CN107049965A

CN107049965A - Nucleic acid lipopolymer compositions

Info

Publication number: CN107049965A
Application number: CN201610978325.6A
Authority: CN
Inventors: 马杰德.马塔; 贾森.费韦尔; 丹尼.H.刘易斯; 库希德.安沃
Original assignee: CLSN LAB Inc
Current assignee: CLSN LAB Inc
Priority date: 2007-08-06
Filing date: 2008-08-06
Publication date: 2017-08-18
Also published as: CN101820864A

Abstract

The invention provides composition, the methods and applications of increase nucleic acid transfection efficiency.On the one hand, it is at least about 0.5mg/ml nucleic acid that a kind of pharmaceutical composition, which can be included with the concentration of cationic lipid polymer condensation being suspended in isotonic solution, wherein described cationic lipid polymer is included with the cholesterol being covalently attached with its main chain and the ionene polymer backbone of polyethylene glycol, and the mol ratio of wherein cholesterol and ionene polymer backbone is about 0.1~about 10, and the mol ratio of polyethylene glycol and ionene polymer backbone is about 0.1~about 10.The composition can also include filler excipient.

Description

Nucleic acid-lipopolymer compositions

The application is the applying date for August in 2008 6 days, Application No. 200880110306.5, denomination of invention " nucleic acid-fat The divisional application of the patent application of polymer composition ".

Technical field

The present invention relates to the concentrated, stable preparation for including nucleic acid and lipopolymer, further relate to the preparation preparation method and Using.Therefore, the present invention relates to molecular biology and biochemical field.

Background technology

Due to its preferably safe compliance, simple chemical and cost-effective manufacture, synthetic gene transport vehicle tool There is advantage more significant than viral vector.However, due to synthetic vectors transfection efficiency relatively low compared with viral vector, thus greatly Most exploitations to synthesizing gene transfer systems are directed to improving efficiency of transmission.As a result, although in preparation stability, expanding scale With administration flexibility in have been found that problem, but to synthesize transmission system drug development be almost not given to concern.From group Fill and usually show poor stability for the medicine containing DNA of nano particle, particularly when preparation is waterborne suspension.At this In class preparation, the DNA with synthetic vectors would generally be carried out with the time and assembled, the optimal administration institute especially in clinical settings During the concentration needed.This kind of preparation is often difficult to prepare concentration>0.3mg/ml DNA is especially right which has limited its business application The local conveying being flexibly administered can be limited in the constraint of wherein volume.DNA aggregations reduction eliminates DNA activity and therefore made Composition is unsuitable for using in the treatment.

The physical instability is one of fundamental cause of transfection activity loss.Due to the higher curvature or lipid of lipid bilayer The breakage of particles or fusion for dissociating and showing from DNA physics have also been assumed that the gene delivery based on cation lipid is answered The bad stability of compound and the possible basic reason of aggregation.The Oxidative hydrolysis chemical modification of such as transport vehicle may also Facilitate particle unstability.

Due to bad stability, early studies in man needs to prepare DNA preparations in beside sickbed.Do not possess preparation and store dense It is main barrier of the viral DNA product in extensive clinical practice and commercialization to spend for the ability of the clinical prods needed for optimal administration Hinder.This will need the doctor of trained pharmaceutical formulation and proposes quality control method on the spot.

Desivac is a kind of method of the useful long-time stability for improving a variety of medicines.However, this method is general It is unsuitable for drying the DNA compounds with synthetic vectors, because being easy to change the physical chemistry of the DNA compounds Property simultaneously causes aggregation and the loss transfected at rehydration (reconstitution).

If having had attempted to drying method to avoid preparation from assembling and damage in freeze-drying process.In some cases, such as Lyophilized dna compound can be provided more product in the presence of the cryoprotectors such as low molecular weight sugar, glucan and polyethylene glycol Good stability, but this method does not appear to improvement administration flexibility.Usual most common method is addition for this purpose Sugar.Have found it is many in test sugar can avoid damage and the particle aggregation of preparation to a certain extent, but the effect quality with The species of sugar and the difference of transport vehicle used and change.

Although the lyophilized certain improvement provided to preparation storage life, the condition required for production lyophilized dna product causes It is simply possible to use in limited pharmaceutical applications.Even if using maximally effective freeze drying protectant sugar, it is also desirable to very high sugar/DNA mol ratios (it is typically larger than 1000:1) to obtain stability.As a result, it is often necessary to freeze-drying prods are diluted into very big multiple isotonic to obtain Preparation, and this causes the DNA concentration that final DNA concentration is down to before freezing.For many cation carriers, final DNA concentration leads to Often it can be about 0.1mg/ml~0.2mg/ml, and often less than 0.1mg/ml.Although low concentration formulation is enough to be used in vitro study, Its clinical practice may be restricted because of the high volume needs being most preferably administered.For example, in the optimal sugared concentration needed for stability Under, 1mg DNA dosage may need to be diluted in keep isotonicity in 5ml~10ml, and this in local volume for applying Volume is excessive.It is that effect is not in human clinical trial for synthetic gene transmission system to inhibit this pharmacy that is flexibly administered to limit Preferably mainly facilitate one of factor, and confirm the DNA preparations more concentrated of needs stabilization and bioactivity.

The content of the invention

The invention provides unexpected stability is shown under high nucleic acid concentration and increases the effect of nucleic acid transfection Rate and the composition of administration flexibility.Composition as described herein can effectively be freezed and rehydration is to include high nucleic acid concentration to exist Interior various nucleic acid concentrations, without losing bioactivity or assembling nucleic acid.

On the one hand, the invention provides include cationic lipid polymer and at least about mixture of 0.5mg/ml nucleic acid Composition, preferably pharmaceutical composition, wherein the mixture is suspended in the aqueous solution.The cationic lipid polymer is included With the cholesterol being independently covalently attached to it and the ionene polymer backbone of polyethylene group.Cholesterol and cation The mol ratio of main polymer chain is about 0.1~about 10, and the mol ratio of polyethylene glycol and ionene polymer backbone be about 0.1~ About 10.The composition can also include filler excipient.In some aspects, the mixture of nucleic acid and lipopolymer forms compound Thing.In some aspects, the composition includes the nucleic acid of condensation.The amount of the nucleic acid of condensation generally depends on the composition structure of nucleic acid Into with condition when preparing composition.

Present invention also offers the method for preparing above-mentioned composition.

On the other hand, the invention provides nucleic acid and the freeze-dried composition of lipopolymer.The freeze-dried composition of the present invention, Preferably freeze-drying medicinal composition includes filler excipient, condensation nucleic acid and cationic lipid polymer.As described above, cationic lipid Polymer is included with the cholesterol being covalently attached to it and the ionene polymer backbone of polyethylene group, and wherein courage is consolidated The mol ratio of alcohol and ionene polymer backbone is about 0.1~about 10, and mole of polyethylene glycol and ionene polymer backbone Than being about 0.1~about 10.

In addition, present invention also offers controlled using composition as described herein for example, by transfecting various cells and tissue The method for treating disease and/or illness.

Brief description of the drawings

Fig. 1 is the schematic diagram of the manufacturing process of the present invention.

Fig. 2 shows the figure of the nucleic acid particle diameter under enrichment stage and non-concentrated state.

Fig. 3 shows the results in electrophoresis with the condensation of show nucleic acid.

Fig. 4 shows the figure of the transfection activity of another embodiment of the invention.

Fig. 5 A and Fig. 5 B are the nerve sections for showing the result treated of lipopolymer using carrying and without IL-12 Photo.

Fig. 6 shows two width figures of the anticancer efficacies of the IL-12 with lipopolymer compared with the control.

Fig. 7 A and Fig. 7 B are the figures of the particle diameter of different nucleic acid/cationic polymer mixtures.

Fig. 8 A and Fig. 8 B are the figures of the luciferase expression as caused by different nucleic acid/cationic polymer mixtures.

Fig. 9 is the figure of show nucleic acid/bioactivity of the cationic lipid polymer composition after long term storage.

Embodiment

Before the present invention is disclosed and described, it will be appreciated that the invention is not restricted to specific structure disclosed herein, side Method step or material, and may extend to affiliated structure, method and step or the material of those of ordinary skill in the related art's accreditation Equivalent.It should also be understood that because the scope of the present invention is only by the limitation of appended claims and its equivalent, therefore use herein Term is only used for describing the purpose of particular implementation and being not intended to be limited.

It must be noted that unless context is clearly it is further noted that as this specification and the appended claims, Singulative " a ", " an " and " the " also includes plural thing.

As used herein, term " nucleic acid of condensation " and " nucleic acid of partial condensates " be used to refer to the present invention sun from The nucleic acid of sub- lipopolymer contact.In some aspects, condensation nucleic acid keeps contacting with cationic lipid polymer phase.Nucleic acid is condensed to lead to Chang Bifei condensation nucleic acid occupies significantly smaller volume.It is recognized, however, that condensation nucleic acid amount may with local environment change And different (for example, lipid in aqueous environments).In the different aspect of the present invention, condensation nucleic acid is that nucleic acid and cationic lipid are poly- Those nucleic acid in the nano particle of compound, the size of the nano particle condensation is about 50nm~about 300nm, is even more preferably about 50nm~200nm and and then even more preferably about 50nm~150nm." nucleic acid of partial condensates " refer to cation with the present invention The nucleic acid of lipopolymer contact, wherein the nucleic acid is not up to complete condensation, but is still occupied substantially more than non-condensation nucleic acid Small volume.

As used herein, term " compound " refers to and lipopolymer, the core that preferably cationic lipid polymer phase is combined Acid.Compound comprising condensation nucleic acid and cationic lipid polymer exists usually as particle preferably as nano particle.

As used herein, term " transfection " and " transfection " refer to nucleic acid from the external environment condition of cell to intracellular environment The transhipment of (particularly relating to cytoplasm and nucleus).It is without being held to a particular theory, it should be appreciated that nucleic acid can be encapsulated in One or more cationic polymer/nucleic acid complexes are interior or attached to it or are transported to after therewith being carried thin Born of the same parents.Nucleic acid is delivered to nucleus by specific transfection example.

As used herein, " study subject " refers to administration or the food in one's mouth of method that can benefit from the pharmaceutical composition of the present invention Newborn animal.The example of study subject includes the mankind, and may also include such as horse, pig, ox, dog, cat, rabbit and aquatic mammals Other animals.

As used herein, " composition " refers to the mixture of two or more compounds, element or molecule.In some sides Face, term " composition " is used to refer to the mixture of nucleic acid and transmission system.

As used herein, " N:P ratios " refer to the phosphate radical in ammonia nitrogen and nucleic acid in functionalization cationic lipid polymer The mol ratio of group.

As used herein, " physicochemical properties " refer to the nucleic acid complexes for such as, but not limited to carrying cationic polymer Particle diameter and the various properties such as surface charge, the pH of particle solution and osmolality (osmolality).

As used herein, term administering ", " being administered " and " conveying " refer to composition being presented to study subject Mode.Using can be completed by various approach known in the art such as such as oral, parenteral, transdermal, suction and implantation.Cause This, orally administering can be realized by swallowing, chewing, sucking the peroral dosage form for including composition.Parenteral administration can lead to Cross in the injectable composition such as intravenous, intra-arterial, intramuscular, intra-articular, intrathecal, intraperitoneal, subcutaneous to realize.The note of used as said purpose The conventionally form as liquid solution or suspension can be prepared as by penetrating agent, or suitable for being prepared as in a liquid before the injection The solid form of solution or suspension, or it is prepared as emulsion.In addition, transdermal administration can be by the way that transdermal composition be applied Deposited, adhesion, spread-coating, attaching, filling apply, press or be applied on skin surface and complete.These and other application process is ability It is known in domain.In a specific aspect, using may include delivery of composition to study subject so as to which the composition is carried out System circulation and being combined with target cell is ingested with will pass through encytosis.

As used herein, term " nucleic acid " refers to DNA and RNA and their synthesis homologue.Nucleic acid it is non-limiting Example can include the DNA of encoding proteins or produce the inhibitory RNA of nucleotide sequence, single-stranded or double-stranded, missense, anti- Switch-mode regulation nucleotides and rate moderating nucleosides prepared by justice, the composition sequence of nonsense and control albumen, peptide and nucleic acid Acid.In addition, nucleic acid can also include but is not limited to genomic DNA, cDNA, siRNA, shRNA, mRNA, tRNA, rRNA, hybridization Sequence or synthesis or semi-synthetic sequence and natural origin or the nucleic acid of artificial source.On the one hand, nucleotide sequence may be used also With those nucleotide sequences encoded including the synthesis to human cytokines or suppression.The human cytokines it is unrestricted Property example can include anticancer, growth factor, Hypoylycemic agents, anti-angiogenic agent, bacterial antigens, viral antigen, tumour resist Former or metabolic enzyme.The example of anticancer can include interleukin 2, interleukin 4, interleukin 7, leucocyte Interleukin -12, IL-15, interferon-' alpha ', interferon-beta, interferon-γ, colony stimulating factor, granulocyte-macrophage are thin Born of the same parents' stimulating factor, anti-angiogenic agent, tumor suppressor gene, thymidine kinase, eNOS, iNOS, p53, p16, TNF-α, Fas match somebody with somebody Body, mutant oncogenes, tumour antigen, viral antigen or bacterial antigens.On the other hand, DNA can encode shRNA points Son, the shRNA molecule is designed to suppress to participate in the albumen of growth or the maintenance of tumour cell or other excessive proliferated cells. In addition, in some aspects, DNA can encode human cytokines and one or more shRNA simultaneously.In other respects, core Acid can also be DNA and the mixture of the synthesis RNA including just RNA, antisense RNA, ribozyme.In addition, nucleic acid Size may be different, can be from oligonucleotides to chromosome.These nucleic acid can be derived from the mankind, animal, plant, bacterium, disease Poison and synthesis.They can be obtained by any technology well known by persons skilled in the art.

As used herein, term " concentration " refers to the composition that its dilution factor is lowered.In certain aspects of the invention, " concentration " composition includes condensation DNA (preferably in isotonic solution).In the particular aspects of the present invention, the combination of concentration Thing includes the condensation DNA at least about 0.5mg/ml being suspended in isotonic solution.

As used herein, term " polymer main chain " is used to refer to a series of polymerization masters of weight average molecular weight within the specified range Chain molecule.Thus, when such as cholesterol equimolecular is described as being covalently attached with certain mol ratio with polymer main chain molecule When, it should be appreciated that the average number for the cholesterol molecule that the proportional representation is connected with the polymer main chain molecular series.For example, It is average by the polymer main chain for having half point if be covalently attached according to description cholesterol with 0.5 mol ratio and main polymer chain Son has connected cholesterol.As another example, if be total to according to description cholesterol with 1.0 mol ratio and main polymer chain Valency is connected, then average each polymer main chain molecule will be connected with a cholesterol molecule.However, actually should be understood that Some polymer main chain molecules in this case may be without connected cholesterol molecule, and other polymer main chain molecules may have Multiple connected cholesterol molecules, and the ratio is the average export by connected cholesterol molecule.Same reasoning is fitted Mol ratio for polyethylene glycol and polymer main chain.

As used herein, term " peptide " can be used to refer to include more than two carboxyls by an amino acid and another The natural or synthetic molecule of the connected amino acid of the alpha-amido of one amino acid.The peptide of the present invention not by length limitation, thus " peptide " can include polypeptide and albumen.

As used herein, term " covalently " and " covalently " refer to the chemical bond that electronics is shared between atom pair.

As used herein, for convenience, multiple projects, structural element, component and/or material can be presented on In one logical table.However, each member in list should be seen as being individually defined as independent and unique member Treat these lists.Therefore, when there is opposite instruction, only it should not be appeared in them in common group and by this kind of list Any individual member is regarded as the actual equivalent of any other member in same list.

Concentration, amount and other numerical datas can be expressed or presented herein with range format.It should be understood that this model Form is enclosed to use merely for convenient and succinct, thus it is bright that it should be neatly read as to the not only extreme value institute comprising the scope The numerical value really listed, but also comprising all individual numerical value or subrange covered in the range of this, such as each numerical value and son Scope is all expressly recited.For example, " about 1~about 5 " number range should be read as not only comprising about 1~about 5 it is bright Value really is listed, also comprising each value and subrange in the specified range.Therefore, such as 2,3 and 4 are included in the number range Deng individual numerical value and the subrange of such as 1~3,2~4 and 3~5 and single 1,2,3,4 and 5.Identical principle is also applied to Only list the scope of a numerical value as minimum value or maximum.No matter in addition, the width of scope or described characteristic are such as What, the Explanation way is all suitable for.

The invention provides can be incited somebody to action when not influenceing the physicochemical properties of nucleic acid or nucleic acid compositions or biological property The highly concentrated technology of low concentration nucleic acid composition (for example, 0.15mg/ml).On the one hand, nucleic acid compositions can be concentrated 33 times without influenceing the property.These highly concentrated nucleic acid compositions allow to using the larger vivo medicine-feeding of scope Scheme, this is extremely stranded in the past due to the related bad stability of the previous trial to attempting to reach greater than about 0.3mg/ml It is difficult.

More specifically, the invention provides concentration and stable pharmaceutical composition, including for preparing and using this kind of The method of composition.On the one hand, for example there is provided pharmaceutical composition include at least about 0.5mg/ml nucleic acid, wherein, nucleic acid It is combined with cationic lipid polymer and the compound is suspended in isotonic solution.It is suspended in the compound in isotonic solution The nucleic acid molecules being condensed comprising partial condensates or completely.The cationic lipid polymer is included to be consolidated with the courage being covalently attached to it The ionene polymer backbone of alcohol and polyethylene group (i.e. molecule).Mole of cholesterol molecule and ionene polymer backbone Than for about 0.1~about 10, and the mol ratio of peg molecule and ionene polymer backbone is about 0.1~about 10.Another Aspect, the mol ratio of peg molecule and the ionene polymer backbone in cationic lipid polymer is about 1~about 10.Again On the one hand, the mol ratio of peg molecule and the ionene polymer backbone in cationic lipid polymer is about 1~about 5. On the other hand, the mol ratio of cholesterol molecule and the ionene polymer backbone in cationic lipid polymer is about 0.3~about 5. It yet still another aspect, the mol ratio of the ionene polymer backbone in cholesterol molecule and cationic lipid polymer be about 0.4~about 1.5。

The composition also includes filler excipient.Resulting composition is suitable to nucleic acid being delivered to target cell so as to basis The function of nucleic acid inducing, suppress or modified biological response.

On the one hand, cholesterol and peg molecule can be independently and directly covalent with ionene polymer backbone Connection.On the other hand, cholesterol and peg molecule are each covalently attached with ionene polymer backbone indirectly.Example Such as, cholesterol molecule can be made directly or indirectly to be coupled with peg molecule by connexon or introns, and the poly- second Glycol molecules are covalently attached with ionene polymer backbone again.Another mode, cholesterol molecule can directly and cation Lipopolymer main chain is connected, and peg molecule is connected with the lipopolymer indirectly by connexon or introns.

A specific connexon between polyethylene glycol and ionene polymer backbone is the alkylidene with end carboxyl, excellent Elect the straight-chain alkyl-sub of 1~20 carbon atom and even more preferably about 2~about 4 carbon atoms as.End carboxyl on the connexon exists The amido link formed when being connected with the amino of ionene polymer backbone between cationic lipid polymer and polyethylene glycol.Suitable for The starting polyethylene glycol of ionene polymer backbone molecule reaction is carried with activated group (for example, n-hydroxysuccinimide Base ester) ending connexon molecule polyethylene glycol.

From polyethyleneimine, cholesteryl chloroformate (ignoring spatial chemistry) and methoxy poly (ethylene glycol)-propionic acid N- hydroxyls The example of a part for the cationic lipid polymer architecture that the reaction of succinimido fat is obtained is following structure.Legend reflects The appropriate distributions of primary amine groups, secondary amine and tertiary amine groups in polyethyleneimine, and herein for clarity, take and do not deposit Regular ethylene imine chain.

In aspects of the present invention, n is typically about 8~about 20, and more specifically about 10~about 15, and and then more specifically About 12；X is typically about 2~about 3, and more specifically about 2.5；Y is typically about 6~about 10, and more specifically about 7~about 9, and then more Body is about 7.5；Z is typically about 0.4~about 0.8, and more specifically about 0.5~about 0.7, and and then more specifically about 0.6.

In addition, in some aspects, proposed technology can be used to have used secondary condensation system before this The nucleic acid of (secondary condensing system) condensation further condensation to realize that nucleic acid is higher steady in higher concentrations It is qualitative.Similarly, before being condensed according to aspects of the present invention, nucleic acid may be at partial condensates form or non-condensation shape Formula.Secondary condensation system can include any condensation material known to persons of ordinary skill in the art or technology, these condensation materials Material or technology include but is not limited to cationic lipid, cationic peptide, cyclodextrin, cationized gelatin, dendrimer (dendrimer), chitosan and combinations thereof.

Various nucleic acid condensation levels can be realized for the composition of the aspect of the present invention.On the one hand, by with sun from Sub- polymer forms compound and is condensed all nucleic acid in composition or significant portion of nucleic acid.On the other hand, combine About 30 weight % nucleic acid is condensed in thing.It yet still another aspect, about 50 weight % nucleic acid is condensed in composition.In another side About 70 weight % nucleic acid is condensed in face, composition.On the other hand, about 90 weight % nucleic acid is condensed.

In addition, the concentration of composition amplifying nucleic acid being expected with according to material therefor in composition, method for concentration and nucleic acid It is different on the way.However, on the one hand, nucleic acid concentration is at least about 0.5mg/ml.On the other hand, nucleic acid concentration is at least About 1mg/ml.It yet still another aspect, nucleic acid concentration is at least about 3mg/ml.In another further aspect, nucleic acid concentration can be at least about 5mg/ml.On the other hand, nucleic acid concentration can be at least about 10mg/ml.It yet still another aspect, nucleic acid concentration can be at least about 20mg/ml.In another further aspect, nucleic acid concentration can be about 10mg/ml~about 40mg/ml.

The condensation level of nucleic acid compositions can be determined using various methods.For example, on the one hand, composition can be made The nucleic acid in composition and the degree added to the cationic polymer formation polymer in composition are determined by electrophoresis.Band The electrostatic attraction of the nucleic acid of negative electricity and the cationic lipid polymer of positively charged suppresses nucleic acid and is moved through Ago-Gel.Then, Carry out after electrophoresis, the nucleic acid being condensed by compound with cationic polymer is remained stationary as in gel, rather than condensation nucleic acid, The nucleic acid not combined with cationic polymer has moved the certain distance relevant with the current strength in gel.At another In example, the condensation of nucleic acid can be determined by the particle diameter in composition.Particle diameter can be determined by dynamic light scattering.It is logical Often, condensation nucleic acid is by with the particle diameter smaller than non-condensation nucleic acid.It is preferred that condensation nucleic acid be to be in nucleic acid and cationic lipid to gather Those nucleic acid in the nano particle of compound, its size is about 50nm~about 300nm, even more preferably about 50nm~200nm and entered And even more preferably about 50nm~150nm.

Any of nucleic acid, including those described above reality can be utilized in the composition and method in terms of the present invention Example.Equally, nucleic acid as described herein should not be considered as a kind of limitation.On the one hand, for example, nucleic acid can include encoding proteins, The plasmid of polypeptide or peptide.It is well known that many peptides have been proved to be when being formulated as pharmaceutical composition according to aspects of the present invention Benefit.The non-limiting examples of some of such peptide can include interleukin 2, interleukin 4, interleukin 7, Interleukin 12, IL-15, interferon-' alpha ', interferon-beta, interferon-γ, colony stimulating factor, granulocyte- Macrophage colony stimulatory factor, angiogenic agent, clotting factor, Hypoylycemic agents, antiapoptotic factors, anti-angiogenic agent, thymidine Kinases, p53, IP10, p16, TNF-α, FasL, tumour antigen, neuropeptide, viral antigen, bacterial antigens and combinations thereof. One specific aspect, nucleic acid can be the plasmid of encoding Interleukin -12.On the other hand, nucleic acid can be that coding suppresses The plasmid of property ribonucleic acid.In another further aspect, nucleic acid can be synthesis short interfering ribonucleic acid.It yet still another aspect, nucleic acid is to set Meter carrys out the antisense molecule of the expression of suppression therapy peptide.

As described above, cationic lipid polymer can be included with the cholesterol and polyethylene glycol being covalently attached with it Ionene polymer backbone.Ionene polymer backbone can be used for condensation including known to persons of ordinary skill in the art With any cationic polymer of the nucleic acid of concentration each aspect of the present invention.However, on the one hand, ionene polymer backbone can With including ionene polymer backbone be selected from polyethyleneimine, poly- (three azomethines), poly- (four azomethines), PPI, Aminoglycoside-polyamine, dideoxy diaminourea-b- cyclodextrin, spermine, spermidine, polymethylacrylic acid (2- dimethylaminos) second Ester, polylysine, polyhistidyl, poly arginine, cationized gelatin, dendrimer, chitosan and combinations thereof.In a tool In terms of body, ionene polymer backbone can be polyethyleneimine.

In a particular aspects, lipopolymer is by the polyethyleneimine that is independently covalently attached with cholesterol and polyethylene glycol (PEI) constitute.The PEG in this aspect, cationic lipid polymer:PEI:The average molar ratio of cholesterol is about 2~3:1:0.25 ~1, and preferably from about 2.25~2.75:1:0.4~0.8, and even more preferably about 2.5:1:0.6.In a particular aspects, the fat The molecular weight (being used as free alkali) of polymer is about 3kD~4kD, preferably from about 3.25kD~3.75kD, and is even more preferably about 3.54kD；The molecular weight of corresponding hydrochloride is about 4kD~5kD, preferably from about 4.5kD.

In addition, the molecular weight of ionene polymer backbone may be according to including properties of nucleic acids, desired use of composition etc. Many factors and it is different.However, on the one hand, the molecular weight of ionene polymer backbone may be about 100~about 500, 000 dalton.In addition, the molecular weight of other various components of cationic lipid polymer may also be different.On the one hand, for example, The molecular weight of polyethylene glycol may be about 50~about 20,000 dalton.

When building the pharmaceutical composition of the present invention, it has been found that ammonia nitrogen and nucleic acid in functionalization cationic lipid polymer In phosphate radical between mol ratio (N:P ratios) degree that nucleic acid may be influenceed to be condensed and/or concentrated.However, optimal N:P ratios may be different to a certain extent according to the chemical property of nucleic acid, on the one hand, in ionene polymer backbone Ammonia nitrogen and nucleic acid in the ratio between phosphate radical be about 0.1:1~about 100:1.On the other hand, in ionene polymer backbone Ammonia nitrogen and nucleic acid in the ratio between phosphate radical be about 3:1~about 20:1.Ammonia in another further aspect, ionene polymer backbone The ratio between phosphate radical in base nitrogen and nucleic acid is about 6:1~about 15:1.In other side, the ratio between phosphate radical in ammonia nitrogen and nucleic acid It is about 3:1~about 100:1, or about 5:1~about 100:1, or about 7:1~about 100:1.It yet still another aspect, the ratio is about 10:1 ~about 100:1, or even more preferably about 10:1~about 20:1.Ammonia nitrogen in a specific aspect, ionene polymer backbone It is about 11 with the ratio between the phosphate radical in nucleic acid:1.

In addition, it is contemplated that filler excipient can be included in described pharmaceutical composition.This kind of filler can provide for preparation Multiple beneficial property, for example, cryoprotection, bonding, isotonic balance, stabilisation during lyophilized and rehydration etc..It should be understood that not With composition between filler material can be different, and specific filler used be not to be construed as it is restricted.On the one hand, example Such as, filler excipient can include various sugar, sugar alcohol, starch, cellulose and combinations thereof.On the other hand, filler excipient Lactose, sucrose, trehalose, dextrose, galactolipin, mannitol, maltitol, maltose, D-sorbite, xylose can be included Alcohol, mannose, glucose, fructose, polyvinylpyrrolidone, glycine, maltodextrin, hydroxymethyl starch, gelatin, sorbose Alcohol, phenanthrene can (ficol), sodium chloride, calcium phosphate, calcium carbonate, polyethylene glycol and combinations thereof.It yet still another aspect, filler excipient can With including lactose, sucrose, trehalose, dextrose, galactolipin, mannitol, maltitol, maltose, D-sorbite, xylitol, Mannose, glucose, fructose, polyvinylpyrrolidone, glycine, maltodextrin and combinations thereof.In a specific aspect, filler Excipient can include sucrose.In another specific aspect, filler excipient can include lactose.

The concentration of filler excipient can be about 0.01%~about 5% in composition, more specifically about 0.1%~about 3.0%, then more specifically about 0.1%~about 0.3%.

In some aspects, it may be beneficial to be by cationic lipid it is functionalized with allow to target study subject or Specific cells or tissue in culture.It is this target be it is known, and example mentioned herein be not to be construed as it is restricted. On the one hand, for example, cationic lipid polymer can include what is be covalently attached with cationic lipid polymer or peg molecule Target part.It is this kind of target part cationic lipid polymer can be allowed systematically to be circulated in study subject with position and it is special Property targets specific cell type or tissue.This kind of example for targeting part can include transferrins, Asialoglycoprotein, Antibody, antibody fragment, low-density lipoprotein, cell receptor, growth factor receptors, cytokine receptor, folic acid, transferrins, Insulin, asialoglycoprotein seromucoid, Man-6-P, mannose, interleukins, GM-CSF, G-CSF, M- It is CSF, stem cell factor, erythropoietin(EPO), EGF (EGF), insulin, asialoglycoprotein seromucoid, sweet Reveal sugar -6- phosphoric acid, mannose, Lewis^xWith sialyl Lewis^x, N- acetyl lactosamines, folic acid, galactolipin, lactose, and thrombus Regulatory protein, such as polymyxin B and hemagglutinin HA2 fusion agents, close lysosome agent (lysosomotropic agent), Such as T antigens nuclear localization signal (NLS) and combinations thereof.The specific selection for targeting part and connection are completely general in this area In the knowledge of logical technical staff.

Present invention also offers can be by long term storage and in the freeze-drying medicinal composition using preceding rehydration.On the one hand, Freeze-drying medicinal composition can include filler excipient and the lyophilized mixture with the nucleic acid of cationic lipid polymer condensation, wherein The cationic lipid polymer is included with the cholesterol being covalently attached to it and the ionene polymer backbone of polyethylene glycol, and The mol ratio of wherein cholesterol and ionene polymer backbone is about 0.1~about 10, and polyethylene glycol and cationic polymerization owner The mol ratio of chain is about 0.1~about 10.Freeze-drying medicinal composition can be a variety of shapes from dry powder to the mixture of part rehydration Formula.

Present invention additionally comprises the method for preparing the various pharmaceutical compositions containing condensation nucleic acid.On the one hand, for example, this hair It is bright to provide the method for preparing the pharmaceutical composition with the condensation nucleic acid concentrated in at least 0.5mg/ml in isotonic solution.Should Method can include with cationic lipid polymer mixing nucleic acid in filler excipient, wherein the cationic lipid polymer bag Containing with the cholesterol being covalently attached to it and the ionene polymer backbone of polyethylene glycol, and wherein, cholesterol gathers with cation The mol ratio of compound main chain is about 0.1~about 10, and the mol ratio of polyethylene glycol and ionene polymer backbone be about 0.1~about 10.The mixture can be freezed for powder with condensed nucleic acid mixture and formed thereafter with diluent rehydration included in etc. The solution of the condensation nucleic acid of at least about 0.5mg/ml in osmometer solution.

Generally, the composition can be mixed by by nucleic acid solution and cationic lipid polymer solution in the presence of disaccharides Merge it is then lyophilized and in isotonic solution rehydration and obtain.This method can be scaled, and (be advised in laboratory so as to produce several milligrams Mould) to hundreds of milligrams (GMP scales) extension storage life highly concentrated nucleic acid preparation.As described above, cationic lipid polymer With the ionene polymer backbone that wherein polyethylene glycol and cholesterol are attached thereto by covalent bond.In the feelings of polyethyleneimine In shape, on the one hand, between the stoichiometric proportion and cholesterol and polyethyleneimine between polyethylene glycol and polyethyleneimine Stoichiometric proportion be respectively 0.5~10 and 0.1~10.The chemical composition of cationic polymer is highly concentrated steady for obtaining Determining nucleic acid preparation may be extremely important.Do not show that the cationic polymer of cholesterol and PEG connections is not allowed to be also easy to produce stabilization Highly concentrated preparation, shown in following article embodiment.

The composition of the aspect of the present invention can also merge with higher nucleic acid concentration with others condensation nucleic acid complexes It is lower to obtain bigger complex stabilities.For example, it is dense in high nucleic acid to improve to add different amounts of PPC The stability of normally unstable other nucleic acid transmission systems under degree.

In all fields, synthesis transmission system is comprising nucleic acid and can be made by various technologies available in the art Standby cation supporting agent.It is known to have multiple nucleic acids cation supporting agent：For example, polyethyleneimine, poly- (three azomethines), poly- (four Azomethine), PPI, aminoglycoside-polyamine, dideoxy diaminourea-b- cyclodextrin, spermine, spermidine, polymethyl Sour (2- dimethylaminos) ethyl ester, polylysine, polyhistidyl, poly arginine, cationized gelatin, dendrimer, shell gather Sugar, such as 1,2-dioleoyloxy-3-trimethylammonio propane (DOTAP), N- [1- (2,3- dioleoyls epoxide) propyl group]-N, N, N- front threes Ammonium chloride (DOTMA), 1- [2- oil trimethylammonium] -2- oleyls -3- (2- ethoxys) imidazolitm chloride quinoline (DOTIM), 2, Oleyl epoxide-the N- of 3- bis- [2- (spermine carboxy and amide groups) ethyl]-N, N- dimethyl -1- propyl group trifluoroacetic acid ammoniums (DOSPA), 3 β-[N- (N', N'- dimethylamino ethane)-carbamoyl] hydrochloric acid cholesterol (DC-Chol HCl), two (heptadecyls) Amide groups (glycyl) spermidine (DOGS), N, N- distearyl acyl groups-N, N- ditallowdimethyl ammonium bromide (DDAB), N- (1,2- Two nutmeg epoxide propyl- 3- yls)-N, N- dimethyl-N-hydroxies ammonium bromide (DMRIE), N, oleyl-N, the N- dimethyl of N- bis- Cation lipids such as ammonium chloride (DODAC) and combinations thereof.When these transmission systems are combined with PPC, nucleic acid is passed The stability of defeated system is increased.

The aspect of the present invention additionally provides the application method of pharmaceutical composition.For example, on the one hand, mammalian cell Transfection can include making mammalian cell contact with composition as described herein, and by mammalian cell under certain condition Incubate so that the composition can enter cell and trigger the bioactivity of nucleic acid.This kind of rotaring dyeing technology is ordinary skill Known to personnel.In addition, on the other hand, can be transfected by the way that composition is transmitted to warm-blooded organisms or study subject Target tissue.This transmission be able to can also include being selected from intra-tumor, intraperitoneal, vein by the composition as described in wherein conveying In interior, intra-arterial, tracheal strips, hepatic portal, oral, encephalic, the administration form such as intramuscular, intra-articular and combinations thereof carry out.The target Tissue can include that any tissue of transfection or the subclass of tissue can be benefited from.For example, the target tissue can be included but not It is limited to ovary, uterus, stomach, colon, rectum, bone, blood, small intestine, pancreas, mammary gland, head, neck, lung, spleen, liver, kidney, brain, first shape Gland, prostate, bladder, thyroid gland, skin, abdominal cavity, thoracic cavity and combinations thereof

Embodiment

Provided herein is following examples to promote the apparent understanding to only certain exemplary embodiments of this invention, but never anticipate Taste any limitation to it.

Preparation A

1g 1800Da branched polyethylenimines (PEI) (0.56mM) are dissolved in 5ml chloroforms and are placed in ml round bottoms burning In bottle, it is stirred at room temperature 20 minutes.By 380mg cholesteryl chloroformates (0.84mM) and 500mg activation methoxy poly (ethylene glycol)s (MPEG-SPA, methoxy poly (ethylene glycol) propionic acid N- hydroxysuccinimides base ester) (550Da) (0.91mM) is dissolved in 5ml chloroforms Middle disease is transferred in dropping funel, and the dropping funel is located at the top of the round-bottomed flask containing PEI solution.In 5~10 minutes The mixture of cholesteryl chloroformate and MPEG-SPA is added slowly in PEI solution in room temperature.Solution is stirred again in room temperature Mix 4 hours.After Rotary Evaporators removing solvent, after-tack material is dissolved in 20ml ethyl acetate under agitation In.By be slowly added 20ml n-hexanes by product the Precipitation from solvent；Liquid is decanted from product and removed.With 20ml's Ethyl acetate/hexane mixture (volume ratio 1/1) washes twice product.Decantation is removed after liquid, and 10 are purged by nitrogen Material was dried in~15 minutes.Material is dissolved in 10ml 0.05N HCl to prepare the salt form of amino.Filter the aqueous solution Pass through 0.2 μm of filter paper.Final product is obtained by lyophilized.

The mol ratio of the embodiment preparation is that 3.0 moles of MPEG-SPA and 1.28 mole of cholesterol and 1 mole of PEI molecule are even Connection.

Preparation B

20g (11.1mmol) side chain PEI (BPEI) and 200mL are dried into chloroform to mix jointly so that BPEI dissolves.It is molten Xie Hou, under agitation in will contain in 20~30 minutes 4g cholesteryl chloroformates and 18.7g (26mmol) activation the poly- second of methoxyl group Glycol (MPEG-SPA, methoxy poly (ethylene glycol) propionic acid N- hydroxysuccinimides base esters, MPEG molecular weight 550, ester molecule amount 719) 200mL dried chloroformic solution and is added dropwise into reactant mixture, followed by the incubation period of 3~4 hours.Then will be mixed Compound is placed under vacuum with concentrate solution and removes residual chloroform.Gained residue is dissolved in 320mL 1M HCl/water solution In and stir.The PPC HCI solutions are concentrated under vacuum again, so as to produce highly viscous material.In order to separate PPC Hydrochloride simultaneously removes byproduct of reaction and unreacted initiation material, by the mixture of concentration and acetone (<0.4% water) and stir Mix, so as to cause PPC hydrochloride precipitates to be the material of free floating.After precipitation, abandoning supernatant.By hygroscopic PPC hydrochloric acid Salt is dried under vacuum.

Polymer DNA is produced by preparing the solution of the debita spissitudo of PPC and DNA in 10% lactose first to be combined Thing.The stock solution of cationic polymer (mg/ml) and DNA (3mg/ml) in water for injection is diluted in 0.3%~3% In lactose solution：This is necessary to obtaining 10% final lactose concn after rehydration.Then DNA is added dropwise under agitation Enter in PPC solution and form compound in incubation at room temperature 15 minutes.

Composition made from 500 μ l is added in 2ml borosilicate glasses bottle and is placed in freeze dryer.By vial Cooled down 4 hours in -34 DEG C, start first drying thereafter.After 24 hours, shelf temperature is risen to 20 DEG C and kept again under vacuo 24 hours.Shelf temperature is finally risen to 4 DEG C and seals vial under vacuo.

Formulation C

180 milligrams of side chain PEI 1800 (0.1mM) are dissolved in 4ml chloro-formates and are stirred at room temperature 30 minutes.By 70 Milligram cholesteryl chloroformate (0.14mM) and 48mg PEG 330 (0.14mM) be dissolved in 1ml chloro-formates, and with inject Device was added slowly in 3~10 minutes in PEI solution.Mixture is stirred at room temperature 4 hours.Add 10ml ethyl acetate with After precipitation, solution is incubated overnight at -20 DEG C, removing is then decanted in liquid from flask.By surplus material 5ml Ethyl acetate/hexane mixture (volume ratio is 1/1) wash 2 times.Purge 10~15 minutes to dry residue by nitrogen Material, was dissolved in 20 minutes in 10ml 0.05N HCl, solution is filtered through 0.2 μm of syringe type mistake Filter.The aqueous solution is freezed to remove water from polymer by being freeze-dried.

The mol ratio of said preparation is 0.85 mole of PEG and 0.9 mole of cholesterol and 1 mole of PEI molecule coupling labeled.

Preparation D

500 milligrams of 25kDa straight chains PEI (0.02mM) is dissolved in 30ml and stirred 30 minutes at 65 DEG C.Will condensation Device and dropping funel are assembled with three-neck flask.By 200mgmPEG-NHS 1000 (0.2mM) and 40mg cholesteryl chloroformates The 5ml chloroformic solutions of (0.08mM) were added slowly in 3~10 minutes in PEI solution.By solution 65 DEG C of uniform stirring 4 again Hour, and volume is reduced into 5ml in Rotary Evaporators.Solution is precipitated in 50ml ether to remove free cholesterol, from Flask decantation removes liquid, and is washed surplus material 2 times with 20ml ether.After being dried with pure nitrogen gas, material is dissolved in In the mixture of 10ml 2.0N HCl and 2ml trifluoroacetic acids.It is with the dialysis tubings of MWCO 15000 that solution is saturating in deionized water Analysis 48 hours, changes fresh water in every 12 hours.Solution is freezed to remove water.

The mol ratio of said preparation is 12.0 moles of PEG and 5.0 mole of cholesterol and 1 mole of PEI molecule coupling labeled.

Preparation E

By 1g PEI (molecular weight：1200 dalton) it is dissolved in 15mL anhydrous methylene chlorides and 100 μ l triethylamine (TEA) in mixture.After stirring 30 minutes on ice, 1.2g cholesteryl chloroformates are added slowly in PEI solution and will Mixture is stirred overnight on ice.Precipitate products therefrom by adding ether, then centrifugation and immediately with extra ether with Acetone is washed.Water-insoluble lipopolymer is dissolved in chloroform to obtain 0.08g/mL ultimate density.Synthesis and after purification, With MALDI-TOFF MS and¹H NMR are characterized to water-insoluble lipopolymer.

The NMR of water-insoluble lipopolymer 1200 determines display, and the amount with the PEI cholesterol being coupled is about 40%.It is non-aqueous The MALDI-TOF mass spectral analyses of dissolubility lipopolymer show that its molecular weight is about 1600.

Preparation F

By 3g PEI (molecular weight：1800 dalton) in the mixture of the anhydrous dichloroethanes of 10ml and 100 μ l triethylamines Stirred 30 minutes on ice.1g cholesteryl chloroformates are dissolved in the anhydrous ice-cold dichloromethane of 5ml and then at 30 minutes Inside it is added slowly in PEI solution.Mixture is being stirred 12 hours and drying products therefrom in Rotary Evaporators on ice. Powder is dissolved in 50ml 0.1N HCl.The aqueous solution is extracted 3 times with 100mL dichloromethane, glass is then filtered through Microfilter.By evaporation solvent enriched product, precipitated with very big excessive acetone, and be dried under vacuum.With MALDI-TOF and 10¹HNMR analyzes product.The NMR results of water-insoluble lipopolymer 1800 are shown, are consolidated with the courage that PEI is coupled The amount of alcohol is about 47%.PEACE MALDI-TOFF mass spectral analyses show that its molecular weight is about 2200.This shows most PEACE 1800 cholesterol and PEI mol ratio are 1/1, but it is some without be coupled or with 2/1 mol ratio (cholesterol/ PEI) it is coupled.

Preparation G

50 milligrams of PEI 1800 are dissolved in 2mL anhydrous methylene chlorides on ice.Then 200 μ L benzyl chloroformates are delayed Slowly it is added in reactant mixture and stirs solution 4 hours on ice.After stirring, add 10mL dichloromethane and use 15mL Saturation NH₄Cl extracts solution.Using magnesium sulfate water is mutually removed from dichloromethane.Liquor capacity is reduced under vacuo and heavy with ether Shallow lake product CBZ protects PEI.50 milligrams of primary amine CBZ protections PEI is dissolved in dichloromethane, addition 10mg chloro-carbonic acids courage is consolidated Alcohol ester simultaneously stirs solution 12 hours on ice.Product CBZ protection lipopolymers are precipitated with ether, washed with acetone, then In the H as hydrogen donor₂Under be dissolved in containing in the DMF as the palladium activated carbon of catalyst.Mixture is stirred at room temperature 15 small When, it is filtered throughAnd reduce liquor capacity by Rotary Evaporators.Final product is obtained with ether precipitation.

Preparation H

In room temperature by 500 milligrams of NH₂- PEG-COOH 3400 (0.15mM) is dissolved in 5ml anhydrous chloroforms 30 minutes.Will Solution (1.5mM) of the 676mg cholesteryl chloroformates in 1ml anhydrous chloroforms is added slowly in PEG solution and then in room temperature It is stirred for 4 hours.Mixture is precipitated 1 hour in 500ml ether on ice, 3 times are then washed with ether to remove non-idol The cholesterol of connection.After being dried with nitrogen purging, powder is dissolved in 5ml 0.05N HCl so that the carboxyl on PEG is acidified.It is logical Cross freeze dryer drying material.100 milligrams of PEI1800 (0.056mM), 50mg DCC and 50mg NHS are dissolved in 5ml in room temperature In chloroform, stir the mixture for 20 minutes, then slowly add solution of the 380mg cholesterol-PEG-COOH in 1ml chloroforms Add in PEI solution.It is stirred at room temperature after 6 hours, organic solvent is removed with Rotary Evaporators.Surplus material is dissolved in Purified in 10ml deionized waters and by FPLC.

Embodiment 1

The preparation of the concentration fat preparation of condensation nucleic acid with cationic lipid polymer

It this example illustrate the preparation of the highly concentrated preparation of condensation nucleic acid completely under laboratory scale production.This is related to Prepare the nucleic acid complexes with cationic polymer and then freeze with rehydration in isotonic solution.Nucleic acid used is coding IL-12 or luciferase gene DNA, and the polymer is comprising common with polyethylene glycol (PEG) and cholesterol (Chol) Polyethyleneimine (PEI) main chain (PEG-PEI-Chol or PPC) of valency connection.PEG and PEI mol ratio and cholesterol and PEI Mol ratio be respectively 0.5~10 and 0.1~10.First, 5mg/ml DNA solution and PPC is prepared respectively in water for injection Solution, is then diluted to 0.15mg/ml (DNA) and 0.554mg/ml (PPC) in 3% lactose.It is with micropipettor that DNA is newborn Sugar juice is added in PPC lactose solutions so that the ratio between nitrogen and phosphate radical (N:P ratios) it is 11:1, and by said preparation in incubation at room temperature 15 minutes to enable compound to be formed.Freezed with the FREEZONE from LABCONCO Corp., Kansas City, MO System freezes the PPC/DNA compounds in 3% lactose.Preparation made from 500 μ l is added to 2ml borosilicate glass tubes In, then use and be lyophilized by the lyophilized program constituted with the next stage：

1) freezing stage (0.25 DEG C/min of alternating temperature (Ramp) is kept for 4 hours at 34 DEG C),

2) first drying stage (being kept for 24 hours at 34 DEG C),

3) redrying stage (alternating temperature is to 20 DEG C and is kept for 24 hours), and

4) with 0.25 DEG C/min of alternating temperature to 4 DEG C.

The various concentration for being 0.1mg/ml~20mg/ml DNA with water for injection rehydration by obtained freeze-drying powder.A collection of allusion quotation The amount of the small-scale preparation of type is the DNA of 100mg~200mg complete preparation.

Embodiment 1A

The step of being summarized essentially according to example 1 above uses N:P ratios are 11:1 cationic lipid polymer and IL-12 Nucleic acid prepares nucleic acid/cationic lipid polymer formulations.The PEG of the cationic lipid polymer:PEI:Cholesterol mol ratio is about 2.5:1:0.6, and molecular weight (being used as free alkali) is about 3.54kD.Gained is lyophilized and can using rehydration as extremely containing lactose formulations Few about 0.5mg/ml nucleic acid concentration is without having nucleic acid aggregation or obvious transfection activity loss.

Embodiment 2

The preparation of the concentrated liquid preparation of condensation nucleic acid with cationic lipid polymer

The preparation of highly concentrated condensation nucleic acid preparation is this example illustrate, as shown in Figure 1.The program, which has been produced, to be exceeded The DNA (compared with 100mg~200mg DNA that the small-scale preparation described in embodiment 1 is produced) of 6000mg complete preparation, And the output of even more high can be extended to.The amplification method be related to peristaltic pump by a large amount of DNA and polymer solution mix from And realize on-line mixing scene to form compound, the lyophilized circulation of heavy load can be coordinated by then implementing.In short, by DNA and PPC is prepared as the solution of the 0.3mg/ml and 1.1mg/ml in 3% lactose respectively.With the silicon sleeve pipe for being 0.89mm with internal diameter The peristaltic pump (WATSON MARLOW, SCI 400) of (WATSON MAPvLOW, Z982-0088) was in the stream of 225 ± 25ml/ minutes Speed merges two components with constant flow rate.Two mixtures are merged by polypropylene T-connection in the end of each pipe.It is poly- The mixing of compound and DNA solution causes to form nano particle immediately.The compound of 40 milliliters of preparations is placed in 100ml glass tubes And freezed with the lyophilized program by being constituted with the next stage：

1) in -50 DEG C of precoolings at most 720 minutes,

2) in 65 μm of Hg, in -40 DEG C of primary dryings at most 180 minutes, then dried at most 1980 minutes at -34 DEG C, and

3) in 65 μm of Hg, in -25 DEG C of redryings at most 720 minutes, then dried at most 3180 minutes at -15 DEG C, Dry at most 1500 minutes, and dried at most 1440 minutes at 4 DEG C at -10 DEG C.

The various concentration for being 0.1mg/ml~20mg/ml DNA with water for injection rehydration by obtained freeze-drying powder.A collection of allusion quotation The amount of the high volume product of type is the DNA of 6000mg complete preparation.

Embodiment 3

The measure of the particle diameter of the concentrated liquid preparation of condensation nucleic acid with cationic lipid polymer

The highly concentrated preparation of the DNA with cationic lipid polymer P PC is prepared as described in Example 1 and 2.To enter Row polymer/nucleic acid particle size determination, with from BROOKHAVEN INSTRUMENTS Corp., Holtsville, NY's 90Plus/BI-MAS Particle Sizer analyze the aliquot of the liquid preparation.Specifically, 50 μ l preparations are added Add in the milli-Q water of 950 μ l in polystyrene cuvette to be analyzed.

Fig. 2 show the pre- lyophilized or DNA/PPC compounds in the preparation (0.15mg/ml DNA) of non-concentrated and with IL-12 plasmids (Fig. 2A) or luciferase plasmids (Fig. 2 B) rehydration are the grain after 0.5mg/ml~10mg/ml higher concentration Footpath.Particle diameter is had no significant effect in the rehydration of higher concentration, this shows that the compound is stable.

Embodiment 4

The analysis of the nucleic acid condensation product of the concentrated liquid preparation of nucleic acid with cationic lipid polymer

The ability of PPC polymer condensation DNAs is have evaluated in this example.Prepare as described in Example 1 and 2 with sun The highly concentrated preparation of ion lipopolymer PPC DNA.Nucleic acid/the polymer complex is entered with 1% Ago-Gel Row electrophoresis.The electrostatic attraction of the PPC polymer of electronegative DNA and positively charged prevents DNA from running by Ago-Gel. As shown in figure 3, all DNA present in the highly concentrated preparation are condensations.

Embodiment 5

The measure of nucleic acid concentration in the concentrated liquid preparation of nucleic acid with cationic lipid polymer

It is right using the spectrophotometers of AGILENT 8453 (AGILENT TECHNOLOGIES, Inc.Santa Clara, CA) DNA is quantified with the nucleic acid amount in the highly concentrated preparation of PPC compounds.With 950 μ l waters for injection (WFI) by described in 50 μ l Preparation dilutes in quartz colorimetric utensil and determines absorbance using 260nm wavelength.Assuming that 1 optical density (at 260nm)=50 μ g/ MlDNA, determines DNA concentration.

Embodiment 6

The measure of the transfection activity of nucleic acid condensation liquid preparation with cationic lipid polymer

The transfection activity of the highly concentrated preparation of DNA and PPC compound is determined in vitro.By itself and non-concentrated preparation Transfection activity is directly compared.Prepared by method described in Examples 1 and 2 containing luciferase or IL-12 plasmids Transfection composite, and rehydration is 0.15mg/ml~10mg/ml DNA concentration.By Cos-1 cells (1.5 × 10⁵Cells/well) connect Plant in the 12 hole tissue culturing plates into 10% hyclone (FBS).By each hole and 4 μ g compound DNA in the presence of no FPS Incubated 6 hours in cumulative volume improves Eagle minimum essential mediums (DMEM) for 500 μ l Dulbecco/Vogt.Work as incubation At the end of phase, culture medium is placed in being supplemented with 10%FBS fresh DMEM 40 hours again for 1ml.At the end of incubation period, Transfection activity is determined in cell culture medium (IL-12) or cell lysate (luciferase).Determine, pass through for IL-12 levels IL-12ELISA tests Direct Analysis cell culture medium.For luciferase assay, it is used in combination with phosphate-buffered salt water washing cell TENT buffer solutions (50mMTris-Cl [pH8.0] 2mM EDTA, 150mM NaCl, 1%Triton X-100) are cracked.Use Orion Microplate Luminometer (BERTHOLD DETECTION SYSTEMS, Oak Ridge, TN) determine cell The uciferase activity as relative light unit (RLU) in lysate.Final fluorescein enzyme values are using RLU/mg total proteins to be single Position report.Total protein water is determined with BCA protein assay reagent kits (PIERCE BIOTECHNOLOGY, Inc., Rockford, IL) It is flat.The IL-12 and Luciferase expression levels of highly concentrated preparation from IL-12 and luciferase plasmids/PPC compounds points Not such as Fig. 4 A and Fig. 4 B.The transfection activity of the nucleic acid complexes of the highly concentrated form of data display is retained.

Embodiment 7

The assessment of various excipient sugar in the prepared product of the concentrated liquid preparation of nucleic acid with cationic lipid polymer And its characterize

It has evaluated as the possibility filler or packing agent during freeze-drying process for preparing the two of highly concentrated preparation Plant conventional sugared (lactose and sucrose).3%, 1.5% and 0.3% PPC/DNA compounds are prepared respectively in lactose and sucrose Solution.These preparations are freezed using scheme used in embodiment 1.With WFI rehydrations it is 0.5mg/ by preparation after freeze-drying process Ml, 1mg/ml and 5mg/ml final DNA concentration.These different preparations are assessed with its particle diameter and outer-gene transfer.Such as the institute of table 1 Show, no matter cryoprotector filler is sucrose or lactose, particle diameter and transfection activity are retained.These results are showed more than one Class sugar can be used for the high concentration nucleic acid with cationic polymer for preparing physical chemistry and bio-stable.

Table 1

The assessment of excipient sugar in the prepared product of the concentration isotonic preparation of nucleic acid with cationic polymer

Embodiment 8

IL- in normal brain parenchym of the concentrated liquid preparation of nucleic acid with cationic lipid polymer after encephalic expression 12 expression

Direct administration of the IL-12 plasmids with cationic polymer PPC in normal cerebral tissue is examined to determine nucleic acid Whether the highly concentrated preparation with cationic lipid polymer is in vivo bioactivity.At 14 days after treatment or January The immunohistochemical staining to IL-12 is carried out in the brain sections of dead animal.The brain parenchym for the animal only treated with PPC does not have Show any IL-12 dyeing (Fig. 5 A).On the contrary, with the brain parenchym pair of the pmIL-12/PPC mouse injected in encephalic In IL-12 be positive staining (Fig. 5 B).The experiment shows that the bioactivity of the nucleic acid complexes with cationic polymer is dense Retained in compression process.Furthermore it is possible to infer that at least one middle of the month of cell factor after injection still has.Moreover, should Presence in the brain for the animal that cell factor is still survived before execution shows IL-12 actual expression without result in brain Fatal toxicity.

Embodiment 9

Effect of the concentrated liquid preparation of nucleic acid with lipopolymer in mouse spongioblastoma model

The highly concentrated preparation of expression IL-12 complete composite nucleic acid is examined in mouse spongioblastoma model Anticancer efficacy.Pass through intracranial injection 1 × 10⁵Individual GL261 spongioblasts oncocyte and 3 μ l's comes from 5mg/ml IL-12 matter Tumour is implanted into Cerebral Cortex by the IL-12/PPC compound co-injection things of the grain highly concentrated preparations of DNA.To animal monitoring Any neurotoxicity sign and may when be autopsied and dead caused with confirming by intracranial tumors.Use Kaplan-Meier survival rate analysises are mapped to survival rate.The list for the pmIL-12/PPC compounds applied with 15 μ g plasmid doses The well-tolerated of secondary intracranial injection, because significant side effect is not observed.The pmIL-12/PPC of 15 μ g plasmid doses The single injection of compound significantly improves animal survival rate.

Embodiment 10

Bioactivity of the concentrated liquid preparation of nucleic acid with cationic lipid polymer in ovarian cancer patients

The highly concentrated preparation of complete condensation nucleic acid of expression IL-12 genes is examined in the trouble with recurrent ovarian carcinoma Bioactivity in person.IL-12 plasmids 4 times a week and PPC highly concentrated isotonic preparation are with recurrent ovarian carcinoma Intraperitoneal in women, which is applied in, produces significant IFN-γ (IL-12 surrogate markers thing) in the peritoneal fluid of treated patient Level.IFN-γ level is 20pg/ml peritoneal fluids~275pg/ml peritoneal fluids.These as shown by data, the height of IL-12 nucleic acid is dense Contraction agent is suitable to clinical practice.

Embodiment 11

The chemical composition of cationic polymer is assessed to the concentrated liquid preparation of the nucleic acid with cationic lipid polymer The influence of property

Trial before this it has been shown that the bad stability and transfection that are caused due to concentration process are lost, thus to It is extremely difficult that the nucleic acid preparation of such as lipid or polymer cation gene supporting agent, which carries out concentration,.In order to determine physical chemistry The successful preparation of nucleic acid is condensed completely whether for test cationic polymer PEG-PEI- courages with the high concentration of bio-stable Sterol (PPC) is unique, the PEI for testing the PEI including being connected with free PEI, with cholesterol or being connected with PEG Cationic polymer and Cationic liposome DOTAP.Prepare 0.15mg/ml DNA compounds and dense as described in Example 1 It is reduced to 0.5mg/ml~5mg/ml.Particle diameter and transfection activity are determined as described in embodiment 3 and 6.As shown in FIG. 7 and 8, with free DNA prepared by PEI (PEI1800, PEI15000, PEI25000) or PEI- cholesterol, PEI-PEG or cation lipid DOTAP Compound does not produce stable compound, because these compounds are assembled after lyophilized and rehydration is 0.5mg/ml~5mg/ml And have lost transfection activity.Stabilization removal effect is more notable than 0.5mg/ml in 5mg/ml.By contrast, PEG-PEI- courages are used DNA compounds prepared by sterol (PPC) are freezing and are maintaining its physicochemical properties and transfection during high DNA concentration rehydration Property (Fig. 7 and 8).These results indicate that with cholesterol and PEG to the covalent modification of cationic polymer in concentration process Activity retain be vital.

Embodiment 12

The lyophilized formulations of nucleic acid with cationic polymer or the long-time stability of concentrated liquid preparation

It is prepared for freezing IL-12/PPC compound on a large scale, and store under cGMP with the method summarized in embodiment 2 In -80 DEG C, -20 DEG C, 4 DEG C and 25 DEG C (60%RH) for stability assessment.When being analyzed, pipe is taken out simultaneously from holder Add 2.4mL WFI.For each sample, pH, DNA concentration, osmolality, particle diameter and bioactivity are determined.Such as Fig. 9 It is shown, DNA concentration, pH, osmolality and the particle diameter of IL-12/PPC compounds during storages in 2 years of assigned temperature all Maintained.As described in Example 6, pIL-12/PPC gene transfer activity is quantified in COS-1 cells.With 4 μ g DNA biomaterial transfection COS-1 cells.48 hours after transfection, with commercially available ELISA kit to the IL- in cell culture medium 12 levels are quantified.During -80 DEG C or -20 DEG C of storage, the bioactivity of biological product does not substantially change.In the time For 0 when, activity be 151 ± 130pg/mL, remainder data is fluctuated in the standard deviation, except that entering at 25 DEG C with the time Row sustained activity declines.At 4 DEG C, the reduction of transfection activity was observed at 360 days, but because sample is not enough, without available Subsequent point in time with drawing a conclusion property assess.

Embodiment 13

The stability of the rehydrated material of the concentrated liquid preparation of nucleic acid with cationic polymer

The stability of rehydrated material is examined in single research.Method according to embodiment 2 prepares lyophilized IL-12 DNAs/PPC compounds, and rehydration is 0.5mg/ml in water for injection.Rehydrated material is stored in 4 DEG C. Take out sample within 60 days and the 90th day and analyze particle diameter, osmolality and gene expression.Analysis is stored in -80 DEG C close simultaneously Lyophilized products in tube sealing are to be compared.As shown in table 2, after with WFI rehydrations, the EGEN-001 of rehydration at least exists at 4 DEG C It is stable in 90 days.Compared with the lyophilized products in the seal pipe for being stored in -80 DEG C, including DNA concentration, particle diameter, infiltration rub That concentration or the stability parameter of gene expression are not significantly changed.

Table 2

The long-time stability of nucleic acid with cationic polymer highly concentrated and the isotonic preparation that is condensed completely at 4 DEG C

Embodiment 14

By the highly concentrated stabilized DNA system that nucleic acid transmission system is prepared with PPC co-formulation Agent

PPC is added in existing nucleic acid transmission system and led to improving under high nucleic acid concentration The stability of often unstable nucleic acid preparation.

In one embodiment, PPC is added to the straight linear polyethylene imines with 25kDa (LPEI25kD) in the DNA preparations prepared.Can be with LPEI25kD with 10:1(N:P ratios) prepared in the presence of 3% lactose it is dense Spend the DNA preparations for 0.15mg/ml.Then can be by PPC with the ratio between the different PPC and DNA of preparation addition Into LPE125kD/DNA compounds.For example, PPC/DNA (N:P ratios) can be (0:1)、(1:1)、(5:1)、(7.5:1)、 (11:1)、(15:1) with (20:1).500 μ l each preparation can be added in 2ml borosilicate glass tubes, then in freezing Freezed in drying system.Lyophilized program with the next stage by being constituted：

1) freezing stage (0.25 DEG C/min of alternating temperature is kept for 4 hours at -34 DEG C),

2) first drying stage (being kept for 24 hours at -34 DEG C),

3) redrying stage (alternating temperature is to -20 DEG C and is kept for 24 hours), and

4) with 0.25 DEG C/min of alternating temperature to 4 DEG C.

With water for injection rehydration can be the suitable concentration of 0.5mg/ml or other by lyophilized formulations.

It should be understood that above-mentioned composition and application model are only the explanations to the preferred embodiment of the present invention.This area Technical staff can dream up a variety of modifications and substitutions without departing from the spirit and scope of the present invention and set, and institute Attached claim is intended to include these modifications and set.Therefore, although above with being presently considered to be most realistic and preferred Embodiments of the present invention specific and detailed description has been carried out to the present invention, but will be apparent to those skilled in the art , a variety of modifications can be carried out in the case of without departing substantially from principle as described herein and concept, these modifications include but do not limited In the change of size, material, shape, form, function and mode of operation.

Claims

1. a kind of composition, the composition is included：

(a) the cationic lipid polymer that is suspended in the aqueous solution and at least about mixture of 0.5mg/ml nucleic acid, wherein the sun Ion lipopolymer includes the ionene polymer backbone being independently covalently attached with cholesterol and polyethylene group, and courage is solid The mol ratio of alcohol and ionene polymer backbone is about 0.1~about 10, and mole of polyethylene glycol and ionene polymer backbone Than being about 0.1~about 10；With

(b) filler excipient.

2. composition as claimed in claim 1, wherein, the mixture formation compound of the nucleic acid and lipopolymer.

3. composition as claimed in claim 1, wherein, the aqueous solution is isotonic solution.

4. composition as claimed in claim 1, wherein, the composition includes condensation nucleic acid.

5. composition as claimed in claim 1, wherein, at least about 30 weight % nucleic acid is condensation.

6. composition as claimed in claim 1, wherein, at least about 90 weight % nucleic acid is condensation.

7. composition as claimed in claim 1, wherein, the composition can be dried and rehydration be at least 0.5mg/ml core Acid concentration and the nucleic acid will not be assembled.

8. composition as claimed in claim 1, wherein, the ionene polymer backbone is to be selected from polyethyleneimine, poly- (three Azomethine), poly- (four azomethines), PPI, aminoglycoside-polyamine, dideoxy diaminourea-b- cyclodextrin, spermine, sub- essence Amine, polymethylacrylic acid (2- dimethylaminos) ethyl ester, polylysine, polyhistidyl, poly arginine, cationized gelatin, tree The member of dendrimer, chitosan and combinations thereof.

9. composition as claimed in claim 1, wherein, the concentration of the nucleic acid is at least 1mg/ml.

10. composition as claimed in claim 1, wherein, the concentration of the nucleic acid is at least 10mg/ml.

11. composition as claimed in claim 1, wherein, ammonia nitrogen and the nucleic acid in the ionene polymer backbone In the ratio between phosphate radical be about 0.1:1~100:1.

12. composition as claimed in claim 1, wherein, the nucleic acid is the matter encoded to the peptide selected from following material Grain：Interleukin 2, interleukin 4, interleukin 7, interleukin 12, IL-15, interferon- α, interferon-beta, interferon-γ, colony stimulating factor, granulocyte-macrophage colony stimutaing factor, angiogenic agent, blood coagulation The factor, Hypoylycemic agents, antiapoptotic factors, anti-angiogenic agent, thymidine kinase, p53, IP10, p16, TNF-α, FasL, tumour Antigen, neuropeptide, viral antigen, bacterial antigens and combinations thereof.

13. composition as claimed in claim 1, wherein, the ionene polymer backbone has about 50 dalton~about The molecular weight of 500,000 dalton.

14. composition as claimed in claim 1, wherein, the polyethylene glycol in the cationic lipid polymer gathers with cation The mol ratio of compound main chain is about 1~about 10.

15. composition as claimed in claim 1, wherein, the filler excipient be selected from sugar, sugar alcohol, starch, cellulose and Its member combined.

16. composition as claimed in claim 1, wherein, the filler excipient is to be selected from lactose, sucrose, trehalose, dextrorotation Sugar, galactolipin, mannitol, maltitol, maltose, D-sorbite, xylitol, mannose, glucose, fructose, polyethylene pyrrole Pyrrolidone, glycine, maltodextrin, hydroxymethyl starch, gelatin, D-sorbite, phenanthrene can, it is sodium chloride, calcium phosphate, calcium carbonate, poly- The member of ethylene glycol and combinations thereof.

17. composition as claimed in claim 1, wherein, the filler excipient is sucrose.

18. composition as claimed in claim 1, wherein, the filler excipient is lactose.

19. composition as claimed in claim 1, wherein, the nucleic acid is the plasmid of Codocyte IL-12 gene, and The ionene polymer backbone is polyethyleneimine (PEI).

20. composition as claimed in claim 19, wherein, ammonia nitrogen and the nucleic acid in the ionene polymer backbone In the ratio between phosphate radical be about 10:1~about 100:1.

21. composition as claimed in claim 1, wherein, ammonia nitrogen and the nucleic acid in the ionene polymer backbone In the ratio between phosphate radical be about 11:1~about 20:1.

22. composition as claimed in claim 1, wherein, the nucleic acid is the plasmid for encoding inhibition ribonucleic acid.

23. composition as claimed in claim 1, wherein, the nucleic acid is the short interfering ribonucleic acid of synthesis.

24. a kind of preparation method of pharmaceutical composition, it is isotonic that described pharmaceutical composition includes being suspended in at least about 0.5mg/ml Nucleic acid in solution, methods described includes：

Nucleic acid, cationic lipid polymer and filler excipient are mixed in an aqueous medium, the cationic lipid polymer is included The cationic polymer being independently covalently attached with cholesterol and polyethylene group, and cholesterol and ionene polymer backbone Mol ratio be about 0.1~about 10, and the mol ratio of polyethylene glycol and ionene polymer backbone is about 0.1~about 10；

The mixture is freezed as powder；With

With powder described in diluent rehydration with formed in isotonic solution comprising at least about 0.5mg/ml condensation nucleic acid solution.

25. method as claimed in claim 22, wherein the phosphorus in ammonia nitrogen and nucleic acid in the ionene polymer backbone The ratio between acid group is about 10:1~about 100:1.

26. a kind of dry pharmaceutical composition, the composition is included：

The mixture of filler excipient, nucleic acid and cationic lipid polymer, the cationic lipid polymer includes cationic polymerization Owner's chain, the ionene polymer backbone has at least one cholesterol molecule and at least one being independently covalently attached with it The ratio between ammonia nitrogen in individual peg molecule, and wherein described ionene polymer backbone and phosphate radical in the nucleic acid are About 10:1~about 100:1.

27. pharmaceutical composition answering in the medicine for transfection mammalian cell is prepared is done as claimed in claim 26 With the transfection includes：

Mammalian cell is in contact with the medicine；With

Mammalian cell is incubated under certain condition with so that the medicine enters the cell and induces the nucleic acid Bioactivity.

28. dry pharmaceutical composition is being prepared for transfecting the application in targeting the medicine of tissue, institute as claimed in claim 26 Stating transfection is included in the drug delivery to warm-blooded organism body.

29. application as claimed in claim 28, wherein convey the medicine to be applied selected from intra-tumor, intraperitoneal apply, it is quiet Apply in arteries and veins, intra-arterial is applied, tracheal strips are applied, apply, orally administer in hepatic portal, encephalic administration, intramuscular administration, intra-articular applied With and combinations thereof administration form carry out.

30. application as claimed in claim 28, wherein it is described target tissue be located at selected from ovary, uterus, stomach, colon, rectum, Bone, blood, small intestine, pancreas, mammary gland, head, neck, lung, spleen, liver, kidney, brain, thyroid gland, prostate, bladder, thyroid gland, skin, abdomen The position of chamber, thoracic cavity and combinations thereof.

31. a kind of composition, the composition is included：

(a) compound formed by nucleic acid and cationic lipid polymer, the compound is suspended in isotonic solution, wherein institute State cationic lipid polymer and include the ionene polymer backbone being independently covalently attached to cholesterol and polyethylene group, rub Your ratio is about 2~3:1:0.25~1 polyethylene glycol:Polyethyleneimine:Cholesterol；With

(b) filler excipient.

32. composition as claimed in claim 31, wherein the nucleic acid is interleukin 12.

33. composition as claimed in claim 32, wherein the lipopolymer with cholesterol and polyethylene glycol by being independently total to Connected polyethyleneimine (PEI) composition of valency, mol ratio average out to 2~3:1:0.25~1 PEG:PEI:Cholesterol.

34. composition as claimed in claim 33, wherein molecular weight of lipopolymer when as free alkali is about 3.5kD。

35. a kind of nucleic acid delivery system, the nucleic acid delivery system includes filler excipient and at least about 0.5mg/ml nucleic acid, Wherein at least some nucleic acid formation compound and the cationic lipid polymer condensation with being suspended in aqueous medium, and the sun Ion lipopolymer includes the ionene polymer backbone being independently covalently attached to cholesterol and polyethylene group, and courage is solid The mol ratio of alcohol and ionene polymer backbone is about 0.1~about 10, and polyethylene glycol and ionene polymer backbone rub Your ratio is about 0.1~about 10.

36. nucleic acid delivery system as claimed in claim 35, wherein the ionene polymer backbone is polyethyleneimine , and the PEG in the cationic lipid polymer (PEI):PEI:Cholesterol mol ratio is about 2~3:1:0.25~1.

37. nucleic acid delivery system as claimed in claim 36, wherein the free alkali molecular weight of the cationic lipid polymer is about For 3kD~4kD.

38. nucleic acid delivery system as claimed in claim 37, wherein ammonia nitrogen and institute in the ionene polymer backbone It is about 10 to state the ratio between phosphate radical in nucleic acid:1~about 100:1.

39. nucleic acid delivery system as claimed in claim 37, wherein ammonia nitrogen and institute in the ionene polymer backbone It is about 11 to state the ratio between phosphate radical in nucleic acid:1~about 20:1.

40. composition as claimed in claim 21, wherein the PEG in the cationic lipid polymer:PEI:Cholesterol mole Than being about 2~3:1:0.25~1.

41. a kind of waterborne compositions comprising nucleic acid and cationic lipid polymer, wherein the composition can be dried and rehydration Be at least 0.5mg/ml nucleic acid concentration and the nucleic acid will not be assembled.

42. composition as claimed in claim 41, wherein the mixture of the nucleic acid and cationic lipid polymer forms compound Thing.

43. composition as claimed in claim 42, wherein the composition includes condensation nucleic acid.

44. composition as claimed in claim 37, wherein the aqueous solution can be isotonic solution by rehydration.

45. composition as claimed in claim 38, the wherein at least about 30 weight % nucleic acid is condensation.

46. composition as claimed in claim 39, wherein ammonia nitrogen and the nucleic acid in the ionene polymer backbone In the ratio between phosphate radical be about 10:1~about 100:1.

47. composition as claimed in claim 40, wherein ammonia nitrogen and the nucleic acid in the ionene polymer backbone In the ratio between phosphate radical be about 11:1~about 20:1.

48. composition as claimed in claim 41, wherein the PEG in the cationic lipid polymer:PEI:Cholesterol mole Than for about 2~3:1:0.25~1.

49. dry composition as claimed in claim 26, wherein the composition can be at least 0.5mg/ml nucleic acid by rehydration Concentration and the nucleic acid will not be assembled.

50. waterborne compositions as claimed in claim 41, the composition also includes filler excipient.

51. a kind of method for making the nucleic acid delivery system comprising nucleic acid and cation supporting agent stable, methods described is described including making Delivery system is contacted with the composition comprising cationic lipid polymer and filler excipient, and the cationic lipid polymer is comprising only The ionene polymer backbone being on the spot covalently attached to cholesterol and polyethylene group, and cholesterol and cationic polymerization owner The mol ratio of chain is about 0.1~about 10, and the mol ratio of polyethylene glycol and ionene polymer backbone is about 0.1~about 10.

52. method as claimed in claim 51, wherein the cation supporting agent be selected from polyethyleneimine, poly- (three azomethines), Poly- (four azomethines), PPI, aminoglycoside-polyamine, dideoxy diaminourea-b- cyclodextrin, spermine, spermidine, poly- first Base acrylic acid (2- dimethylaminos) ethyl ester, polylysine, polyhistidyl, poly arginine, cationized gelatin, dendroid point Son, chitosan, 1,2-dioleoyloxy-3-trimethylammonio propane (DOTAP), N- [1- (2,3- dioleoyls epoxide) propyl group]-N, N, N- trimethyl ammonium chlorides (DOTMA), 1- [2- (oleoyl epoxide) ethyl] -2- oleyls -3- (2- ethoxys) imidazolitm chloride quinoline (DOTIM), the oleyl epoxide-N- of 2,3- bis- [2- (spermine carboxy and amide groups) ethyl]-N, N- dimethyl -1- propyl group trifluoroacetic acids Ammonium (DOSPA), 3 β-[N- (N', N'- dimethylamino ethane)-carbamoyl] hydrochloric acid cholesterol (DC-Chol HCl), two (heptadecyl) amide groups (glycyl) spermidine (DOGS), N, N- distearyl acyl groups-N, N- ditallowdimethyl ammonium bromide (DDAB) oil of, N- (the nutmeg epoxide propyl- 3- yls of 1,2- bis-)-N, N- dimethyl-N-hydroxies ammonium bromide (DMRIE), N, N- bis- Alkenyl-N, N- alkyl dimethyl ammonium chloride (DODAC) and combinations thereof.