size (nm) Counts (kcps) SUC. Chit-CDM12 10.5 182 325 0214) Approximatly 30% of the resulting residue was Chit-CDM12-CDM 10.5 160 210 purified by semi-preparative HPLC on a Beta Basic Cyano Chit-O1 10.5 252 147 column (150 A, 5 tim, 250x21 mm, Keystone Scientific, Chit-O1-SA 10.5 152 264 Chit-O1-CDM 10.5 875 12 Inc.) with acetonitrile/HO/trifluoroacetic acid eluent. Three Chit-LBA-CDM12 1O.O 258 393 compounds were isolated from the column and verified by MC791-CDM12 1O.O 183 344 mass spectroscopy (Sciex API 150EX). MC791-CDM12-CDM 1O.O 170 248 MC787-CDM12 1O.O 293 24OO MC787-CDM12-CDM 1O.O 160 556 MC763 1-(3-lauroylaminopropyl),4-(3-oleoylaminopropyl) piperazine (MW = 647) MC762 1,4-bis(3-lauroylaminopropyl)piperazine (MW = 564) MC798 1,4-bis(3-oleoylaminopropyl)piperazine (MW = 729.25) 0207. The particle size data indicates particles are present when both the polymer and the modified DNA are present. US 2003/023.5916 A1 Dec. 25, 2003

Example 28 Example 31 0215 Synthesis of 1(3-myristoylaminopropyl), 4-(3-ole 0222 Synthesis of 1,4-bis(3-thioctoylaminopropyl)pip oylaminopropyl)piperazine (MC765), 1,4-bis(3-myristoy erazine (MC777), 1(3-linolenoylaminopropyl), 4-(3-thio laminopropyl)piperazine (MC764), and 1,4-bis(3-oleoy ctoylaminopropyl)viperazine (MC778), 1,4-bis(3-linole laminopropyl)piperazine (MC798). noylaminopropyl)piperazine (MC779). 0223) To a solution of benzotriazole-1-yl-oxy-tris-pyrro 0216) To a 25 mL flame dried flask was added oleoyl lidino-phosphonium hexafluorophosphate (PyBOP, 1.300 g, chloride (freshly distilled, 1.0 ml, 3.0 mmol) and myristoyl 2.500 mmol, NovaBiochem) in dichloromethane (8 ml) was chloride (0.83 ml, 3.0 mmol, Aldrich Chemical Company) in added thioctic acid (0.248 g, 1.20 mmol, Aldrich Chemical 15 ml dichloromethane under N. The resulting solution was Company) and linolenic acid (365 ul, 1.20 mmol, Aldrich cooled to 0°C. in an ice bath. N,N-Diisopropylethylamine Chemical Company). To the resulting Solution was added 1,4-bis(3-aminopropyl)-piperazine (206 ul, 1.00 mmol) fol (1.1 ml, 6.1 mmol) was added followed by 1,4-bis(3- lowed by N,N-Diisopropylethylamine (610 ul, 3.5 mmol). aminopropyl)piperazine (0.50 ml, 2.4 mmol). The ice bath After 16 hrs at ambient temperature, the Solution was was removed and the Solution Stirred at ambient temperature washed with water (2x20 ml), and concentrated under for 15 hr. The solution was washed twice with 1N NaOH (10 reduced pressure to afford 1,800 g of crude material. A 85 ml), twice with water (10 ml), and concentrated under mg portion of the crude material was dissolved in 2 ml of reduced pressure. acetonitrile (0.1% trifluoroacetic acid)/1 ml of water (0.1% trifluoroacetic acid), and purified by reverse phase HPLC 0217 Approximatly 30% of the resulting residue was (10-90% B over 40 min) on a Beta Basic Cyano column to purified by semi-prerarative HPLC on a Beta Basic Cyano afford 31.8 mg MC777, 1.3 mg MC778, and 1.5 mg MC779. column with acetonitrile/HO/trifluoroacetic acid eluent. Three compounds were isolated from the column and Veri Example 32 fied by mass spectroScopy. 0224 Synthesis of 1,4-bis(3-(trans)-retinoylaminopropy l)piperazine (MC780), 1-(3-(cis-11)-eicosenoylaminopro pyl), 4-(3-(trans)-retinoylaminopropyl)piperazine (MC781), MC765 1-(3-myristoylaminopropyl),4-(3-oleoylaminopropyl) piperazine 1,4-bis(3-(cis-11)-eicosenoylaminopropyl)piperazine (MW = 674) (MC782). MC764 1,4-bis(3-myristoylaminopropyl)piperazine (MW = 620) MC798 1,4-bis(3-oleoylaminopropyl)piperazine (MW = 729.25) 0225 Compounds MC780, MC 781, and MC782 were made using a similar synthesis to compounds MC777, MC778, and MC779. The crude material from the synthesis was dissolved in 2 ml of acetonitrile (0.1% trifluoroacetic Example 29 acid)/1 ml of water (0.1% trifluoroacetic acid), and purified by reverse phase HPLC (10-90% B over 40 min) on a Beta 0218 Synthesis of 1,4-bis(3-decanoylaminopropyl)pip Basic Cyano column to afford 7.1 mg MC780, 13.0 mg erazine MC774. MC781, and 18.0 mg MC782. 0219. To a solution of 1,4-bis(3-aminopropyl)piperazine Example 33 (10 ul, 0.049 mmol, Aldrich Chemical Company) in dichlo 0226 Synthesis of bis(2-aminoethyl)-(2-oleoylaminoet romethane (1 ml) cooled to 0° C., was added decanoyl hyl)amine (MC753), 2-aminoethyl-bis-(2-oleoylaminoethy chloride (25 ul, 0.12 mmol, Aldrich Chemical Company) l)amine (MC754), and tris-(2-oleoylaminoethyl)amine and N,N-Diisopropylethylamine (21 ul, 0.12 mmol). After (MC755). 30 min, the Solution was allowed to warm to ambient 0227 Tris(2-aminoethyl)amine (0.2 mL, 1.4 mmol, Ald temperature. After 12 hrs, the Solution was washed with rich Chemical Company) as taken up in dichloromethane (3 water (2x2 ml), and concentrated under reduced pressure to mL, 0.5 M) and cooled in a dry ice/acetone bath. To the afford 1,4-bis(3-decanoylaminopropyl)piperazine (MC774) resulting solution was added oleoyl chloride (0.18 mL, 0.45 (21.6 mg, 87%) of sufficient purity by TLC. mmol, Aldrich Chemical Company), dropwise, while under a blanket of N. The reaction was warmed to ambient Example 30 temperature with Stirring. The reaction was stirred at rt for 1 hr and analyzed by TLC and mass spectrometry (Sciex API 0220 Synthesis of 1,4-bis(3-palmitoylaminopropyl)pip 150EX) to verify that the reaction was complete. The erazine (MC775). reaction was concentrated under reduced pressure to afford a beige film (330 mg). A portion of the material was purified 0221) To a solution of 1,4-bis(3-aminopropyl)piperazine by reverse phase HPLC on a Diphenyl column (Vydac), (10 ul, 0.049 mmol) in dichloromethane (1 mL), was added 40-90% B over 20 min (A=0.1% TFA/HO, B=0.1% TFA/ palmitoleic acid (30.8 mg, 0.12 mmol, Aldrich Chemical acetonitrile). Four HPLC runs were performed. Fractions Company), N,N-Diisopropylethylamine (21 uL, 0.12 were collected at 210 nm and analyzed by mass Spectrom mmol), and dicyclohexylcarbodiimide (25 mg, 0.12 mmol). etry (Sciex API 150EX). Fractions for MC753 (5,6,15-17, After 12 hrs, the Solution was filtered and washed with water 25-31,42-44), MC754 (7,18,19,32.33,45,46) and MC755(8- (2x2 mL), and concentrated under reduced pressure to afford 10,20-3.34-7.47-50) were pooled together, concentrated 1,4-bis(3-palmitoylaminopropyl)piperazine (MC775) (26.5 under reduced pressure, froze and lyophilized. Yielded mg, 81%) of Sufficient purity by TLC. MC753 (26 mg), MC754 (8.6 mg) and MC755 (150 mg). US 2003/023.5916 A1 Dec. 25, 2003

Example 34 diisopropylethylamine (0.02 mL, 17 mg, 0.13 mmol, Aldrich Chemical Company). The reaction Solution was cooled in an 0228 Synthesis of S-oleoyl-N-acetyl-L-cysteine-3-(dim ice bath (0° C.) and oleoyl chloride (39 mg, 0.13 mmol, ethylaminopropylamine)-amide (MC909). Aldrich Chemical Company) was added dropwise by 0229 Preparation of Di-(Dimethylamino)propylamino Syringe with Stirring. The reaction was allowed to warm to Cystine: To a flame dried round bottom flask (25 mL) was ambient temperature naturally. The reaction was analyzed by added N,N-Di-Boc-L-Cystine (500 mg, 1.1 mmol, Aldrich TLC and mass spectrometry (Sciex API 150EX) to verify Chemical Company) and taken up in THF (6 mL, 0.2 M) that the reaction was complete. The reaction Solution was with stirring. To the resulting solution was added N-hydrox precipitated in cold ether (5 mL), washed with ether (2x5 ySuccinimde (260 mg, 2.3 mmol, Aldrich Chemical Com mL), dried under N and placed on high vacuum. TLC and pany) and dicyclohexylcarbodiimide (520 mg, 2.5 mmol, mass spectrometry (Sciex API 150EX) showed sufficient Aldrich Chemical Company). After 2 min, 3-(Dimethylami purity. Yielded S-oleoyl-N-acetyl-L-cysteine-3-(dimethy no)propylamine (0.28 mL, 230 mg, 1.1 mmol, Aldrich laminopropylamine)-amide (MC909) as a yellow oil (45.3 Chemical Company) was added to the reaction mixture. The mg, 68%). reaction was stirred at rt for 30 min and analyzed by TLC and mass spectrometry (Sciex API 150EX) to verify that the Example 35 reaction was complete. The reaction was filtered to remove the DCU and concentrated under reduced pressure. The 0233 Preparation of S-oleoyl, N-acetvyl 3-(dimethy resulting resudue was precipitated in cold ether (10 mL) and laminopropylamine)-L-cysteine-amide (MC909) Hydro washed with ether (2x10 mL). The precipitate was dried chloride. under N2 then placed on high vacuum to afford di-(dimethy 0234 MC909 (9 mg, 0.018 mmol) was taken up in lamino)propylamino-di-Boc-cystine as a white powder (670 ethanol (0.09 mL) and added dropwise to cold HCl/ether (1 mg, 97%). mL of 1N, Aldrich Chemical Company). The precipitate was 0230 Di-(dimethylamino)propylamino-di-Boc-L-cystine spun down by centrifugation, washed with ether (2x1 mL), (670 mg, 1.1 mmol) was taken up in TIPS/HO/TFA (0.5/ dried under N and placed on high vacuum. Yielded white 4.5/95% vol., 5 mL, 0.2 M) covered with N and stirred at rt crystalline material (6 mg, 62%). for 1 hr. Deprotection of the material was verified by TLC and mass spectrometry (Sciex API 150EX). The material Example 36 was precipitated in cold ether (10 mL), washed in ether (2x10 mL), dried under N and placed under vacuum to 0235 Synthesis of B-D-Glucopyranosyl Decane Disul afford di-(dimethylamino)propylamino-cystine as a white fide and O-Glycine-f-D-Glucopyranosyl Decane Disulfide. powder (670 mg, 100%). (MC749). 0231. To a solution of di-(dimethylamino)propylamino 0236 To a solution of decanethiol (0.59 mL, 2.9 mmol, cystine (670 mg, 1.6 mmol) in 1-methyl-2-pyrrolidinone (8 Aldrich Chemical Company) chloroform (11 mL) was added mL, 0.2 M, VWR), was added diisopropylethylamine (0.58 sulfuryl chloride (0.46 mL, 5.7 mmol, Aldrich Chemical mL, 430 mg, 3.3 mmol, Aldrich Chemical Company) with Company), and the resulting mixture was stirred at room stirring. The reaction was cooled in an ice bath (0°C) and temperature for 18 hr. Removal of solvent (aspirator), acetic anhydride (1.5 mL, 1.7g, 16 mmol, Aldrich Chemical afforded decansulfenyl chloride. Company) was slowly added. The reaction mixture was 0237) To a solution of decansul?enyl chloride (190 mg, warmed to ambient temperature naturally and continued to 0.92 mmol) in 4 mL acetonitrile was added 1-thio-f-D- Stir for 1 hr. Amine capping was verified by TLC and mass glucose sodium salt hydrate (200 mg, 0.92 mmol, Aldrich spectrometry (Sciex API 150EX). The reaction solution was Chemical Company) and 15-crown-5 (0.18 mL, 0.899 added dropwise to cold ether (25 mL) and a white precipitate mmol, Aldrich Chemical Company). The resulting mixture formed. The precipitate was washed with ether (2x25 mL), was stirred at ambient temperature for 16 hr, filtered, and dried under N then placed under vacuum. Yielded a dark precipitated in EtO. The residue was triturated with EtO yellow oil (200 mg, 25%). and purified by reverse phase HPLC on an Aquasil C18 0232 The yellow oil (83 mg, 0.17 mmol) was taken up in column (Keystone Scientific Inc.), 10-90% B, 20 min THF (2 mL, 0.2 M) with stirring. Dithiol threitol (35 mg, (A=0.1% TFA in HO, B=0.1% TFA in Acetonitrile). Lyo 0.23 mmol, 0.1 M, Sigma Chemical Company) was added philization afforded 10 mg (3%) of B-D-glucopyranosyl with stirring. The reaction stirred at rt for 16 hr. The material decane disulfide as a fine white Solid. was analyzed by TLC and mass spectrometry (Sciex API 0238 To a solution of B-D-glucopyranosyl decane disul 150EX) to verify that the disulfide cleavage was complete. fide (8 mg, 0.02 mmol) in 80 uL THF was added N-Boc A portion of the material (54 mg) was purified by reverse glycine (15 mg, 0.09 mmol, Sigma Chemical Company), phase HPLC on an Aquasil C18 column (Keystone Scientific DCC (18 mg, 0.09 mmol, Aldrich Chemical Company), and Inc.), 10-40% B, 0-10 min, 4-60% B 10-20 min, 60-90% B a catalytic amount of dimethylaminopyridine (Aldrich 20-30 min (A=0.1% TFA in HO, B=0.1% TFA in acetoni Chemical Company). The resulting Solution was stirred at trile). Fractions were collected at 210 nm and analyzed by ambient temperature for 12 hr, and centrifugated to remove mass spectrometry (Sciex API 150EX). Fractions (3, 430, the Solid. The resulting Solution was concentrated under 31) that contained desired product from two HPLC runs reduced pressure, resuspended in dichloromethane, filtered were pooled together, concentrated under reduced pressure, through a plug of Silica gel, and concentrated (aspirator). froze and lyophilized. Afforded 32 mg (60%) as a clear oil. The Boc protecting group was removed by taking the The oil (32 mg, 0.13 mmol) was taken up with THF (1 mL, residue up in 200 uL of 2.5% TIPS/50% TFA/dichlo 0.13 M) with stirring. To the resulting solution was added romethane for 12 hr. Removal of solvent (aspirator), fol US 2003/023.5916 A1 Dec. 25, 2003 lowed by purification by reverse phase HPLC on a Aquasil (76 uL, 0.38 mmol, Aldrich Chemical Company) were C18 column (Keystone Scientific Inc.), 10-90% B, 20 min added. After 1 hr at ambient temperature the Solvent was (A=0.1% TFA in HO, B=0.1% TFA in Acetonitrile) removed (aspirator) and the residue was partitioned in afforded 0.7 mg (5%) of O-glycine-B-D-glucopyranosyl EtOAc/H2O. The organic layer was concentrated and the decane disulfide (MC749) as a fine white solid following resulting residue was purified by flash column chromatog lyophilization. raphy on silica gel (5-10% MeOH/0.1% TFA/dichlo romethane eluent) to afford 19 mg (8%) f-D-glucopyranosyl Example 37 N-dodecanoyl-cysteine-dodecanoate disulfide. 0239 Synthesis of B-D-Glucopyranosyl Cholesterol Dis 0245) To a solution of B-D-glucopyranosyl N-dode ulfide. canoyl-cysteine-dodecanoate disulfide (3.9 mg, 0.0045 mmol) in 100 uL dichloromethane was added N-Boc glycine 0240. By similar methodology as described in example (3.2 mg, 0.018 mmol, Sigma Chemical Company), DCC 36, B-D-glucopyranosyl cholesterol disulfide was isolated (3.8 mg, 0.018 mmol, Aldrich Chemical Company), and a (12% yield). catalytic amount of dimethylaminopyridine (Aldrich Chemi cal Company). The resulting Solution was stirred at ambient Example 38 temperature for 4 hr, and filtered. The Boc protecting group was removed by taking the residue up in 2 mL of 1% 0241 Synthesis of Two Tailed B-D-Glucopyranosyl Dis TIPS/50% TFA/dichloromethane for 2 hr. Removal of Sol ulfide Derivatives. B-D-Glucopyranosyl N-Dodecanoyl vent (aspirator), followed by purification by reverse phase Cysteine-Dodecanoate Disulfide and O-Glycine-B-D-Glu HPLC on a Diphenyl column (Vydaq), 20-90% B, 20 min copyranosyl N-Dodecanoyl-Cysteine-Dodecanoate (A=0.1% TFA in HO, B=0.1% TFA in Acetonitrile) Disulfide. afforded 3.6 mg (90%) of O-glycine-B-D-glucopyranosyl 0242) To a solution of N-FMOC-S-Trt-Cysteine (585 mg, decane disulfide as a fine white Solid following lyophiliza 1.0 mmol, NovaBioChem) in dichloromethane (4mL) was tion. added 1-dodecanol (240 mg, 1.3 mmol, Aldrich Chemical Company), DCC (260 mg, 1.3 mmol, Aldrich Chemical Example 39 Company), and a catalytic amount of dimethylaminopyri dine (Aldrich Chemical Company). The resulting solution 0246 General Preparation of Peptides. was stirred at ambient temperature for 30 min, filtered, and 0247 Peptides were prepaired by standard solid phase purified by flash chromatography on silica gel (10-20% peptide synthesis using an ABI433A Peptide Synthesizer EtOAc/hexane eluent). Removal of solvent (aspirator) (Applied BioSystems), employing FastMoc chemistry. Pep afforded 572 mg (76%) of the protected cysteine-dode tides were sysnthesized on the 0.1 or 1.0 mmol scale. Canoate. Deprotections and cleavage of the resin were accomplished 0243 To a solution of protected cysteine-dodecanoate utilizing Standard deprotection techniques. Peptides were (572 mg, 0.76 mmol) was added 3 mL of 20% piperidine in purified by reverse phase HPLC to at least a 90% purity DMF. The resulting solution was stirred at ambient tem level, and verified by mass spectroscopy (Sciex API perature for 1 hr., and partitioned in EtOAc/H2O. The 150EX). aqueous layer was extracted 2xEtOAc. The combined Example 40 organic layer was washed 2x1N HCl, dried (NaSO), and concentrated to afford S-Trt-cysteine-dodecanoate. The resi 0248 Synthesis of the Mixed disulfide of HN-CR due was Suspended in dichloromethane (2 mL), and cooled RRRRRRRR-OH (SEQ ID 11) and N-dodecanoyl-cys to -20° C. Diisopropylethylamine (0.16 mL, 0.92 mmol, teine-dodecanoate (MC756). Aldrich Chemical Company) was added followed dode 0249. The peptide HN-CRRRRRRRRR-OH canoyl chloride (0.26 mL, 1.1 mmol, Aldrich Chemical (LH168-100) (5.0 mg, 0.0033 mmol) was taken up with Company), and the Solution was allowed to slowly warm to isopropyl alcohol (0.5 mL) and trifluoroacetic acid (0.5 mL). ambient temperature. After 1 hr, the Solvent was removed To the resulting solution was added aldrithiol (0.79 mg, (aspirator), and the residue partitioned in EtOAC/HO. The 0.0036 mmol of 10 mg/mL isopropyl alcohol, Aldrich organic layer was washed 2x1 N HCl, 1.x brine, dried Chemical Company). The reaction was stirred at rt for 2 hr (NaSO), and the solvent was removed (aspirator). The and analyzed by mass spectrometry to verify (Sciex API resulting residue was suspended in 2% TIPS/50% TFA/ EX150) that product formed. The material was precipitated dichloromethane to remove the trityl protecting group. After in cold ether (2 mL) and washed with ether (2x2 mL) and 4 hr the Solution was concentrated, and the resulting residue dried under N. The dried precipitate was taken up with was purified by flash column chromatography on Silica gel isopropyl alcohol (0.25 mL) and trifluoroacetic acid (0.25 (10-20% EtOAc/hexanes eluent) to afford 180 mg (42%) mL). To the resulting solution was added N-dodecanoyl N-dodecanoyl-cysteine-dodecanoate (M--1=472.6). cysteine-dodecanoate (0.15 mL, 1.5 mg, of 10 mg/mL 0244. To a solution of N-dodecanoyl-cysteine-dode ethanol, 0.0033 mmol). The reaction was stirred at rt for 2 canoate (180 mg, 0.38 mmol) in 0.5 mL chloroform was hr and analyzed by mass spectrometry (Sciex API EX150) to added sulfuryl chloride (62 uL, 0.76 mmol, Aldrich Chemi verify that the product formed. The material was precipitated cal Company). The resulting Solution was stirred at ambient in cold diethyl ether (4 mL) and washed with diethyl temperature for 2 hr and the Solvent was removed (aspira ether(2x4 mL). The precipitate was purified by reverse tor). The resulting residue was Suspended in 1 mL acetoni phase HPLC on a Aquasil C18 column (Keystone Scientific trile, and 1-thio-f-D-glucose Sodium salt hydrate (85 mg, Inc), 5-90% B (A=0.1% TFA/HO, B=0.1% TFA/acetoni 0.39 mmol, Aldrich Chemical Company) and 15-crown-5 trile) over 20 min. Fractions were collected at 210 nm and US 2003/023.5916 A1 Dec. 25, 2003 20 analyzed by mass spectrometry (Sciex API EX150). Frac and stirred at rt for 1 hr. The reaction was monitored by TLC tions (9 and 18) were pooled together from the two HPLC and after 1 hr, the deprotection was not complete. TFA (1.5 runs, concentrated, froze and lyophilized to afforded MC756 mL) was added to the solution and after 10 min the solution (0.5 mg) as white fluffy crystalline material. turned from yellow to clear. The deprotected product was verified by TLC. The reaction was concentrated under Example 41 reduced pressure and yielded 3-thiol-propionic acid (3-(dim ethylamino)propylamine)amide, as a crystalline slurry. 0250 Preparation of 1 Decanethiochloride. 0251 1-Decanethiol (0.59 mL, 500 mg, 2.9 mmol, Ald Example 44 rich Chemical Company) was transferred to a flame dried round bottom flask and taken up with chloroform (11 mL, 0256 Synthesis of the Mixed disulfide of 3-thiol-propi 0.25 M). To the resulting solution was added sulfuryl onic acid (3-(dimethylamino)propylamine)amide and chloride (0.46 mL, 770 mg, 5.7 mmol, Aldrich Chemical decanethiol (MC744). Company) dropwise over 5 min with Stirring. The reaction 0257 3-Thiol-propionic acid (3-(dimethylamino)propy was stirred at rt under N for 16 hr. The reaction mixture was lamine)amide (82 mg, 0.43 mmol) was taken up in dichlo analyzed by TLC to verify that the reaction was complete. romethane (1.8 mL, 0.25 M). To the resulting solution was The reaction was concentrated under N2 then placed on the added 1-decanethiochloride (90 mg, 0.43 mmol) with stir speed vacuum. Material was stored at -20° C. until use. ring. The reaction was stirred at rt under N2 and instantly turned orange in color. After 20 min, the reaction mixture Example 42 was analyzed by TCL and mass spectrometry (Sciex API 150EX) to verify that the reaction was complete. The 0252 Trityl Protection of 3-Mercaptopropionic acid. material was stored at -20° C. for 16 hr. Crystals formed 0253 3-Mercaptopropionic acid (0.41 mL, 500 mg, 4.7 (PyBop urea) and were filtered and washed with dichlo mmol, Aldrich Chemical Company) was transferred to a romethane. The resulting Solution was concentrated under flame dried round bottom flask and taken up with dichlo reduced pressure and taken up with dichloromethane (1 mL) romethane (18 mL, 0.26 M). To the resulting solution was and purified by flash chromatography (10% MeOH/CH.Cl added diisopropylethylamine (0.82 mL, 610 mg, 4.7 mmol, eluent) and fractions were collected. The fractions were Aldrich Chemical Company) followed by the addition of analyzed by TLC and mass spectrometry (Sciex API trityl chloride (1.4g, 4.9 mmol, Aldrich Chemical Com 150EX). The desired product was in the final fraction and pany). The solution was stirred at rt under N for 16 hr. The yielded MC 744 (17 mg). reaction mixture was analyzed by TLC and mass spectrom etry (Sciex API 150EX) to verify that the reaction was Example 45 complete. The reaction mixture was concentrated under reduced pressure and yielded white crystalline material. The 0258 Preparation of 1-Dodecanethiolchloride. white crystalline material was slurried in EtOAc (50 mL) 0259 1-Dodecanethiol (0.59 mL, 500 mg, 2.5 mmol, and partitioned in HO (50 mL). The reaction was neutral Aldrich Chemical Company) was transferred to a flame ized with 0.1 M NaHCO. The organic layer was washed dried round bottom flask and taken up with chloroform (10 with HO (2x50 mL) and brine (50 mL). The organic layer mL, 0.25 M). To the resulting solution was added sulfuryl was transferred and concentrated under reduced pressure. chloride (0.40 mL, 670 mg, 4.9 mmol, Aldrich Chemical The crystals were dried on the high vacuum to afford Company) dropwise over 5 min with Stirring. The reaction trityl-mercaptopropionic acid (1.7 g) as white crystalline was stirred at rt under N for 3 hrs. The reaction mixture was material. The product was verified by mass spectrometry analyzed by TLC to verify that the reaction was complete. (Sciex API 150EX). The reaction was concentrated under N2 then on the Speed vacuum. Material was stored at -20° C. until use. Example 43 0254 Acylation of 3-(Dimethylamino)propylamine with Example 46 Trityl-mercaptopropionic acid. Synthesis of 3-thiol-propi 0260 Acylation of 3-(Dimethylamino)propylamine with onic acid (3-(dimethylamino)propylamine)amide. Trityl-mercaptopropionic acid. Synthesis of 3-thiol-propi 0255 Trityl-mercaptopropionic acid (300 mg, 0.86 onic acid (3-(dimethylamino)propylamine)amide. mmol) was taken up in dichloromethane (3.5 mL, 0.25M). 0261 Trityl-mercaptopropionic acid (500 mg, 1.4 mmol) To the resulting solution was added PyBOP (450 mg, 0.86 was taken up in dichloromethane (6 mL, 0.25 M). To the mmol, Novabiochem) and stirred at rt for 5 min. 3-(Dim resulting solution was added PyBOP (750 mg, 1.4 mmol, ethylamino)propylamine (0.11 mL, 0.86 mmol, Aldrich Novabiochem) and stirred at rt for 5 min. 3-(Dimethylami Chemical Company) was added with Stirring. The reaction no)propylamine (0.18 mL, 1.4 mmol, Aldrich Chemical mixture was stirred at rt under N for 16 hr. The reaction Company) was added with stirring. The reaction mixture mixture was analyzed by TLC and mass spectrometry (ScieX was stirred at rt under N for 16 hr. The reaction mixture was API 150EX) to verify that the reaction was complete. The analyzed by TLC and mass spectrometry (Sciex API 150EX) reaction mixture was concentrated under reduced pressure to Verify that the reaction was complete. The reaction and taken up in EtOAc (4 mL) and partitioned in HO (4 mixture was concentrated under reduced preSSure and taken mL), washed with HO (2x4 mL) and brine (4 mL). The up in EtOAc (20 mL) and partitioned in HO (20 mL), material was dried over Na2SO, filtered and concentrated washed with HO (2x20 mL) and brine (20 mL). The under reduced preSSure. The concentrated material was material was dried over Na2SO, filtered and concentrated taken up with TIPS/TFA/CHCl (2.5/47.5/50% vol, 3 mL) under reduced pressure. The concentrated material was US 2003/023.5916 A1 Dec. 25, 2003 taken up in dichloromethane (10 mL) and formed a white Example 49 precipitate. The material was spun down by centrifugation 0266 Preparation of Trimethylaminododecaneamine. and the precipitate was washed with dichloromethane (10 mL). The precipitated material was taken up with TIPS/ 0267 Boc-aminedodecaneamine (745 mg, 2.5 mmol) was taken up in acetonitrile (12 mL, 0.2M). To the resulting TFA/CHCl (2.5/50/47% vol, 6 mL) and stirred at rt for 1.5 solution was added diisopropylethylamine (0.43 mL, 370 hr. The mixture immediately turned from clear to bright mg, 2.5 mmol, Aldrich Chemical Company) and methyl yellow (release of the trityl cation) and back to clear. iodide (0.77 mL, 1.8 g., 12.4 mmol, Aldrich Chemical Deprotection of the material was verified by TLC and mass Company). The reaction was stirred under Nat rt for 16 hr. spectrometry (Sciex API 150EX). The reaction mixture was The reaction was analyzed by TLC and mass spectrometry concentrated under reduced pressure and yielded 3-thiol (Sciex API 150EX) to verify that the reaction was complete. propionic acid (3-(dimethylamino)propylamine)amide as a The reaction mixture was heated with a heat gun and white crystalline solid (245 mg, 91% yield). allowed to cool to ambient temperature naturally. Crystals formed and were analyzed by TLC and mass spectrometry Example 47 (Sciex API 150EX). The crystals were determined to be 1,12-dodecaneamine. The crystals were removed by filtra 0262 Synthesis of Synthesis of the Mixed Disulfide of tion and the Supernatant was concentrated under reduced 3-thiol-propionic acid (3-(dimethylamino)propylamine)am preSSure. The concentrated material was taken up with ide and dodecanethiol (MC745). TFA/HO/TIPS (95/4.5/1.5% vol, 10 mL) and stirred at rt under N. Complete deprotection was verified by TLC. The 0263 3-Thiol-propionic acid (3-(dimethylamino)propy reaction Solution was precipitated in cold ether (25 mL), lamine)amide (100 mg, 0.52 mmol) was taken up in dichlo spun by centrifugation and washed with ether (2x25 mL). romethane (2 mL, 0.25 M). To the resulting solution was The precipitate was dried under N2 then on high vacuum. added 1dodecanethiochloride (120 mg, 0.52 mmol) with Yielded white crystalline material (370 mg). The product Stirring. The reaction was stirred at rt under N2 and instantly was verified by TLC and mass spectrometry (Sciex API turned orange in color. After 1 hr, the reaction mixture was 150EX). analyzed by TLC and mass spectrometry (Sciex API 150EX) to Verify that the reaction was complete. The resulting Example 50 Solution was concentrated under reduced pressure, yielded a 0268 Boc Protection of imidazoleacetic acid. yellow oil (300 mg). The oil (160 mg) was taken up with dichloromethane (0.5 mL) and purified by flash chromatog 0269) 4-Imidazoleacetic hydrochloride (250 mg, 1.5 mmol, Aldrich Chemical Company) was taken up in aceto raphy (10% MeOH/CHCl, 0.1% TFA) and fractions were nitrile (5 mL, 0.3 M). To the resulting solution was added collected. The percentage of MeOH was slowly increased to triethylamine (0.22 mL, 160 mg, 1.5 mmol, Aldrich Chemi 20% and fractions were collected. The fractions were ana cal Company). Di-tert-butyl dicarbonate (400 mg, 1.8 mmol, lyzed by TLC and mass spectrometry (Sciex API 150EX). Aldrich Chemical Company) was added followed by a The desired product was in the final fraction. The final catalytic amount of 4-dimethylaminopyridine (Kodak fraction was concentrated under reduced pressure and Chemical Company). After 30 min, the reaction was ana yielded MC745 (22.4 mg, 22% yield) as a yellow oil. lyzed by TLC and mass spectrometry (Sciex API 150EX) to Verify that the reaction was complete. The reaction mixture Example 48 was concentrated under reduced preSSure, taken up in H2O (50 mL) and the pH was adjusted to pH 5 with NaOH (1N). 0264 Mono Protection of 1,12-Diaminododecane with The solution was partitioned with EtOAc (50 mL) and BOC. washed with brine (2x50 mL). The material was dried over 0265 1,12-Diaminododecane (1.0 g, 10 mmol amine, MgSO, filtered, concentrated under reduced pressure and Aldrich Chemical Company) was taken up with dichlo yielded an oil (82 mg, 23% yield). romethane (25 mL) and HO (25 mL). Sodium hydroxide (2 pellets, Fisher Scientific) were added and the reaction was Example 51 stirred vigorously. Di-tert-butyl dicarbonate (500 mg, 2.3 0270 Synthesis of Imidazoleacetic acid (trimethylamin mmol, Aldrich Chemical Company) was taken up in dichlo ododecaneamine)amide (MC933). romethane (10 mL) and added dropwise to the Stirring 0271 Trimethylaminododecaneamine (53 mg, 0.14 reaction over 5 min. After 1 hr, the reaction was analyzed by mmol) was taken up in DMF (0.7 mL, 0.2 M). To the TLC and mass spectrometry (Sciex API 150EX) to verify resulting Solution was added Boc-imidazoleacetic acid (32 that the reaction was complete. The reaction was transferred mg, 0.14 mmol) followed by dicyclohexylcarbodiimide (30 to a Separatory funnel and the layers were separated. The mg, 0.17 mmol, Aldrich Chemical Company). The reaction organic layer was concentrated under reduced pressure. Half was stirred at rt under N for 36 hr. The reaction mixture was of the concentrated material was brought up in hot acetoni cooled to -20° C. and the urea was removed via centrifu trile (100 mL), and crystallized. The material was spun down gation in the form of a pellet. The Supernatant was anlalyzed by centrifugation and washed with acetonitrile (2x25 mL). by TLC and mass spectrometry (Sciex API 150EX) to verify The material was dried on high Vacuum and yielded crys that it contained product. The Supernatant was concentrated talline material (745 mg, 99% yield). The product, Boc on the speed vacuum and taken up with TIPS/TFA/H2O aminedodecaneamine was verified by TLC and mass Spec (0.1/97.4/2.5% vol, 1.5 mL). The reaction was stirred at rt trometry (Sciex API 150EX). under N for 1 hr. The reaction was analyzed by TLC and US 2003/023.5916 A1 Dec. 25, 2003 22 mass spectrometry (Sciex API 150EX) to verify complete was stirred at rt for 30 min, concentrated on the Speed deprotection. The reaction mixture was added dropwise to vacuum afford N,N,N-trimethylaminopropyl-dimethylocta cold ether (10 mL) and formed a white precipitate. The decylsilazane (MC927) without further purification. The precipitate was washed with ether (2x10 mL), dried under resulting residue was taken up with DMF (100 uL, 20 N, then placed on high vacuum. Yielded crystalline material Aug?u). (39.2 mg). The material was taken up in acetonitrile (2 mL) with heat. Impurities crystallized and were filtered. The Example 55 mother liquor was analyzed by TLC and mass spectrometry (Sciex API 150EX) and verified to be product. The mother 0278 Preparation of the Mixed disulfide of thiopopionic liquor was concentrated under reduced preSSure and afford 3-dimethylaminopropanoate and decanethiol (MC746). MC933 as an oil (20 mg, 33% yield). 0279 To a solution of S-trityl-thiopropionic acid (0.36 g, 1.0 mmol, Aldrich Chemical Company) in dichloromethane Example 52 (4.0 mL) was added PyBOP (0.54g, 1.0 mmol, NovaBio Chem). The mixture was stirred at ambient temperature for 0272 Preparation of N,N,N-Trimethylaminopropy 5 min before the addition of dimethylaminopropanol (0.12 lamine. mL, 1.0 mmol, Aldrich Chemical Company). The reaction 0273 N-Boc-1,3-diaminopropane (0.25 mL,250 mg, 1.4 was continuously stirred at room temperature for 18 hr, and mmol, Fluka Chemical Company) was taken up with anhy concentrated under reduced preSSure. The residue was drous acetonitrile (7 mL, 0.2 M). To the resulting solution brought up in EtOAC and partitioned in H2O. The organic was added diisopropylethylamine, (0.25 mL, 190 mg, 1.4 layer was washed 2xH2O, 1x brine, dried (NaSO4), and the mmol Aldrich Chemical Company) followed by methyl Solvent removed (aspirator). The resulting residue was Sus iodide (0.31 mL, 720 mg, 5.0 mmol, Aldrich Chemical pended in 2% TIPS/50% TEA/CHCl (3 mL) to remove the Company) and the solution was stirred with heat (70C) trityl protecting group. After 2 hr the Solution was concen under N for 16 hr. The reaction mixture was analyzed by trated to afford thiopopionic-3-dimethylaminopropanoate. TLC and mass spectrometry (Sciex API 150EX) to verify 0280. To a solution of thiopopionic-3-dimethylaminopro that the reaction was complete. The reaction mixture was panoate (0.10g, 0.52 mmol) in dichloromethane (2 mL) was precipitated in cold ether (20 mL), washed with ether (2x20 added decanethiolchloride (0.11g, 0.52 mmol). The result mL) and dried under N. The precipitate was diisopropyl ing Solution was stirred at ambient temperature for 20 min. ethylamine Salt (174 mg). The ether layer was concentrated The Solvent was removed and a portion of the resulting under reduced pressure and taken up in TFA/HO/TIPS residue (25 mg) was purified by plug filtration on Silica gel (97.5/2/0.5% vol. 10 mL). The reaction mixture was stirred (10% MeOH/CHCl eluent) to afford 20.9 mg (84%) of the at rt under N for 16 hr. The reaction mixture was added mixed disulfide of decanethiol and thiopopionic-3dimethy dropwise to cold ether (20 mL) yielding a white precipitate. laminopropanoate (M+1=364.4). The precipitate was washed with ether (2x20 mL), dried under N and placed on high vacuum. Yielded light orange Example 56 hydroscopic solid (370 mg, 75% yield). The product, N.N., N-trimethylaminopropylamine was analyzed by TLC and 0281 Preparation of the Mixed disulfide of thiopopionic mass spectrometry (Sciex API 150EX). 3-dimethylaminopropanoate and dodecanethiol (MC747). 0282) To a solution of thiopopionic-3-dimethylaminopro Example 53 panoate (0.10g, 0.52 mmol) in dichloromethane (2 mL) was 0274 Synthesis of N,N,N-trimethylaminopropylamine added dodecanethiolchloride (0.11 g, 0.52 mmol). The resulting Solution was stirred at ambient temperature for 20 CDMNC12 (MC928). min. The Solvent was removed and a portion of the resulting 0275) To N.N.N-trimethylaminopropylamine (0.50 mg, residue (150 mg) was purified by flash column chromatog 25 uL of 20 tug/uL 75% THF/DMF, 4.3 umol), was added raphy on silica gel (1%TFA/10% MeOH/CHCl eluent) to diisopropylethylamine (0.55 mg, 5.5 till of 100 ug?u THF, afford 38 mg (25%) of the mixed disulfide of decanethiol Aldrich Chemical Company) and vortexed. To the resulting and thiopopionic-3-dimethylaminopropanoate (M-1= solution was added CDMNC12 (1.5 mg, 15 uL of 100 ug?uL 392.4). THF) and vortexed. The reaction stirred at rt for 30 min, concentrated on the speed vacuum to afford N,N,N-trim Example 57 ethylaminopropylamine-CDMNC12 (MC928) without fur ther purification. The resulting residue was taken up with 0283 Amidation of L-Lysine with Laurylamine. DMF (100 ul, 20 tug?u). 0284 Di-N-Boc-L-Lysine dicyclohexylammonium salt (1.0g, 1.9 mmol, Sigma Chemical Company) was taken up Example 54 with EtOAc (50 mL) and partitioned with HO (50 mL), washed with HCl (3x50 mL of 1N) and brine (1x50 mL). 0276 Synthesis of N,N,N-trimethylaminopropyl-dim The organic layer was dried over MgSO, filtered and ethyloctadecylsilazane (MC927). concentrated under reduced pressure. The material was 0277 To N,N,N-trimethylaminopropylamine (0.81 mg, verified by mass spectrometry (Sciex API 150EX) (M+1= 41 uL of 20 tug/uL 75% THF/DMF, 7.0 umol), was added 347.7). The concentrated material was taken up with dichlo diisopropyldiethylamine (0.90 mg, 9.0 uL of 100 ug?uL romethane (10 mL). To the resulting solution was added THF, Aldrich Chemical Company). To the resulting solution dicyclohexylcarbodiimide (0.78 g., 3.8 mmol, Aldrich was added chlorodimethyloctadecylsilane (1.4 mg, 14 till of Chemical Company) and laurylamine (0.4 g, 2.2 mmol, 100 ug?ul THF, Aldrich Chemical Company). The reaction Aldrich Chemical Company). The reaction was covered US 2003/023.5916 A1 Dec. 25, 2003

with a blanket of N and stirred at rt for 1 hr. The reaction and reconstituted in HO (2 mL, 5 mg/mL). Material (5 mg, was analyzed by TLC to verify that the reaction was 1 mL) was loaded onto a Sephadex G50 column, eluted with complete. The reaction mixture was filtered to remove the HO and collected fractions (1 mL). The fractions were DCU and concentrated under reduced pressure. The con analyzed by fluorescence (Varian Cary Eclipse spectrofluo centrated material was taken up with 5% MeOH/CHCl (1 rometer, Ex 495 nm and Em 530 nm). Fractions 24-37 were mL) and ran on a silica gel column with 5% MeOH/CH.Cl pooled together, froze and lyophilized. Yielded fluffy yel (100 mL). The fraction was concentrated under reduced low/orange material (5.0 mg) and was taken up in H2O (0.5 pressure and yielded an oil (410 mg). The oil was taken up mL, 10 mg/mL). with TFA/CH.Cl/TIPS (5 mL/2 mL/0.05 mL) and stirred at rt for 1 hr under N. The reaction was analyzed by TLC to Example 60 Verify that the reaction was complete. The material was concentrated under reduced pressure and taken up with 0290 Preparation of Rh-DNA-NC-PyrDet. acetonitrile (1 mL) and precipitated in H2O. The precipitate 0291 Rh-DNA (100 pg, 50 uL of 2 ug?u L HO solution, was dried under vacuum to afford the laurylamine amide of 0.30 umol phosphate) was taken up in HO (50 u , 1 lug/ull L-lysine ad off white crystals (77 mg). pIDNA) and gently mixed. In a separate tube, dodecylamine hydrochloride (13 ug, 13 uL of 10 uguL HO solution, 0.61 Example 58 umol) and PyrDet (9.5 lug, 9.5 uL of 1 tug/ull THF, 0.015 0285 Preparation of Pyrenedodecanoic-3-Trimethylami almol) were mixed together. The detergents were added to nopropylamine Carboxamide Iodide. the Rh-DNA solution and gently mixed. The material pre cipitated and was spun down by centrifugation. The Super 0286 Pyrenedodecanoic acid (10 mg, 0.025 mmol, natant was removed and the pellet was washed with HO Molecular Probes) was taken up in THF (0.13 mL, 0.2 M). (2x100 uL). The pellet was lyophilized over POs for 72 hr To the resulting Solution was added 3-dimethylaminopropy and reconstituted in THF (100 uL, 1 lug?u). lamine (0.004 mL, 3.2 mg, 0.031 mmol, Aldrich Chemical Company) followed by the addition of dicyclohexylcarbo Example 61 diimide (6.4 mg., 0.031 mmol, Aldrich Chemical Company). The reactions were stirred at rt under N protected from light 0292 Ultracentrifugation of Hydrophobic pl)NA Com for 16 hr. The reaction mixture was analyzed by TLC and plexes. mass spectrometry (Sciex API 150EX) to verify that the reaction was complete. The reaction mixture was concen trated under reduced pressure, taken up in EtOAc (0.5 mL), partitioned in HO (0.5 mL), washed with HC1 (2x0.5 mL Sample Amt (ug) HO (uL) of 1N) and brine (0.5 mL). The organic layer was concen Rh-DNA 50 1OOO trated under reduced pressure to afford pyrenedodecanoic Rh-DNA-NC-PyrDet 50 1OOO 3-dimethylaminopropylamine carboxamide. Rh-DNA/FITC-Chit-O1 50/105 1OOO PyrDet 4.7 1OOO 0287 Pyrenedodecanoic-3-dimethylaminopropylamine Rh-DNA-NC-PyrDet/FITC-Chit-O1 50/105 1OOO carboxamide was taken up in acetonitrile (0.1 mL, 0.25M). FITC-Chit-O1 105 1OOO To the resulting Solution was added a catalytic amount of KCO, followed by methyl iodide (0.03 mL, 7.1 mg, 0.050 mmol, Aldrich Chemical Company). The reaction mixture 0293) To HO (1000 uL) was added either polymer or was covered with N and heated (55C.) with stirring for 16 detergent and Vortexed. To the resulting Solution was added hr. The reaction mixture was analyzed by TLC and mass RhDNA or Rh-DNA-NC-PyrDet (50 ug) and vortexed. spectrometry (Sciex API 150EX) to verify that the reaction The samples were loaded into tubeS prepared with a Sucrose was complete. The reaction was concentrated under reduced gradient (18-54%, 10 mM Hepes, 1 mM EDTA, 9.5 mL) and preSSure. The concentrated material was taken up in dichlo trizamide as the base (cushion). The samples were centrifu romethane (1 mL), filtered through a plug of Silica (6 cm, gated at 35 krpm at 4C. under vacuum for 20 hr. Each sample 10% MeOH/CHCl (50 mL) eluent). The solution was was separated into 750 till fractions, fractionating from the concentrated under reduced pressure, to afford the pyrene bottom of the tube to the top. To each fraction was added dodecanoic-3-trimethylaminopropylamine carboxamide NaCl (150 uL, of 5 M, 1 M final) and Triton (37.5 ul of 20%, 1% final) with vortexing. The fractions were analyzed iodide an oil (8.7 mg, 56% yield). on a spectrofluorometer (Varian Cary Eclipse) at the Example 59 rhodamine (Ex 555 nm and Em 585 nm), FITC (Ex 495 nm and Em 530 nm), and pyrene (EX 340nm and Em 377 nm) 0288 Preparation of FITC-Chit-Ol. channels. 0289. To Chit-O1 (10 mg, 10 mL of 1 mg/mL in 80% 0294 Rhodamine fluorescence results (FIG. 1) indicate DMF solution, 0.026 mmolamine) was added NaCO (10 that both Rh-DNA-NC-PyrDet and Rh-DNA/FITC-Chit mL of 0.1M) to raise the pH to 9. Fluorescein isothiocyanate Ol form particles/aggregates based on the observed fluores (2 mg, 0.2 mL of 10 mg/mL DMF solution, 0.0053 mmol, cence of the densest fractions (fractions 12-14). FITC fluo Aldrich Chemical Company) was added to the solution. The rescence results indicates that most of the FITC-Chit-Ol reaction was stirred at rt and protected from light for 16 hr. remains in the least dense fractions (top fractions). A Small The reaction was analyzed by TLC to verify that the reaction amount of FITC fluorescence is observed in fraction 7 for was complete. The reaction was concentrated under reduced the complex (Rh-DNA-NC-PyrDet/FITC-Chit-Ol) and pressure, precipitated in cold ether (10 mL) and washed with overlaps with the rhodamine fluorescence complex and thus ether (2x10 mL). The material was dried on the high vacuum indicates an interaction between the Rh-DNA-NC-PyrDet US 2003/023.5916 A1 Dec. 25, 2003 24 with the FITC-Chit-Ol. Pyrene fluorescence results indicate ug?u. DMF solution, 0.45 umol) and vortexed. To the that all pyrene detergent remains in the top fractions. Inten resulting solution was added DMF (12 u.L., 2 ug?uL plDNA). sities for the Rh-DNA-NC-PyrDet and Rh-DNA-NC PyrDet/FITC-Chit-Ol are most likely lower as a result of Example 68 adding PyrDet during formulation of Rh-DNA-NC-Pyr Det. If the PyrDet was not incorporated into the Rh-DNA 0307 Preparation of pDNA-MC933 (1:1). NC particle, it would have been washed out during the HO washes during formulation. This would explain the 0308 plDNA (pCI Luc, 50 lug, 25uL of 2 tug/ull solution, observed lower fluorescence intensity. 0.15 umol phosphate) was taken up in HO (25 uL) and vortexed. To the resulting solution was added MC933 (7 uL, Example 62 70 ug of 10 ug?ul HO Solution pH 8, 0.15umol) and gently mixed. The Solution was froze and dried for 16 hr on the 0295 Preparation of Geranylamine Hydrochloride. Speed vacuum. 0296 Geranlyamine (0.6 mL, 500 mg, 3.3 mmol, Aldrich Chemical Company) was taken up with methanol (0.4 mL) Example 69 and gently mixed with Vortexing. The Solution was added dropwise to cold HCl/ether (5.5 mL of 1M, Aldrich Chemi 0309 Preparation of pDNA-MC933 (1:2). cal Company) and formed a white precipitate. The precipi tate was spun down under centrifugation and washed with 0310 plDNA(pCI Luc, 50 lug, 25 uLof 2 ug?u LHO, 0.15 ether (2x6 mL). The material was dried under N then on almol phosphate) was taken up in H2O (25 uD) and Vortexed. high vacuum. Yielded white crystalline material (550 mg, To the resulting solution was added MC933 (15uL, 150 lug of 10 ug?u.L HO solution pH 8, 0.30 umol) and gently 89%) mixed. The Solution was froze and dried for 16 hr on the Example 63 Speed vacuum. 0297 Preparation of pDNA-CPB. 0311. By a similar procedure as described for MC933 0298 pDNA (pCI Luc, 50 uL,100 lug of 2 ug?ul HO, (1:2) the following preparations were formed: plDNA-NCs, 0.30 umol phosphate) was added cetylpyridinium bromide pDNA-NC, plDNA-CHAPS, plDNA-Lys-NC12, plDNA (24.4 u , 244 ug of 10 ug?u L H2O, 0.61 umol, Aldrich MC753, plDNA-MC754, plDNA-MC744, plDNA-MC745, Chemical Company) and gently mixed. The precipitate was pDNA-MC746, plDNA-MC747, plDNA-MC909. Spun down by centrifugation and lyophilized over P-Os. Example 70 Example 64 0312 Preparation of pDNA-MC933 (1:3). 0299 Preparation of pDNA-NC (1:1). 0313 plDNA (pCI Luc, 50 lug, 25uL of 2 tug/ull solution, 0300. In triplicate, pIDNA (pCI Luc, 50 lug, 25 uL of 2 0.15 umol phosphate) was taken up in HO (25 uL) and Aug?u LH2O, 0.15 umol phosphate, pMIR48) was taken up in vortexed. To the resulting solution was added MC933 (220 HO (25 uL) and vortexed. To the resulting solution was AiL, 22 ug of 10 ug?u L HO Solution pH 8, 0.45 umol) and added NC HCl (1.4 uL, 14 lug of 10 ug?ul HO solution, gently mixed. The solution was frozen and dried 16 hr on the 0.15 umol, Aldrich Chemical Company) and gently mixed. Speed vacuum. The solution was froze and dried for 16 hr on the speed WCUU. Example 71 Example 65 0314 Preparation of Ol-Mel-CDM. 0301 Preparation of pDNA-MC927 (1:1). 0315 Ol-Mel (40 uL, 400 ug of 10 ug?u LDMF solution, 0302) The dried material, plDNA-NC (50 ug, 0.15umol 0.52 umol amine) was added diisopropylethylamine (1.8 ul, phosphate) was taken up with MC927 (4 uL, 40 tug of 10 18 tug of 10 ug?u LDMF solution, 0.14tuol, Aldrich Chemi ug/ull DMF solution, 0.15 umol) and vortexed. To the cal Company) and vortexed. CDM (2.6 uL, 26 lug of 10 resulting solution was added DMF (21 uL, 2 ug/ull plDNA). ug?u. DMF solution, 0.14 umol) was added and vortexed. The reaction mixture was vortexed at rt for 30 min. Final Example 66 concentration was at 10 ug?u L Ol-Mel. 0303 Preparation of pDNA-MC927 (1:2). Example 72 0304) The dried material, plDNA-NC (50 ug, 0.15umol phosphate) was taken up with MC927 (9 uL, 90 tug of 10 0316 Preparation of Ol-Mel-CDM(2.5). ug/ull DMF solution, 0.30 umol) and vortexed. To the resulting solution was added DMF (16 uL, 2 ug/ull plDNA). 0317 Ol-Mel (25 uL, 250 ug of 10 ug?u LDMF solution, 0.32 umol amine) was added diisopropylethylamine (1.1 u, Example 67 11 tug of 10 ug?u L DMF solution, 0.085 utol, Aldrich Chemical Company) and vortexed. CDM (3.7 uL, 37 lug of 0305 Preparation of pDNA-MC927 (1:3). 10 ug/ull DMF solution, 0.20 umol) was added and vor 0306 The dried material, plDNA-NC (50 ug, 0.15umol texed. The reaction mixture was vortexed at rt for 30 min. phosphate) was taken up with MC927 (13 uL, 130 ug of 10 Final concentration was at 8.4 tug/ul Ol-Mel. US 2003/023.5916 A1 Dec. 25, 2003

Example 73 0318) Hepa Cell Transfection. -continued 0319) 10% FBS and 24 hr Harvest (starting confluency # Complex wt Ratio (ug) RLU Mean 55%). Seventeen Samples were formulated as follows: 8 pDNA+ O1-Mel-CDM 1/5 76,320 9 pDNA+ O1-Mel-CDM 1/10 637,307 0320 1) To Opti (300 ul) was added plDNA (pCI 10 pDNA-MC927(1:1) + O1-Mel-CDM 1/5 56,349 Luc, 1.5 till, 3 ug of 2 ug?u, H2O) and Vortexed. Lt-1 11 pDNA-MC927(1:2) + O1-Mel-CDM 1/5 81,021 (9 uL, Mirus Corporation) was added and vortexed. 12 pDNA-MC927(1:1) + O1-Mel-CDM 1/10 9,007 13 pDNA-MC927(1:2) + O1-Mel-CDM 1/10 29,503 0321) 2-7) To HO (300 ul) was added plDNA 14 pDNA-MC933(1:1) + O1-Mel-CDM 1/5 149,091 15 pDNA-MC933(1:2) + O1-Mel-CDM 1/5 594,391 MC927 formulations (pCI Luc, 1.5 uL, 3 ug of 2 16 pDNA-MC933(1:1) + O1-Mel-CDM 1/10 213,776 ug/ull DMF solution) and vortexed. 17 pDNA-MC933(1:2) + O1-Mel-CDM 1/10 98.898 0322 8) To HO (300 u) was added plDNA (pCI Luc, 1.5 ul, 3 ug of 2 ug?u L H2O) and Vortexed. 0330 Results indicate that the pDNA/cationic surfactant Ol-Mel-CDM (1.5 uL, 15 lug of 10 ug?u. DMF complex is expressible in vitro when interacted with the Solution) was added and Vortexed. ol-mel derivatives. 0323 9) To HO (300 u) was added plDNA (pCI Luc, 1.5 ul, 3 ug of 2 ug?u L HO) and Vortexed. Example 74 Ol-Mel-CDM (3 uL, 30 tug of 10 ug/ull DMF solu tion) was added and Vortexed. 0331 Hepa Cell Transfection. 0324 10-11) To HO (300 uL) was added plDNA 0332) 10% FBS, 24 hr harvest. MC927 (pCI Luc, 1.5 ul, 3 ug of 2 ug?u. DMF 0333 Twenty-three samples were formulated for cell solution) and vortexed. Ol-Mel-CDM (1.5ull, 15ug transfections. of 10 ug/ull DMF solution) was added and vortexed. 0334) 1) To Opti (200 uL) was added plDNA (pCI 0325 12-13) To HO (300 uL) was added plDNA Luc, 1 u , 2 ug of 2 ug/ull H2O) and the Solution was MC927 (pCI Luc, 1.5 ul, 3 ug of 2 ug?u. DMF vortexed. Lt-1 (6 ul, Mirus Corporation) was added solution) and vortexed. Ol-Mel-CDM (3 uL, 30 ug of and the Solution was again Vortexed. 10 ug/uL DMF solution) was added and vortexed. 0335 2-25) To HO (200 uL) was added plDNA or 0326) 14-15) To HO (300 uL) was added plDNA pIDNA/cationic Surfactant complex (pCI Luc, 1 u , 2 MC933 (pCI Luc, 1.5 ul, 3 ug of 2 ug?u. DMF Aug of 2 ug?u L DMF) and the Solution was vortexed. solution) and vortexed. Ol-Mel-CDM (1.5ull, 15ug Ol-Mel or Ol-Mel-CDM was added and the Solution of 10 ug/ull DMF solution) was added and vortexed. was again Vortexed. 0327) 16-17) To HO (300 uL) was added plDNA 0336 Hepa cells were maintained in DMEM. Approxi MC933 (pCI Luc, 1.5 ul, 3 ug of 2 ug?u. DMF mately 24 h prior to transfection, cells were plated at an solution) and vortexed. Ol-Mel-CDM (3 uL, 30 ug of appropriate density in 48-well plates and incubated over 10 ug?uL DMF solution) was added and vortexed. night. Cultures were maintained in a humidified atmosphere containing 5% CO2 at 37 C. The cells were transfected at 0328 Hepa cells were maintained in DMEM. Approxi a starting confluency of 73% by combining 100 lull sample mately 24 h prior to transfection, cells were plated at an (1 ug plDNA per well) with the cells in 1 mL of media. Cells appropriate density in 48-well plates and incubated over were harvested after 24 h and assayed for luciferase activity night. Cultures were maintained in a humidified atmosphere using a Lumat LB 9507 (EG&G Berthold, Bad-Wildbad, containing 5% CO2 at 37 C. The cells were transfected at Germany) luminometer. The amount of luciferase expres a starting confluency of 73% by combining 100 lull sample Sion was recorded in relative light units. Numbers are the (1 ug plDNA per well) with the cells in 1 mL of media. Cells average for two separate wells. were harvested after 24 h and assayed for luciferase activity using a Lumat LB 9507 (EG&G Berthold, Bad-Wildbad, Germany) luminometer. The amount of luciferase expres Sion was recorded in relative light units. Numbers are the wt Ratio average for two separate Wells. # Complex (ug) RLU Mean 1 ploNA + Lt-1 1/3 uL 18,598,949 0329 Hepa Cell Transfection Results: 2 pDNA + O1-Mel 1/5 22,822 3 pDNA + O1-Mel 1/10 68,954 4 pDNA + O1-Mel 1/15 2,686 5 pDNA-MC933(1:2) + O1-Mel 1/5 481,520 # Complex wt Ratio (ug) RLU Mean 6 pDNA-MC933(1:2) + O1-Mel 1/10 193,520 7 pDNA-MC933(1:2) + O1-Mel 1/15 4,876 1 ploNA + Lt-1 1/3 uL 16,024,685 8 pDNA + O1-Mel-CDM 1/5 12,917 2 pDNA-MC927 (1:1) 1. 103 9 pDNA + O1-Mel-CDM 1/10 119,281 3 pDNA-MC927 (1:2) 1. 96 10 pDNA+ O1-Mel-CDM 1/15 12,310 4 pDNA-MC927 (1:3) 1. 81 11 pDNA-MC927(1:2) + O1-Mel-CDM 1/5 64,128 5 pDNA-MC933 (1:1) 1. 91 12 pDNA-MC927(1:2) + O1-Mel-CDM 1/10 86,395 6 pDNA-MC933 (1:2) 1. 77 13 pDNA-MC927(1:2) + O1-Mel-CDM 1/15 1,409 7 pDNA-MC933 (1:3) 1. 121 14 pDNA-MC933(1:2) + O1-Mel-CDM 1/5 424,049 US 2003/023.5916 A1 Dec. 25, 2003 26

Example 77 -continued 0342 Mouse Tail Vein Injections. wt Ratio # Complex (ug) RLU Mean 0343 HP Tail Vein Dual Luciferase: siRNAdelivery Five complexes were prepared as follows: 15 pDNA-MC933(1:2) + O1-Mel-CDM 1/10 298,644 16 pDNA-MC933(1:2) + O1-Mel-CDM 1/15 9,358 0344) Complex I-V: To Ringers (10 mL, 1x) was 17 pDNA+ O1-Mel-CDM(2.5) 1/5 20,570 18 pDNA+ O1-Mel-CDM(2.5) 1/10 78,689 added pMIR116 (pCI Luc, 40 ug, 20 uL of 2 uguL 19 pDNA+ O1-Mel-CDM(2.5) 1/15 9,137 HO) and vortexed. pMIR122 (pCI Ren, 4 ug, 2 ul 20 pDNA-MC927(1:2) + O1-Mel-CDM(2.5) 1/5 69,485 of 2 ug/ull H2O) was added to the Solution and 21 pDNA-MC927(1:2) + O1-Mel-CDM(2.5) 1/10 111,287 vortexed. 22 pDNA-MC927(1:2) + O1-Mel-CDM(2.5) 1/15 14,218 23 pDNA-MC933(1:2) + O1-Mel-CDM(2.5) 1/5 722,841 0345 Complex II: To the plasmid solution was 24 pDNA-MC933(1:2) + O1-Mel-CDM(2.5) 1/10 129,665 added GL3-153 (20 ug, 1.5 u1L of 13.3 tug/ull 10 25 pDNA-MC933(1:2) + O1-Mel-CDM(2.5) 1/15 5,611 mM NaCl, Dharmacon) and vortexed. 0337 Results indicate that the pDNA/cationic surfactant 0346 Complex III: To the plasmid solution was complex is expressible in vitro when interacted with the added EGFP-64 (20 ug, 1.5ull of 13.4 ug/ull 10 mM modified peptide derivatives. NaCl, Dharmacon) and vortexed. 0347 Complex IV: To the plasmid solution was Example 75 added GL3-153-NC (20 ug, 10 ul of 2 uguL 0338 Preparation of GL3-153-NC.. DMF) and vortexed. 0339) To HO (46.2 uL) was added GL3-153 (3.8 uL, 50 0348 Complex V: To the plasmid solution was lug of 13.3 tug/ull 10 mM NaCl, 0.16 umol phosphate, added EGFP-64-NC (20 ug, 10 uIl of 2 uguL 2'OH-CWUACG CUGAGUACU UCGAdTdT (SEQ ID DMF) and vortexed. 12) and its compliment 2'OH-UCG AAGUACUCA GCG UAA GdTdT (SEQ ID 13), Dharmacon) and vortexed. To 0349 Tail vein injections of 1.0 mL per 10 g body weight the resulting solution was added dodecylamine HCl (7 uL, (-2.5 ml) were preformed on ICR mice (n=2) using a 30 70ug, of 10 ug?u, HO, 0.31 umol) and gently mixed. The gauge, 0.5 inch needle. Injections were done manually with reaction mixture was incubated at rt for 30 min, froze and injection times of 4-5 sec Zhang et al. 1999; Liu et al. lyophilized over POs 16 hr. The dried material was recon 1999). One day after injection, the livers were harvested and stituted in DMF (25 uL, 2 ug/ull GL3-153) homogenized in lysis buffer (0.1% Triton X-100, 0.1 M K-phosphate, 1 mM DTT, pH 7.8). Insoluble material was Example 76 cleared by centrifugation and 10 ul of the cellular extract or 0340 Preparation of EGFP-64-NC. extract diluted 10x was analyzed for luciferase activity as 0341) To HO (46.3 uL) was added EGFP-64 (3.7 uL, 50 previously reported Wolfetal 1990). lug of 13.4 ug?u L 10 mM NaCl, 0.16 umol phosphate, 0350 Results:

Complex # Formulation Luc RLU Ren RLU Lucf Ren Complex I pMIR116/pMIR122 12,986,650 42,206,060 31 Complex I pMIR116/pMIR122 56,986,540 153,988,270 37 Complex II pMIR116/pMIR122/GL3-153 16,886,310 62,075,320 27 Complex II pMIR116/pMIR122/GL3-153 12,171,210 165,623,300 7 Complex III pMIR116/pMIR122/EGFP-64 1,730,890 41,515,930 4 Complex III pMIR116/pMIR122/EGFP-64 6,513,560 128,718,130 5 Complex IV pMIR116/pMIR122/GL3-153- 49,854,070 143,555,100 35 NC12 Complex IV pMIR116/pMIR122/GL3-153- 13,225,000 26,978,760 49 NC12 Complex V pMIR116/pMIR122/EGFP-64- 22,132,210 57,071,140 39 NC12 Complex V pMIR116/pMIR122/EGFP-64- 6,517,230 113,748,060 6 NC12

2'OH-GAC GUAAACGGC CACAAG UGC AdTdT (SEQ 0351 Results indicate that the hydrophobic siRNA (GL3 ID 14) and its compliment 2'OH-CG CUG CAU UUG 153) inhibits luciferase expression. CCG GUG UUC A GdTdT (SEQ ID 15, Dharmacon) and Vortexed. To the resulting Solution was added dodecylamine Example 78 HCl (7 ul, 70 ug, of 10 ug?ul HO, 0.31 umol) and gently mixed. The reaction mixture was incubated at rt for 30 min, 0352 Preparation of CDM-Mel-Si(Me) Cs. froze and lyophilized over POs 16 hr. The dried material 0353 Melittin (15 uL, 300 lug, of 20 tug/ul DMF, 0.11 was reconstituted in DMF (25 uL, 2 tug/uL EGFP-64) umol) was taken up in DMF (2.5 uL) and vortexed. To the US 2003/023.5916 A1 Dec. 25, 2003 27 resulting Solution was added diisopropylethylamine (1.5 ul, 0364 Complex V: To HO (9.7 mL) was added 15 lug of 10 ug?u L DMF, 0.11 limol, Aldrich Chemical pDNA-NC (pCI Luc, 20 uL, 40 ug of 2 ug/uLDMF) Company) and vortexed. Chlorodimethyloctadecylsilane (4 and Vortexed. till, 40 tug of 10 ug?u THF, 0.11 limol, Aldrich Chemical 0365 Complex VI: To HO (9.7 mL) was added Company) was added to the solution and vortexed. The pDNA (pCI Luc, 20 u, 40 tug of 2 ug?ul HO) and reaction was heated (70° C.) for 5 min and diisopropylethy vortexed. NaCl (300 uL of 5 M, 150 mM final) was lamine (2 uL, 20 tug of 10 ug?u. DMF, 0.16 umol, Aldrich added to each complex prior to injection. Tail vein Chemical Company) was added and vortexed. CDM (5uL, injections of 1.0 mL per 10 g body weight (-2.5 ml) 50 ug of 10 ug?u. DMF, 0.27 umol) was added and vortexed. were preformed on ICR mice (n=2) using a 30 gauge, The reaction mixture was mixed with shaking for 30 min at 0.5 inch needle. Injections were done manually with rt. The resulting mixture was used without purification. injection times of 4-5 sec Zhang et al. 1999; Liu et Example 79 al. 1999). One day after injection, the livers were harvested and homogenized in lysis buffer (0.1% 0354) Preparation of CDM-Mel. Triton X-100, 0.1 M K-phosphate, 1 mM DTT, pH 0355 Melittin (15 uL, 300 lug, of 20 tug/uL DMF, 0.11 7.8). Insoluble material was cleared by centrifuga umol) was taken up in DMF (6.5 uL) and vortexed. To the tion and 10 ul of the cellular extract or extract diluted resulting Solution was added diisopropylethylamine (3.5 ul, 10x was analyzed for luciferase activity as previ 35 lug of 10 ug?u. DMF, 0.27 umol, Aldrich Chemical ously reported Wolff et al 1990). Company) and vortexed. CDM (5 uL, 50 tug of 10 ug?ul 0366 Results: Mean RLU (n=3) DMF, 0.27 umol) was added and vortexed. The reaction mixture was mixed with shaking for 30 min at rt. The resulting mixture was used without purification. Liver 100x Example 80 Complex # dilution Spleen Lung Heart Kidney 0356. Preparation of CDM-Mel-CDMNC12. Complex I 3.268,842 704,378 1553,333 216,703 471,391 Complex II 1,395,436 144,766 176,723 37,294 757,639 0357 Melittin (1582 L, 300 ug, of 20 tug/uL DMF, 0.11 Complex III 6,000,523 646,471 843,741 115,154 579,530 Complex IV 791,962 197,011 78.403 26,309 74,368 umol) was taken up in DMF (3.5 uL) and vortexed. To the Complex V 2,425,812 125,634 201,128 65,457 250,216 resulting Solution was added diisopropylethylamine (1.5 ul, Complex VI 1,753,888 140,723 211857 209,652 599,696 15 lug of 10 ug?u L DMF, 0.11 limol, Aldrich Chemical Company) and vortexed. CDMNC12 (4 uL, 40 ug of 10 ug/ull DMF, 0.11 umol) was added and vortexed. The reaction mixture was mixed with shaking for 30 min at rt. To Example 82 the reaction mixture was added diisopropylethylamine (2 0367 Low Pressure Tail Vein Mouse. till, 20 tug of 10 ug?u. DMF, 0.16 umol, Aldrich Chemical Company) and vortexed. CDM (5 uL, 50 tug of 10 ug?ul 0368 Four complexes were made as follows: DMF, 0.27 umol) was added to the reaction and vortexed. 0369 Complex I: To HO (600 uL) was added The reaction was mixed with shaking for 30 min at rt. The pDNA-NC (pCI Luc, 15 uL, 30 ug of 2 ug/uLDMF) resulting mixture was used without purification. was added and Vortexed. Immediately before injec tion, CDM-Mel-SiMe2Cs (11.2 u , 112 lug of 10 Example 81 ug/ull DMF) was added and vortexed. 0358 Mouse Tail Vein Injections HP. 0370 Complex II: To HO (600 uL) was added 0359 Six complexes were made as follows: pDNA-NC (pCI Luc, 15 uL, 30 ug of 2 ug/uLDMF) was added and vortexed. Ol-Mel-CDM (11.2 u , 112 0360 Complex I: To HO (9.7 mL) was added ug of 10 ug/ull DMF) was added and vortexed. pDNA-NC, (pCILuc, 20 uL, 40 ug of 2 ug/ul DMF) and vortexed. CDM-Mel-SiMeCs (15 uL, 150 ug 0371 Complex III: To HO (600 uL) was added of 10 ug/ull DMF) was added immediately before pDNA-NC (pCI Luc, 15 uL, 30 ug of 2 ug/uLDMF) injection. was added and vortexed. CDM-Mel-CDMNC12 (11.2 uL, 112 lug of 10 ug/ull DMF) was added and 0361 Complex II: To HO (9.7 mL) was added vortexed. pDNA-NC, (pCILuc, 20 uL, 40 ug of 2 ug/ul DMF) 0372) Complex IV: To HO (600 uL) was added and vortexed. Ol-Mel-CDM (15 u , 150 lug of 10 pDNA-NC (pCI Luc, 15 uL, 30 ug of 2 ug/uLDMF) ug/ull DMF) was added and vortexed. was added and vortexed. CDM-Mel (11.2 uL, 112 tug 0362 Complex III: To HO (9.7 mL) was added of 10 ug?uL DMF) was added and vortexed. 200 uL pDNA-NC (pCILuc, 20 uL, 40 ug of 2 ug/uL DMF) tail vein injections of 200 lull of the complex were and vortexed. CDM-Mel-CDMNC12 (15ulL, 150 lug preformed on ICR mice (n=2) using a 30 gauge, 0.5 of 10 ugul DMF) was added and vortexed. inch needle, with the total solution injected by hand within 10 seconds. One day after injection, the 0363 Complex IV: To HO (9.7 mL) was added animal was Sacrificed, and a luciferase assay was pDNA-NC, (pCILuc, 20 ul, 40 ug of 2 ug/ul DMF) conducted on the liver, Spleen, lung, heart, and and vortexed. CDM-Mel (15uL, 150 ug of 10 ug?uL kidney. Luciferase expression was determined as DMF) was added and vortexed. previously reported (Wolff, J. A., Malone, R. W., US 2003/023.5916 A1 Dec. 25, 2003 28

Williams, P., Chong, W., Acsadi, G., Jani, A. and ug?u. DMF) and vortexed. Ol-Mel-CDM (15 uL, Felgner, P. L. Direct gene transfer into mouse muscle 150 ug of 10 ug?u. DMF) was added and vortexed. in vivo. Science, 1465-1468, 1990.). A Lumat LB 0385 Complex II: To isotonic mannitol (800 uL) 9507 (EG&G Berthold, Bad-Wildbad, Germany) was added plDNA-NC (pCI Luc, 20 uL, 40 ug of 2 luminometer was used. ug?u. DMF) and vortexed. Ol-Mel-CDMC12 (15 0373) Results: Mean RLU (n=2) uL, 150 lug of 10 ug/4L DMF) was added and vortexed. 0386 Complex III: To isotonic mannitol (800 uL) Complex # Liver Spleen Lung Heart Kidney was added plDNA-NC (pCI Luc, 20 uL, 40 ug of 2 ug?u. DMF) and vortexed. CDM-Mel (15 uL, 150 Complex I 1,155,276 1879 1,071 1,339 3,733 Complex II 51,508 3.287 474 954 2,400 ug of 10 ug/ull DMF) was added and vortexed. Complex III 907 2,504 860 274 632 0387 Complex IV: To isotonic mannitol (800 uL) Complex IV 1,685 416 32O 247 292 was added plDNA-NC (pCI Luc, 20 uL, 40 ug of 2 ug?u. DMF) and vortexed. Ol-Mel-CDM (15 uL, 0374. The results indicate that hydrophobic modified 150 ug of 10 ug?u. DMF) was added and vortexed. peptide ternary complexes deliver p)NA predominatly to MC894 (3.5uL, 70 tug of 20 tug/ul DMF) was added the liver, where the pDNA is able to be transcribed. and Vortexed. 0388 Bile duct injections on ICR mice were performed Example 83 using a Harvard Apparatus PH 2000 programmable pump with a 30-gauge, 72 inch needle and 1 ml Syringe. The pump 0375 Bile Duct Injections. was programmed to deliver 200 till over 4 seconds. A 5x1 0376 Two complexes were made as follows: mm, Kleinert Kutz microVessel clip was used to occlude the bile duct downstream from the point of injection in order to 0377 Complex I: To isotonic mannitol (800 uL) was prevent flow to the duodenum and away from the liver. The added plDNA-MC933 (1:2) (pCI Luc, 20 uL, 40 ug gallbladder inlet was not occluded. In these injections, the of 2 ug/ull DMF) and vortexed. junction of the hepatic vein and caudal vena cava were not 0378 Complex II: To isotonic mannitol (800 uL) was clamped. Additionally, the portal vein and hepatic artery added plDNA-MC933 (1:2) (pCI Luc, 20 uL, 40 tug of 2 were not clamed for the injection. ug/ull DMF) and vortexed. Ol-Mel-CDM (15uL, 150 lug of 0389 Results: Mean RLU (n=3) 10 ugul DMF) was added and vortexed. 0379 Bile duct injections on ICR mice were performed using a Harvard Apparatus PH 2000 programmable pump Complex # Liver 10x Diluted with a 30-gauge, 72 inch needle and 1 ml Syringe. The pump Complex I 76,485 was programmed to deliver 200 till over 4 seconds. A 5x1 Complex II 31,771 mm, Kleinert Kutz microVessel clip was used to occlude the Complex III 91,396 bile duct downstream from the point of injection in order to Complex IV 92,118 prevent flow to the duodenum and away from the liver. The gallbladder inlet was not occluded. In these injections, the junction of the hepatic vein and caudal vena cava were not 0390 The results indicate that the described binary and clamped. Additionally, the portal vein and hepatic artery ternary complexes are able to deliver pl)NA to the liver via were not clamed for the injection. the bile duct. 0380 Results: Mean RLU Example 85 0391 plDNA-NC Degradation with DNAse I via Gel Electrophoresis. Complex # = Liver 0392 Samples were formulated (2-13) as described Complex I 3 403,002 below (total volume 10 uD) and incubated at 37° C. for 30 Complex II 2 508,318 min. Loading buffer was added to all Samples and mixed. The Samples were loaded onto 0.8% agarose gel containing 0381. The results indicate that the described binary and ethidium bromide. The gel was run at 95V for 45 min and ternary complexes are able to deliver pl)NA to the liver via analyzed by UV light. the bile duct.

Example 84 Lane Description 0382 Bile Duct Injections. 1. 1 kb Ladder (500 ng) 2 pDNA (500 ng) 0383 Four Complexes were made as follows: 3 pDNA (500 ng) + DNase I (1.25 u) 4 pDNA (500 ng) + DNase I (2.5 u) 0384 Complex I: To isotonic mannitol (800 uL) was 5 pDNA (500 ng) + DNase I (5 u) added pdNA-NC (pCI Luc, 20 uL, 40 tug of 2 US 2003/023.5916 A1 Dec. 25, 2003 29

Example 87 -continued 0402 NaCl Stability of RhDNA+NC Complexes. Lane Description 0403. After the 20 hr incubation, samples were titrated 6 pDNA (500 ng) + pI L (315 ng) with NaCl and analyzed by fluorescence (Varian Cary 7 pDNA (500 ng) + pI L (315 ng) + Eclipse Spectrofluorometer). FIG. 4. Five complexes were DNase I (1.25 u) made in quadruplicate as follows: 8 pDNA (500 ng) + pI L (315 ng) + DNase I (2.5 u) 9 pDNA (500 ng) + pI L (315 ng) + 04.04 Complex I: To HO (500 uL) was added DNase I (5 u) RhDNA (2.5ull, 5ug of 2 tug/uL HO, 0.015umol) 1O pDNA-NC (500 ng) and vortexed. Dodecylamine hydrochloride (6.7 uL, 11 pDNA-NC (500 ng) + DNase I 6.7 ug, of 1 tug/uL HO, 0.030 umol) was added to (1.25 u) the Solution and vortexed. The final concentration of 12 pDNA-NC (500 ng) + DNase I (2.5 u) RhDNA was 0.1 tug/ul. 13 pDNA-NC (500 ng) + DNase I (5u) 04.05 Complex II: To HO (468 uL) was added 14 empty RhDNA (25 uL, 50 tug of 2 tug/ul H2O, 0.15umol) and vortexed. Dodecylamine hydrochloride (6.7 uL, 67 ug, of 10 ugul, 0.030 mol) was added to the 0393 Results (FIG. 2) indicate that the pDNA in the Solution and vortexed. The final concentration of pDNA-NC complex is still accessible to DNAseI. RhDNA was 0.1 tug/ul. 0406 Complex III: To HO (218 uL) was added Example 86 RhDNA (25 uL, 50 tug of 2 tug/ul HO, 0.15umol) and vortexed. Dodecylamine hydrochloride (6.7 uL, 0394 Condensation of RhDNA with NC Concentration 67 lug, of 10 ug?ul, 0.030 umol) was added to the and Temperature Dependence. Solution and vortexed. The final concentration of 0395. Five complexes were made in quadruplicate as RhDNA was 0.2 tug/ul. follows: 04.07 Complex IV: RhDNA-NC (2 tug/uL DMF) 0396 Complex I: To HO (500 uL) was added 0408 Complex IV: RhDNA-NC (2 ug?u.L benzyl RhDNA (2.5 uL, 5ug of 2 tug/uL HO, 0.015umol) and vortexed. Dodecylamine hydrochloride (6.7 uL, alcohol) 6.7 ug, of 1 tug/uL HO, 0.030 umol) was added to 04.09 Complexes I-III were incubated at several different the Solution and vortexed. The final concentration of temperatures (RT, 37° C., 50° C. and 70° C.). After the 20 RhDNA was 0.1 tug/ul. hr incubation, samples were titrated with NaCl and analyzed by fluorescence (Varian Cary Eclipse Spectrofluorometer). 0397) Complex II: To HO (468 ul) was added FIG. 4 RhDNA (25 uL, 50 tug of 2 tug/ul H2O, 0.15umol) and vortexed. Dodecylamine hydrochloride (6.7 uL, 0410 Results. The results indicate that the sample heated 67 ug, of 10 ug?ul, 0.030 umol) was added to the to 70° C. decondenses in the presence of NaCl. The 50° C. Solution and vortexed. The final concentration of sample is stable in salt, and the 37 C. indicates a small RhDNA was 0.1 tug/ul. amount of decondensation, whereas the rt Samples indicate greater decondensation. For the 37 C. and RT samples, the 0398 Complex III: To HO (218 uL) was added more dilute the Sample, the greater the Stability, indicating RhDNA (25 uL, 50 tug of 2 tug/ul H2O, 0.15umol) that the binary complex can be Stabilized based on time, and vortexed. Dodecylamine hydrochloride (6.7 uL, temperature, and concentration. 67 ug, of 10 ug?ul, 0.030 umol) was added to the Solution and vortexed. The final concentration of Example 88 RhDNA was 0.2 tug/ul. 0411 RhDNA-NC Stability at 37° C. with 150 mM 0399 Complex IV: RhDNA-NC (2 ug?u L DMF) NaCl. Complex IV: RhDNA-NC (2 tug/ull benzyl alco 0412 Four complexes were made in duplicate as follows: hol) 0413 Complex I: To HO (500 uL) was added 04.00 Complexes I-III were incubated at different tem peratures (RT, 37° C., 50° C. and 70° C). Their fluorescence RhDNA-NC (2.5 uL, 5 ug of 2 ug?u.L DMF, 0.015 (Varian Cary Eclipse Spectrofluorometer) was monitored at almol) and Vortexed. Several time points (t=0, 1, 2, 4 and 20 hr) and compared to 0414) Complex II: To HO (500 uL) was added Complexes IV and V. RhDNA (2.5ull, 5ug of 2 tug/uL HO, 0.015umol) and vortexed. Dodecylamine hydrochloride (6.7 uL, 04.01 Results: The results indicate that the pdNA in the 6.7 ug, of 1 tug/uL HO, 0.030 umol) was added to binary complex is more condensed when the Sample is the Solution and Vortexed. heated relative to a room temperature Sample. The more dilute samples indicate the greatest amount of condensation 0415 Complex III: To HO (500 uL) was added relative to time, and the closer in overall condensation to RhDNA (2.5ull, 5ug of 2 tug/uL HO, 0.015umol) controls that were dried under vacumn. and vortexed. p-L-Lysine HBr (9.5ull, 9.5 lug, of 1 US 2003/023.5916 A1 Dec. 25, 2003 30

Aug?u LH), 0.030 limol, Sigma Chemical Company) of 10 ug?uL HO) was added to the solution and was added to the Solution and Vortexed. vortexed. Methanol (15ul ) was added to the solu tion and Vortexed. 0416) Complex IV: To HO (500 uL) was added RhDNA (2.5ull, 5ug of 2 tug/uL HO, 0.015umol) 0429 Complex III: To HO (1685 uL) was added and Vortexed pDNA+NC+37C. (300 uL, 60 ug of 0.2 uguL HO) and vortexed. Dodecylamine hydrochloride (8 0417. To one set of the complexes, NaCl (15 uL of 5M, AiL, 80 ug of 10 ug?u L H2O) was added to the 150 mM) was added to each complex and the solutions were solution and vortexed. Methanol (15ul ) was added vortexed. To the second set of complexes, no NaCl was to the Solution and vortexed added. All complexes were incubated at 37C. The fluores cence of each Sample was analyzed over Several hours (t=0, 0430 Complex IV: To HO (1985 uL) was added 4, 24 hr) (Varian Cary Eclipse Spectrofluorometer). FIG. 5. pDNA-NC (1582 L, 60 ug of 4 ug/ul methanol) Results: The results indicate that samples heated in the and Vortexed. presence of Salt show leSS condensation of the pDNA, than 0431 Results: The results indicate that there is a shift in Samples heated with no Salt present. the CD spectra in the presence of a cationic detergent. The results also indicate a lowering of the Signal intensity for a Example 89 complex that has been dried. 0418 RhDNA-NC Stability in Serum. Example 91 0419 Three complexes were made as follows: 0432 Liposomal Incorporation of a Binary Complex. 0420 Complex I: To HO (500 uL) was added 0433 Rhodamine labeled pCNA/lauryl amine hydro RhDNA-NC (2.5 uL, 5 ug of 2 uguL DMF, 0.015 chloride binary complex (RhDNA-NC12) was prepared as almol) and Vortexed. previously described, and the dried solid was dissolved in 0421 Complex II: To HO (500 ul) was added DMF as a 2 mg/mL solution. To phosphatidylcholine RhDNA (2.5ull, 5ug of 2 tug/uL HO, 0.015umol) (Avanti Polar Lipids, InS.)/cholesterol hemisuccinate (Sigma and vortexed. Dodecylamine hydrochloride (6.7 uL, Chemical Company) (2 mg, 1:1 wit, in chloroform) was 6.7 ug, of 1 tug/uL HO, 0.030 umol) was added to added 20 tug of RhDNA-NC12 (based on DNA wt). The the Solution and Vortexed. Solution was dried under reduced preSSure and then under high vacuum. The resulting residue was hydrated with 2 mL 0422 Complex III: To HO (500 uL) was added water, and Sonicated to form liposomes. The Solution was RhDNA (2.5ull, 5ug of 2 tug/uL HO, 0.015umol) passed through a size exclusion column (Sepharose CL-4B and vortexed. p-L-Lysine HBr (9.5ull, 9.5 lug, of 1 200, 150 mM NaCl eluent, 1 mL fractions). Aug?u LH2O, 0.030 limol, Sigma Chemical Company) 0434) Results: The results indicate that the RhDNA was added to the Solution and Vortexed. eluded with the lipoSome fractions, and Separate from 0423 Samples were analyzed by fluorescence (Varian RhDNA blank. Cary Eclipse Spectrofluorometer). Serum (5ull, 10%) was added to each complex with Vortexing. The fluorescence of Example 92 each Sample was analyzed over Several hours (t=0,0.2,0.3, 0435) In Vivo Tail Vein Injections into Mice. 4, 24 hr) (Varian Cary Eclipse Spectrofluorometer). FIG. 6 0436 Complexes were made as follows: 0424) Results: The results indicate that serum causes a decrease in the level of condensation over time, indicated 0437 Complex I: To HO (1600 uL) was added that the pl)NA is being released in the presence of Serum pDNA-NC (pCI Luc, 32 uL, 80 ug of 2.5 uguL over time. benzyl alcohol) was added and vortexed. 0438 Complex II: To HO (1600 uL) was added Example 90 Chit-Ol-LBA (168 ug, 252 uL of 0.67 ug?u L DMF/ H) and vortexed. plDNA-NC (pCI Luc, 32 uL, 80 0425 Circular Dichroism of Hydrophobic DNA. Aug of 2.5 ug?u L benzyl alcohol) was added and 0426 Preparation of pDNA+NC+37C. (0.2 tug/uL): vortexed. pDNA(pCI Luc, 100 uL,200 ug of 2 ug?u, 0.61 umol) was taken up in HO (873 uL) and vortexed. To the resulting 0439 Complex III: To HO (1600 uL) was added solution was added dodecylamine hydrochloride (27 u , 270 Chit-Ol-LBA (168 ug, 252 uL of 0.67 ug?u L DMF/ Aug of 10 ug/ull H2O, 1.2 umol). The Solution was incubated H) and vortexed. plDNA-NC (pCI Luc, 32 u , 80 at 37C. for 16 h. Four complexes were made as follows: Aug of 2.5 ug?u L benzyl alcohol) was added and vortexed. 0427 Complex I: To HO (1955 uL) was added pDNA (pCI Luc, 30 ul, 60 tug of 2 uguL HO) and 0440 200 till tail vein injections of 200 lull of the com vortexed. Methanol (15 uL) was added to the solu plex were preformed on ICR mice (n=4) using a 30 gauge, tion and Vortexed. 0.5 inch needle, with the total solution injected by hand within 10 Seconds. Three days after injection, the animal 0428 Complex II: To HO (1947 uL) was added from group A was Sacrificed, and a luciferase assay was pDNA (pCI Luc, 30 ul, 60 tug of 2 uguL HO) and conducted on the liver, Spleen, lung, and kidney. Seven days vortexed. Dodecylamine hydrochloride (8 uL, 80 ug after injection, the animal from group B was Sacrificed, and US 2003/023.5916 A1 Dec. 25, 2003 31 a luciferase assay was conducted on the liver, Spleen, lung, of liposome-mediated DNA transfection. Nucleic Acids heart, and kidney. Luciferase expression was determined as Res. 22, 536-537 (1994). Wagner, E., Curiel, D. & previously reported (Wolff et al. 1990.). A Lumat LB 9507 Cotten, M. Delivery of drugs, proteins and genes into (EG&G Berthold, Bad-Wildbad, Germany) luminometer cells using transferrin as a ligand for receptor-mediated was used. endocytosis. Advanced Drug Delivery Reviews 14, 113 0441 Results: Mean RLU (n=2) Harvest Day 3, Group A 135 (1994) 0448. 3. Duzgunes, N., Straubinger, R. M., Baldwin, P. A. & Papahadjopoulos, D. PH-sensitive liposomes. (eds Wilsehub, J. & Hoekstra, D.) p. 713-730 (Marcel Complex # Liver Spleen Lung Kidney Deker INC, 1991) Complex I 20,599 433 521 512 Complex II* 5,306 471 449 679 0449 4. Melnikov, 1995 Complex III* 4,517 336 686 347 0450) 5. Sergeyev, 1999 n = 1 0451 6. Sergeyev, 1999 0442. Results: Mean RLU (n=2) Harvest Day 7, Group B 0452 7. Sukhorukov, 2000 0453 8. Tanaka, 1996 0454) 9. Ijiro, 1992 Complex # Liver Spleen Lung Kidney 0455 10. Melnikov, 1995 Complex I 2,783 3,803 4,068 3,105 0456 11. Clamme J. P. Bemacchi S, Vuilleumier C, Complex II* 943 1,670 1,299 2,373 Duportail G, Mely Y. Gene transfer by cationic surfac Complex III* 1597 1,667 1,407 1,507 tants is essentially limited by the trapping of the n = 1 surfactant/DNA complexes onto the cell membrane: a fluorescence investigation. Biochim BiophyS Acta. 0443) The results indicate that the plDNA in the plDNA Aug. 25, 2000;1467(2):347-61. NC12 complexes is accessible for transcription. 0457) 12. Trubetskoy VS, et al. (1999). “Quantitative 0444 The foregoing is considered as illustrative only of assessment of DNA condensation.’Anal Biochem the principles of the invention. Furthermore, Since numerous 267(2): 309-313. modifications and changes will readily occur to those skilled 0458) 13. Wolff, J. A., Malone, R. W., Williams, P., in the art, it is not desired to limit the invention to the exact Chong, W., Aesadi, G., Jani, A. and Felgner, P. L. Direct construction and operation shown and described. Therefore, gene transfer into mouse muscle in Vivo. Science, all suitable modifications and equivalents fall within the 1465-1468, 1990. Scope of the invention. 0459 14. Zhang, G., Vargo, D., Budker, V., Armstrong, 0445) References: N., Knechtle, S., Wolff, J. A. Human Gene Therapy, 8, 0446 1. Legendre, J. & Szoka, F. Delivery of plasmid 1763-1772, 1997. DNA into mammalian cell lines using pH-sensitive 0460 15. Zhang et al. 1999 liposomes: Comparison with cationic liposomes. Phar maceut. Res. 9, 1235–1242(1992) 0461) 16. Liu et al. 1999 0447 2. Kamata, H., Yagisawa, H., Takahashi, S. & 0462) 17. Calbiochem, 2000-2001 Hirata, H. Amphiphilic peptides enhance the efficiency 0463

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS : 15 <210> SEQ ID NO 1 &2 11s LENGTH 13 &212> TYPE PRT <213> ORGANISM: Simian virus 40

<400 SEQUENCE: 1 Cys Gly Tyr Gly Pro Llys Lys Lys Arg Lys Val Gly Gly 1 5 10

<210> SEQ ID NO 2 &2 11s LENGTH 39 &212> TYPE PRT US 2003/023.5916 A1 Dec. 25, 2003 32

-continued <213> ORGANISM: Simian virus 40

<400 SEQUENCE: 2 Cys Lys Lys Lys Ser Ser Ser Asp Asp Glu Ala Thr Ala Asp Ser Glin 1 5 10 15 His Ser Thr Pro Pro Lys Lys Lys Arg Lys Val Glu Asp Pro Lys Asp 2O 25 30

Phe Pro Ser Glu Lieu. Leu Ser 35

<210> SEQ ID NO 3 &2 11s LENGTH 37 &212> TYPE PRT <213> ORGANISM: Simian virus 40

<400 SEQUENCE: 3 Cys Lys Lys Lys Trp Asp Asp Glu Ala Thr Ala Asp Ser Glin His Ser 1 5 10 15 Thr Pro Pro Llys Lys Lys Arg Lys Val Glu Asp Pro Lys Asp Phe Pro 2O 25 30

Ser Glu Lieu Lleu Ser 35

<210> SEQ ID NO 4 &2 11s LENGTH: 31 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 4 Cys Tyr Asn Asp Phe Gly Asn Tyr Asn. Asn Glin Ser Ser Asn. Phe Gly 1 5 10 15 Pro Met Lys Glin Gly Asn Phe Gly Gly Arg Ser Ser Gly Pro Tyr 2O 25 30

<210 SEQ ID NO 5 &2 11s LENGTH 10 &212> TYPE PRT <213> ORGANISM: Human adenovirus type 1 <400 SEQUENCE: 5 Cys Lys Arg Gly Pro Lys Arg Pro Arg Pro 1 5 10

<210> SEQ ID NO 6 <211& LENGTH 22 &212> TYPE PRT <213> ORGANISM: Xenopus laevis <400 SEQUENCE: 6 Cys Lys Lys Ala Wall Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Glin 1 5 10 15 Ala Lys Lys Lys Lys Lieu 2O

<210 SEQ ID NO 7 <211& LENGTH: 14 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 7 US 2003/023.5916 A1 Dec. 25, 2003 33

-continued

Cys Lys Lys Lys Gly Pro Ala Ala Lys Arg Val Lys Lieu. Asp 1 5 10

<210 SEQ ID NO 8 <211& LENGTH 4 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 8 Lys Asp Glu Lieu 1

<210 SEQ ID NO 9 &2 11s LENGTH 10 &212> TYPE PRT <213> ORGANISM: Human immunodeficiency virus <400 SEQUENCE: 9 Gly Arg Lys Lys Arg Arg Glin Arg Arg Arg 1 5 10

<210> SEQ ID NO 10 &2 11s LENGTH 16 &212> TYPE PRT <213> ORGANISM: Drosophila melanogaster <400 SEQUENCE: 10 Arg Glin Ile Lys Ile Trp Phe Glin Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15

<210> SEQ ID NO 11 &2 11s LENGTH 10 &212> TYPE PRT <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: polyarginine synthetic peptide <400 SEQUENCE: 11 Cys Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 10

<210> SEQ ID NO 12 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Photinus pyralis <400 SEQUENCE: 12 cuuacgcuga guacuucg at t 21

<210> SEQ ID NO 13 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Photinus pyralis <400 SEQUENCE: 13 ucgaaguacu cagcguaagt t 21

<210> SEQ ID NO 14 <211& LENGTH 24 &212> TYPE DNA <213> ORGANISM: Aequorea victoria US 2003/023.5916 A1 Dec. 25, 2003 34

-continued <400 SEQUENCE: 14 gacguaaacg gcc acaagug catt 24

<210 SEQ ID NO 15 <211& LENGTH 24 &212> TYPE DNA <213> ORGANISM: Aequorea victoria <400 SEQUENCE: 15

CgCugCauluu gcc.gguguuc agtt 24

We claim: 19. The process of claim 15 wherein the amphipathic 1. A proceSS for delivering a polynucleotide to a cell in compound is Selected from the list consisting of polymer Vivo comprising: associating the polynucleotide with a cat containing one or more hydrophobic moieties, peptide con ionic Surfactant to form a binary complex, Stabilizing the taining one or more hydrophobic moieties, targeting group complex, and delivering the complex to a cell in a mammal. containing one or more hydrophobic moieties, Steric Stabi 2. The process of claim 1 wherein the cationic Surfactant lizer containing one or more hydrophobic moieties, Surfac consists of a detergent. tants and lipids. 3. The process of claim 1 wherein Stabilizing the complex 20. The process of claim 19 wherein the polymer con comprises: incubating the complex at elevated temperature. taining one or more hydrophobic moieties consists of modi 4. The process of claim 1 wherein Stabilizing the complex fied chitosan. consists of drying the complex. 21. The process of claim 15 wherein delivering the 5. The process of claim 1 wherein delivering the complex complex to a cell in a mammal comprises injecting the to a cell in a mammal comprises injecting the complex in a complex in a Solution into a tissue in a mammal. Solution into a tissue in a mammal. 22. The process of claim 15 wherein delivering the 6. The process of claim 1 wherein delivering the complex complex to a cell in a mammal comprises inserting the to a cell in a mammal comprises inserting the complex in a complex in a Solution into a vessel in a mammal. Solution into a vessel in a mammal. 23. The process of claim 22 wherein the vessel is selected 7. The process of claim 6 wherein the vessel is selected from the listing consisting of: portal vein, hepatic vein, from the listing consisting of portal vein, hepatic vein, inferior Vena cava, tail vein, hepatic artery, and bile duct. inferior Vena cava, tail vein, hepatic artery, and bile duct. 24. The process of claim 15 wherein the cell consists of 8. The process of claim 1 wherein the cell consists of a a liver cell. liver cell. 25. The process of claim 24 wherein the liver cell consists 9. The process of claim 8 wherein the liver cell consists of a hepatocyte. of a hepatocyte. 26. The process of claim 15 wherein the polynucleotide 10. The process of claim 1 wherein the polynucleotide consists of DNA. consists of DNA. 11. The process of claim 10 wherein the DNA comprises 27. The process of claim 26 wherein the DNA comprises an expression cassette. an expression cassette. 12. The process of claim 11 wherein the expression 28. The process of claim 27 wherein the expression cassette encodes a protein. cassette encodes a protein. 13. The process of claim 1 wherein the polynucleotide 29. The process of claim 15 wherein the polynucleotide consists of a polynucleotide containing 18-30 nucleotide consists of a polynucleotide containing 18-30 nucleotide monomeric Subunits. monomeric Subunits. 14. The process of claim 13 wherein the polynucleotide 30. The process of claim 29 wherein the polynucleotide consists of a polynucleotide that induces RNA interference. consists of a polynucleotide that induces RNA interference. 15. A proceSS for delivering a polynucleotide to a cell 31. A proceSS for forming a Small Salt Stable polynucle comprising: associating a polynucleotide with a cationic otide-containing complex comprising: associating the poly Surfactant to from a binary complex, Stabilizing the binary nucleotide with a cationic Surfactant in a Solution and complex, associating the binary complex with an amphip incubating the complex in the Solution at elevated tempera athic compound to form a ternary complex, and delivering ture. the complex to the cell. 32. The process of claim 31 wherein the cationic surfac 16. The process of claim 15 wherein the cationic surfac tant is Selected from the list consisting of detergent and tant is Selected from the list consisting of detergent and lipid. lipid. 33. A proceSS for forming a Small Salt Stable polynucle 17. The process of claim 15 whereinstabilizing the binary otide-containing complex comprising: associating the poly complex comprises: incubating the complex at elevated nucleotide with a cationic Surfactant in a Solution to form a temperature. binary complex, Stabilizing the binary complex, and asso 18. The process of claim 15 whereinstabilizing the binary ciating the binary complex with an amphipathic compound complex consists of drying the complex. to form a ternary complex. US 2003/023.5916 A1 Dec. 25, 2003 35

34. The process of claim 34 wherein the cationic surfac containing one or more hydrophobic moieties, peptide con tant is Selected from the list consisting of detergent and taining one or more hydrophobic moieties, targeting group lipid. containing one or more hydrophobic moieties, Steric Stabi 35. The process of claim 34 whereinstabilizing the binary lizer containing one or more hydrophobic moieties, deter complex comprises: incubating the complex at elevated gent and lipid. temperature. 38. The process of claim 38 wherein the polymer con 36. The process of claim 34 whereinstabilizing the binary taining one or more hydrophobic moieties consists of modi complex consists of drying the complex. fied chitosan. 37. The process of claim 34 wherein the amphipathic compound is Selected from the list consisting of polymer