CN101180400A

CN101180400A - Nanoparticles comprising rna ligands

Info

Publication number: CN101180400A
Application number: CNA2005800240377A
Authority: CN
Inventors: T·W·拉德迈克; K·古玛; M·马丁-洛马斯; S·佩纳德斯; R·欧杰达; A·G·巴雷特斯
Original assignee: Midatech Ltd
Current assignee: Midatech Ltd
Priority date: 2004-05-24
Filing date: 2005-05-24
Publication date: 2008-05-14
Anticipated expiration: 2025-05-24
Also published as: ES2533238T3; ES2655069T3; CN102872463A; CN101180400B; CN102872463B; GB0411537D0

Abstract

Materials and methods are provided for making nanoparticles having a core including metal and/or semiconductor atoms, which core is covalently linked to a plurality of ligands comprising a RNA ligand. The RNA ligands may include siRNA or miRNA. Also provided are uses of these nanoparticles in therapy and diagnosis.

Description

The nano particle that contains the RNA part

Technical field

The present invention relates to nano particle, relate more specifically to contain the nano particle of RNA part such as siRNA (siRNA) and microRNA (miRNA), and the purposes in various application.

Background technology

Have been found that small RNA molecular plays multiple effect in regulatory gene is expressed.These effects comprise the orientation degraded of siRNA (siRNA) to mRNA, PTGS (PTG), and microRNA (miRNA) is to growth controllable type sequence-specific translation restraining effect and the directed transcriptional gene silencing of mRNA.The RNAi activity has limited transposon and has shifted and provide antiviral defense (Pal-Bhadra etc., 2004).Also verified RNAi mechanism and little RNA are in the target-seeking of heterochromatin mixture and the effect (Verdel etc., 2004) in the epigenetic gene silencing on the specific chromosomal foci.Reticent behind double-stranded RNA (dsRNA) dependent transcription, be also referred to as little inhibitions RNA (siRNA) or RNA and disturb (RNAi), be wherein the dsRNA mixture can the accurate specific homologous gene of target with the phenomenon of silence in short time period.It has the signal of the mRNA degraded of sequence identity as promotion.The common sufficiently long of 20-nt siRNA is with induced gene specificity silence, and enough short in to avoid host response (Elbashir etc., 2001).The reduction that the directed gene product is expressed can be on a large scale, has by several siRNA molecule inductive 90% silences.

Because sending of small molecules oligonucleotide can be walked around the difficulty relevant with gene therapy, so use siRNA may have the advantage that is better than traditional gene therapy.Up to now, effectively send obstacle in the gene therapy that remains success in the body based on the therapeutic gene of carrier.Although having observed by siRNA is not that nonvolatil, independent siRNA transfection can cause the proteic inhibition that hits of parent and daughter cell to prolong (Tuschl, 2001) to the rejecting of target gene.Yet sending in the art of siRNA still has problems.

WO 02/32404 (Consejo Superior de InvestigacionesScientificas) discloses the nano particle that is formed by metal or semiconductor atom, and the core that wherein comprises the part of carbohydrate and nano particle is covalently bound.These nano particles are used to regulate the interaction of carbohydrate mediation and are solvable and nontoxic.The PCT application of enjoying GB-A-0313259.4 (Consejo Superior de InvestigacionesScientificas and Midatech Limited) right of priority discloses the magnetic nanoparticle with core, this core comprises passive magneticmetal atom, and core and part are covalently bound.

Summary of the invention

Say that loosely the present invention relates to have the nano particle of core, this core comprises metal and/or semiconductor atom, core and RNA part are covalently bound.The RNA part normally designs the short rna sequence of simulating siRNA (siRNA) and microRNA (miRNA) sequence.Nano particle can be used to send the RNA part and have purposes widely, can use in vitro system and be used for the treatment of or diagnose.For example, nano particle of the present invention can be used for (1) directed transcriptional gene silencing, (2) directed mRNA degraded, (3) mRNA imaging, (4) by using the multiple RNA part on the identical or different nano particle to suppress various approach, (5) aerosol is sent, and for example to lung, (6) are used for target siRNA resistance mRNA and (7) instrument as functional genome in conjunction with the mRNA silence.

In the art, according to its source with the short rna sequence be called " short interfering rna " (siRNA) or " microRNA " (miRNA).Two types sequence can be by in conjunction with complementary RNA (nmRNA) with cause mRNA and eliminate (RNAi) or stop mRNA to translate into protein and be used for down-regulation of gene expression.SiRNA produces by the processing of long dsrna, and external source normally when in fact finding.Little RNA interfering (miRNA) is the little non-coding RNA of interior source code, and the processing of pressing from both sides by bob produces.SiRNA and miRNA can suppress to have the part complementary target sequence mRNA translation and do not have RNA to shear, and can degrade and have the mRNA of fully-complementary sequence.The RNAi approach also acts on genome, as is described in Science, 301:1060-1061,2003.

The RNA that links to each other with nano particle can be strand or double-stranded (duplex).As in the situation of part, the RNA sequence can be a hair clip with miRNA sample sequence, promptly comprises the complementary district of part, and can anneal near them forms the end of hair clip.Nano particle can randomly comprise the part of other type, forms sugared nano particle as carbohydrate, and/or more than a kind of siRNA.Below will discuss nano particle and uses thereof in more detail.Advantageously, siRNA makes it not to be subjected in blood, the tissue culture medium (TCM) or the influence of the exoribonuclease that exists in the cell with the protection to siRNA can be provided being connected of nano particle.

Therefore, in first aspect, the invention provides the nano particle that contains core, this core comprises metal and/or semiconductor atom, and wherein covalently bound the and part of core and multiple part comprises the RNA part.

The siRNA part that forms nano particle can be strand or double-stranded (duplex).Yet, validity for the target gene function downward modulation of optimizing RNA mediation, preferably select the length of siRNA molecule to guarantee of the correct identification of RISC mixture to siRNA, when the compound-mediated mRNA target of described RISC is used nano particle in the identification of siRNA and the preferred body, the enough short host response that reduces of siRNA.

The miRNA part normally strand and have a complementary district of the part that can make part form hair clip.MiRNA is the form of single stranded RNA normally, and thinks and regulate other expression of gene.MiRNA transcribes from DNA, but does not translate into proteinic rna gene.The dna sequence dna of coding miRNA gene is longer than miRNA.This dna sequence dna comprises that miRNA sequence and approximate reverse are to complement.Divide the period of the day from 11 p.m. to 1 a.m when this dna sequence dna is transcribed into single stranded RNA, miRNA sequence and reverse complemental body base pair thereof form the double-stranded RNA sections; Generally speaking, the structure of this RNA is called hairpin structure (' short hairpin RNA ' or shRNA).The Dicer enzyme cuts away double stranded region from hairpin structure then, and discharges ripe miRNA.

By under the control of rna plymerase iii promotor such as people H1 or 7SK promotor, using the DNA construct transfectional cell of coding shRNA sequence, can in cell, produce shRNA.Perhaps, can externally synthesize shRNA also directly introduces in the cell.

Usually, the RNA part that is intended to simulate siRNA and miRNA effect has 10 to 40 ribonucleotides (or its synthetic analogues), more preferably 17 to 30 ribonucleotides, more preferably 19 to 25 ribonucleotides, most preferably 21 to 23 ribonucleotides.In embodiments more of the present invention of using double-stranded siRNA, this molecule can have symmetric 3 ' overhang, for example, and the 3 ' overhang of one or two (ribose) Nucleotide, the normally UU3 ' overhang of dTdT.

Produce in the situation of miRNA in the shearing by shRNA, 40 to 100 bases of shRNA sequence preference are long, and more preferably 40 to 70 bases are long.Preferred 19 to 30 base pairs in the stem district of hair clip are long.The stem district can contain G-U and match to stablize hairpin structure.

In using the embodiment of double-stranded RNA, justice and the antisense strand formation duplex of can annealing is arranged.Form nano particle by comprise duplex in reaction mixture, RNA can be connected to core in the process that particle assembles automatically.Quantity according to double-stranded siRNA derivation end (four 5 ' and 3 ' possible ends of every chain), four nano particles can be connected on the single siRNA duplex at the most, and for each double-stranded siRNA, be formed up to many 15 possible constructs in theory, one in these has four nano particles (each terminal two), four have a nano particle, and six have two nano particles and four and have three nano particles.In the situation of using strand siRNA, the nano particle core can be connected on any or two the derivation ends (that is, 5 ' or 3 ' end) of siRNA, for example, produces three kinds of different nano particles.These nano particles can use or this method can randomly comprise the complementary strand annealing that makes the siRNA that contains nano particle and siRNA with this form, thereby original position forms the other step of duplex on preformed nano particle.In the forming process of miRNA sample part, one or two nano particle is connected on the end of RNA sequence usually.

Therefore, in another aspect, the invention provides the production method of said nano particle.Usually, this method comprises puts together the core of RNA part and nano particle, by with connector derivation RNA chain and the RNA of derivation is contained in the reaction mixture of synthesis of nano granular core thus.In the process that nano particle assembles automatically, the nano particle core is connected on the RNA by connector.Preferably, connector is a disulfide linkers, and blended disulfide linkers for example is although also can use ethylidene connector or peptide connector.The example linking group is by general formula HO-(CH ₂) _n-S-S-(CH ₂) _m-OH represents that wherein n and m are 1 to 5 independently.RNA, by a terminal hydroxyl, can be connected with spacer in the situation of preferred mixed disulfide connector easily by the terminal phosphate group.When the synthesis of nano particle, connector-S-S-breaks to form two sulfur-bearing connectors, separately by-the S-group can be covalently bound to the core of nano particle.Use the blended disulfide linkers to help avoid the formation of RNA dipolymer.

As mentioned above, be in the situation of strand at the RNA that is connected with the nano particle core, this method can comprise making with the first chain complementary RNA molecule anneals so that the other step of the double-stranded RNA part that is connected on the nano particle to be provided.Can also prepare the nano particle with annealed double-stranded RNA, one of this RNA or two chains are by the disulfide linkers functionalization.Alternatively or additionally, this RNA's has justice and antisense strand can be connected on the different nano particles and anneals together.

In the preferred embodiments of the invention,, can come one or two end of this RNA of derivation with blended disulphide for double-stranded RNA being mixed in the nano particle in the automatic assembling.After mixing duplex in the nano particle, the chemical ingredients of RNA and mixed disulfide is all mixed in the bead.Therefore, the chemical ingredients of mixed disulfide can comprise important information (for example, specificity is in conjunction with right member) as the target characteristic with the character of itself, other physical property is provided maybe might for the final nano particle that forms.Blended disulphide can be connected on the 3 ' end or 5 ' end of justice or antisense strand.

In one embodiment, use and can synthesize the nano particle with core in the method described in the WO 02/32404 first, this core comprises gold atom, wherein uses disulfide linkers to come the derivation part and make the part and the HAuCl of derivation in the presence of reductive agent ₄(tetra chlorauric acid) reaction generates nano particle.According to this method, the RNA of the protection of the disulphide in methyl alcohol or the water can be added in the aqueous solution of tetra chlorauric acid.Preferred reductive agent is a sodium borohydride.These and other feature descriptions of this method are in WO 02/32404.

Some can use many different RNA molecules in using.Can be used as the different ligands of puting together with one group of nano particle, these different RNA molecules or different RNA molecules are provided can be independent nano particle group, and randomly mixes.In first kind of situation, those skilled in the art will recognize that by RNA and nano particle and put together in the situation that forms product mixture that one group of nano particle can comprise multiple aforesaid product.In the situation of using a plurality of parts, can use the part of simulation siRNA and miRNA.

Except the RNA molecule, part and/or RNA part that nano particle can comprise one or more other types can also comprise one or more dissimilar groups or structural domains except the RNA component.For example, other part, or the group of part or structural domain can comprise one or more peptides, protein domain, nucleic acid molecule, lipid groups, carbohydrate group, any organic or negatively charged ion or cation group.Carbohydrate group can be a polysaccharide, oligosaccharides or monose group.Preferred part comprises glycoconjugate, thereby forms sugared nano particle.As following pointed in the discussion of nano particle purposes, part (RNA or other part) can be that specificity is in conjunction with right member and be used for wherein there be the target position of specificity in conjunction with another right member in the nano particle target.Also exist except the RNA part in the situation of nucleic acid molecule, this nucleic acid molecule can comprise list or double-stranded DNA or RNA.Particle can have the part that is fixed thereon more than a kind of, for example, and 2,3,4,5,10,20 or 100 kind of different ligands.Alternatively or additionally, can use the nano particle of number of different types together.In the preferred embodiment, the mean number that is connected to the whole parts on the particle single metal core is at least one part, more preferably 50 parts, most preferably 60 parts.

Preferably, it is 0.5 to 50nm that nano particle has mean diameter, and more preferably 0.5 to 10nm, and more preferably 1.0 to 5nm, more more preferably 3.0 to 7.0nm core.When also considering part except core, the overall average diameter of preferred particulates is 5.0 to 100nm, and more preferably 5 to 50nm, and most preferably 10 to 30nm.Can use technology well known in the art such as transmission electron microscopy to measure mean diameter.

Core material can be metal or semi-conductor and can be formed by the atom more than a type.Preferably, core material is to be selected from Au, the metal of Fe or Cu.The nano particle core can also be formed by alloy, and described alloy comprises Au/Fe, Au/Cu, and Au/Gd, Au/Fe/Cu, Au/Fe/Gd and Au/Fe/Cu/Gd, and can be used for the present invention.Preferred core material is Au and Fe, and most preferred material is Au.The core of nano particle preferably comprises about 100 to 500 atoms (for example, gold atom) provides nano level core diameter.The active atom of one or more NMR of doping in other useful especially core materials makes and can use NMR to detect nano particle in vitro and in vivo.The example of NMR active atomic comprises Mn ^{+ 2}, Gd ^{+ 3}, Eu ^{+ 2}, Cu ^{+ 2}, V ^{+ 2}, Co ^{+ 2}, Ni ^{+ 2}, Fe ^{+ 2}, Fe ^{+ 3}And lanthanon ^{+ 3}, or the described quantum dot in the application other places.

Can detect the nano particle core that contains semiconductor atom, because the nano semiconductor crystal can be as quantum dot, be that they can absorb light, thus with the electron excitation in the material to higher energy level, discharge the photon of light subsequently with the characteristic frequency of material.The example of semiconductive core material is a cadmium selenide, Cadmium Sulfide, cadmium telluride.Also comprise zn cpds, as zinc sulphide.

In some embodiments, nano particle of the present invention or RNA molecule comprise detectable mark.This mark can be the composition of nano particle core or RNA part or another kind of part.But because the natural characteristics of this composition of nano particle or by being connected with the another kind test section, put together or continuous, this mark is detectable.Preferred mark example is included as fluorophor, radionuclide, the mark of magnetic mark or dyestuff.Fluorophor comprises fluorescein, rhodamine or tetramethylrhodamin, Texas-is red, Cy3, Cy5 etc. can detect (Y.C.Cao by the fluorescently-labeled light that excites and use the detection of raman scattering spectrum assay method to be launched, R.Jin, C.A.Mirkin, Science 2002,297:1536-1539).

In some embodiments, nano particle can comprise radionuclide, is used to use the radioactivity of radionuclide emission to detect nano particle, for example, uses PET, SPECT, or be used for the treatment of, promptly be used for kill target cell.Commonly used being easy to adjusted and comprised to be suitable for radionuclide example of the present invention in this area ^99mTC, it exists with multiple oxidation state, although the most stable be TcO ^4- ³²P or ³³P; ⁵⁷Co; ⁵⁹Fe; Usually with Cu ²⁺Salt uses ⁶⁷Cu; Usually with Ga ³⁺Salt uses ⁶⁷Ga, for example, gallium citrate; ⁶⁸Ge; ⁸²Sr; ⁹⁹Mo; ¹⁰³Pd; Usually with In ³⁺Salt uses ¹¹¹In; Usually use with sodium iodide ¹²⁵I or ¹²¹I; ¹³⁷Cs; ¹⁵³Gd; ¹⁵³Sm; ¹⁵⁸Au; ¹⁸⁶Re; Usually with Tl ⁺Salt uses as thallium chloride ²⁰¹Tl; ³⁹Y ³⁺ ⁷¹Lu ³⁺With ²⁴Cr ²⁺Radionuclide is with marking and the general use of tracer agent is well known in the art and those skilled in the art can easily adjust to be suitable in the each side of the present invention.By it being doped in the core of nano particle or comprising that they as the mark that a part part that is fixed on the nano particle exists, can use radionuclide easily.

Additionally or replacedly, can use multiple technologies well known in the art, use the aforesaid mark that links to each other with nano particle or detect nano particle of the present invention by the characteristic of using them, or the result of they and other component interaction.The gathering that these methods that detect nano particles can take place when detecting nano particle in conjunction with another kind of component, for example, by simple range estimation or use scattering of light (transmissivity that contains nanoparticles solution), to using sophisticated technology such as transmission electron microscopy (TEM) or atomic force microscopy (AFM) to observe nano particle.The other method that detects metallic particles is to use plasmon resonance, promptly in the electron excitation of metallic surface, is caused by rayed usually.Surface plasmon resonance (SPR) phenomenon is present on the interface of metal (as Ag or Au) and insulating material such as air or water.Along with analyte is bonded to the specific refractory power that the part that is fixed on the nano grain surface has changed the interface, change has taken place in SPR.Another advantage of SPR is to be used to monitor real-time interaction.As mentioned above, if nano particle comprises or the NMR active atomic that mixed, this technology can be used to use technology well known in the art to detect this particle in external or body so.Can also use the system of amplifying, utilize the also original nano particle that detects of the promoted silver of nano particle (I) based on quantitative signal.If nano particle comprises the part as fluorescent probe, then can use spectrofluorimetry.In addition, the isotopic labeling of carbohydrate can be with the detection of helping them.

The invention provides the method for the ball array that presents part, this array has the advantage of the other types array that is better than proposing in the prior art.Especially, nano particle especially is soluble in water in most of organic solvents.This can be used for their purifying and importantly mean and can use in solution, is used to present the part that is fixed in particle surface.The fact is that soluble nano particle has the advantage that presents the native conformation part.Use for treatment, nano particle is nontoxic under physiological condition, and is soluble and be stable.

In some embodiments, the core of nano particle can be magnetic and comprise the magneticmetal atom, randomly combine with the passive metal atom.For example, passive metal can be gold and platinum, silver or copper, and magneticmetal can be iron or gadolinium.In the preferred embodiment, passive metal is that gold and magneticmetal are iron.In this case, the suitable proportion of passive metal atom and magneticmetal atom is about 5: 0.1 to about 2: 5 in the core.More preferably, ratio is about 5: 0.1 to about 5: 1.As used herein, term " passive metal " refers to and does not demonstrate magnetic properties and be chemically stable metal for oxidation.Passive metal of the present invention can be diamagnetic.Diamagnetism refers to the material with whole paired electronss, so its each atom does not have permanent Net magnetic moment.Magneticsubstance have some not paired electronics and for the external magnetic field be absolute responsive-promptly, the external magnetic field induces electronics along with externally-applied magnetic field is lined up, so electronic magnetic moment obtains adjusting.Magneticsubstance can be paramagnetic, and is superparamagnetism or ferromagnetic.Paramagnetic material is not very responsive to the external magnetic field, and no longer keeps their magnetic properties when removing the external magnetic field.Ferromagnetic substance is extremely sensitive to the external magnetic field, even also contains magnetic domain when not having the external magnetic field to exist, because contiguous atom cooperation makes that their electron spinning is parallel.The magnetic moment of adjacent domain is adjusted in the external magnetic field, has enlarged magnetic effect.Usually the very little particle that has the ferromagnetic characteristics material is not ferromagnetic, because do not produce cooperative effect in 300nm or littler particle, makes material not have permanent magnetic.Yet particle is still very responsive and have intensive paramagnetism characteristic to the external magnetic field, is called superparamagnetism.Preferably, nano particle of the present invention is a superparamagnetism.

Example with the nano particle that comprises the paramagnetic metal core comprises and contains Mn ^{+ 2}, Gd ^{+ 3}, Eu ^{+ 2}, Cu ^{+ 2}, V ^{+ 2}, Co ^{+ 2}, Ni ^{+ 2}, Fe ^{+ 2}, Fe ^{+ 3}And lanthanon ^{+ 3}Those.

Can form other magnetic nanoparticles from the material that can form nano particle (magnetic fluid) such as MnFe (ferrospinel) or CoFe (vectolite), add or do not add other aforesaid core material.Biotechnol.Prog., 19:1095-100 (2003), J.Am.Chem.Soc.125:9828-33 (2003) has provided the chemical example of automatic assembling connection that is used to produce such nano particle among the J.ColloidInterface Sci.255:293-8 (2002).

On the other hand, the invention provides composition, comprise the group of one or more above institutes definitions particles.In some embodiments, the nano particle group can have the identical or different part that is connected in core of different densities.In the certain situation, it is desirable to nano particle is incapsulated a plurality of nano particles can be delivered to target site.The examples of suitable technology is well known to a person skilled in the art.Encapsulated nano particle group can be a kind of, two kinds, and three kinds or multiple different type.In one embodiment, the invention provides aerosol combination at this defined nano particle.Aerosol combination can comprise nano particle and optional thinner.The example of these composition purposes below is discussed.

Unrestricted mode provides the example of following nano particle application to support the broad applicability of said technology by explanation.Dorseet ﹠amp; Tuschl, Nature Reviews, 3:318-329 provides siRNA general purpose summary in 2004.

On the other hand, the nano particle that the invention provides above definition be used for the treatment of or diagnose in purposes.

On the other hand, the nano particle that the invention provides above definition is used to prepare the purposes of medicine, and this medicine is used for the treatment of by giving the illness that nano particle is eased.Below described according to the present invention the example of treatable concrete purposes, other application with nano particle comprise external and intravital purposes.For example, nano particle or derivatives thereof described herein can be formulated in the pharmaceutical composition, and delivers medicine to the patient with various forms, especially for treating by giving the illness that the RNA part is alleviated.For example, this can be used for the treatment of the illness of alleviating by by the expression of RNA down-regulated gene, wherein comes down-regulated gene by RNA, or is used for the treatment of and the relevant illness of overexpression of gene by RNA target and downward modulation.

The adjusting of genetic expression

The nano particle that comprises the RNA part can be used for regulatory gene in many ways expresses, comprise orientation degraded by siRNA (siRNA) mRNA, PTGS (PTG) is by growth controllable type sequence-specific translation inhibition and the directed transcriptional gene silencing of little-RNA (miRNA) mRNA.

Generally speaking, the invention provides the purposes that nano particle described herein is used to reduce target gene.Among the application, downward modulation can be external, for example, study genetic expression, or it is intravital, in the target experimental system or be used for medical usage, promptly nano particle can be used for preparing the medicine of illness that treatment alleviates by RNA downward modulation target gene expression or the treatment illness relevant with the target gene overexpression.For example, this illness can comprise cancer, for example, breast cancer, it can use the RNA based on the Her2/Neu sequence to treat.As described herein, because in fact the downward modulation that is provided by RNA may be temporary transient, so, in some embodiments, preferred nano particle further comprises radionuclide, and medicine or other medicaments, described medicament are used for the treatment of or kill the wherein cell of nano particle downward modulation target gene.

RNA disturbs the disease that can be used for the treatment of any with too active gene-correlation, and for example the cancer of most of forms for example, is used RNA to be used for oncogene and suppressed.Specific gene such as hepatitis or too active genetic expression help that shutting down of necrocytosis acceptor also is the target of RNA treatment in other illnesss of nosopathology.The other example that uses the suitable illness of nano particle treatment of the present invention is the macular degeneration in the eyes, or utilize the natural action conduct of RNA to lose the method that ability is resisted pathogenic virus by the RNA that makes virus, these viruses are particularly including HIV, third liver or influenza (Check, 2003; Zamore etc., 2003; Song etc., 2003; Matzke ﹠amp; Matzke, 2003).

The downward modulation of approach

It should be noted that especially the present invention allows to send the RNA molecule more than a kind of, this is impossible thing in the past in the art always.Therefore, in some embodiments, the invention provides the Nanoparticulate compositions that contains at least two kinds of different RNA sequences, this RNA sequence is puted together or is present in the composition of at least two kinds of dissimilar nano particles with identical nano particle, can be used for expression in the down-regulated gene approach.The number of the RNA part that exists in the composition will depend on the complicacy of approach and the number gene that needs target to be used to reduce.The example of approach that can target is used for research or treatment, comprises the approach that causes inflammation, antiviral pathway, the signal pathway in the cancer, route of metastasis or pathways metabolism.For example, this comprises the adjusting of the new sugared generation approach that produces for glucose in the type ii diabetes.

In another relevant embodiment, come structural domain conservative between the target family member by designated rna, the RNA-nano particle can be used for the expression of down-regulated gene family.

Be used for using any purposes of RNA inhibition of gene expression, nano particle can also comprise the siRNA sequence and the reticent sequence of mRNA is come target mRNA at nano particle.These nano particles can be used to suppress siRNA resistance mRNA.This can be used for wherein that target cell is the situation of resistance to the siRNA silence, because their express the protein that blocking-up siRNA suppresses mechanism.In this case, can express the gene product of induction of resistance to improve the siRNA resistance by siRNA is aimed at.

Use other parts or use the target of RNA to use

In a kind of application, Nanoparticulate compositions of the present invention can be used to give the target characteristic, is used for RNA is delivered to target cell.This can by provide have an other type with close part that the nano particle core puts together or the nano particle that can make RNA nano particle and target cell group specificity interactive domains that links to each other with the RNA part and realize.For example, the nano particle that the contains RNA cell mass that can preferentially lead, by the nano particle with part is provided, this part be can specificity in conjunction with the right member of particular combination who is present in target cell surface or inner binding partners, for example, target specific cells structure is as nuclear.The right example of particular combination that is suitable for use as the part of puting together with the nano particle that is used for target comprises part and acceptor, and many alternatives are that those skilled in the art are conspicuous.For example, the nano particle of glucose derivation can be used for the cell (18) that target contains protein G LUT family member.Can use other the carbohydrate ligands that is used for nano particle, as Glc β 4GlcNAc or Glc β 4GlcNH ₂The former can be used at first will containing the GLUT translocator on the nano particle targeted cells surface of siRNA, (shears the back at Polyglucosidase) then when entering cell, and GlcNAc will further examine siRNA nano particle target.Back one construct is added into the surface of nano particle with positive charge, further promotes to adhere to also to absorb to cell.Because the nano particle of assembling can be used to mix allos part such as lipid automatically, peptide or any other chemical ingredients are (for example, described in as above part is discussed), it is connected with disulphide by spacer, and multiple other targeted moleculars can be used for the specific cell type of RNA nano particle target.For example, these technology can be used for and will carry the nano particle target tumor cell of RNA.

Nano particle is used for another example of targeted cells type purposes, and the RNA part of nano particle can be as providing the entity of nano particle target, the cell that the nano particle guiding is wherein expressed with the mRNA of RNA ligand interaction.The example of the target cell type of target is the cell of tumour cell or virus infection by this way, uses RNA to come the expression of virogene in the target target cell or tumor marker or oncogene.In this embodiment, preferred RNA-nano particle can permeate through cell membranes, this be provide by their small size and can be randomly by coming a kind of effect of enhanced (referring to NatureBiotechnology 18:410-414,2000) with film transposition signal derivation nano particle.The RNA-nano particle has the advantage of reducing said target mrna in the cell selective mode to the target of the cell of wherein expressing corresponding mRNA.Yet,,, make and use the cell of RNA target to be killed by selectivity so the nano particle with radionuclide or medicine preferably is provided because this effect is normally temporary transient.Target cell and treating processes imaging can be followed marked with nanometer granule, the nano particle of for example aforesaid use radioactivity or magnetic.

Make the mRNA imaging

On the other hand, the invention provides the method for mRNA detection and/or imaging, it has used nano particle described herein.Especially, before the present invention, there is not the known mRNA of making image formation method in the prior art.This method can be included in and make in the body under the RNA part that wherein is present on the nano particle and the interactional condition of said target mrna or the RNA contact nanometer particle in the sample, and detects nano particle-RNA-mRNA mixture.Detect the step of mixture and can use the natural characteristics of nano particle or the mark that links to each other with nano particle by detection.The preferred embodiment that is applicable to the mark in this respect of the present invention comprises the nano particle that contains the group that is magnetic, quantum dot or radionuclide.Quantum dot, for example, the nanocrystal by cadmium selenide provides, or can also use other negatively charged ion such as sulfide except selenide, and it has the potential purposes in the screening of biological imaging, electronics and optical device, quantum computer and drug candidate.

The RNA-nano particle is as the instrument of functional genome

Genome screening utilizes the RNA sequence that produces at random usually, then with its transfection to test cell and monitor the variation of protein expression.Nano particle of the present invention has two significant advantages that are better than the genome screening method of these prior aries.At first be that in fact many sequences of siRNA at random do not have effect, but test cell needs single screening at present, increased the artificial and cost of these experiments of this form.The present invention allows to comprise a plurality of siRNA sequences as the part on the nano particle, thereby the possibility of accelerating screening speed is provided.Therefore, if in cell, observe effect, then can screen the RNA molecule that exists on the bead and determine which sequence to cause this effect by.In addition, because nano particle provides the delivery system that is used for RNA, so can avoid the use of the transfection reagent that needs in the prior art.This is needed advantage, because transfection reagent self can cause the change of protein expression in the test cell.Therefore, on the other hand in, nano particle of the present invention can be as the instrument of functional genome, for example as Nature Reviews, the 3rd rolls up, in April, 2004 is described in the 318-329 page or leaf.Nano particle can have each particle more than two, more than 5, more than 10 or more than 20 or study intravital gene function and as being used for the instrument of the screening of full genome range more than 100 RNA sequence.

Aerosol is sent

On the other hand, the invention provides the purposes of nano particle described herein in aerosol.This is that small size because of nano particle becomes possible.Aerosol combination can be used to send the RNA part, particularly is delivered to lung, is used for imaging and/or therepic use, for example, infects in the illness of lung in treatment.

In the above-mentioned either side, nano particle can be connected with therapeutic active substance, as antibody or tumor-killing medicine.The magnetic properties of nano particle can also be used for target tumor, comes guided nano granule to tumour cell by using magnetic field.Yet, use separately magnetic field with nano particle lead tumour cell always unfeasible or accurately, therefore the invention provides a kind of advantage, make nano particle pass through the tumour-specific part tumour cell that can lead specifically.The possibility that this will allow to use less medicine and reduce side effect is because medicine is only at its cell of needs and not at the cell of health.

Another advantage of nano particle of the present invention is their especially little sizes, and this makes them preferablyly be absorbed by cell, even when being connected with target or treatment molecule.

On the other hand, wherein part is that antigenic nano particle can be used as vaccine administration, for example, by trajectory, uses and to send rifle and quicken them and penetrate through skin by epidermis is outer field.Nano particle can for example absorb by dendritic cell then, and maturation causes the adjusting of immunne response and resists antigenic vaccine inoculation by lymphoid migration along with them.

Nano particle of the present invention can be mixed with the pharmaceutical composition of solid or forms of liquid compositions.Such composition comprises certain carrier usually, for example solid carrier such as gelatin or auxiliary agent or inert diluent, or liquid vehicle such as water, oil, animal or plant oil, mineral oil or synthetic oil.Can comprise normal saline solution, or glycols such as ethylene glycol, propylene glycol or polyoxyethylene glycol.Such composition and preparation contain the compound of 0.1wt% at least usually.

Can Nanoparticulate compositions be delivered medicine to the patient by many different approaches.Parenterai administration comprises the administration by following approach: intravenously, and skin or subcutaneous, nose, intramuscular, intraocular, through epithelium, intraperitoneal and part (comprise skin, eye, rectum, nose sucks and aerosol) and rectum whole body approach.For intravenously, skin or subcutaneous injection, or in the injection at ailing position, activeconstituents will be the acceptable aqueous solution form of non-enteron aisle, and it is pyrogen-free and has suitable pH, isotonicity and stability.This area relevant technologies personnel can prepare suitable solution fully, use for example solution of compound or derivatives thereof, and for example, the solution in physiological saline is with the dispersion of glycerine, liquid macrogol or oil preparation.

Except one or more compounds, randomly in conjunction with other activeconstituentss, composition can also comprise acceptable vehicle on one or more pharmacology, carrier, buffer reagent, stablizer, isotonic agent, sanitas or antioxidant or well known to a person skilled in the art other materials.Such material should be nontoxic and the effect of interferon activity composition not.The definite character of carrier or other materials can depend on route of administration, for example, and oral or non-enteron aisle.

Usually that it is had is about 3.0 to 9.0 for the obtaining liq pharmaceutical composition, and more preferably from about 4.5 to 8.5,5.0 to 8.0 pH more preferably from about again.Can be by use buffer reagent such as acetate, Citrate trianion, phosphoric acid salt, succinate, Tris or Histidine are kept the pH of composition, and common use range is about 1mM to 50mM.Can regulate the pH of composition in addition by acceptable acid or alkali on the use physiology.

Generally include sanitas in the pharmaceutical composition and stop microorganism growth, prolong the preservation period of composition and allow multiple purposes packing.The example of sanitas comprises phenol ,-cresols, benzylalcohol, right-hydroxy-benzoic acid and ester thereof, methyl p-hydroxybenzoate, propylparaben, benzalkonium chloride, Solamin.The common use range of sanitas is about 0.1 to 1.0% (w/v).

Preferably, give individuality with prevention significant quantity or treatment significant quantity (deciding on concrete situation, is treatment although prevention can be looked) with pharmaceutical composition, this is enough to demonstrate the benefit to individuality.Usually, this will cause providing activity useful in the treatment of benefit to individuality.The actual amount of institute's administered compound, medicine-feeding rate and time course will depend on the character and the severity of illness to be treated.The prescription of treatment for example, to the decision of dosage etc., is in general practitioner and other physician's responsibilities, and considers disease to be treated usually, the situation of individual patient, site of delivery, known other factors of medication and doctor.The example of above-mentioned technology and experimental program can be at Handbook of Pharmaceutical Additives (medicated premix handbook), the 2nd edition (editor M.Ash and I.Ash), 2001, (Synapse InformationResources, Inc., Endicott, New York, USA), Remington ' s PharmaceuticalScience (Lei Mingdun pharmaceutical science), the 18th edition, Mack Publishing Company, Easton, Pa., 1990; With Hankbook of Pharmaceutical Excipients (handbook of pharmaceutical excipients), the 2nd edition, find in 1994.For example, preferably with about 0.01 to the 100mg active compound of every kg body weight, and more preferably from about the dosage of 0.5 to 10mg/kg body weight delivers medicine to the patient with composition.

Referring now to accompanying drawing embodiment of the present invention are described for example and not limitationly.

Description of drawings

Fig. 1 has shown the transmission electron micrograph of RNA-Au-Glc nano particle.

Fig. 2 has shown the existence of testing RNA in the nano particle that makes.(a) no UV light: 1.RNA-Au-Glc nano particle+EtBr; 2.Glc-Au+EtBr; 3.Glc-Au; 4. resistates+the EtBr of washing soln.(b) use UV light: 1.RNA-Au-Glc nano particle+EtBr; 2.Glc-Au+EtBr; 3.Glc-Au; 4. resistates+the EtBr of washing soln.

Fig. 3 a has shown after the Her-2/neu siRNA transfection 48 hours from the proteic western blotting of the Her-2/neu of isopyknic SKBR3 cell lysate.C=contrasts (being untreated) cell; The cell that Au=handles with the siRNA that is attached on the gold nano grain, no RNAiFectamine; S=has the reticent siRNA of RNAiFectamine; NS=has the non-reticent siRNA of RNAiFectamine.

Fig. 3 b has shown after the Her-2/neu siRNA transfection 72 hours from the proteic western blotting of the Her-2/neu of isopyknic SKBR3 cell lysate.C=contrasts (being untreated) cell; The cell that Au=handles with the siRNA that is attached on the gold nano grain, no RNAiFectamine.

Fig. 4 has shown the diagram of the preferred nano particle of siRNA of comprising of the present invention and carbohydrate ligand.

Fig. 5 has shown with siRNA separately (A) or with per 1000 cells, 0.25 μ g (rhombus), 0.5 μ g (square), 1.0 μ g (trilateral), the effect of the cell proliferation of the OVCAR cell of siRNA-nano particle (B) transfection of 1.5 μ g (grey fork) and 2.0 μ g (black fork) siRNA-nano particle.X-axis=fate; Y-axis=cell count (log ₁₀).

Fig. 6 shown the effect with the cell proliferation of the OVCAR cell of siRNA-nano particle transfection, with and without transfection reagent.Used the nano particle of three kinds of concentration:

1. use transfection reagent (square) and do not use transfection reagent (rhombus);

2. use transfection reagent (grey fork) and do not use transfection reagent (trilateral);

3. use transfection reagent (circle) and do not use transfection reagent (black fork);

X-axis=fate; Y-axis=cell count (log ₁₀).

Embodiment

The product p185Her-2/neu of Her-2/neu oncogene and coding thereof belongs to epithelial growth factor receptor Tyrosylprotein kinase (Bargmann etc., 1986).The HER receptor family is striden film Tyrosylprotein kinase: EGFR (being also referred to as Her-1 or erbB-1) by four kinds, erbB-2 (Her-2), and erbB-3 (Her-3) and erbB-4 (Her-4) form.The known Her-2/neu signal pathway (Yarden﹠amp that in cell growth and differentiation, vicious transformation and resistance, plays a crucial role to chemotherapeutics; Sliwkowski, 2001).In about 1/3rd people's breast cancer or ovarian cancer case, Her-2/neu is an overexpression, and its overexpression and poor prognosis relevant (Berchuck etc., 1990).

Having made many trials comes the Her-2/neu in the anticancer to express as the potential methods of treatment.Humanized monoclonal antibodies (trastuzumab or Trastuzumab) at Her-2/neu is effective (Mendelsohn ﹠amp in Her-2/neu-overexpression metastatic cancer; Baselga, 2000; Baselga etc., 1996) but the expression of Her-3 is raised in discovery.Shown the apoptosis (Roh etc., 2000) of inducing the human breast cancer cell system of overexpression Her-2/neu at the antisense oligonucleotide of Her-2/neu.Use the gene therapy of E1A, send by liposome or by adenovirus carrier, the mortality ratio that has mice with tumor in the Her-2/neu-overexpression ovarian cancer model can be reduced and breast cancer model medium and long distance metastasis rate (Chang etc., 1996) can be reduced.

Find that the downward modulation that Her-2/neu expresses causes PI3K, the minimizing of Akt and phosphorylation Akt, the expression that it causes cyclin D1 to reduce, cyclin D1 are (the cyclin Sherr ﹠amp of the adjusting of abortion of a kind of G0/G1 of participation cell and oncogene conversion; Robert, 1999).Relatively antisense oligonucleotide and siRNA effect the latest study proves that siRNA is to making at least 10 times of acceptor gene silences more effective (Miyagishi etc., 2003) on the nM basis.Several researchs have in the past proved that Her-2/neu promotes transcribing of VEGF, and VEGF is effectively short angiogenesis factor (Kumar ﹠amp; Yarmand-Bagheri, 2001), its level significantly reduces after the Her-2/neu expression silencing.Reverse transcription siRNA has improved the level of thrombospondin-1, thrombospondin-the 1st, strong angiogenesis inhibitor (Izumi etc., 2002) to the downward modulation of Her-2/neu.Vitro data has proved the HLA I class surface expression (Choudhur y etc., 2004) in the also remarkable mediator's of the going up tumour of HER2 siRNA treatment.

A. tactful: silence

Her2/Neu cDNA target sequence: AAG CCT CAC AGA GAT CTT GAA

A) justice is arranged: 5 '-G CCU CAC AGA GAU CUU GAAdTdT-3 '

B) antisense: 3 '-dTdTC GGA GUG UCU CUA GAA CUU-5 '

C) justice is arranged: 5 '-G CCU CAC AGA GAU CUU GAAdTdT-3 ' SS

D) antisense: 3 ' SS-dTdTC GGA GUG UCU CUA GAA CUU-5 '

B. annealing

Reticent: 5 ' (a)3 '

3’ (b) 5’

5’ (a) 3’

3’SS (d) 5’

5’ (c) 3’SS

3’ (b) 5’

5’ (a) 3’SS

3’SS (d) 5’

C. possible GNP combination

X=is used in should studying.

D. method

1. clone

SK-BR-3 human breast carcinoma (catalog number (Cat.No.) HTB-30) from ATCC.OVCAR-3 people's ascites gland cancer from NCI-Frederick cancer DCTD tumour/clone storer (bottle 0502296).

2.siRNA stock solution

The Her-2/neu DNA target sequence of selecting is AAGCCTCACAGAGATCTTGAA.

Have adopted siRNA to have sequence r (GCCUCACAGAGAUCUUGAA) d (TT) 3ThSS (MW 7416.25 of K-salt), antisense sequences r (UUCAAGAUCUCUGUGAGGC) d (TT) 3ThSS (MW 7409.57 of K-salt) obtains from Qiagen.

The contrast double-stranded sequence of (non-silence) siRNA (catalog number (Cat.No.) 1022076) is from Qiagen, it is that r (UUC UCC GAA CGU GUC ACG U) d (TT) and antisense sequences are r (ACG UGA CAC GUU CGG AGA A) d (TT) that adopted sequence is wherein arranged, and the MW of annealed K-salt is 14839.5.

An inclusion (296.65 μ g) that adopted siRNA pipe arranged is dissolved in the 1ml sterile buffer, and (the 2mM magnesium acetate forms 40 μ M liquid storages in pH7.4) for 100mM Potassium ethanoate, 30mM Hepes-KOH.Every μ l contains 0.297 μ g siRNA.

The inclusion (296.38 μ g) of an antisense siRNA pipe is dissolved in the 1ml sterile buffer, and (the 2mM magnesium acetate forms 40 μ M liquid storages in pH7.4) for 100mM Potassium ethanoate, 30mM Hepes-KOH.Every μ l contains 0.296 μ g siRNA.

In order to anneal, with the RNA oligomer solution of 3 μ l and the 5X annealing buffer of 15 μ l merge separately.The concentration of final damping fluid is 50mM Tris in the water handled of DEPC-, pH7.5-8.0,100mM NaCl.Final volume is 75 μ l, and the final concentration of siRNA duplex is 16 μ M.

With solution incubation 1 minute in 90-95 ℃ water-bath, and make it be cooled to room temperature (that is, being lower than 30 ℃).With the of short duration centrifugal all liquid of collecting the pipe bottom of pipe.Slowly cool to room temperature and need 45-60 minute.Resulting solution is stored in-20 ℃ of standby and tolerance freeze thawing repeatedly.

3.siRNA nanometer gold stock solution

General method

Buy HAuCl from Aldrich chemical company ₄(99.999%) and NaBH ₄In the cur laboratory, use standard method to come Synthetic 2-thio-ethyl-β-D-glucopyranose glycosides.For all experiments and solution, use the nanopure water of handling with DEPC (diethylpyrocarbonate) (18.1m Ω).All Eppendorf tubes, spatula and bottle all are no RNA enzymes.

Buy the double-stranded siRNA of annealed from Qiagen-Xeragon Inc.Specification is:

DNA target sequence AAGCCTCACAGAGATCTTGAA.

3 '-sulfydryl on adopted si RNA r (GCCUCACAGAGACUUGAA) d (TT) 3 '-(SS)-C3-connector is arranged

(MW 7416.25 of K-salt)

Antisense siRNA r (UUCAAGAUCUCUGUGAGGC) d (TT)

(MW 7409.57 of K-salt)

The preparation of e.RNA-Au-Glc nano particle

To 100mM, in 2-thio-ethyl-β-D-glucopyranose glycosides in the TRIS damping fluid of pH7.7 (250 μ L) (0.9mg, 3.75 μ mol) and siRNA (0.148mg, the 0.01 μ mol) solution, add HAuCl ₄The aqueous solution (22 μ L, 0.025M).Then, with 1N NaBH ₄The aqueous solution (30 μ L) divides several parts of addings, and quick oscillation.Formed brown suspension was vibrated 1 hour in addition at 4 ℃.(AMICON MW 10000,30 minutes, 14000rpm) comes purifying suspension by 4 ℃ by centrifuging.This process is repeated twice, wash with 125 μ L TRIS damping fluids.Resistates in the AMICON filter is dissolved in the TRIS damping fluid of 250 μ L and freeze-drying, obtains the RNA-Au-Glc nano particle (resuspension of solid in 1mL water should obtain 6 ± 1 μ M solution of RNA in 20mM TRIS damping fluid) of 4mg.(3000,4 ℃ of MW are 14000rpm) with filtrate desalination and freeze-drying to use AMICON.The weight of resistates＜50 μ g.Transmission electron micrograph shown in Fig. 1 (TEM) shows that the particulate mean particle size is 2.8nm, and average 807 gold atom/particles, the glucose-derivative of siRNA and 100 molecules are as shown in Figure 4 and have proximate MW＞160,000.

F. check the existence of RNA in the nano particle

The resistates that RNA-Au-Glc nano particle, Glu-Au nano particle and washing may be contained the RNA-Au-Glc nano particle of RNA oligonucleotide and glucose-derivative is dissolved in the 30 μ L water separately.(1 μ L 0.1%v/v) mixes with the aqueous solution of ethidium bromide (EtBr) with the sample aliquot (1 μ L) of these solution.Observe fluorescence (referring to Fig. 2) under the UV lamp, the nano particle that has proved such preparation has the siRNA (Fig. 2 b, pipe 1) that mixes, and the nano particle that only contains glucose does not demonstrate any fluorescence (Fig. 2 b, pipe 2).

4mg is dissolved in 6 ± 1 μ M liquid storages that obtain the 1ml water among the 20mM tris from the siRNA/ nano-Au composite that 148 μ g siRNA produce.Every μ l solution contains the Equivalent of 0.078 μ gsiRNA.

Cell is dull and stereotyped to be cultivated

1. in transfection preceding 24 hours, with 6 * 10 ⁴Individual cell is inhaled to move in the 24 hole flat boards and with suitable medium volume is complemented to 0.5ml.

2. make cell reach 50-80% and converge, probably need 24 hours.

3. remove substratum and alternative by 300 μ l fresh culture/holes.

With siRNA mixture transfectional cell

1. the double-stranded siRNA liquid storage that 3.3 μ l are suitable (or 12.8 μ l nano-Au composites) is dispensed in the 24 hole flat boards that correspondence contains cell.

2. in each hole, add 96.7 μ l suitable medium (or for nano-Au composite 87.2 μ l) and move 5 times and come thorough mixing by inhaling up and down.

3. in each hole (except the nano-Au composite hole) adds 6 μ l RNAiFect and moves 5 times and come thorough mixing by inhaling up and down.

4. solution was made mixture form at room temperature incubation 10-15 minute.

5. be covered on the cell in the 300l substratum with the suitable transfection composite of 100 μ l.

6. flat board is slightly shaken and mix to avoid vortex.

With flat board at CO ₂In the incubator in 37 ℃ of following incubation 48-72 hours.

8. remove substratum and with ice-cold PBS with cell washing three times.

9. with lysis, and measure the protein content of lysate.

10. by the SDS-PAGE isolated protein, then use the Her2/ErbB2 multi-clone rabbit antibody (catalog number (Cat.No.) 2242) of Cell SignallingTechnology to carry out western blot analysis.

11. handle trace with anti-rabbit igg-HRP conjugate, then ECL colour developing.

G. result

Shown the preliminary observed result that uses 1 μ g siRNA/ hole among Fig. 3 a and the 3b.The siRNA-gold nano grain is added in the cell RNAiFectamine useless.The SKBR3 cell reach 80% converge slower than OVCAR cell.SKBR3 result from transfection after 48 hours lysate and OVCAR result from transfection after 72 hours lysate.The synoptic diagram of nano particle is shown among Fig. 4.

SiRNA-Au-Glc nano particle pair cell nontoxicity

Separately with siRNA with use the siRNA that puts together with the sugared nano particle of gold to come transfectional cell.Fig. 5 has shown that the siRNA that nano particle is puted together is effective and free of toxic effects.Observe the dose-dependent effects of pair cell number, show that the siRNA nano particle has improved cell proliferation.

The SiRNA-Au-Glc nano particle enters cell

With siRNA-nano particle transfection OVCAR cell, with or need not use the needed transfection reagent of siRNA transfectional cell usually.Fig. 6 has shown that entering cell for the siRNA-nano particle does not need transfection reagent.The result shows that the siRNA nano particle is delivered in the cell effectively, even under the non-existent situation of transfection reagent; In fact, transfection reagent useless send it seems more effective.The dose-dependent effects of pair cell number has shown the real response to the siRNA-nano particle.

Reference

Reference all specially is incorporated herein by reference referred in this.

Pal-Bhadra etc., RNAi-Mediated targeting of Heterochromatinby the RITS complex (the heterochromatin target of the RNAi-mediation by the RITS mixture) .Science (2004) vol.303,669.

Verdel etc., Heterochromatic silencing and HP1 localizationin Drosphila are dependent on the RNAi Machinery (heterochromatin silence and HP1 location depend on RNAi mechanism in the fruit bat) .Science (2004) vol.303,672.

Elbashir etc., Duplexes of 21-nucleotide RNAs mediate RNAinterference in cultured mammalian cells (RNA in the mammalian cell of the duplex mediation cultivation of the RNA of 21 Nucleotide disturbs) .Nature 2001; 411:494-8.

Tuschl, RNA interference and small interfering RNAs (RNA disturbs and siRNA) .Chem Biochem 2001; 2:239-45.

Bargmann etc., The neu oncogene encodes an epidermal growthfactor receptor-related protein (neu oncogene coding schedule skin growth factor receptor associated protein(RAP)). (1986) Nature 319,226-230.

Yarden ﹠amp; Sliwkowski, Untangling the ErbBsignalling network (untiing ErbB signal net). (2001) Nat Rev Mol Cell Biol 2,127-137.

Berchuck etc., Overexpression of HER-2/neuisassociated withpoor survival in advanced epithelial ovarian cancer (overexpression of HER-2/neu is relevant with the survival of advanced epithelial ovarian carcinoma difference). (1990) Cancer Res 50,4087-4091.

Mendelsohn ﹠amp; Baselga, The EGF receptor family as targetsfor cancer therapy (as the EGF receptor family of cancer therapy target). (2000) Oncogene 19,6550-6565.

Baselga etc., Phase II study of weekly intravenous recombinanthumanized anti-p185HER2 monoclonal antibody in patients withHER2/neu over expressing metastatic breast cancer (have the II phase of the anti-p185HER2 monoclonal antibody of intravenously recombinant humanized is studied weekly among the HER2/neu patient of overexpression transitivity breast cancer) .J Clin Oncol 1996; 14:737-44.

Roh etc., Down regulation of HER2/neu expression inducesapoptosis in human cancer cells that over-express HER2/neu (the human cancer cell apoptosis that overexpression ER2/neu is induced in the downward modulation that HER2/neu expresses). (2000) Cancer Res 60,560-565.

Chang etc., Inhibition of intratracheal lung cancerdevelopment by systemic delivery of E1A (suppressing lung cancer development in the tracheae) (1996) Oncogene 13,1405-1412 by systemic delivery E1A.

Sherr ﹠amp; Roberts, and CDK inhibitors:positive and negativeregulators of G1-phase progression (CDK inhibitor: the positive and negative instrumentality that the G1-phase makes progress). (1999) Genes Dev 13,1501-1512.

Miyagishi etc., Comparison of the suppressive effects ofantisense oligonucleotides and siRNA sdirected against the sametargets in mammalian cells (antisense oligonucleotide with at the inhibition effect of the siRNA of identical target in the mammalian cell comparison) .Antisense Nucleic Acid DrugDev 2003; 13:1-7.

Kumar ﹠amp; Yarmand-Bagheri, The role of HER2 in angiogenesis (effect of HER2 in vasculogenesis). (2001) Semin Oncol 28,27-32.

Izumi etc., Tumour biology:herceptin acts as anantiangiogenic cocktail (oncobiology: Trastuzumab is as the mixture of angiogenesis inhibitor). (2002) Nature 416,279-280.

Choudhury etc., Small interfering RNA (siRNA) inhibits theexpression of the Her2/Neu gene, (siRNA (siRNA) suppresses the Her2/Neu expression of gene to upregulates HLA Class I andinduces apoptosis of Her2/Neu positive tumour cell lines, the apoptosis that raises I class HLA and induce Her2/Neu positive tumor cell system) .Int J Cancer 108,71-77,2004.

Overbaugh, HTLV sweet-talksits way into cells (HTLV enters in the cell dexterously) .Nat.Med (2004) vol.10,20.

Check, RNA to the rescue (RNA that is used to save) .Nature (2003) vol 425,10-12.

Zamore etc., siRNAs knock down hepatitis (siRNA suppresses hepatitis) .Nature (2003) vol 9,266-267.

Song etc., RNA interference targeting Fas protects mice fromfulminant hepatitis (RNA of target Fas disturbs and makes mouse avoid fulminant hepatitis) .Nat.Med. (2003) vol.9,347-351.

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WO 02/32404, enjoys the PCT application of GB-A-0313259.4 right of priority.

Claims

1. the nano particle that contains core, this core comprises metal and/or semiconductor atom, wherein covalently bound the and part of core and a plurality of part comprises the RNA part.

2. the nano particle of claim 1, wherein the RNA part is siRNA or miRNA part.

3. the nano particle of claim 1 or claim 2, wherein nano particle further comprises the part that contains carbohydrate group.

4. each nano particle of claim 1 to 3, wherein nano particle and siRNA molecule lead to that to connect group covalently bound.

5. the nano particle of each claim before, wherein linking group is a mercapto groups, ethylidene group or peptide group.

6. the nano particle of each claim before, wherein the RNA part is that 17 to 30 ribonucleotides are long.

7. the nano particle of each claim before, wherein part is siRNA part and the 3 ' overhang that comprises 2 ribonucleotides.

8. the nano particle of each claim before, wherein first of the RNA molecule has justice or antisense strand covalently bound with the nano particle core by its 5 ' and/or 3 ' end.

9. the nano particle of claim 8 wherein will be annealed with second chain of the first chain complementary RNA molecule and first chain of RNA molecule.

10. the nano particle of claim 9, wherein the 2nd RNA chain is covalently bound with the nano particle core by its 5 ' and/or 3 ' end.

11. the nano particle of claim 9 or claim 10, wherein first and second chains of RNA molecule connect with the nano particle core respectively and annealing subsequently.

12. each nano particle of claim 1 to 6, wherein the RNA part is the miRNA part and comprises hair clip.

13. the nano particle of each claim before, wherein the RNA part is based on the Her2 gene order.

14. the nano particle of each claim before, wherein nano particle comprises mark.

15. the nano particle of claim 14, wherein mark is a fluorophor, radionuclide, magnetic mark, dyestuff, NMR active atomic, the atom that maybe can use surperficial plasmon resonance to detect.

16. the nano particle of claim 15, wherein fluorophor is a fluorescein, rhodamine or tetramethylrhodamin, and Texas-is red, Cy3 or Cy5.

17. the nano particle of claim 15, wherein radionuclide is ^99mTc, ³²P, ³³P, ⁵⁷Co, ⁵⁹Fe ³⁺, ⁶⁷Cu ²⁺, ⁶⁷Ga ³⁺, ⁶⁸Ge, ⁸²Sr, ⁹⁹Mo, ¹⁰³Pd, ¹¹¹In ³⁺, ¹²⁵I, ¹³¹I, ¹³⁷Cs, ¹⁵³Gd, ¹⁵³Sm, ¹⁵⁸Au, ¹⁸⁶Re, ²⁰¹Tl ⁺, ³⁹Y ³⁺, ⁷¹Lu ³⁺Or ²⁴Cr ²⁺

18. the nano particle of claim 15, wherein magnetic mark is to comprise Mn ^{+ 2}, Gd ^{+ 3}, Eu ^{+ 2}, Cu ^{+ 2}, V ^{+ 2}, Co ^{+ 2}, Ni ^{+ 2}, Fe ^{+ 2}, Fe ^{+ 3}Or lanthanon ^{+ 3}The paramagnetic group.

19. the nano particle of claim 15, wherein the NMR active atomic is Mn ^{+ 2}, Gd ^{+ 3}, Eu ^{+ 2}, Cu ^{+ 2}, V ^{+ 2}, Co ^{+ 2}, Ni ^{+ 2}, Fe ^{+ 2}, Fe ^{+ 3}Or lanthanon ^{+ 3}The paramagnetic group.

20. the nano particle of each claim before, wherein nano particle is water miscible.

21. the nano particle of each claim before, wherein the core of nano particle has 0.5 to 10nm mean diameter.

22. the nano particle of each claim before, wherein the core of nano particle has 1 to 5nm mean diameter.

23. the nano particle of each claim before has 10 to 30nm mean diameter comprising the nano particle of its part.

24. the nano particle of each claim before, wherein core is a metallic core.

25. the nano particle of each claim before, wherein metallic core comprises Au, Ag or Cu.

26. the nano particle of claim 24 or claim 25, wherein metallic core is to be selected from Au/Ag, Au/Cu, Au/Ag/Cu, Au/Pt, Au/Pd, Au/Ag/Cu/Pd, Au/Fe, Au/Cu, Au/Gd, Au/Fe/Cu, the alloy of Au/Fe/Gd or Au/Fe/Cu/Gd.

27. each nano particle of claim 24 to 26, wherein the core of nano particle is a magnetic.

28. the nano particle of claim 26, wherein nano particle comprises the passive metal atom and the magneticmetal atom of about 5: 0.1 to about 2: 5 ratios in core.

29. the nano particle of claim 28, wherein passive metal is gold, platinum, silver or copper, and magneticmetal is iron or cobalt.

30. the nano particle of claim 27, wherein core comprises MnFe (ferrospinel) or CoFe (vectolite).

31. each nano particle of claim 1 to 23, wherein core comprises semiconductor atom.

32. the nano particle of claim 31, wherein semiconductor atom can be as quantum dot.

33. the nano particle of claim 31 or claim 32, wherein semi-conductor is cadmium selenide, Cadmium Sulfide, cadmium telluride or zinc sulphide.

34. the nano particle of each claim before, wherein nano particle comprises a plurality of dissimilar parts.

35. the nano particle of each claim before, wherein part further comprises peptide, protein domain, nucleic acid sections or carbohydrate group.

36. the nano particle of each claim before, wherein part comprises polysaccharide, oligosaccharides or monose group.

37. the nano particle of each claim before, wherein part is sugared nanometer conjugate.

38. the nano particle of claim 37, wherein sugared nanometer conjugate comprises glycolipid or glycoprotein.

39. the nano particle of each claim before, wherein part comprises DNA or RNA.

40. comprise one or more compositions of the nano particle group of each claim before.

41. each the method for nano particle of preparation claim 1 to 39, it is by RNA being conjugated to the core of nano particle, and this method comprises:

Produce with 5 ' and/or the first and/or the 2nd RNA chain of 3 ' end connector derivation with first and/or second chain of connector derivation RNA; With

With the core that the RNA and the reactant reaction of connector derivation is used to produce nano particle, make that at nano particle the nano particle core is connected to RNA by connector automatically in the process of assembling.

42. the method for claim 41, wherein connector is the sulfydryl connector, ethylidene connector or peptide connector.

43. the method for claim 41 or claim 42, wherein reaction mixture comprises the siRNA of derivation, and the salt and the reductive agent of metal and/or semiconductor atom produce nano particle.

44. by each the obtainable nano particle of method of claim 41 to 43.

45. the purposes that each nano particle of claim 1 to 40 is used for the treatment of or diagnoses.

46. each nano particle of claim 1 to 40 be used to prepare illness that treatment can alleviate by down-regulation of gene expression or with the purposes of the medicine of gene overexpression associated conditions, wherein come target and down-regulated gene by the RNA part.

47. each nano particle of claim 1 to 40 is used to prepare the purposes of the medicine for the treatment of cancer, virus infection or eyes macular degeneration.

48. the purposes of claim 46 or claim 47, wherein cancer is that breast cancer or virus are HIV, hepatitis or influenza.

49. each purposes of claim 46 to 48, wherein RNA is based on the Her2/Neu gene order.

50. each purposes of claim 46 to 49, wherein nano particle comprises the other part puted together with the nano particle core or the structural domain relevant with the RNA molecule, and wherein other part or structural domain can be specifically and the cell interaction of expressing target gene.

51. the purposes of claim 50, wherein other part or structural domain be present on the target cell surface or inner can specificity in conjunction with the specificity of its binding partners in conjunction with right member.

52. each purposes of claim 46 to 51, wherein the RNA molecule with the nano particle targeted expression can with the cell of the mRNA of RNA interaction of molecules.

53. each purposes of claim 46 to 52, wherein nano particle further comprises radionuclide, medicine or is used for the treatment of or kills other medicaments by the molecular targeted cell of RNA.

54. each purposes of claim 46 to 53, wherein nano particle comprises film transposition signal, makes that they can permeate through cell membranes.

55. comprising, the using method of downward modulation target gene, this method make each the nano particle of cells contacting claim 1 to 40 that contains gene.

56. the method for claim 55, wherein this method is carried out external.

57. the method for claim 55 or claim 56, wherein this method causes the instantaneous rejecting of target gene.

58. each method of claim 55 to 57; wherein this method is used the nano particle with at least two kinds of different RNA parts; described part is conjugated to identical nano particle or is present in the composition of at least two kinds of dissimilar nano particles, comes at least two kinds of expression of gene in the downward modulation approach.

59. the method for claim 58, wherein approach is a pathways of inflammation, antiviral pathway, the signal pathway in the cancer, route of metastasis or pathways metabolism.

60. each method of claim 55 to 59, wherein the RNA part comes a plurality of members' of down-regulated gene family expression based on the conserved domain of gene family.

RNA detects or the purposes of imaging 61. each nano particle of claim 1 to 40 is used for.

62. use each the mRNA of nano particle of claim 1 to 40 to detect and/or image formation method, this method be included in the RNA that wherein is present on the nano particle can with the interactional condition of said target mrna under, make the nano particle contact contain the sample/cell of said target mrna, and detect nano particle-RNA-mRNA mixture.

63. the method for claim 62 wherein detects the step of mixture and uses the inwardness of nano particle or the mark that links to each other with nano particle by detection.

64. the method for claim 63, wherein mark is magnetic group, quantum dot or radionuclide.

65. each method of claim 61 to 64, wherein this method is containing external carrying out on the sample of mRNA.

66. each method of claim 61 to 65 is wherein carried out this method in vivo and is used to detect intracellular mRNA.

67. each nano particle of claim 1 to 40 is used for the RNA molecule is delivered to the purposes of target cell.

68. the purposes of claim 67, wherein this purposes is to be used to prepare the RNA molecule is delivered to the medicine that is subjected to the target cell that disease or illness influence.

69. each nano particle of claim 1 to 40 is as the purposes of functional genomics instrument.

70. contain the purposes that each the aerosol combination of nano particle of claim 1 to 40 is used to prepare the medicine that is delivered to lung, wherein nano particle comprises the mark that is used for imaging or is used for the treatment of the disease that influences the Mammals lung.