CN102178957A - Respiratory tract homing siRNA atomizing nanometer drug delivery system and preparation method thereof - Google Patents
Respiratory tract homing siRNA atomizing nanometer drug delivery system and preparation method thereof Download PDFInfo
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- CN102178957A CN102178957A CN2011100405428A CN201110040542A CN102178957A CN 102178957 A CN102178957 A CN 102178957A CN 2011100405428 A CN2011100405428 A CN 2011100405428A CN 201110040542 A CN201110040542 A CN 201110040542A CN 102178957 A CN102178957 A CN 102178957A
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Abstract
The invention discloses a respiratory tract homing siRNA atomizing nanometer drug delivery system which uses salbutamol guanidinated chitosan treated by ultrasonic atomization as a carrier material, and is prepared by wrapping siRNA which interferes with target pathogenic genes. The invention also discloses a preparation method of the respiratory tract homing siRNA atomizing nanometer drug delivery system. The drug delivery system disclosed by the invention can be used to deliver siRNA which interferes with target pathogenic genes so as to play an RNA interference role and inhibit the replication of the pathogenic genes, can deliver siRNA with the target of lung respiratory tract epithelia in vivo, and is a non-virus delivery system which exerts corresponding drug effect by aiming at silence the target genes.
Description
Technical field
The present invention relates to a kind of drug-supplying system, especially relate to a kind of respiratory tract siRNA atomizing administration nano-drug administration system of going back to the nest.The present invention also relates to the go back to the nest preparation method of siRNA atomizing administration nano-drug administration system of above-mentioned respiratory tract simultaneously.
Background technology
RNA disturbs that (interfering RNAs RNAi) is the field that tool application prospect is studied in gene therapy.With regard to biological universal rule, RNAi can make any gene silencing, simultaneously, because high special, the efficient and simplification of RNAi mediated gene silencing, invade by pathogen at some and to have obvious potential applicability in clinical practice aspect the disease treatment that causes as viral infection or gene mutation.There are some researches show,, can suppress to infect the virus replication in the sars coronavirus Rhesus Macacus lung with the siRNA of some structural protein gene of sars coronavirus.In addition, utilize siRNA to suppress the overexpression of oncogene, cancer associated gene or mutant gene specifically, make these genes remain on silent or resting state, be expected to reach antitumor action.RNA disturbs also can be applied to the airway inflammation treatment of diseases, Shao etc. studies show that, suppress reduction and the mucous secretion (ShaoMX of minimizing airway epithelia that the TGF α among people's air flue epithelium born of the same parents NCI-H292 can cause GFR to express with the siRNA specificity, NakanagaT, NadelJA.Cigarette smoke induces MUC5ACmucin overproduction via tumor necrosis factor-α-converting enzyme in human airwayepithelial (NCI-H292) cells.Am J Physiol Lung Cell Mol Physiol, 2004,287 (2): L420-L427).Yet siRNA is in vivo easily by nuclease degradation, thus its poor stability and half-life short, lack targeting, need carry out sending of gene in conjunction with carrier system.Gene delivery method at present commonly used mainly is divided into two classes, i.e. viral vector and non-virus carrier leading-in technique, and wherein, viral vector transfection efficiency height is the main tool of present vivo gene treatment.Yet although viral vector has very high transfection efficiency, there are safety hidden danger such as immunogenicity and potential oncogenicity in it.Non-virus carrier mainly contains liposome, cation superpolymer etc., but non-viral gene vector is because of lacking the natural gene delivery mechanism of viral vector, and transfection efficiency is low, has greatly limited its practical application.Therefore,, enter fully effectively performance RNA interference effect of target cell, become the bottleneck problem of present clinical practice siRNA treatment how with the safe and effective pathological changes target organ that is transported to of siRNA.
Chitosan is a kind of novel non-viral gene transport vehicle that development in recent years is got up, and also is unique a kind of natural cationic polysaccharide.It has excellent biological compatibility, biodegradability, and safety is good.Yet the same with other non-virus carriers, chitosan self leap cell membrane barrier ability is lower, need modify it, strides the film ability to improve it.(cell penetrating peptide is that a class can be carried macromolecular substances and entered cell CPP) to cell-penetrating peptide, has the small peptide of wearing the film ability.Discoveries such as Wender, arginine plays crucial effect striding of cell-penetrating peptide in the film, its characteristic group guanidine radicals is central factor (Wender P.A, Mitchell D.J, the Pattabiraman K that promotes to stride film, et al.Thedesign, synthesis, and evaluation of molecules that enable or enhance cellular uptake:peptoid moleculartransporters, Proc.Natl.Acad.Sci.USA, 2000,97:13000-13008).The inventor has successfully synthesized guanidinated chitosan early stage, and confirms that this method of modifying can obviously improve the ability of tegument polysaccharide cell in vitro transfection DNA nucleic acid.Guanidine radicals is modified simply nontoxic, can fully dissolve under the chitosan physiology PH environment after guanidine radicals is modified.
In order further to improve the ability of guanidinated chitosan to the cell traffic nucleic acid substances, according to part and receptor principle of combining, a kind of albuterol grafting guanidinated chitosan and preparation method thereof is disclosed among inventor's the Chinese patent application CN 101781373A, be clinical medicinal β 2 adrenoreceptor agonists albuterol (air flue spasmolytic anti-inflammatory preparation) to be grafted to constitute albuterol grafting guanidinated chitosan on the guanidinated chitosan molecule, and studies confirm that by cell in vitro this carrier can further improve the gene transfection efficient of cell.But, and do not mean that Application feasibility in the body because therefore human body or animal internal milieu complexity only prove that in external direct transfection cultured cells this carrier has gene transfection efficient preferably.
The carrier particle diameter is the important parameter of gene microparticulate delivery system, its size and physical characteristics be except meeting influences carrier particles the absorption on target cell surface and picked-up, also can influence the time multiplexed cell compound from endosome successfully escape, the processes such as release of nucleic acid substances.And the uniformity of nanometer particle size is another important parameter of nano-carrier, the distribution of particle diameter is wide, it is all less to be illustrated in the nanoparticle that distributes in arbitrary subrange, so no matter how many suitableeest particle diameters is, though the suitableeest transfection particle diameter of this nano-carrier near mean diameter, but because of near the ratio of the nanoparticle mean diameter little, most of particle grain size is away from mean diameter, perhaps too big, perhaps very little, so can be effectively gene be carried out the carrier ratio of transfection and few.From a of Fig. 9 as seen, the particle size distribution of previous albuterol guanidinated chitosan (Sal-guanidinated chitosan) is inhomogeneous, and the particle diameter after Sal-guanidinated chitosan and nucleic acid substances are compound is less, between 20～60nm scope, the particle size distribution dispersion is big, and the ratio few (Figure 10) of the most suitable particle diameter Sal-guanidinated chitosan nanoparticle that helps cell transfecting is described.Importing Target organ tissue and cell and how to realize transporting in the chitosan nano carrier body nucleic acid substances, is the key scientific problems that the clinical practice of siRNA mediated gene silencing need solve.
Adopting the inhalation mode is the clinical effective administering mode of treatment respiratory tract disease, can realize the targeting Target organ.But the granular size of inhalation aerosol only between 4～6um, is brought into play drug effect thereby the medicine droplet is deposited in lung.With respect to clinical other suction, ultrasonic atomizatio sucks more simple.This suction by adopt a ultrasonic atomizing device with the water solublity medicinal liquid under the effect of the higher-order of oscillation, form the aerosol that constitutes by 4～6um ultramicron and suck treatment.But problem is the higher-order of oscillation can damage the structure of nucleic acid substances, therefore clinical be difficult to adopt suck the treatment pattern and carry out respiratory system disease microRNA interference treatment.
Summary of the invention
First purpose of the present invention is by a kind of respiratory tract siRNA atomizing administration nano-drug administration system of going back to the nest is provided, this drug-supplying system can be used for transporting the siRNA of jamming target Disease-causing gene, with performance RNA interference effect, suppressing Disease-causing gene duplicates, can transport microRNA (siRNA) targeting pulmonary respiration road epithelium in vivo, be to be that the non-virus that purpose is brought into play corresponding drug action is transported system with reticent target gene.
Second purpose of the present invention provides the go back to the nest preparation method of siRNA atomizing administration nano-drug administration system of above-mentioned respiratory tract.
First purpose of the present invention is achieved through the following technical solutions: a kind of respiratory tract siRNA atomizing administration nano-drug administration system of going back to the nest, this drug-supplying system is a carrier material with the albuterol guanidinated chitosan after handling through ultrasonic atomizatio, and the siRNA of parcel jamming target Disease-causing gene makes.
Have β 2 adrenergic receptor part albuterol in the albuterol guanidinated chitosan (Sal-guanidinated chitosan), promptly had the device of going back to the nest, for airway epithelial cell, smooth muscle cell and mastocyte etc. has the targeting effect.And before ultrasonic atomizatio is handled, the particle diameter of Sal-guanidinated chitosan is less, the particle size distribution dispersion is big, size and distribution are all very undesirable, and the ratio of the suitableeest particle diameter Sal-guanidinated chitosan nanoparticle is few, cause the intake of the cell of target organ in the human body few, be difficult to bring into play effective RNA interference effect.No matter through discovering of the inventor is independent Sal-guanidinated chitosan nanoparticle or Sal-guanidinated chitosan and nucleic acid substances composite nano-granule, and after handling through ultrasonic atomizatio, its particle diameter all corresponding increase can occur.The particle diameter of the Sal-guanidinated chitosan nanoparticle after the ultrasonic atomizatio physical modification is big before than ultrasonic atomizatio, and the particle size distribution dispersion is little, and the ratio of the suitableeest particle diameter Sal-guanidinated chitosan nanoparticle is many.The experiment proved that, siRNA is compound with the Sal-guanidinated chitosan nano-carrier after the ultrasonic atomizatio processing, form in vivo the version that can be absorbed by the target organ cell high-efficient (specifically be exactly can in vivo specifically by the version of airway epithelia cellular uptake in the lung), then can fully discharge the effect of performance RNA interference treatment from endosome.As seen, the compound atomizing nanometer of Sal-guanidinated chitosan after ultrasonic atomizatio is handled and nucleic acid substances formation can further improve picked-up and the gene transfection efficient of cell to nucleic acid substances.
Described respiratory tract of the present invention is gone back to the nest the particle diameter of siRNA atomizing administration nano-drug administration system between 65～85nm scope.
The siRNA of jamming target Disease-causing gene of the present invention is that specificity disturbs intrusive viruses gene or interference to cause the siRNA of the gene of inflammation or oncosis, as the siRNA of some structural protein gene of sars coronavirus, siRNA, the siRNA of hepatitis virus of influenza virus A type conservative region encoding gene, and the siRNA of the siRNA of tumor-related gene or mutant gene etc.The siRNA of jamming target Disease-causing gene can design the screening preparation according to the structure of objectives Disease-causing gene according to a conventional method and get final product.
Mass ratio between the described albuterol guanidinated chitosan among the present invention and the siRNA of jamming target Disease-causing gene is 1～5: 1.
Second purpose of the present invention can realize by following technical measures: the go back to the nest preparation method of siRNA atomizing administration nano-drug administration system of a kind of above-mentioned respiratory tract, and it may further comprise the steps:
(I) getting the albuterol guanidinated chitosan is dissolved in water, preparation obtains albuterol guanidinated chitosan aqueous solution, the siRNA mixing that adds the jamming target Disease-causing gene then is compound, and the siRNA of albuterol guanidinated chitosan parcel jamming target Disease-causing gene forms the complex solution of the siRNA of albuterol guanidinated chitosan and jamming target Disease-causing gene;
(II) complex solution with the siRNA of described albuterol guanidinated chitosan and jamming target Disease-causing gene places ultrasonic atomizing device, be atomized into droplet, obtain the compound atomizing nanoparticle of siRNA of albuterol guanidinated chitosan and jamming target Disease-causing gene, drug-supplying system promptly of the present invention.
When the complex solution of the siRNA of described albuterol guanidinated chitosan and jamming target Disease-causing gene touches the atomizing piece of ultrasonic atomizing device, but moment is atomized into droplet, be that the ultrasonic atomizatio of complex solution of the siRNA of described albuterol guanidinated chitosan and jamming target Disease-causing gene is handled and promptly to be finished in moment, destroy the structure of albuterol guanidinated chitosan to avoid the long higher-order of oscillation.
Mass ratio between the siRNA of albuterol guanidinated chitosan described in the step of the present invention (I) and jamming target Disease-causing gene is 1～5: 1.
Between power 115KHz～140KHz scope that ultrasonic atomizatio is handled in the described step (II).
As the go back to the nest preparation method of siRNA atomizing administration nano-drug administration system of the feasible respiratory tract of another kind of the present invention, its concrete operations are:
(1) get the albuterol guanidinated chitosan and be dissolved in water, preparation obtains albuterol guanidinated chitosan aqueous solution, carries out ultrasonic atomizatio then and handles, and collects the droplet of ultrasonic atomizatio gained;
(2) siRNA that adds the jamming target Disease-causing gene in step (1) in the droplet of collecting carries out compound, the siRNA of albuterol guanidinated chitosan parcel jamming target Disease-causing gene forms the compound atomizing nanoparticle of siRNA of albuterol guanidinated chitosan and jamming target Disease-causing gene, promptly obtains drug-supplying system of the present invention.
The ultrasonic atomizatio of albuterol guanidinated chitosan solution is handled and is adopted ultrasonic atomizing device in the described step (1), when albuterol guanidinated chitosan solution touches the atomizing piece of ultrasonic atomizing device, but moment is atomized into droplet, the ultrasonic atomizatio processing that is described albuterol guanidinated chitosan was promptly finished in moment, destroyed the structure of albuterol guanidinated chitosan to avoid the long higher-order of oscillation.
Mass ratio between the siRNA of albuterol guanidinated chitosan described in the step of the present invention (1) and jamming target Disease-causing gene is 1～5: 1.
Between power 115KHz～140KHz scope that ultrasonic atomizatio is handled in the described step (1).
Set forth the present invention's beneficial effect compared with prior art below in conjunction with test:
(1) the present invention handles by the albuterol guanidinated chitosan being carried out ultrasonic atomizatio, and the compound atomizing administration nano-drug administration system that constitutes with nucleic acid substances can further improve picked-up and the gene transfection efficient of cell to nucleic acid substances again.
The chitosan nano of albuterol guanidine radicals modification is compound with siRNA in proportion, utilize fluorescent dye YOYO1 labeling nucleic acid, the interior experiment of external and body is all found: the modification of albuterol guanidine radicals can increase cell endocytosis (Fig. 2 to the chitosan nucleic acid complexes, Fig. 3), albuterol guanidinated chitosan nanometer in proportion with the siRNA of targeting green fluorescent protein (GFP) compound after, through hatching altogether with cultured cell, can be gulped down drink by cell (cell strain of a stably express green fluorescent protein) rapidly, and discharge from endosome immediately, effectively mediate the specific interference effect of this siRNA, the expression of reduction cell GFP (Fig. 4, Fig. 5).Simultaneously, this interference effect also is verified (Fig. 6) in animal body.
The Beta adrenoreceptor is one of super family of G-protein coupled receptor (GPCRs), has 7 and strides the film district, is divided into β 1,3 three kinds of hypotypes of β 2 and β, and wherein beta 2 receptor is distributed widely in smooth muscle and the lung tissue.GPCRs and corresponding part in conjunction with after, can bring out the internalization of the ligand/receptor complex that clathrin mediates, the cell that has corresponding receptor can increase being grafted with the carrier specificity picked-up of beta 2 receptor part.Because 293 cells can be expressed the beta 2 receptor of certain level, and the 16HBE cell is not expressed, relatively confirm by these two kinds of cells are carried out transfection experiment: than the 16HBE cell of not expressing beta 2 receptor, the gene transfection efficient of Sal-guanidinated chitosan nano-carrier will be apparently higher than simple guanidinated chitosan carrier (Fig. 7) at 293 cells that have beta 2 receptor.The compound atomizing nanoparticle of albuterol guanidinated chitosan and siRNA is after intratracheal administration, and the expression that as seen reduces GFP transgenic mice lung inner structure cell GFP is (Fig. 8) the most effectively.Therefore can prove from experiment in vivo and vitro that albuterol guanidinated chitosan nucleic acid nano grain has the interior targeting airway epithelial of body and transports nucleic acid substances, the treatment ability of siRNA is disturbed in performance.
After the present invention carries out physical modification by the employing ultrasonic atomizatio to the albuterol guanidinated chitosan, make the particle diameter of albuterol guanidinated chitosan nanoparticle and albuterol guanidinated chitosan and nucleic acid composite nano-granule handle preceding big than ultrasonic atomizatio, and the particle size distribution dispersion of nanoparticle obviously reduces, the increasing proportion of the suitableeest grain diameter nano grain, uniformity significantly increases (referring to Fig. 9 and Figure 10), proves that the ultrasonic atomizatio effect can further optimize albuterol guanidinated chitosan nano-carrier.
And be better than albuterol guanidinated chitosan (referring to Figure 12 and Figure 13) without the ultrasonic atomizatio processing through the ability that ultrasonic atomizatio is handled the intracellular transport siRNA of back albuterol guanidinated chitosan, above experimental result illustrates the respiratory tract of the present invention version of version for being absorbed by the target organ cell high-efficient in vivo of siRNA atomizing administration nano-drug administration system of going back to the nest, and can fully discharge the effect of performance RNA interference treatment from endosome.
(2) ultrasonic atomizatio effect of the present invention is primarily aimed at carrier itself, and is related little with the order of ultrasonic atomizatio processing and compound transfectional cell, and promptly earlier compound ultrasonic atomizatio is again handled and can be improved picked-up and the gene transfection efficient of cell to nucleic acid substances equally.
No matter albuterol guanidinated chitosan nanometer is handled through ultrasonic atomizatio earlier forms compound atomizing nanoparticle with nucleic acid substances again; or carry out ultrasonic atomizatio again after albuterol guanidinated chitosan nanometer and nucleic acid substances are compound; the parcel of albuterol guanidinated chitosan nanometer all can effectively protect nucleic acid substances to avoid the degraded destruction (Figure 11) of mechanical shear stress; the albuterol guanidinated chitosan just can obviously strengthen carrying the ability of nucleic acid substances as long as handle through ultrasonic atomizatio.
And it is through the albuterol guanidinated chitosan of ultrasonic atomizatio the efficient higher (Figure 12,13) with rotaring redyeing gene of transporting than not modified carriers such as chitosan.Thereby aforesaid two kinds prepare order and all can improve picked-up and the gene transfection efficient of cell to nucleic acid substances.
(3) the present invention adopts the siRNA compound mode of again carrying out ultrasonic atomizatio processing of elder generation with albuterol guanidinated chitosan and jamming target Disease-causing gene, more helps simplifying the preparation process of drug-supplying system, be suitable for the clinical administration operation more, and effect can be better.
The experiment proved that, no matter be earlier the albuterol guanidinated chitosan to be carried out the siRNA that ultrasonic atomizatio handles with the jamming target Disease-causing gene again to carry out compound, or carrying out ultrasonic atomizatio after the siRNA of albuterol guanidinated chitosan and jamming target Disease-causing gene is compound again handles, the albuterol guanidinated chitosan of gained and the compound atomizing nanometer of the siRNA of jamming target Disease-causing gene all have higher cell transfecting efficient, the effect that ultrasonic atomizatio has been described is only had an effect to the albuterol guanidinated chitosan, does not destroy the structure of siRNA.But the siRNA of albuterol guanidinated chitosan and jamming target Disease-causing gene mixes the compound ultrasonic atomizatio that carries out again earlier to be handled, carry out being atomized into when ultrasonic atomizatio is handled can clinical use droplet, make that the preparation process of drug-supplying system of the present invention is simpler, be suitable for the clinical administration operation more, and better effects if.
Description of drawings
Fig. 1 is that fluorescent dye Yoyo1 combines situation with carrier.
A:yoyo1; The B:yoyo1+Sal-guanidinated chitosan; C:yoyo1+siRNA; D:yoyo1+siRNA+lipofectamine; The E:yoyo1+siRNA+ chitosan; The F:yoyo1+siRNA+Sal-guanidinated chitosan.Separately Yoyo1 (A) and Yoyo1 carrier mixture (B) all do not show fluorescence under the situation of bind nucleic acid not.When with nucleic acid substances siRNA in conjunction with then sending strong green fluorescence (C, D, E, F) later on
Fig. 2 is the situation of each carrier intracellular transport nucleic acid.
A:Lipofectamine; B: chitosan; The C:Sal-guanidinated chitosan.Fig. 2 A shows in most of cell visible green fluorescence, Fig. 2 B only sees a small amount of gfp positive cell, Fig. 2 C is presented at Sal-guanidinated chitosan vehicle group, and the ratio of gfp positive cell is lower than matched group, but fluorescence intensity is higher than matched group in the individual cells.
Fig. 3 is various carriers are transported nucleic acid in the middle of mouse lung a situation.
A, B, C:Lipofectamine; D, E, F: chitosan; G H, the I:Sal-guanidinated chitosan
A, D, G:yoyo1 spike; B, E, H:DAPI dyeing; C, F, I: green fluorescence (yoyo1) is compound with blue-fluorescence (DAPI).
Figure A and figure B show that mouse lung is not observed the male lung airway cell of green fluorescence basically.At Sal-guanidinated chitosan and siRNA complex group mouse lung tissue, the equal visible green fluorescence of its alveolar and airway epithelia place positive cells is as figure C.
Fig. 4 is that fluorescence microscope shows that various carriers carry the situation that microRNA is expressed at cell internal interference GFP.
The complex of A:Sal-guanidinated chitosan and negative-control siRNA; B: the complex of tegument polysaccharide and GFPsiRNA; The complex of C:Sal-guanidinated chitosan and GFP siRNA.
The average fluorescent strength of figure B showed cell is weaker than the cell in the negative control group among the figure A slightly.The complex nanometer of Sal-guanidinated chitosan and GFP siRNA can significantly weaken the average fluorescent strength of transfected cell, and its cell fluorescence intensity is starkly lower than figure A negative control group cell shown in the figure C.Annotate: this cytotostatic is expressed GFP.
Fig. 5 is that the various carriers of flow cytometer quantitative analysis carry the situation that siRNA expresses at cell internal interference GFP.
1:Sal-guanidinated chitosan and negative-control siRNA composite nano-granule;
2: tegument polysaccharide and GFP siRNA composite nano-granule (mass ratio: tegument polysaccharide: GFP siRNA=5: 1);
3: tegument polysaccharide and GFP siRNA composite nano-granule (mass ratio: tegument polysaccharide: GFP siRNA=3: 1);
4: tegument polysaccharide and GFP siRNA composite nano-granule (mass ratio: tegument polysaccharide: GFP siRNA=1: 1);
5:Sal-guanidinated chitosan and GFP siRNA composite nano-granule (mass ratio: Sal-guanidinated chitosan: GFPsiRNA=5: 1);
6:Sal-guanidinated chitosan and GFP siRNA composite nano-granule (mass ratio: Sal-guanidinated chitosan: GFPsiRNA=3: 1);
7:Sal-guanidinated chitosan and GFP siRNA composite nano-granule (mass ratio: Sal-guanidinated chitosan: GFPsiRNA=1: 1);
Annotate: this cytotostatic is expressed GFP; * P＜0.05.
Fig. 6 is that fluorescence microscope shows Sal-guanidinated chitosan interferential situation of mediate rna in the mice body.
Neg:Sal-guanidinated chitosan and negative-control siRNA composite nano-granule; CS: tegument polysaccharide and GFP siRNA composite nano-granule; Sal-Gua-CS:Sal-guanidinated chitosan and GFP siRNA composite nano-granule.
Wherein, Neg and CS group visible airway epithelia cell of mouse lung tissue slice and the equal strongly expressed green fluorescent protein of alveolar epithelial cells.By comparison, give the mouse lung tissue that Sal-guanidinated chitosan and GFP siRNA composite nano-granule are handled, the GFP expression significantly descends.
Annotate: this mice is the GFP transgenic mice.
Fig. 7 is two kinds of carriers bar diagram of the efficient in HEK293 cell and 16HBE cell respectively.
The A:HEK293 cell, flow cytometer detects; The B:16HBE cell, the cell quantity of expressing green fluorescent protein in the middle of average each visual field of microscopically counting.*v.s.Chitosan?P＜0.01
Fig. 8 is that fluorescence microscope shows Sal-guanidinated chitosan interferential situation of targeting mediate rna in the mice body.
CS: tegument polysaccharide and GFP siRNA composite nano-granule; Gua-CS: guanidinated chitosan and GFP siRNA composite nano-granule; Sal-Gua-CS:Sal-guanidinated chitosan and GFP siRNA composite nano-granule.
CS group visible airway epithelia cell of mouse lung tissue slice and the equal strongly expressed green fluorescent protein of alveolar epithelial cells.By comparison, give the mouse lung tissue of the composite nano-granule processing of guanidinated chitosan and GFP siRNA, the GFP expression descends to some extent, and gives the mouse lung tissue of the composite nano-granule processing of Sal-guanidinated chitosan and GFP siRNA, and the GFP expression descends more obvious.
Fig. 9 has an X-rayed Electronic Speculum figure before albuterol guanidinated chitosan and the compound atomizing administration nano-drug administration system of the DNA ultrasonic atomizatio and behind the ultrasonic atomizatio;
The a:Sal-guanidinated chitosan; B:Sal-guanidinated chitosan and DNA composite nano-granule;
C: the Sal-guanidinated chitosan after ultrasonic atomizatio is handled; D: Sal-guanidinated chitosan and the compound atomizing nanoparticle of DNA after ultrasonic atomizatio is handled.
Figure 10 is before albuterol guanidinated chitosan and the compound atomizing administration nano-drug administration system of DNA ultrasonic atomizatio are handled and the scattergram of ultrasonic atomizatio processing back nanometer particle size.
Chi/DNA: albuterol guanidinated chitosan and DNA composite nano-granule before ultrasonic atomizatio is handled;
A (chi/DNA): albuterol guanidinated chitosan and DNA composite nano-granule after ultrasonic atomizatio is handled;
Before ultrasonic atomizatio was handled, the particle diameter of Sal-guanidinated chitosan and Sal-guanidinated chitosan and DNA composite nano-granule was less, the distribution broad; Ultrasonic atomizatio is handled its particle diameter of back and is increased to some extent, and particle size distribution range narrows down, the more preceding obvious improvement of the uniformity.
* P＜0.01; Little square frame representative diameter wherein distributes.
Figure 11 be naked DNA and DNA by multi-form with Sal-guanidinated chitosan carrier the electrophoretogram after compound;
Lane 4: the naked DNA behind the ultrasonic atomizatio; Lane 5: behind the naked DNA ultrasonic atomizatio with the complex of Sal-guanidinated chitosan; Lane 6: the complex of the Sal-guanidinated chitosan behind naked DNA behind the independent ultrasonic atomizatio and the independent ultrasonic atomizatio; The complex of Lane 7:DNA and Sal-guanidinated chitosan is through the product of ultrasonic atomizatio;
Lane4, Lane5 and Lane6 show that all naked DNA is subjected to the tangible mechanical shearing effect of ultrasonic atomizatio and is degraded, the product distribution that becomes band; Lane7 carries out ultrasonic atomizatio and atomizing after showing carrier parcel DNA again, and complex intactly is trapped in the well.
Figure 12 is the influence (HEK293 cell) of ultrasonic atomizatio to each carrier complexes transfection efficiency.
1.Sal-guanidinated chitosan and DNA (pEGFP-N2 plasmid) composite nano-granule group;
2.Sal-the composite nano-granule group of the naked DNA behind guanidinated chitosan and the ultrasonic atomizatio;
3. the composite nano-granule group of the naked DNA behind Sal-guanidinated chitosan and the ultrasonic atomizatio behind the ultrasonic atomizatio (Sal-guanidinated chitosan and naked DNA ultrasonic atomizatio after compound again) respectively;
4. the compound atomizing nanoparticle of ultrasonic atomizatio Sal-guanidinated chitosan and DNA group (the Sal-guanidinated chitosan carries out ultrasonic atomizatio earlier to be handled, compound with DNA again);
5.Sal-the compound atomizing nanoparticle group of guanidinated chitosan and DNA (after Sal-guanidinated chitosan and DNA are compound droplet that handle to collect of ultrasonic atomizatio) again.
*v.s?group?1，P＜0.01.
Figure 13 is the variation (16HBE cell) of transfection efficiency before and after Sal-guanidinated chitosan and the DNA complex ultrasonic atomizatio;
Gua:Sal-guanidinated chitosan and DNA (pEGFP-N2 plasmid) composite nano-granule group;
The compound atomizing nanoparticle group of Aerosol:Sal-guanidinated chitosan and DNA (pEGFP-N2 plasmid);
*P＜0.01。
The specific embodiment
Describe respiratory tract provided by the invention the go back to the nest preparation and the application of siRNA atomizing administration nano-drug administration system by the following examples, embodiment only is used to illustrate purpose of the present invention, does not limit protection scope of the present invention.
Experimental example one: the ability of transhipment nucleic acid in the Sal-guanidinated chitosan nanometer body
Main experiment material
Natural chitosan nano: U.S. Sigma company
Sal-guanidinated chitosan: according to " salbutamol modified guanidinated chitosan and preparation method and application " (application number: the preparation of method 200910245193.6)
MEM culture medium: U.S. Gibico company
Hyclone: Hangzhou Ilex purpurea Hassk.[I.chinensis Sims company
Plasmid extraction test kit: U.S. Promega company
Lipofectamin 2000 (nucleic acid transfection reagent): American I nvitrogen company
SiRNA: U.S. AMBION company
Yoyo1 (fluorescent dye is used for the labeling nucleic acid material): American I nvitrogen company
Experimental technique
1.Sal-the guanidinated chitosan nanometer is to intracellular transport nucleic acid
1.1 it is compound to get siRNA and the Yoyo1 fluorescent dye lucifuge of 1ug, (siRNA: mass ratio carrier) carried out compound by 1: 3 with tegument polysaccharide, Sal-guanidinated chitosan and Lipofectamine2000 (reference substance) respectively later in 2 hours, the described compound process that is meant carrier parcel siRNA such as tegument polysaccharide, Sal-guanidinated chitosan and Lipofectamine2000, and tegument polysaccharide and Sal-guanidinated chitosan are all positively charged, and siRNA is electronegative, mixes natural chitosan in back and Sal-guanidinated chitosan and can wrap up siRNA.Observe the fluorescence intensity of each complex Yoyo1 after half an hour at laser confocal microscope.
1.2 get the HEK293 cell with 2 * 10
5Cell/ hole concentration is inoculated in 24 orifice plates, and routine is incubated at 37 ℃, 5%CO
2In the incubator, treat that cell density reaches to use the various carriers of stating preparation at 60～70% o'clock respectively and the complex of nucleic acid substances carries out transfection (siRNA1ug/ hole).Under Laser Scanning Confocal Microscope, observe the situation that fluorescence indicator Yoyo1 distributes after 24 hours in cell.
2.Sal-the guanidinated chitosan nanometer is transported nucleic acid in vivo
2.1 get siRNA and Yoyo1 fluorescent dye lucifuge is compound, (siRNA: mass ratio carrier) carried out compound half an hour by 1: 3 with tegument polysaccharide, Sal-guanidinated chitosan and Lipofectamine 2000 (reference substance) respectively later in 2 hours.Through trachea various carriers/nucleic acid substances complex is injected into mouse lung (every mice of siRNA 2ug/), put to death mice in 6 hours later on, separate the freezing embedding of lungs and carry out tissue freezing section, under laser confocal microscope, observe the distribution situation of green fluorescence dyestuff yoyo1 at the lung inner cell.
Experimental result
1. independent Yoyo1, Yoyo1 and carrier mixture be can't see fluorescence substantially under the situation of bind nucleic acid material siRNA not.After Yoyo1 and nucleic acid substances siRNA combination, send strong green fluorescence at once, but being a silk wadding sample, major part distributes.Add after the carrier, most of fluorescent material is graininess and distributes, and fraction is assembled agglomerating, referring to Fig. 1.Laser confocal microscope is observed three kinds of carriers import ability from siRNA to cell, show: in Lipofectamin2000 group (reference substance group: liposome), visible green fluorescence (seeing Fig. 2 A) all in nearly all cell, in Sal-guanidinated chitosan vehicle group, though the ratio of gfp positive cell is lower than contrast liposome group, but fluorescence intensity is higher than matched group (seeing Fig. 2 C) in the individual cells, and the siRNA amount of prompting individual cells picked-up increases than matched group.At the only visible a small amount of gfp positive cell (seeing Fig. 2 B) of tegument polysaccharide vehicle group.
2. in each experimental mice lung tissue, laser confocal microscope shows: give Lipofectamin 2000 and do not observe the male lung airway cell of green fluorescence basically with the mouse lung of siRNA composite nano-granule, the nucleic acid transfection reagent Lipofectamin liposome of prompting transfection isolated cells can not be transported siRNA into mouse lung cell (seeing Fig. 3 A, D and G) effectively in vivo.Can not transport nucleic acid substances siRNA (seeing Fig. 3 B, E and H) equally effectively in vivo through the natural chitosan nano carrier of modifying.On the contrary, at the mouse lung tissue of Sal-guanidinated chitosan and siRNA complex group, the equal visible green fluorescence of its alveolar and airway epithelia place positive cells (seeing Fig. 3 C, F and I).Show through trachea and give the Sal-guanidinated chitosan and the siRNA composite nano-granule can effectively be absorbed by the cell of mouse lung in vivo.
Conclusion: Sal-guanidinated chitosan and siRNA composite nano-granule can effectively be transported siRNA the cell into In vitro culture, and can transport siRNA in vivo to the lung inner cell through air flue under condition of living organism.
Experimental example two: Sal-guanidinated chitosan and siRNA composite nano-granule are brought into play the RNA interference effect in vivo
Main experiment material
Sal-guanidinated chitosan: according to " salbutamol modified guanidinated chitosan and preparation method and application " (application number: the preparation of method 200910245193.6)
Natural chitosan nano: U.S. Sigma company
Green fluorescent protein (GFP) siRNA: U.S. AMBION company
Negative Control siRNA: U.S. AMBION company
GFP stably express HEK293 cell strain: this laboratory screening and cultivation
EGFP transgenic mice: match industry Bioisystech Co., Ltd.The EGFP transgenic mice is the C57BL/6 background, and expression way is a whole body expression.EGFP is gene constructed on the pCAGGS carrier, and this carrier has chicken β-actin promoter, cmv enhancer.
Ultrasonic atomizatio head (Aeroneb
Lab Nebulizer): U.S. Aerogen company
Microatomization device: U.S. Penn-Century company
Experimental technique
1.Sal-the interferential effect of guanidinated chitosan mediate rna (cell of In vitro culture)
The Sal-guanidinated chitosan by with 3: 1 mass ratio of Negative Control siRNA compound half an hour; Tegument polysaccharide and GFP siRNA be by 5: 1, the compound half an hour of mass ratio of 3: 1 and 1: 1; Sal-guanidinated chitosan and GFPsiRNA be by 5: 1, the compound half an hour of mass ratio of 3: 1 and 1: 1.Inoculation HEK293 cell (stably express GFP)) with 2 * 105cell/ hole concentration in 24 orifice plates, Sal-guanidinated chitosan and Negative Control siRNA composite nano-granule, tegument polysaccharide and GFP siRNA composite nano-granule and Sal-guanidinated chitosan and GFP siRNA composite nano-granule are added into respectively in the hole of inoculating cell (the siRNA/ hole of 1ug).Laser confocal microscope is observed the change that each processed group cell GFP expresses after 24 hours, respectively organizes the average fluorescent strength of cell GFP simultaneously with the flow cytometer detection by quantitative.
2.Sal-guanidinated chitosan and siRNA composite nano-granule be the interferential effect of mediate rna in the mice body
Sal-guanidinated chitosan and Negative Control siRNA press 3: 1 compound half an hour of mass ratio, obtain Sal-guanidinated chitosan and Negative Control siRNA composite nano-granule; Tegument polysaccharide and GFP siRNA press 3: 1 compound half an hour of mass ratio, obtain tegument polysaccharide and GFP siRNA composite nano-granule; Sal-guanidinated chitosan and GFP siRNA press 3: 1 compound half an hour of mass ratio, obtain Sal-guanidinated chitosan and GFP siRNA composite nano-granule; Again the using ultrasound atomising head (
Lab Nebulizer) respectively three kinds of composite nano-granules is carried out ultrasonic atomizatio, connect the atomized liquid outlet of atomising head then with 10ml glass centrifuge tube, collect droplet (aerosol of formation drips about 4～6 microns of diameter Distribution, is fit to clinical nebulae inhalation).The transgenic mice (the equal expressing green fluorescent protein of each organs and tissues in the body) of stably express GFP is adopted in experiment, the various complex nanometers of utilizing the microatomization device of Penn-Century company to collect give the GFP transgenic mice respectively by the mode that atomizes in the air flue, once a day, each 100ul (siRNA that contains 2ug), for three days on end, put to death mice on the 4th day, frozen section is carried out in freezing embedding, and laser confocal microscope is observed the influence that each processing factor organizes GFP to express to mouse lung.
The result
1. laser confocal microscope and flow cytometer detect and to show: the HEK293 cell of the stably express GFP of tegument polysaccharide and GFPsiRNA composite nano-granule transfection In vitro culture, can slightly reduce the average fluorescent strength of cell; By contrast, Sal-guanidinated chitosan behind the ultrasonic atomizatio and GFPsiRNA composite nano-granule can significantly weaken the average fluorescent strength of transfected cell, show Sal-guanidinated chitosan behind the ultrasonic atomizatio and GFP siRNA composite nano-granule can with GFP siRNA effectively transhipment bring in the cell, and the interferential effect of performance RNA, the expression of GFP in the reduction cell.(seeing Fig. 4 and Fig. 5).
2. laser confocal microscope is observed lung tissue section and shown: the expression that the mouse lung that gives Sal-guanidinated chitosan and GFPsiRNA composite nano-granule is organized GFP is handled remarkable decline (see figure 6) than negative control or tegument polysaccharide and GFPsiRNA composite nano-granule.
Conclusion: can under condition of living organism, in lung, bring into play effective RNA interference effect through air flue through Sal-guanidinated chitosan and siRNA composite nano-granule that ultrasonic atomizatio is handled.
Experimental example three: targeting transhipment nucleic acid carries out the RNA interference capability under the composite nano-granule condition of living organism of Sal-guanidinated chitosan and nucleic acid
Material and method
Main experiment material
Guanidinated chitosan: according to " salbutamol modified guanidinated chitosan and preparation method and application " (Chinese patent application, application number: the preparation of method 200910245193.6).
Albuterol (Sal): U.S. Sigma company.
Sal-guanidinated chitosan nano-carrier: according to " husky fourth limb alcohol modified guanidinated chitosan and preparation method and application " (Chinese patent application, application number: the preparation of method 200910245193.6).
16HBE cell (human bronchial epithelial cell strain): professor Steven.Holgate of university of Britain Southampton (Southampton) is so kind as to give.
PEGFP-N2 plasmid: U.S. Clontech company.
Green fluorescent protein (GFP) siRNA: U.S. AMBION company.
GFP transgenic mice: with experimental example two.
The ultrasonic atomizatio head (
Lab Nebulizer): U.S. Aerogen company.
Microatomization device: U.S. Penn-Century company.
Method
1, because the HEK293 cell can be expressed beta 2 receptor, and the 16HBE cell is not expressed beta 2 receptor, and at first the guanidinated chitosan that has compared grafting beta 2 receptor agonist albuterol not at cultured cell in vitro and the guanidinated chitosan (Sal-guanidinated chitosan) of grafting albuterol are in the different abilities to above-mentioned two kinds of cell transfecting nucleic acid.
Get the HEK293 cell with 2 * 10
5Cell/ hole concentration is inoculated in 24 orifice plates, and routine is incubated at 37 ℃, 5%CO
2In the incubator, treat to carry out when cell density reaches 60%-70% transfection, experiment divides two groups, and simple guanidinated chitosan transfection group and Sal-guanidinated chitosan transfection group are carried out transfection (plasmid/hole of 1ug) with 5: 1 mass ratio and pEGFP-N2 plasmid after compound respectively.Changed liquid once in 24 hours, after the transfection 72 hours, flow cytometer calculate and relatively guanidinated chitosan and pEGFP-N2 plasmid composite nano-granule and Sal-guanidinated chitosan and pEGFP-N2 plasmid composite nano-granule at the HEK293 cell by the positive cell ratio of successful transfection pEGFP-N2 plasmid.Meanwhile, guanidinated chitosan and different (according to expressing green fluorescent protein cell number/on average each visual field cell number measuring and calculating) of Sal-guanidinated chitosan under the fluorescence microscope have been compared in the 16HBE cell transfecting pEGFP-N2 efficient of not expressing beta 2 receptor.
2, checking Sal-guanidinated chitosan and pEGFP-N2 plasmid composite nano-granule under condition of living organism by the influence of device targeting air flue cell of going back to the nest to the siRNA interference performance.
Tegument polysaccharide, guanidinated chitosan and Sal-guanidinated chitosan respectively with GFP siRNA by compound half an hour of mass ratio of 3: 1, again the using ultrasound atomising head (
Lab Nebulizer) carries out ultrasonic atomizatio, connect the atomized liquid outlet of atomising head then with 10ml glass centrifuge tube, collect droplet (aerosol of formation drips about 4～6 microns of diameter Distribution, is fit to clinical nebulae inhalation).The transgenic mice (the equal expressing green fluorescent protein of each organs and tissues in the body) of stably express GFP is adopted in experiment, three kinds of complex nanometers utilizing the microatomization device of Penn-Century company to collect give the GFP transgenic mice respectively by the mode that atomizes in the air flue, once a day, each 100ul (siRNA that contains 2ug), for three days on end, put to death mice on the 4th day, frozen section is carried out in freezing embedding, and laser confocal microscope is observed the influence that each processing factor organizes GFP to express to mouse lung.
The result
1, fluorescence microscope and flow cytometer detect and show: the HEK293 cell of expressing beta 2 receptor in isolated culture, the efficient of Sal-guanidinated chitosan nano-carrier transfection pEGFP-N2 plasmid is significantly higher than the not guanidinated chitosan nano-carrier of grafting Sal, and the cell proportion of expressing green fluorescent protein significantly increases (seeing Fig. 7 A).And do not expressing the 16HBE cell of beta 2 receptor, have or not Sal-to go back to the nest device to the not obviously influence of the efficient of carrier transfection plasmid.(seeing Fig. 7 B).This result can prove that going back to the nest of guanidinated chitosan grafting Sal modify and to have targeting, can improve HEK293 cell with beta 2 receptor picked-up ability to nucleic acid.
2, laser confocal microscope shows: after live body gives the composite nano-granule of tegument polysaccharide and GFP siRNA composite nano-granule, guanidinated chitosan and GFP siRNA composite nano-granule and Sal-guanidinated chitosan GFP siRNA, can see from each group mouse lung tissue slice: than tegument polysaccharide and GFPsiRN A composite nano-granule group or guanidinated chitosan and GFPsiRNA composite nano-granule group, it is the most obvious to give the degree that the mouse lung histofluorescence of Sal-guanidinated chitosan and GFPsiRNA composite nano-granule express to descend, and shows that the interferential efficient of siRNA is best.(see figure 8).
Conclusion: Sal-guanidinated chitosan and the siRNA composite nano-granule per nasal under condition of living organism handled through ultrasonic atomizatio give mice, have targeting air flue cell traffic microRNA, realize and have improved the RNA interference capability.
Experimental example four: the go back to the nest preparation of nucleic acid atomizing nanoparticle and of respiratory tract to the transhipment effect of nucleic acid substances
Material and method
Main material and device
Sal-guanidinated chitosan carrier: according to " salbutamol modified guanidinated chitosan and preparation method and application " (Chinese patent application, application number: the preparation of method 200910245193.6)
PEGFP-N2 plasmid: U.S. Clontech company
The ultrasonic atomizatio head (
Lab Nebulizer): U.S. Aerogen company
Experimental technique
Utilized frequency for the ultrasonic atomizatio head of 128KHz ± 10% (
Lab Nebulizer drives reconfiguration by original-pack alternating current power supply and becomes portable aneroid battery to drive) the complex nanometer is carried out ultrasonic atomizatio.Be that 5: 1 ratio is compound in mass ratio earlier with Sal-guanidinated chitosan and pEGFP-N2 plasmid, use the Aeroneb atomising head again and under the frequency of 128KHz ± 10%, carry out ultrasonic atomizatio, connect the atomized liquid outlet of atomising head then with 10ml glass centrifuge tube, (aerosol of formation drips about 4～6 microns of diameter Distribution to collect droplet, be fit to clinical nebulae inhalation), atomized nanoparticle.
1, the atomizing nanoparticle that will collect prepares electron microscope specimen and carries out transmission electron microscope observing nanometer particle size size and the variation that distributes.
2, gel electrophoresis carries out the DNA-gel retardation assasy and observes the integrity of DNA in the atomizing nanometer and the chitosan nano package action to nucleic acid.Deposition condition: 1% agarose gel 60V electrophoresis half an hour, be divided into 7 swimming lanes, Lane1 and Lane2 are naked DNA, Lane3 is the complex of Sal-guanidinated chitosan and DNA, Lane4 is the naked DNA behind the individual atomization, Lane5 is the naked DNA behind the individual atomization and the complex of Sal-guanidinated chitosan, and Lane6 is the naked DNA behind the individual atomization and the complex of the Sal-guanidinated chitosan behind the individual atomization, and Lane7 is the product of the complex ultrasonic atomizatio of Sal-guanidinated chitosan and naked DNA.
3, carry out the nucleic acid substances of its parcel of cell transfecting laboratory observation with the complex atomizing nanoparticle of collecting, i.e. the transfection efficiency of pEGFP-N2 plasmid.
The HEK293 cell is with 8 * 10
5/ hole concentration is inoculated in 6 orifice plates, and routine is incubated at 37 ℃, 5%CO
2Incubator treats to carry out when cell density reaches 60-70% transfection.Carry out transfection when cell density reaches 60%-70%, the experiment of transfection HEK293 cell divides five groups: 1.Sal-guanidinated chitosan and DNA (pEGFP-N2 plasmid) composite nano-granule group; 2.Sal-the naked DNA composite nano-granule group behind guanidinated chitosan and the ultrasonic atomizatio; 3. the composite nano-granule group of the naked DNA behind Sal-guanidinated chitosan and the ultrasonic atomizatio behind the ultrasonic atomizatio (Sal-guanidinated chitosan and naked DNA ultrasonic atomizatio after compound again) respectively; 4. ultrasonic atomizatio Sal-guanidinated chitosan and DNA composite nano-granule group (the Sal-guanidinated chitosan carries out ultrasonic atomizatio earlier to be handled, compound with DNA again); 5.Sal-the compound atomizing nanoparticle group of guanidinated chitosan and DNA (after Sal-guanidinated chitosan and DNA are compound droplet that handle to collect of ultrasonic atomizatio) again.Collect and respectively organize cells transfected, flow cytometer calculates egfp expression positive cell ratio.The experiment of transfection 16HBE divides two groups, is respectively Sal-guanidinated chitosan and DNA (pEGFP-N2 plasmid) composite nano-granule group and Sal-guanidinated chitosan and the compound atomizing nanoparticle group of DNA (pEGFP-N2 plasmid).Fluorescence microscope calculates the quantity of the 16HBE cell of expressing green fluorescent protein in average each visual field down.
The result
1. transmission electron microscope shows: after ultrasonic atomizatio is handled, the more preceding increase of particle diameter of carrier S al-guanidinated chitosan nanometer particle size and Sal-guanidinated chitosan and DNA composite nano-granule, between 65～85nm scope, particle size distribution range narrows down, and the particle size distribution dispersion of Sal-guanidinated chitosan nanoparticle obviously reduces, the increasing proportion of the suitableeest grain diameter nano grain, uniformity significantly increases (referring to Fig. 9 and Figure 10), proves that the ultrasonic atomizatio effect can further optimize Sal-guanidinated chitosan nano-carrier.
2. gel retardation assasy shows: ultrasonic atomizatio produces tangible mechanical shearing effect to naked DNA, and naked DNA is degraded (Figure 11 swimming lane 4,5,6), the naked DNA aerosol after electrophoresis in the swimming lane distribution that becomes band.Atomize again behind the carrier S al-guanidinated chitosan parcel DNA; the electrophoresis showed complex intactly is trapped in (Figure 11 swimming lane 7) in the well, and prompting Sal-guanidinated chitosan nano-carrier can effectively protect DNA to avoid the destruction of ultrasonic atomizatio to nucleic acid substances.
3. cell transfecting experiment shows: still be the 16HBE cell at the HEK293 cell no matter, the Sal-guanidinated chitosan after the ultrasonic atomizatio processing and the cell transfecting efficient of the compound atomizing nanoparticle of DNA (pEGFP-N2 plasmid) all before significantly improve.In addition, earlier carrier S al-guanidinated chitosan is carried out ultrasonic atomizatio and modify, compound with nucleic acid substances again, can make cell transfecting efficient obtain the raising (seeing Figure 12 and Figure 13) of similar level equally.
Conclusion: Sal-guanidinated chitosan nano-carrier or Sal-guanidinated chitosan and nucleic acid composite nano-granule carry out physical modification through the ultrasonic atomizatio of certain frequency, significantly improved its performance as the carrier transport nucleic acid substances, suitable aerosol drips meanwhile can to form clinical atomization inspiration treatment, provides through respiratory tract and has treated the new system that uses the nucleic acid medicine.
Embodiment one
(1) get the albuterol guanidinated chitosan and be dissolved in water, preparation obtains albuterol guanidinated chitosan aqueous solution, places ultrasonic atomizing device to carry out ultrasonic atomizatio then under the 128KHz frequency and becomes 4um～6um droplet, collects the droplet of ultrasonic atomizatio gained;
(2) siRNA that adds the jamming target Disease-causing gene in step (1) in the droplet of collecting carries out compound, mass ratio between the siRNA of albuterol guanidinated chitosan and jamming target Disease-causing gene is 1: 1, the siRNA of albuterol guanidinated chitosan parcel jamming target Disease-causing gene forms the compound atomizing nanoparticle of siRNA of albuterol guanidinated chitosan and jamming target Disease-causing gene, i.e. the respiratory tract siRNA administration nano-drug administration system that atomizes of going back to the nest.Collect droplet and make electron microscope specimen, atomize the particle diameter of administration nano-drug administration system between 65～85nm to the gained respiratory tract siRNA that goes back to the nest through transmission electron microscope observing.
Embodiment two
(I) getting the albuterol guanidinated chitosan is dissolved in water, obtain albuterol guanidinated chitosan aqueous solution, the siRNA that adds the jamming target Disease-causing gene then mixes compound, the siRNA of albuterol guanidinated chitosan parcel jamming target Disease-causing gene forms the complex solution of the siRNA of albuterol guanidinated chitosan and jamming target Disease-causing gene, and wherein the siRNA of albuterol guanidinated chitosan and jamming target Disease-causing gene is mass ratio 3: 1.
(II) the albuterol guanidinated chitosan places ultrasonic atomizing device to carry out ultrasonic atomizatio under the 128KHz frequency with the complex solution of the siRNA of jamming target Disease-causing gene to become 4um～6um droplet, obtain the compound atomizing nanoparticle of siRNA of albuterol guanidinated chitosan and jamming target Disease-causing gene, i.e. the respiratory tract siRNA atomizing administration nano-drug administration system of going back to the nest.Collect droplet and make electron microscope specimen, atomize the particle diameter of administration nano-drug administration system between 65～85nm to the gained respiratory tract siRNA that goes back to the nest through transmission electron microscope observing.
Embodiment three
(I) getting the albuterol guanidinated chitosan is dissolved in water, obtain albuterol guanidinated chitosan aqueous solution, the siRNA that adds the jamming target Disease-causing gene then mixes compound, the siRNA of albuterol guanidinated chitosan parcel jamming target Disease-causing gene forms the complex solution of the siRNA of albuterol guanidinated chitosan and jamming target Disease-causing gene, and wherein the siRNA of albuterol guanidinated chitosan and jamming target Disease-causing gene is mass ratio 5: 1.
(II) the albuterol guanidinated chitosan places ultrasonic atomizing device to carry out ultrasonic atomizatio under the 128KHz frequency with the complex solution of the siRNA of jamming target Disease-causing gene to become 4um～6um droplet, obtain the compound atomizing nanoparticle of siRNA of albuterol guanidinated chitosan and jamming target Disease-causing gene, i.e. the respiratory tract siRNA atomizing administration nano-drug administration system of going back to the nest.Collect droplet and make electron microscope specimen, atomize the particle diameter of administration nano-drug administration system between 65～85nm to the gained respiratory tract siRNA that goes back to the nest through transmission electron microscope observing.
The siRNA of the jamming target Disease-causing gene among the above embodiment is meant siRNA, the siRNA of oncogene, the siRNA of tumor-related gene or the siRNA of mutant gene etc. of the viral gene that causes inflammation or oncosis, as siRNA, the siRNA of influenza virus A type conservative gene, the siRNA of hepatitis virus etc. of some structural protein gene of sars coronavirus.The siRNA of above jamming target Disease-causing gene prepares according to a conventional method according to the structure of objectives Disease-causing gene and gets final product.
The present invention can summarize with other the concrete form without prejudice to spirit of the present invention or principal character.Above-mentioned embodiment of the present invention all can only be thought explanation of the present invention rather than restriction, therefore every foundation essence technology of the present invention all belongs in the scope of technical solution of the present invention any trickle modification, equivalent variations and modification that above embodiment did.
Claims (9)
1. the respiratory tract siRNA atomizing administration nano-drug administration system of going back to the nest is characterized in that this drug-supplying system is a carrier material with the albuterol guanidinated chitosan after handling through ultrasonic atomizatio, and the siRNA of parcel jamming target Disease-causing gene makes.
2. the respiratory tract according to claim 1 siRNA atomizing administration nano-drug administration system of going back to the nest is characterized in that, described respiratory tract is gone back to the nest the particle diameter of siRNA atomizing administration nano-drug administration system between 65～85nm scope.
3. the respiratory tract according to claim 1 siRNA atomizing administration nano-drug administration system of going back to the nest is characterized in that the mass ratio between the siRNA of described albuterol guanidinated chitosan and jamming target Disease-causing gene is 1～5: 1.
4. the described respiratory tract of the arbitrary claim of claim 1～3 preparation method of siRNA atomizing administration nano-drug administration system of going back to the nest is characterized in that it may further comprise the steps:
(I) getting the albuterol guanidinated chitosan is dissolved in water, preparation obtains albuterol guanidinated chitosan aqueous solution, the siRNA mixing that adds the jamming target Disease-causing gene then is compound, and the siRNA of albuterol guanidinated chitosan parcel jamming target Disease-causing gene forms the complex solution of the siRNA of albuterol guanidinated chitosan and jamming target Disease-causing gene;
(II) complex solution with the siRNA of described albuterol guanidinated chitosan and jamming target Disease-causing gene places ultrasonic atomizing device, be atomized into droplet, obtain the compound atomizing nanoparticle of siRNA of albuterol guanidinated chitosan and jamming target Disease-causing gene, drug-supplying system promptly of the present invention.
5. the go back to the nest preparation method of siRNA atomizing administration nano-drug administration system of respiratory tract according to claim 4, it is characterized in that the mass ratio between the siRNA of albuterol guanidinated chitosan described in the described step (I) and jamming target Disease-causing gene is 1～5: 1.
6. the go back to the nest preparation method of siRNA atomizing administration nano-drug administration system of respiratory tract according to claim 5 is characterized in that, between power 115KHz～140KHz scope that ultrasonic atomizatio is handled in the described step (II).
7. the described respiratory tract of the arbitrary claim of claim 1～3 preparation method of siRNA atomizing administration nano-drug administration system of going back to the nest is characterized in that it may further comprise the steps:
(1) get the albuterol guanidinated chitosan and be dissolved in water, preparation obtains albuterol guanidinated chitosan aqueous solution, carries out ultrasonic atomizatio then and handles, and collects the droplet of ultrasonic atomizatio gained;
(2) siRNA that adds the jamming target Disease-causing gene in step (1) in the droplet of collecting carries out compound, the siRNA of albuterol guanidinated chitosan parcel jamming target Disease-causing gene forms the compound atomizing nanoparticle of siRNA of albuterol guanidinated chitosan and jamming target Disease-causing gene, promptly obtains drug-supplying system of the present invention.
8. the go back to the nest preparation method of siRNA atomizing administration nano-drug administration system of respiratory tract according to claim 7, it is characterized in that the mass ratio between the siRNA of albuterol guanidinated chitosan described in the described step (1) and jamming target Disease-causing gene is 1～5: 1.
9. the go back to the nest preparation method of siRNA atomizing administration nano-drug administration system of respiratory tract according to claim 8 is characterized in that, between power 115KHz～140KHz scope that ultrasonic atomizatio is handled in the described step (1).
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Application publication date: 20110914 |