CN101437544A - Chemically modified polycation polymer for siRNA delivery - Google Patents

Abstract

The present invention provides a unique non-viral carrier for nucleic acid delivery in vitro and in vivo, and methods of using thereof.

Description

The polycationic polymer that is used for the chemical modification of siRNA transmission

The application requires the U.S. Provisional Patent Application submitted on January 23rd, 2006 number 60/761, the U.S. Provisional Patent Application of submitting on March 29th, 182 and 2006 number 60/787,057 rights and interests, its disclosure separately all is incorporated herein by reference with it at this, is used for all purposes.

Background technology

The attention that the transfer system of the non-virus of gene has been increased is because human gene therapy's realization day by day.Cationic lipoid is mixed with liposome, and soluble cationic polymer has proved and has been easy to complexation and transmits it effectively and enter cell based on the medicine of nucleic acid and external.Major obstacle on the cellular level is the endosome film, its can be by overcome by the cationic lipoid that triggers (flip-flop) mechanism (people such as Xu, Biochemistry Vol.35 (18), the 5616th page (1996)) or by the cationic polymer of so-called proton sponge mechanism overcome ( Boussif, PNAS Vol. 92 (16), the 7297th page (1995)).Proton sponge hypothesis explanation, between the pH decrement phase, polymer may be used as buffer agent in endosome, and therefore the more protons of needs are to reach about 5 final pH.This causes increasing the input of chloride counter ion and water, and it causes the explosion of phlycten and its inclusions to discharge into Cytoplasm at last.External usually is relevant with cationic polymer and lipoid with the interior significant toxicity of body.At cellular level, cationic electric charge causes membrane damage, and carrier may cause necrosis and apoptosis.Level in vivo, cationic charge causes in conjunction with cellular blood component such as erythrocyte and/or non-specific relevant with serum albumin and blood vessel endothelium.The clearance rate very fast of RES prevents that reagent from arriving the desired anatomical site of intervention.Development tactics solve these not desired characteristics as with protein and stealthy molecule such as PEG shielding electric charge and is connected and expects the cell activity targeting part that is used for the treatment of.

One of polymer that the most acceptable and widely used gene transmits is polymine (PEI).Described have a lot of desirable propertieses be used for the degradable PEI derivant that gene transmits (D.W.Pack, Bioconjugate Chemistry, the 14th volume, the 934th page (2003)), to compare with 25,000 molecular weight PEI of non-degraded, the gene with 16 times stronger transmits activity, and has hypotoxicity.It is synthetic like this, by using branched PEI 800, make itself and 1, the 6-hexanediyl ester reacts with the Michael form under the 1:1 molar ratio.The molecular weight that obtains is about 30,000, and based on proton N MR meter, structure has a lot of biodegradable ester bonds.When pH5, the half-life of degraded is 30 hours.In relevant research, use the polymine generation of acetic anhydride partial acetylation to be up to 21 times of enhanced genes transmission activity, do not change the cytotoxicity (D.W.Pack of polymer Pharmaceutical Research, the 21st volume,The 365th page (2004)).Be used for the other forms of degradable polymer that transmits based on the therapeutic of polynucleotide medicine such as DNA, used Michael addition to the different diacrylate of different amine to obtain (people such as Langer, JACS, the 123rd volume, 8155-8156 page or leaf, (calendar year 2001); JACS, the 122nd volume, 10761-10768 page or leaf, (2000)).In the case, final vector product has ester bond, does not have to participate in the primary amine or the secondary amine of N-acylation reaction; The existence of ester bond has improved biodegradability in the polymer backbone.

People such as Klibanov ( Pharmaceutical Res. the 22nd volume373-380 page or leaf, (2005)) research use two kinds of different cross-linking agent, promptly, suberic acid two succinimido esters (DSS) and ethylene glycol bisthioglycolate [succinimido succinate] (EGS) are connected to 423-Da and 2kDa PEI on the more high molecular carrier of DNA.Cross-linking agent DSS produces amido link and also keeps aliphatic skeleton after the amine with PEI reacts, and it connects the part of two amine reactive groups (active ester) as carrier structure originally.This has just produced amido link, and keeps aliphatic skeleton as part-structure.Use EGS and PEI also to produce amido link, but because its structure, this carrier also has ester bond.People's requirements such as Klibanov, the material that makes with EGS are 30 times of effectiveness of the material that made by DSS in gene transmits.People such as Klibanov propose " promote the release (carrier is not filled) of DNA based on the higher biological degradability of the conjugate of EGS, therefore strengthened the availability of transcribing of DNA ".

Ideally, Zui Jia non-virus carrier should be the strength polymer with hypotoxicity and high gene transmission efficiency.The major advantage of polymer of the present invention is to transmit the ability of the nucleic acid of wide region.As standard polymers such as PEI25kDa when being effective in plasmid DNA transmits, it is invalid in siRNAs transmits, even does not observe actual gene expression knock out (people such as Kim when high polymer dosage more Bioconjugate ChemistryThe 17th volume, 241-244 page or leaf, 2006).For linear PEI, document is to be a little (the people such as Hassani of contradiction J Gene Medicine, The 7th volume,The 198-207 page or leaf, 2005; People such as Urban-Klein Gene Therapy, the 12ndVolume, 2005), however the data of the experiment of carrying out from document and applicant point out that linear PEI22kDa is suitable for knocking out the gene of transfection momently; Yet, be unsuitable in the cell line of stable transfection, producing strong knocking out.Opposite with these standard reagents, polymer of the present invention can be realized knocking out in of short duration and stably transfected cell line, and shows efficient in plasmid transmits.

In a word, the nucleic acid carrier system that needs a kind of non-virus, it has as the increase of commercially available prod stability, hypotoxicity, transfection effective, if molecular weight keeps lowly by the ability of the potential removing of kidney, the advantage of biodegradability by amido link, and make targeting and stealthy part can carry out numerous primary amine and secondary amine that chemistry is connected by it.

Summary of the invention

Summary of the invention

According to an aspect of the present invention, provide chemical compound with general formula I:

Wherein:

Polycation is a polymine;

L is the connector part of non-ester;

A is the integer of about 1-about 20;

B is the integer of about 1-about 10;

C is the integer of about 1-about 20; With

D is the integer of about 1-about 1000.

Preferably, PEI is oligomerisation of ethylene imines (OEI).The polycation of formula I can be chosen wantonly to have identical polycation repetition or also can have the multiple combination of different polycations.

The connector of " L " or formula I is the connector part that contains non-ester.The connector that contains non-ester that is fit to includes, but are not limited to acylamino-connector part, amino connector part, carbonyl connector part, carbamate connector part, urea connector part, ether connector part, succinoamino (succinamidyl) connector part and combination thereof.Connector partly is attached to amido contained in the polycation.In preferred embodiments, the connector that contains non-ester partly is the propiono unit, is defined as the chemical group by following representative :-CH ₂-CHR '-CO-N-, wherein R ' is H or alkyl.In a preferred embodiment, non-ester connector is a beta-amino propiono amide connector part.

In another embodiment, the chemical compound of formula I further comprises the biomolecule of complexation to chemical compound.Biomolecule can have one or more anionic groups and can with formula I or Ia compound formation ionic bond.Example with biomolecule of one or more anionic groups comprises nucleic acid (for example, DNA, single stranded RNA, double-stranded RNA, ribozyme, DNA-RNA bybridizer, siRNA, anitosence DNA and antisense oligonucleotide (antisence ligo)), protein, peptide class, lipoidis and carbohydrate.

And further embodiment provides a kind of method of transfecting eukaryotic cells, and this method comprises with the chemical compound of such formula I and biomolecule exposing cell, transmits biomolecule whereby to cell.The method can relate to the treatment mammal, comprises and identifies the mammal that needs gene therapy, to the such chemical compound of this mammal administration.In preferred embodiments, biomolecule is siRNA, and wherein siRNA effectively reduces justice (interest) expression of gene is arranged.

Another embodiment provides Pharmaceutical composition, and it comprises chemical compound and the biomolecule of formula I.

Another embodiment further provides the chemical compound with general formula I a:

Wherein:

Polycation is a polymine;

L is non-ester connector part;

S is basic at interval or does not exist;

A is reagent or does not exist;

A is the integer of about 1-about 20;

B is the integer of about 1-about 10;

C is the integer of about 1-about 20; With

D is the integer of about 1-about 1000.

The connector of L or formula I is the connector part that contains non-ester.The connector that contains non-ester that is fit to includes, but are not limited to acylamino-connector part, amino connector part, carbonyl connector part, carbamate connector part, urea connector part, ether connector part, succinoamino connector part and combination thereof.This connector partly is incorporated on the amine groups contained in the polycation.In preferred embodiments, the connector that contains non-ester partly is the propiono unit, is defined as the chemical group by following representative :-CH ₂-CHR '-CO-N-, wherein R ' is H or alkyl.In a preferred embodiment, non-ester connector is a beta-amino propiono amide connector part.

S is basic at interval or does not exist.Base can be that for example replace or unsubstituted, saturated or undersaturated hydrocarbon chain and replacement or unsubstituted, saturated or undersaturated by the hydrocarbon chain of at least one hetero atom such as oxygen, nitrogen and sulfur interruption at interval.Preferably, hydrocarbon chain comprises 2-20 carbon atom, is more preferably 2-10 carbon atom, most preferably is 2-6 carbon atom.The interval base that is fit to also can include, but are not limited to Polyethylene Glycol (PEG).

A be can promote for example receptor identification of one or more functions in the eukaryotic cell, inherent effect, biomolecule from intracellular escape, nucleus is fixed (nucleus localization), biomolecule discharges and the reagent of system stability.Therapeutic agent can include, but are not limited to cytotoxic agent such as paclitaxel, Inclusion lytic agent (endosomolytic agent), hydrophobic polymer includes but not limited to benzoyl and lauroyl (lauryl), targeting moiety and screener.Screener can include but not limited to hydrophilic entity, includes but not limited to Polyethylene Glycol (PEG), lactose, sugar and polypropylene-base amide (polyacrylaminde).The targeting part includes, but are not limited to transferrin, epidermal growth factor, folate, peptide class, antibody or its fragment, saccharide and integrin-binding entity such as RGD peptide class.

In another embodiment, the chemical compound of formula Ia further comprises the biomolecule of complexation to this chemical compound.Biomolecule can have one or more anionic groups and can with formula Ia compound formation ionic bond.Example with biomolecule of one or more anionic groups comprises nucleic acid (for example, DNA, single stranded RNA, double-stranded RNA, ribozyme, DNA-RNA bybridizer, siRNA, anitosence DNA and antisense oligonucleotide), protein, peptide class, lipoidis and carbohydrate.

In another embodiment, the chemical compound of formula Ia comprises that further biomolecule and complexation are to the reagent of this chemical compound (that is, screener, targeting agent and/or transmit reinforcing agent), the optional base at interval that comprises.

And another embodiment provides a kind of method of transfecting eukaryotic cells, and this method comprises with such formula Ia chemical compound and biomolecule exposing cell, chooses wantonly further to comprise a kind of reagent, transmits biomolecule whereby to cell.The method can relate to the treatment mammal, comprises and identifies the mammal that needs gene therapy, to the such chemical compound of this mammal administration.In preferred embodiments, biomolecule is siRNA, and wherein siRNA can effectively reduce adopted expression of gene.

Another embodiment provides a kind of chemical compound of formula Ia and pharmaceutical composition of biomolecule of containing, and it can further contain the reagent of complexation to polymer.

And further embodiment provides a kind of treatment mammiferous method, this method comprises identifies the mammal that needs gene therapy, to the chemical compound of the formula I of this mammal administration complexation biomolecule, wherein biomolecule is effectively to reduce the siRNA that adopted expression of gene is arranged.

And further embodiment provides a kind of treatment mammiferous method, this method comprises identifies the mammal that needs gene therapy, to the chemical compound of the formula Ia of this mammal administration complexation biomolecule, wherein said biomolecule is effectively to reduce the siRNA that adopted expression of gene is arranged.

Brief description of drawings

Fig. 1 a-1b. proposes the mechanism that polycation is modified.

The infrared spectrum of Fig. 2 .OEI-HD product.

Fig. 3 a-3b. structural element and IR of the PEI-800 of suberoyl chlorine modification.

Fig. 4. use OEI-HD-1siRNA to transmit the HUH7/EGPLuc cell.

Fig. 5. in different medium, use the siRNA of OEI-HD-1 to knock out (knockdown).

Fig. 6 a-6b. uses the siRNA of OEI-HD-1 to knock out in containing the medium of serum.

Fig. 7. use OEI-SUB-1 to fail siRNA and knock out.

Fig. 8. the polycation of use chemical modification is used for the result of RAN-siRNA in the body.

Fig. 9 a-9b. beta-amino propiono amide connector example.

Detailed Description Of The Invention

The present invention relates to a kind of non-virus carrier of the uniqueness that transmits for biomolecule, wherein carrier is the polymer with the polycation that connects with propiono amide units chemistry. The invention further relates to the compound of following formula I and Ia, the method for the described compound of preparation, and the method for using the compound of formula I and Ia.

I. the compound of formula I

A kind of embodiment provides uses the polycation that connects suc as formula the chemistry of the propiono amide units described in the I:

[[polycation]_a-[L] _b-[polycation]_c] _d

In formula I, when being defined as in being placed on the aqueous solution, polycation can obtain the molecule of two above cationic charges. For example, in certain embodiments, the polycation of formula I can comprise, but be not limited to polymine 400Da-750kDa, dendrimer (dendrimer) structure (the PPI dendrimer or the PAMAM dendrimer that for example, have different structure and molecular weight), spermine, spermidine, trien, tetren and penten. Preferably, the polycation of formula I be polymine (PEI) and more preferably the polycation of formula I be oligomerisation of ethylene imines (OEI). The polycation of formula I can be chosen wantonly has identical polycation repetition, perhaps also can have the combination that different polycations repeat.

The connector of " L " or formula I is the connector part that contains non-ester. The connector that contains non-ester that is fit to includes, but are not limited to acylamino-connector part, amino connector part, carbonyl connector part, carbamate connector part, urea connector part, ether connector part, succinoamino connector part and combination thereof. Connector partly is incorporated into amine groups contained in the polycation. In preferred embodiments, the connector that contains non-ester partly is the propiono unit, is defined as the chemical group by following representative :-CH₂-CHR '-CO-N-, wherein R ' is H or alkyl. In a preferred embodiment, non-ester connector is beta-amino propiono acid amides connector part.

Polycation can contain the combination of repetitive or the different polycations of identical polycation, and wherein a and c are the integers of about 1-about 10. In addition, L can be repetition, also have the combination of identical connector part or different connector part, so b is the integer of about 1-about 10. All the compound of formula I also can be repetition, and d is the integer of about 1-about 100, preferably the integer in about 30 scopes.

II. the compound of formula Ia

Further embodiment provides and has used the polycation that connects suc as formula the chemistry of the propiono amide units described in the Ia:

Wherein:

" S " is the interval base or do not exist. The interval base can be that for example replace or unsubstituted, saturated or undersaturated hydrocarbon chain and replacement or unsubstituted, saturated or undersaturated by the hydrocarbon chain of at least one hetero atom such as oxygen, nitrogen and sulphur interruption. Preferably, hydrocarbon chain comprises 2-20 carbon atom, more preferably 2-10 carbon atom, most preferably 2-6 carbon atom. The interval base that is fit to also can include, but are not limited to polyethylene glycol (PEG).

In further embodiment, the compound of formula Ia also can comprise a kind of reagent (" A "). " A " is reagent or do not exist. Preferably, " A " be can promote in the eukaryotic one or more functions for example Receptor recognition, internalization, biomolecule from intracellular escape, nucleus is fixed, biomolecule discharges and the reagent of system stability. Therapeutic agent can include, but are not limited to cytotoxic agent such as taxol, inclusion body lytic agent, hydrophobic polymer includes but not limited to benzoyl and lauroyl, targeting moiety and screener. Screener can include but not limited to hydrophilic entity, including but not limited to polyethylene glycol (PEG), lactose, sugar and polypropylene-base acid amides (polyacrylaminde). The target part includes, but are not limited to transferrin, EGF, folate, antibody or its fragment, peptide class, carbohydrate and integrin-binding entity such as RGD peptide class.

Should be appreciated that S and A must be present among the formula Ia one of at least.

Polycation can contain the combination of repetitive or the different polycations of identical polycation, and wherein a and c are the integers of about 1-about 20. In addition, L can be repetition, also have identical connector part or the combination with different connector parts, so b is the integer of about 1-about 10. All the compound of formula Ia also can be repetition, and d is the integer of about 1-about 100, preferably the integer in about 30 scopes.

The molecular weight ranges of formula I compound can be about 800 dalton-Yue 1,000,000 dalton, and preferably about 20,000 dalton-Yue 200,000 dalton most preferably are about 20,000 dalton-Yue 30,000 dalton.

The molecular weight ranges of the compound of formula Ia can be about 800 dalton-Yue 1,000,000 dalton, and preferably about 20,000 dalton-Yue 200,000 dalton most preferably are about 20,000 dalton-Yue 30,000 dalton.

The mol ratio of polycation and L is 20-50. Unhindered amina on the polycation and the mol ratio of reagent can change according to reagent simultaneously, and can be from about 1000-2.

III. the compound of the formula I of complexing biomolecule or Ia

The compound of formula I can form complex compound with biomolecule, and therefore is used as transmitting biomolecule to the carrier of cell. The example that forms the biomolecule of complex compound with formula I compound comprises nucleic acid, protein, peptide class, lipoidis and carbohydrate. The example of nucleic acid comprises for example ASON (antisense oligo) of DNA, single stranded RNA, double-stranded RNA, ribozyme, DNA-RNA hybridizer and antisense (antisense) DNA. Preferred nucleic acid is siRNA. Contain complexing to the cationic lipopolymer of the biomolecule of polymer and can be by in mutual solvent mutually the lipopolymer of mixed-cation and biomolecule form, be more preferably by the method described in following examples and form.

IV. also choose wantonly with the biomolecule complexing and have the formula I of reagent or the compound of Ia

Polymer of the present invention also can form ionic complex or with the covalent bond of particular therapeutic agent, include but not limited to cytotoxic agent such as taxol, inclusion body lytic agent, hydrophobic polymer and other targeting moieties.

The OEI-HD carrier can adopt shielding ligand modified, and it will reduce afterwards undesirable non-specific appearance that reacts to each other of vivo medicine-feeding, and therefore improves the cycle life (lifetime) after the administration. The shielding part comprises hydrophilic entity, including but not limited to polyethylene glycol (PEG), lactose, sugar and polypropylene-base acid amides. In addition, in order to improve picked-up target approach tissue, carrier can be the OEI-HD of OEI-HD or shielding, can have the target part with its combination. The example of target part is including but not limited to transferrin, EGF, folate, antibody or its fragment, sugar and integrin-binding entity such as RGD peptide.

V. the preparation of the compound of formula I and Ia

Polycation is polymine (PEI) crosslinked preferably, and the part Michael addition of the amine by polymer to the vinyl groups of crosslinked group occurs, and forms the N-acidylate of side (pendant) ester group. Crosslinked group can be acrylate or methacrylate monomers. Should be appreciated that acrylate and methacrylate monomers comprise several groups, include but not limited to diacrylate, dialkyl group acrylate, dimethylacrylate, diacrylate monomer. Acidylate is to be defined as the molecule of acyl group being introduced the organic compound with hydroxyl (O-acidylate) or amino (N-acidylate). In addition, the N-acidylate of polymer can realize by ester such as ethyl acetate and the reaction that contains the acid anhydrides of polymer. Chemical modification can occur at primary amine and the secondary amine place of polymer architecture, has therefore reduced the net number of ionogen. If poly functional reagent is for modification, the mean molecule quantity that polymer then occurs increases. These mechanism and the chemical unit of introducing are to illustrate in Fig. 1.

Resulting polymer has the application as the non-viral synthetic vectors of multiple entity, and described entity has opposite electric charge, includes but not limited to the peptide class of nucleic acid and treatment. The major advantage of polymer of the present invention is to transmit the ability of wide region nucleic acid. Although standard polymers such as PEI 25 kDa are to be that effectively it can not effectively transmit siRNAs in DNA transmits, in addition than high polymer dosage the time, do not observe substantive gene expression knock out (people such as Kim,Bioconjugate ChemistryThe 17th volume, 241-244 page or leaf, 2006). For linear PEI, document be to a certain extent contradiction (people such as Hassani,J Gene Medicine, the 7th volume, 198-207 page or leaf, 2005; The people such as Urban-Klein,Gene Therapy the 12nd volume,2005), yet, point out that from the data of document and applicant's experiment linear PEI 22 kDa are suitable for knocking out of temporary transient rotaring redyeing gene, yet it is unsuitable for producing strong knocking out in the clone of stable transfection. Opposite with these standard reagents, polymer of the present invention is can obtain to knock out in the clone of of short duration and stable transfection, and shows efficient in plasmid transmits. Therefore, double stranded rna molecule such as siRNA s (siRNA) are suitable for transmitting with polymer support of the present invention uniquely. It can be to be used for the treatment of purpose or target spot affirmation that siRNA transmits, and, identifies the potential target of new therapeutic purposes that is.

Polymer support of the present invention can make from various oligomeric amines, (for example include but not limited to the dendrimer structure, PPI dendrimer or PAMAM dendrimer with different structure and molecular weight), spermine, spermidine, trien, tetren and penten or from 400-25, the alkaline matter that the branched polyethylenimine of 000MW scope consists of. Branched polyethylenimine be chemical modification or with single-, two-or multi-functional dose crosslinked. Polymine contains superfluous primary amine, secondary amine and tertiary amine, and those amine account for about 30% of polymer quality. Primary amine and secondary amine are obtainable for the reaction with crosslinking agent. Crosslinking agent is defined as such molecule, and it has at least 2 active groups and is used at least 2 polymer molecules of chemistry connection. Can be used as the reagent of crosslinking agent, include but not limited to glycol diacrylate, ethylene glycol dimethacrylate, 1,6-hexanediyl ester, Macrogol 600 diacrylate and other two-or many acrylate or two-or many-methacrylic acid ester molecule. Being used for preferred reagent of the present invention is diacrylate, namely 1, and the 6-hexanediyl ester is abbreviated as HD. HD has four possible reaction site, that is, and and 2 vinyl groups and 2 ester groups. Because polymine has low-molecular-weight, usually be called the oligomerisation of ethylene imines, be abbreviated as OEI. When OEI and HD are combined heating during a couple of days, reacting amines adds in the Michael mode, intersects to be added in undersaturated two sites.

Used HD and the ratio of OEI normally 1 to 1 is based on the mole meter. When ratio greater than 1 to 1 the time, figure denote reflects mol ratio so, for example to refer to the ratio of HD and OEI be that the ratio that 1 to 1, OEI-HD-5 refers to HD and OEI is 5 to 1 to OEI-HD-1. When reaction was finished, proton N MR had shown that all vinyl groups consume, and resulting product is dissolved in water usually. The crosslinked of OEI chain do not have to produce to the degree that forms the water swellable gel so. The molecular-exclusion chromatography of typical sample (size exclusion chromatography) obtains chromatogram, and it allows to calculate number average and weight average molecular weight and polydispersity (weight average molecular weight is divided by number-average molecular weight or Mw/Mn) therefore. These numerical value are respectively 3000,16,000 and 5.3. Polydispersity value is the single dispersing molecule amount of 1 expression. Numerical value 2-3 is narrow distribution in a way, and 4 and the wider distribution of above expression.

This polymer is that to be designed to compare with the polymine (PEI) 25,000 of higher molecular weight be toxicity still less, its level be use in the body that transmits of gene transfection or siRNA unacceptable. Low-molecular-weight PEI ' s such as OEI 800 as initiation material of the present invention, are relatively nontoxic, but are not very effective when transmitting nucleic acid by cell membrane. The product of above-mentioned reaction is relatively nontoxic for cell, is very effective when transmitting siRNA and DNA by cell membrane. Also discharge them and enter cytoplasm, they can produce biological effect in cell so. The other feature of above-mentioned idealized structure is, it should be to be biodegradable by ester bond, and it can be part-structure. In theory, hexylene glycol can be when ester is hydrolyzed obtains as accessory substance and the OEI 800 that contains 1-alkyl amino-propionic acid end group.

Infrared spectrum is to carry out at such carrier at synthetic and after separating, but we are surprisingly found out that and have considerably less ester that strong acid amides peak is dominant. As if carboxylic acid does not point out in infrared spectrum that it gets rid of too early ester hydrolysis (Fig. 2) yet. Point out not exist the methylene of aliphatic series of the part that should be the HD key with the proton N MR of the product after the distill water dialysis. The reaction that can explain these results is ester bond by the upper residual amine acidylate of OEI, and it should enrich based on the molar ratio computing of initiation material. Such acidylate also will provide accessory substance 1, the 6-hexylene glycol, its be water-soluble and with the dialysis remove. The further evidence of the ester acidylate by the upper amine of OEI is to be that the ethyl acetate cleaning product of non-solvent provides by using for polymer. In the case, analyze the limited amount N-acidylate of pointing out to exist ethyl acetate.

Similar Poly-cation can obtain by two-stage process. In this alternative two-stage process, the first step comprises that nuclear core polycation is to modify with crosslinking agent, and second step comprises further modification, by adding similar or dissimilar polycations, and for example spermine and penten.

The specific embodiment

Embodiment

Further by following examples explanation, it only is intended to illustrate the present invention rather than will limits the scope of the invention in the present invention.

Embodiment 1. carriers synthetic, OEI-HD-1

5.0g the polymine of (0.0063 mole) (weight average molecular weight 800) is dissolved in the DMSO of 7.5ml.In the container that separates, 1 of the DMSO of adding 3.3ml and 1.4ml=1.4g (0.0063 mole), 6-hexanediyl ester.Mix two kinds of solution.The 50ml round-bottomed flask immerses in 60 ℃ of homothermic oil baths and assembles magnetic stirring bar.The flask loosely clogs, and makes its reaction 4 days.Reaction solution drops in the ethyl acetate solution of quick stirring of 200ml then, forms thick substances whereby on drag and sidewall.Drain solvent and add the aliquot ethyl acetate of fresh 200ml, compounding substances.Drain again, add other 100ml aliquot again, mix, drain, stay heavy-gravity material.In the evaporating dish that this substance transfer to aluminium foil is made, place in vacuum tank in room temperature and to spend the night.This vacuum fails to remove all ethyl acetate, because the low surface area of this material and high viscosity also need further purification.

Embodiment 2. is by dialysis purification OEI-HD-1

Weigh up the OEI-HD-1 of 0.80g and it is added scintillation vial, then add the Dulbucceos PBS buffer agent of 10ml.It dissolves after the short time jolting.By boiling in the distilled water of a large beaker about 10 minutes, the spectrum 3500 of about 1 linear feet of preaging (preconditioned) (linear foot) cuts dialyzer (length measurements of 0.4ml/cm).Then, the knotting of an end of Dialysis tubing adds OEI-HD-1 solution, ties up a knot sealing at the other end.Pipe is positioned in about 3 gallons of distilled water, stirs water lightly 4 days.After this, material is removed from pipe, about 30% of the polymer quality in the lyophilizing acquisition adding pipe.Carry out proton and carbon 13NMR for this product.

Precipitate in the embodiment 3.OEI-HD-1 Zai diox

Repeat embodiment 1, but be to use diox to replace ethyl acetate to be used for washing.Use diox to avoid owing to the acetylizad probability of ethyl acetate to unhindered amina.

Embodiment 4.OEI-HD-5's is synthetic

In the 125ml vial, in the DMSO of 7.5ml, dissolve the polymine (weight average molecular weight 800) of 5.0g (0.0063 mole).In the container that separates, 1 of the DMSO of adding 17ml and 7.0ml=7.0g (0.0315 mole), the 6-hexanediyl ester also mixes.Merge two kinds of solution in the bottle of 125ml, loosely covers, and is placed in 60 ℃ of homothermic baking ovens.After about 30 minutes of reaction, see the formation gel.Make reaction continue to amount to 3 days.Then, decantation DMSO from gel, the gel broken in bulk of scraper.Little piece is put into the 20ml scintillation vial of the water that contains the 10ml that has an appointment, so most gel is seemingly dissolved.

Embodiment 5.OEI-HD-10's is synthetic

5.0g the polymine of (0.0063 mole) (weight average molecular weight 800) is dissolved among the DMSO of the 7.5ml in the 125ml vial.In the container that separates, 1 of the DMSO of adding 34ml and 14ml=14g (0.0630 mole), the 6-hexanediyl ester also mixes.Merge two kinds of solution in the bottle of 125ml, loosely covers, and places it in 60 ℃ of homothermic baking ovens.After about 30 minutes of reaction, see the formation gel.Make reaction continue to amount to 3 days.Then, decantation DMSO from gel, the gel broken in bulk of scraper.Little piece was put into the 20ml scintillation vial of the water that contains the 10ml that has an appointment and jolting 2 days.There is not substance dissolves.

Embodiment 6.OEI-Sub-1's is synthetic

Weigh up the PEI-800 (0.0013 mole) of 1.0g and the DMSO that the 5ml of magnesium chloride standing and drying is passed through in adding.PEI dissolves fully, but forms muddy suspension.The suberoyl chlorine that drips 0.22ml (0.26g, 1.172,0.0013 mole of density) enters among the anhydrous DMSO of 5ml.All glass drying ovens are with burning dry-off moisture.Be added dropwise to suberoyl solution in the PEI solution with the hands jolting under room temperature.Form insoluble gel-like substance immediately.Gel is with excessive fresh anhydrous DMSO washing 1 time, by using Liang diox washing 2 times.The solvent that decantation is residual, gel chamber relaxing the bowels with purgatives of warm nature were placed 19 hours under indoor (house) vacuum.Even material has significant foul smell after vacuum drying.Sample carries out IR (microscope) and D ₂Proton N MR among the O.Proton N MR shows except desired water, has residual DMSO with diox.From files (file) sample, take out 50mg, remaining sample is dissolved in fully in the distilled water of about 10ml.Sample is placed into Dialysis tubing (cut-out of 3500 molecular weight) and dialysed 5 days under the gentle agitation of magnetic stirring bar with about 12 liters distilled water.Sample divides in two bottles and lyophilizing.After lyophilizing, be recovered to the sample of about 100mg.The correction of the 50mg that keeps as impure files sample, the forfeiture of about 90% material, most of may be by passing dialyzer.The structure of the proposal of PEI-800-Sub-1 is as shown in Fig. 3 a, and the IR of product is as in Fig. 3 b graphic extension.

Embodiment 7. adopts the SiRNA of OEI-HD-1 to knock out

The result who uses OEI-HD-1 to transmit siRNA for the HUH7/EGFPLuc cell summarizes in Fig. 4.Transfection is to carry out in the 96-orifice plate, uses 5,000 cells/well in the culture medium that does not contain serum (OptiMEM).The OEI-HD-1/siRNA preparation is to make in 20 μ l HBS (20mMHEPES, 150mM NaCl), and joins in the culture medium that does not contain serum (100 μ l cumulative volume) of 80 μ l.Transmitted back four hours, transfection media replaces with growth medium, measures luciferase activity two days later.Using 0.10 μ gsiRNA (40nM) and C/P ratio is 8/1 (OEI-HD-1/siRNA:w/w), and luciferase activity is up to 50% knock out to compare with the infection of using non-specific MutsiRNA and obtain.Be clarifying purpose, the C/P ratio refers to the weight ratio of carrier and plasmid, and it can be DNA or siRNA for purpose of the present invention.MutsiRNA is with comparing, and is the toxic good measure of carrier.If so adopt MutsiRNA to see the signal of reduction, it should not have biologic activity, then adopting being seen the knocking out of specific siRNA under same concentrations should be to the correction of carrier for the toxic effect of cell.

Use 0.25 μ gsiRNA (100nM) and C/P ratio 4/1, luciferase activity is up to 60%, and to knock out be the realization of comparing with the transfection of using non-specific MutsiRNA.For 0.50 μ g siRNA (200nM), be up to 80% and knock out and be observed.The siRNA (being up to 1.00 μ g siRNA (400nM)) of use higher concentration does not cause any further reduction of luciferase activity.Therefore, for transmitting among the HUH7/EGFPLuc that uses OEI-HD-1 (complexation among the HBS does not contain in the blood serum medium and infects), the maximum of luciferase expression knocks out can lead to 200nM (0.50 μ g siRNA/5,000 cell) and 2/1 acquisition of C/P ratio.

Embodiment 8. carries out SiRNA with OEI-HD-1 and knocks out in different buffer agent media

The ability that knocks out the OEI-HD-1 of luciferase expression is at different serum complexation medium (HBS:20mM HEPES, the 150mM NaCl of not containing; HBG:20mM HEPES, 5% glucose; OptiMEM: the culture medium that does not contain serum that salt reduces, Gibco) middle test.Do not rely on used complexation medium, the OEI-HD-1/siRNA preparation can knock out luciferase activity and be up to 80%, does not have significant difference between the complexation medium.The preparation of OptiMEM is efficiently at 100nM (C/P:2/1).This possibility is owing to the particulate very fast gathering of OEI-HD-1/siRNA causes.The result is as shown in Figure 5.

Embodiment 9. uses the SiRNA of OEI-HD-1 to knock out in containing serum medium

SiRNA transmits and uses OEI-HD-1 to carry out in the presence of serum (10% FCS) among the HUH7/EGFPLuc.The OEI-HD-1/siRNA preparation is to make at HBS (20 μ l), and complex adds in the culture medium that contains serum of 80 μ l on cell.Carry out two kinds of different processing; At first, the common conversion of culture medium was 4 hours after siRNA transmitted, and secondly, not conversion of culture medium reaches two days.After culture medium changed, the maximum of luciferase expression knocked out and is to use 200nM siRNA and C/P ratio 6/1 to obtain, and it is opposite with the best jump condition that reaches in the culture medium that does not contain serum: 200nM siRNA, C/P ratio 2/1.

Do not change culture medium, when the best jump condition that OEI-HD-1/siRNA transmits does not exist with serum identical (200nM siRNA, C/P ratio 2/1).In addition, do not change culture medium, OEI-HD-1 is that toxicity is much bigger, when C/P ratio 6/1 and 200nM, and cell death.What is interesting is that the w/o culture medium changes, 100nM siRNA and C/P ratio 4/1 are efficiently for the knocking out of expression of luciferase genes.

In a word, it also is effective that the siRNA under OEI-HD-1 carrier (vehicle) exists for serum transmits.Similar with the result in the culture medium that does not contain serum, being up to of luciferase expression 80% knocks out in the presence of 10% FCS and also observes.In addition, do not change culture medium, lower siRNA concentration is to be enough to obtain ceiling effect.The result sums up in Fig. 6 a and 6b.

Embodiment 10. adopts OEI-Sub-1 to fail SiRNA and knocks out

Use OEI (800Da) and suberoyl acid (suberoyl acid) (C ₆H ₁₂O ₂Cl ₂) synthetic OEI-Sub-1, the siRNA for the HUH7/EGFPLuc cell that uses different polymer/siRNA ratio transmits as cross-linking agent (as described in example 6 above) test.Carry out transfection in the 96-orifice plate, 5,000 cells/well are triplicate, use LucsiRNA (GL3) and MutsiRNA (IX) (Dharmacon).The OEI-complex prepares in HBS, and it is to carry out in the culture medium that does not contain serum 4 hours that siRNA transmits.Transfection media is to replace with growth medium then, 2 days measurement luciferase expression after siRNA transmits.The amount of siRNA is from 0.1 to 0.50 μ g/5,000 cell, and the OEI-Sub-1/siRNA ratio is also to change; Yet the silence of not observing the luciferase genes expression is shown in Fig. 7.

Embodiment 11.K5 simulating peptide (peptide mimetic) coupling agent OEI-HD-1

OEI-HD (2.8mg) is dissolved in the reaction buffer that contains 150mM sodium chloride and 20mM4-(2-ethoxy)-1-piperazine ethyl sulfonic acid (HEPES) of 1mL, and pH is 7.5.In this solution in 100% ethanol of N-succinimido 3-(2-pyridine radicals dithio) propionic ester (SPDP) (80 μ g) adding 80 μ L, stir simultaneously.Make reaction continue 90 minutes.The activated OEI-HD of SPDP-is then by using the gel filtration purification of Sephadex G-25.In second step, the K5 peptide that contains cysteine at the N-end of 3-times of molar excess joins in the identical buffer.Reaction was at room temperature carried out 12 hours, yet by using the gel filtration purification of Sephadex G-25.

Embodiment 12.RGDC (cysteine end) peptide coupling agent OEI-HD-1

OEI-HD (2.8mg) is dissolved in the reaction buffer of the 4-that contains 150mM sodium chloride and 20mM (2-ethoxy)-1-piperazine ethyl sulfonic acid (HEPES) of 1mL, pH is 7.5.In this solution in 100% ethanol of N-succinimido 3-(2-pyridine radicals dithio) propionic ester (SPDP) (80 μ g) adding 80 μ L, stir simultaneously.Make reaction continue 90 minutes.The activated OEI of SPDP-is then by using the gel filtration purification of Sephadex G-25.In second step, the RGDC peptide of 3-times of molar excess joins in the identical buffer.Reaction was at room temperature carried out 12 hours, yet by using the gel filtration purification of Sephadex G-25.

The coupling of embodiment 13. antibody fragments (Fab ')

Use is derived from people's such as Merdan Bioconjugate ChemistryThe 14th volume, the method for the 989th page (2003), OEI-HD (2.8mg) is dissolved in the reaction buffer of the 4-that contains 150mM sodium chloride and 20mM (2-ethoxy)-1-piperazine ethyl sulfonic acid (HEPES) of 1mL, and pH is 7.5.N-succinimido 3-(2-pyridine radicals dithio) propionic ester (SPDP) (80 μ g) is in this solution that adds in 100% ethanol of 80 μ L, stirs simultaneously.Use reaction to continue 90 minutes.The activated OEI of SPDP-is then by using the gel filtration purification of Sephadex G-25.

In second step, the fresh reductive Fab ' of 1.4-times of molar excess joins in the identical buffer.Reaction was at room temperature carried out 12 hours, and purification is by using 0.9%NaCl, 10mMHEPES pH7.4 to finish as the GEC method of buffer B and MacroPrep HighS (from Amersham Pharmacia) as buffer A and 3M NaCl, 10mM HEPES pH7.4.

Embodiment 14. arrives OEI-HD by the HMDI activation with polyethylene glycol conjugation

PEG monomethyl ether (0.5-20kDa) is dissolved in anhydrous chloroform (200g/L), uses 60 ℃ the doubly excessive hexamethylene diisocyanate of 10-, HMDI activation 24 hours.Unreacted HMDI extracts repeatedly by light gas and oil (light petrol) and carefully removes.Subsequently, being reflected in 60 ℃ the anhydrous chloroform of the amino of the PEG of isocyanates-end and OEI-HD carried out 24 hours.The degree of PEGization can be regulated by the ratio that changes activatory PEG and OEI-HD.Reaction solution precipitates the product vacuum drying in diethyl ether or other non-solvents that is fit to.

Embodiment 15. by PEG-NHS with polyethylene glycol conjugation agent OEI-HD

PEG-N-N-Hydroxysuccinimide base ester with desired molecular weight is dissolved in DMSO and is added in the aqueous solution of OEI-HD-1.Reaction was stirred 24 hours, and the PEG fluidized polymer is to separate by for example molecular-exclusion chromatography.

Embodiment 16. ring-type RGD peptides are by the targeting of the PEG-basic coupling in interval with formation PEG shielding Carrier

According to Nucleic Acids Research, 32 volumes,149 pages of disclosed methods in 2004 are with RGD coupling agent Polyethylene Glycol.The chemical combination (conjugate) of Polyethylene Glycol and RGD (H-ACRGDMFGCA-OH) is at first synthetic.This is to form the ring-type 10-mer RGD peptide with disulfide bridge bond by two cysteine residues of oxidation to realize.Then, the cyclic peptide of 60mg is dissolved in the dimethyl sulfoxine (DMSO) of 600 μ l.The triethylamine (TEA) that is dissolved in 8.54 μ l in the 20 μ l oxolanes in advance adds in the peptide under nitrogen protection gas.After stirring 1 minute, activatory PEG is that (212mg is in THF:DMSO for NHS-PEG-VS; 300 μ l:100 μ l) the disposable adding of solution is so that the amino terminal reaction of n-N-Hydroxysuccinimide (NHS) group on the PEG and peptide.Reactant mixture at room temperature stirred 4 hours, used trifluoroacetic acid (TFA) quencher with the TEA equivalent.RGD-PEG-VS is by using the distill water dialysis purification, then lyophilizing.In second step, the RGD-PEG-VS intermediate of the purification of 100mg (21.7 μ mol) is dissolved in the anhydrous DMSO of 1ml.In this solution, add the six normal TEA and the mixing that are dissolved among the 0.5ml THF.The aliquot OEI-HD that is dissolved in the 9.4mg (218 μ mol are in amine) of dimethyl formamide (0.5ml) adds so far in the solution, and at room temperature stirs 12 hours so that the Michael addition takes place for amine on the OEI and the vinyl sulfone on the PEG (VS).Product passes through the HPLC purification as tfa salt.

Embodiment 17.OEI-HD prodrug

With an end of paclitaxel coupling agent peg molecule, as ( Materials Research Innovations the 9th volume, 13-14 page or leaf, 2005) and described.Other C-terminals of PEG chain are to react to prepare activatory PEG end group with three chloro-s-triazines.Activatory PEG is that the OEI-HD in the water with pH9.0 combines, so that PEG-paclitaxel coupling agent OEI-HD carrier.This OEI-HD-PEG-paclitaxel part has the targeting part with other and the OEI-HD polymer of the specific siRNA of target cell is combined, to form polycation complex (polyplex).This polycation complex has the transmission agent of interference cell biography record (siRNA) and transmits the agent of minicell toxin is the character of paclitaxel.

Purposes in the body of the polycation of the chemical modification that embodiment 18.RAN-siRNA transmits

RAN siRNA is the siRNA of facedown RAN GTPase.This kind of enzyme is that essential knocking out with expression causes poisonous effect for most cells.15 (15) mices generate local tumor at back subcutaneous injection 1,000,000 Neuro 2A tumor cells, and it allows to grow to the size of 3mm diameter when using slide calliper rule by skin measurement.Mice is divided into 3 groups, every group of 5 animals, that is, and treatment group, matched group and vehicle group.The treatment winding is subjected to polycation to be complexed to the RAN-siRNA of OEI-HD carrier, and weight ratio is 0.6 to 1, and carrier contains the OEI-HD-PEG-Tf of 10% weight.Transferrin (Tf) is that at interval base is covalently bound to OEI-HD by PEG, and as the targeting agent because target tumor known be the receptor of expressing transferrin.Matched group is accepted the siCONTROL (contrast siRNA is deactivation biologically) that polycation is complexed to OEI-HD, and part by weight is 0.6 to 1, and carrier contains the OEI-HD-PEG-Tf of 10% weight.Last winding is cushioned excipient, the buffered glucose of hepes.Each mice was accepted three (3) 200-microlitre injections in three days, by the tail vein.Each treatment injection contains the siRNA of 35 micrograms, and each contrast injection contains the siCONTROL of 40 micrograms.Vehicle group is only accepted the buffer of 200 microlitres.All animals are measured tumor size and the weight of animals every day.

After mice is injected first, make its life 8 days.Do not see losing weight at any experimental group owing to research.The tumor growth curve of siControl and buffer group is substantially the same; Yet, change in the positivity mode at the growth curve of the 3rd day RANsiRNA treatment and to slow down the speed shown in Fig. 8.

Purposes in the body of the polycation of the chemical modification that embodiment 19.RAF-1-siRNA transmits

Repeat the experimental program described in the embodiment 20, but replace the SCID-mice, 5,000,000 HuH7 tumor cell of liver are to produce tumor and RAF-1-siRNA in the treatment group.The direct anti-RAF-1 of RAF-1-siRNA-siRNA is for the important biological molecule of a lot of cancerous cell.

Embodiment 20. is used for using in the body of polycation of the chemical modification that PLK-1-SiRNA transmits On the way

Experimental program described in the repetition embodiment 19, but known expression PLK-1 and verified replaced at tumor cell with showed cell death after using PLK-1 siRNA transfection in the cell culture.The direct anti-polo sample kinases 1 of PLK-1-siRNA-siRNA (a kind of important regulator of cell cycle growth).Expression knocks out and causes poisonous effect.

The formation and the sign of the polycation complex of embodiment 21. embodiment 19

The sign of polycation complex size is to use Malvern Zetasizer Nano ZS to carry out.This instrument can be measured the commercial measurement granularity that Zeta potential also passes through quasielastic laser light scattering by conventional means.1.1 the OEI-PEG-transferrin of the OEI-HD of microgram and 0.12 microgram is the HBG that is mixed into cumulative volume 25 microlitres (the buffered glucose of HEPES contains 5% glucose solution (weight/volume) of HEPES of the pH7.3 of 20mM).The siRNA of each amount (2.0 microgram) uses HBG to be diluted to 25 microlitres in another bottle.Subsequently, polymer solution adds siRNA solution, mixes for 10 times by container inverted.The suspension of gained polycation complex has the granularity of 200-300nm and-1.3 millivolts Zeta potential.Zeta potential be with particle stabilized sheath and moving iron diffusion layer between colloidal solid in the electric field on surface of shearing force move relevant current potential.

Embodiment 22. activatory bifunctional PEG are coupled to nucleic acid carrier OEI-HD-1, then transport the ferrum egg Be coupled to the activatory PEG end group of side chain in vain

OEI (34K), 300mg (～9 x 10 ^-6Mole) is dissolved in the 100mM HEPES buffer of 5ml pH8.6.In the bottle that separates, (NMR counts 66% purity to 75mg ,～1 x 10 ^-5Mole) OPSS-PEG (5K)-SPA (neighbour-pyridyl disulfide-Polyethylene Glycol-succinyl phosphorons amino propyl acid ester) is dissolved in 4ml ethanol.Two kinds of solution merge, and at room temperature stir 2 hours.Reaction solution carries out ion exchange chromatography subsequently, collects the fraction that contains OPSS-PEG-OEI.The volume of purified product uses the Centricon concentrator to be reduced to 1ml.Product uses PD10 pillar (pre-saturated W/OEI) to remove freshen.This is to finish by the water that the sample solution of 1ml adding pillar is then added 1.5ml.Abandon initial effluent (flow-through), sample is eluting in 2ml water.This solution lyophilizing also uses proton N MR to analyze, and it shows that per 714 OEI repetitives have a pyridine radicals dithio.This intermediate called after OEI-HD-1-Polyethylene Glycol-2-pyridine radicals dithio-propionic acid amide. or abbreviate " OEI-PEG-OPSS " as.

OEI-PEG-OPSS is reduced into OEI-PEG-SH

The lyophilizing OEI-PEG-OPSS of 25mg is dissolved in 1.5 100mM HEPES buffer.Subsequently, 40mg (～2.6 x 10 ^-4Mole) DL-dithiothreitol, DTT (DTT) adds sample solution, stirs 30 minutes.For the process of monitoring reaction, the sample solution of 10 μ l is diluted to 500 μ l with 100mM HEPES buffer, uses the UV/VIS photometer to absorb scanning.Local maximum is observed at Zai @343nm place, confirms the progress of this reaction.Use the PD10 post, purification reaction solution.This be by.The reaction solution of 1.5ml is added pillar (with W/OEI pre-saturated and with HBS buffer balance) add then that the HBS buffer (before the use, HBS buffer with W/ argon bubble) of 1ml carries out.Abandon initial effluent, sample 2ml HBS eluting.The concentration of OEI-PEG-SH sample solution is 5mg/ml, as measuring by copper complexing algoscopy.In order to preserve, before the sealing, sample solution was to use the argon bubble 5 minutes.

Use N-succinimido 3-(2-pyridine radicals dithio)-propionic ester (SPDP) to modify transferrin

Transferrin (60mg) is dissolved in the HBS buffer of 3ml.Subsequently, the SPDP of 3.5mg is dissolved in the ethanol (careful heated solution is used for dissolving fully) of 7ml.Immediately the SPDP drips of solution of 0.5ml was added transferrin solution and gentle agitation 1 hour.

Reaction solution is transferred to YM-10 Centricon concentrator to reduce volume to 1ml.Add the 1ml buffer-exchanged again, on the Centricon concentrator, finish.The transferrin of purification-SPDP solution is used argon bubble 5 minutes.

With transferrin-SPDP coupling OEI-PEG-SH

The OEI-PEG-SH of 10mg that is dissolved in the HBS buffer of 2ml is merging in new scintillation vial with the purification of 2.7ml and activatory transferrin solution, and at room temperature gentle agitation whole night.Then, unreacted sulfydryl (sulfhydryl) is with 5mg (4 x 10 ^-5Mole) N-ethyl-maleimide quencher stirred 30 minutes.

The OEI-PEG-transferrin is by the ion exchange chromatography purification.Use the Centricon concentrator to reduce volume, according to the previous described salinity of removing product.

Concentration by polyamines (OEI) among the bioconjugate of copper compleximetric determination method mensuration is 1.3mg/ml, and according to the mensuration of UV spectrographic method, transferrin is measured under 2.9mg/ml.

Ferrum is mixed transferrin

Is the ferrum transferrin of packing into to add a sample and carry out by the ferrum buffer that carries every milligram of transferrin content 1.25 μ l, according to Kursa M, and Walker GF, Roessler V, Ogris M, Roedl W, Kircheis R, Wagner E. is described in the Bioconjugate Chem.2003.

Use the displacement algoscopy to measure the binding affinity of siRNA in conjunction with OEI-PEG 5K-transferrin

0.01mg/ml siRNA solution be to make from non-specific siRNA stock solution.The siRNA solution of 20 μ l is transferred to some little plastic bottles, then increases the amount of OEI-PEG5K-transferrin in the HBG buffer, keep the cumulative volume in each bottle constant at 40ul.The weight ratio scope of OEI-PEG 5K-transferrin and siRNA is 0-10.After moving liquid, fully thing in the hybrid bottle left standstill 10 minutes.Then, its that is transferred to agarose gel plate from the 20 μ l aliquots of each bottle is in should the hole.Agarose gel plate is positioned in 75 volts the electrophoresis apparatus.After 15 minutes, remove plate, photoelectric measurement.We find, do not observe siRNA migration band in weight ratio 1.5 or higher hole.This shows, all siRNA are 1.5 with during the Geng Gao weight ratio and OEI (34K)-PEG (5K)-transferrin complexation.

Embodiment 23: the chemical compound of benzoyl benzoic acid coupling agent formula I or Ia

(the relative flat light emission of RLU=; C:P+ carrier and plasmid or siRNA ratio w/w; The anti-luciferase siRNA of luc=; Scr=scrambler siRNA contrast; With lip=fat transfection amine transfection agents)

Use benzoyl benzoic acid-succinimido ester (Invitrogen # 1577), in HBS (Hepes buffer saline, 10mM HEPES, 150mM NaCl, pH7.3), finish reaction.20mg polymer (following) is dissolved in 2ml HBS, regulates pH to 7.3.Then, not commensurability benzoyl benzoic acid-NHS ester joins among the anhydrous DMSO of 1ml.

Before the use deionized water was diluted to about 6ml, total overall reaction was carried out 12 hours.Subsequently, dialyse conjugate lyophilizing after this.

Table 1

Used dialyzer:

Table 2

The link coupled practical extent of synthetic conjugate

Sample lyophilization and be dissolved in HBS (Hepes buffered saline, 10mMHEPES, 150mM NaCl, pH7.3) once more.These solution concentrations are that unmodified relatively polymer uses the copper algoscopy to determine.

By dynamic laser diffuse transmission measuring size

0.5 the OEI complexation of each amount in the siRNA of microgram and the 50 microlitre cumulative volumes.

Table 3

C:P	PEI 2Kbenzo-1 is low	Among the PEI 2Kbenzo-2	PEI 2Kbenzo-3 height
				0.3	254	370	392
0.6	202	239	204
				1.2	178	198	194
2.0	182	195	189

Table 4

C:P	OEI?5Kbenzo-1	OEI?5Kbenzo-2	OEI?5Kbenzo-3
				0.3	370	72	mult
0.6	303	350	195
				1.2	196	210	260
2.0	190	121	99

The mult=multiplet

Table 5

C:P	OEI?30Kbenzo-1	OEI?30Kbenzo-2	OEI?30Kbenzo-3
				0.3	436	457	342
0.6	151	mult	249
				1.2	mult	mult	116
2.0	mult	mult						120

Table 6

Knock out efficient H1299 cell, 0.5 microgram siRNA/ hole

Table 7

Table 8

Table 9

Table 10

Embodiment 24: lauric acid is coupled to the chemical compound of formula I or Ia

Use lauric acid N-hydroxyl-succinimido ester (Sigma-Aldrich # OL3900-5g, Lot # 087H5174), (150mMNaCl reacts in pH7.3) for Hepes buffered saline, 10mM HEPES at HBS.

The 20mg polymer dissolution is regulated pH to 7.3 in 2ml HBS.Then in the anhydrous DMSO of 1ml, add not commensurability lauric acid NHS ester.

Table 11

PEI 800 Da and PEI 2 kDa

Table 12

OEI5kDa, 9kDa and 30kDa

20mg mg lauric acid NHS acid/PEI comment

5kDa 2.4 2 does not have

5kDa 11.9 10 does not have

5kDa 29.7 25 muddinesses

9kDa 2.7 4 does not have

9kDa 10 15 does not have

9kDa 27 40 precipitations

30kDa 1.2 6 does not have

30kDa 5 25 does not have

30kDa 20 100 precipitations

Before the use deionized water was diluted to about 6ml, total overall reaction was carried out 12 hours.Subsequently, dialyse conjugate lyophilizing after this.

Table 13

Subsequently, conjugate is dissolved among the D2O, carries out ¹H-NMR.

Following actual coupling degree is measured by NMR.

Coupling degree based on NMR calculating:

Table 14

Sample lyophilization and be dissolved in HBS (Hepes buffered saline, 10mMHEPES, 150mM NaCl, pH7.3) once more.

The unmodified relatively polymer of these solution concentrations uses the copper algoscopy to determine.

By dynamic laser diffuse transmission measuring size

0.5 the siRNA of microgram be with 50 microlitre cumulative volumes in the OEI complexation of each amount.Experiment is carried out in HBS.

Table 15

Table 16

Table 17

Pure (plain) OEI 9k obtains multiplet in gamut!

Table 18

Knock out efficient H1299 cell, 0.5 microgram siRNA/ hole.

PEI 800 and PEI2k and derivant thereof

Table 19

Table 20

Table 21

OEI 5k and derivant thereof

Table 22

Table 23

Table 24

OEI 9k derivant

Table 24

Table 25

OEI 30 k derivants

Table 26

Table 27

Embodiment 23: lauric acid coupling PEIs/OEIs, and the physical chemistry of conjugate and commenting biology Valency

Use lauric acid N-hydroxyl-succinimido ester (Sigma-Aldrich # OL3900-5g, Lot # 087H5174), reaction is that (Hepes buffered saline, 10mM HEPES, 150mM NaCl carry out in pH7.3) at HBS.

The 20mg polymer dissolution is regulated pH to 7.3 in 2ml HBS.Then, not commensurability lauric acid NHS ester adds among the anhydrous DMSO of 1ml.

PEI 800 Da and PEI 2 kDa

Table 28

OEI 5kDa, 9 kDa and 30 kDa

Table 29

The every PEI comment of 20mg mg lauric acid NHS acid

5kDa 2.4 2 does not have

5kDa 11.9 10 does not have

5kDa 29.7 25 muddinesses

9kDa 2.7 4 does not have

9kDa 10 15 does not have

9kDa 27 40 precipitations

30kDa 1.2 6 does not have

30kDa 5 25 does not have

30kDa 20 100 precipitations

Before the use deionized water was diluted to about 6ml, total overall reaction was carried out 12 hours.Subsequently, dialyse conjugate lyophilizing after this.

Table 30

Subsequently, conjugate is dissolved among the D2O, is used for ¹H-NMR.

Following actual coupling degree is to measure by NMR.

Coupling degree based on NMR calculating:

Table 31

Sample lyophilization and be dissolved in HBS (Hepes buffered saline, 10mMHEPES, 150mM NaCl, pH7.3) once more.

These solution concentrations are that unmodified relatively polymer uses the copper algoscopy to determine.

By dynamic laser diffuse transmission measuring size

0.5 the OEI complexation of each amount in the siRNA of microgram and the 50 microlitre cumulative volumes.Experiment is finished in HBS.

Table 32

Table 33

Table 34

Pure OEI9k obtains multiplet in gamut!

Table 35

Pure OEI 30k obtains multiplet in gamut!

Knock out efficient H1299 cell, 0.5 microgram siRNA/ hole.

PEI 800 and PEI2k and derivant thereof

Table 36

Table 37

Table 38

OEI 5k and derivant thereof

Table 39

Table 40

Table 41

OEI 9k derivant

Table 42

Table 43

OEI 30 k derivants

Table 44

Table 45

Embodiment 24: benzoylbenzoic acid is coupled to PEIs/OEIs, the physical chemistry of conjugate and biology Learn assessment

Using benzoylbenzoic acid-succinimido ester (Invitrogen # 1577), reaction is that (150mM NaCl carries out in pH7.3) for Hepes buffered saline, 10mM HEPES at HBS.

The 20mg polymer dissolution is regulated pH to 7.3 in 2ml HBS.Then, not commensurability benzoylbenzoic acid-NHS ester adds among the anhydrous DMSO of 1ml.

Before the use deionized water was diluted to about 6ml, total overall reaction was carried out 12 hours.Subsequently, dialyse conjugate lyophilizing after this.

Table 46

Used dialyzer:

Table 47

The link coupled practical extent of synthetic conjugate

Table 48

Sample lyophilization and be dissolved in HBS (Hepes buffered saline, 10mMHEPES, 150mM NaCl, pH7.3) once more.

The unmodified relatively polymer of these solution concentrations uses the copper algoscopy to determine.

By dynamic laser diffuse transmission measuring size

0.5 the OEI complexation of each amount in the siRNA of microgram and the 50 microlitre cumulative volumes.

Table 49

C:P	PEI 2Kbenzo-1 is low	Among the PEI 2Kbenzo-2	PEI 2Kbenzo-3 height
				0.3	254	370	392
0.6	202	239	204
				1.2	178	198	194
2.0	182	195	189

Table 50

C:P	OEI?5Kbenzo-1	OEI?5Kbenzo-2	OEI?5Kbenzo-3
				0.3	370	72	mult
0.6	303	350	195
				1.2	196	210	260
2.0	190	121	99

The mult=multiplet

Table 51

C:P	OEI?30Kbenzo-1	OEI?30Kbenzo-2	OEI?30Kbenzo-3
				0.3	436	457	342
0.6	151	mult	249
				1.2	mult	mult	116
2.0	mult	mult						120

Knock out efficient H1299 cell, 0.5 microgram SiRNA/ hole

Table 52

Table 53

Table 54

Table 55

Table 56

Claims (46)

1, a kind of polymer, it forms by the polycation that propiono amide units chemistry connects, and wherein said polymer is used as the non-virus carrier that nucleic acid transmits.

2, the polymer of claim 1, wherein polycation is polymine (PEI).

3, the polymer of claim 1, it is used for siRNA and transmits.

4, the polymer of claim 1, it further contains the shielding part.

5, the polymer of claim 4, wherein shielding part is Polyethylene Glycol (PEG).

6, the polymer of claim 5, it further comprises the targeting part.

7, the polymer of claim 6, wherein the targeting part is a transferrin.

8, the polymer of claim 4, it further comprises and the polynucleotide coupling.

9, the polymer of claim 5, it further comprises and the polynucleotide coupling.

10, the polymer of claim 6, it further comprises and the polynucleotide coupling.

11, the polymer of claim 7, it further comprises and the polynucleotide coupling.

12, a kind of method for preparing the polymer of claim 1, wherein polymer be the part Michael addition of the amine by polymer to the vinyl groups of cross-linking agent and crosslinked, and further come modification by the N-acidylate of side chain ester group.

13, the method for claim 12, wherein polymer is further modification by the addition of free ester, anhydride or carboxylic acid halides.

14, the method for claim 12 is on the wherein crosslinked primary amine and secondary amine two places that can occur in polymer architecture.

15, the method for claim 12, wherein cross-linking agent comprises the ester monomer of following reagent: acrylate, methacrylate, glycol diacrylate, ethylene glycol dimethacrylate, 1,6-hexanediyl ester and Macrogol 600 diacrylate.

16, the method for claim 15, wherein cross-linking agent is 1, the 6-hexanediyl ester.

17, a kind of method that transmits gene to target tissue in vivo, it uses the polymer of claim 1.

18, a kind of method that siRNA is sent to cell in culture, it uses the polymer of claim 1.

19, a kind of method that in vivo siRNA is sent to tissue, it uses the polymer of claim 1.

20, the method for claim 19, wherein the transmission of siRNA is to be used for the treatment of purpose or to be used for target spot to confirm.

21, a kind of method of using the polymer of claim 1, but the chelating agent of polymer formation targeting wherein, described chelating agent can and be sent to target tissue in conjunction with the entity of opposite charges.

22, a kind of polymer that uses claim 1 transmits the method for justice treatment entity to the patient, and wherein polymer forms ionic complex or covalent bond with justice treatment entity is arranged.

23, the method for claim 22 wherein has justice treatment entity to comprise cytotoxic agent, Inclusion lytic agent and hydrophilic polymer.

24, formula I chemical compound, it comprises:

[[polycation] _a-[L] _b-[polycation] _c] _d

Wherein:

Polycation is the polyethyleneimine: amine unit;

L is non-ester connector;

A is the integer of about 1-about 20;

B is the integer of about 1-about 10;

C is the integer of about 1-about 20; With

D is the integer of about 1-about 1000.

25, the chemical compound of claim 24, wherein polycation is the oligomerisation of ethylene imines.

26, the chemical compound of claim 24, wherein L is selected from acylamino-connector part, amino connector part, carbonyl connector part, carbamate connector part, urea connector part, ether connector part, disulphide connector part and succinoamino connector part.

27, the non-ester connector of claim 26, wherein L is a beta-amino propiono amide connector part.

28, the chemical compound of claim 24, it further comprises biomolecule.

29, the chemical compound of claim 28, wherein said biomolecule is siRNA.

30, the chemical compound of claim 24, its weight average molecular weight range that has are that about 800 dalton are to about 1,000,000 dalton.

31, the chemical compound of claim 24, its weight average molecular weight range that has are that about 20,000 dalton are to about 200,000 dalton.

32, the chemical compound of claim 24, its weight average molecular weight range that has are that about 20,000 dalton are to about 30,000 dalton.

33, formula I chemical compound, it comprises:

Wherein:

Polycation is a polymine;

L is non-ester connector part;

S is basic at interval or does not exist;

A is reagent or does not exist;

A is the integer of about 1-about 20;

B is the integer of about 1-about 10;

C is the integer of about 1-about 20; With

D is the integer of about 1-about 1000.

34, the chemical compound of claim 33, wherein polycation is the oligomerisation of ethylene imines.

35, the chemical compound of claim 33, wherein L is selected from acylamino-connector part, amino connector part, carbonyl connector part, carbamate connector part, urea connector part, ether connector part, disulphide connector part and succinoamino connector part.

36, the non-ester connector of claim 33, wherein L is a beta-amino propiono amide connector part.

37, the chemical compound of claim 33, it further contains biomolecule.

38, the chemical compound of claim 37, wherein said biomolecule is siRNA.

39, the chemical compound of claim 33, it further comprises reagent and optional S.

40, claim 38 or 39 chemical compound, wherein said reagent be selected from promote receptor identification, internalization, biomolecule from intracellular escape, nucleus is fixed, biomolecule discharges and the reagent of system stability.

41, claim 38 or 39 chemical compound, wherein said reagent is selected from cytotoxic agent, hydrophobic group, screener and targeting part.

42, the chemical compound of claim 11, wherein said reagent is transferrin.

43, a kind of non-viral transfer system, it comprises: (a) biomolecule; (b) be coupled to the chemical compound of biomolecule, wherein chemical compound-biomolecule conjugate comprises the chemical compound of claim 24 or 33.

44, the non-viral transfer system of claim 45, wherein said biomolecule is siRNA.

45, the mammiferous method of a kind of treatment, this method comprise identifies the mammal need gene therapy, and to the chemical compound of mammal administration claim 28, wherein said biomolecule is effectively to reduce the siRNA that adopted gene expression is arranged.

46, the mammiferous method of a kind of treatment, this method comprise identifies the mammal need gene therapy, and to the chemical compound of mammal administration claim 28, wherein said biomolecule is effectively to reduce the siRNA that adopted gene expression is arranged.

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