CN101180400B - Nanoparticles comprising RNA ligands - Google Patents
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- A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
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- B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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- C12N2310/00—Structure or type of the nucleic acid
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- C12N2310/14—Type of nucleic acid interfering N.A.
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- C12N2320/00—Applications; Uses
- C12N2320/10—Applications; Uses in screening processes
- C12N2320/12—Applications; Uses in screening processes in functional genomics, i.e. for the determination of gene function
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
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 siRNA (siRNA) to the orientation degraded of 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-ntsiRNA 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 90% silence of being induced by several siRNA molecules.
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 the rejecting of having observed by the target gene of siRNA is not that nonvolatil, independent siRNA transfection can cause the inhibition of target protein in parent and the daughter cell to prolong (Tuschl, 2001).Yet sending in the art of siRNA still has problems.
WO02/32404 (Consejo Superior de Investigaciones Scientificas) 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 for regulating 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 the magneticmetal atom of passive state, 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 to simulate the short rna sequence of siRNA (siRNA) and microRNA (miRNA) sequence.Nano particle can be used for sending the RNA part and having 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 as the instrument of functional genome in conjunction with the reticent target siRNA resistance mRNA of being used for of mRNA and (7).
In the art, according to its source the short rna sequence is 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 hair clip with miRNA sample sequence, namely 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 that the RISC mixture is to the correct identification of siRNA, when the compound-mediated mRNA target of described RISC was used nano particle in the identification of siRNA and the preferred body, siRNA is the short host response that reduces enough.
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 the rna gene of protein.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 3 ' overhang of symmetry, 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 disulfide linkers, and the disulfide linkers of Hun Heing 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 disulfide linkers that mixes 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 makes the other step of annealing to provide the double-stranded RNA part that is connected on the nano particle with the RNA molecule of the first chain complementation.The nano particle that can also prepare the double-stranded RNA with annealing, 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, for double-stranded RNA being mixed in the nano particle in the automatic assembling, can come one or two end of this RNA of derivation with the disulphide that mixes.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.The disulphide that mixes 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 WO02/32404 first, this core comprises gold atom, wherein uses disulfide linkers to come the derivation part and make 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 sodium borohydride.These and other feature descriptions of this method are in WO02/32404.
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 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 for wherein existing specificity in conjunction with another right member target position 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 atom of one or more NMR activity of mixing 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
+ 3And 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 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 launch, R.Jin, C.A.Mirkin, Science2002,297:1536-1539).
In some embodiments, nano particle can comprise radionuclide, is used for using the radioactivity of radionuclide emission to detect nano particle, for example, uses PET, SPECT, or be used for the treatment of, namely 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
131I;
137Cs;
153Gd;
153Sm;
158Au;
186Re; Usually with T1
+Salt uses as thallium chloride
201T1;
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 aspect 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, namely 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 can be used for monitoring to interact in real time.As mentioned above, if nano particle comprises or the NMR active atomic that mixed, this technology can be used for using technology well known in the art to detect this particle in external or body so.Can also use the system of amplifying based on quantitative signal, the silver (I) that utilizes nano particle to promote also detected nano particle originally.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 for presenting 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 be combined 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 that about 5:0.1 is to about 2:5 in the core.More preferably, ratio is that about 5:0.1 is 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-namely, 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 strong paramagnetism characteristic to the external magnetic field, is called superparamagnetism.Preferably, nano particle of the present invention is 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
+ 3And lanthanon
+ 3Those.
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 example that connects chemistry for the production of the automatic assembling of 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 the 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 invention provides the nano particle of above definition for the preparation of the purposes of medicine, 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 the purposes in external and the body.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 the illness relevant with the overexpression of the gene of also reducing by the RNA target.
The adjusting of genetic expression
The nano particle that comprises the RNA part can be used for regulatory gene in many ways expresses, comprise the 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 for the downward modulation target gene.Among the application, downward modulation can be external, for example, study genetic expression, or in the body, in the target experimental system or be used for medical usage, namely 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 and identical nano particle are puted together or are present in the composition of at least two kinds of dissimilar nano particles, 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.
For any purposes of using the 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 for suppressing 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 for giving the target characteristic, is used for RNA is delivered to target cell.This can by provide have an other type with close the 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 apparent.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 for mixing 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 carrying 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 the entity that the nano particle target is provided, 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 provided by their small size and a kind of effect (referring to NatureBiotechnology18:410-414,2000) by strengthening with film transposition signal derivation nano particle randomly.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, because this effect is normally temporary transient, so the nano particle with radionuclide or medicine preferably is provided, makes and use the cell of RNA target to be killed by selectivity.Target cell and treating processes imaging can be followed marked with nanometer granule, for example nano particle of 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 image formation method that makes 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 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 more than 100 RNA sequence study in the body gene function and as the instrument of the screening that is used for full genome range.
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 for sending 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 magnetic field that the nano particle tumour cell that leads is always unfeasible or accurately separately, 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 the nano particle of antigen can be used as vaccine administration, for example, by trajectory, uses and sends rifle and accelerate them and penetrate through skin by epidermis is outer field.Nano particle can for example absorb by dendritic cell then, along with they by lymphoid migration maturation, cause immunne response adjusting and the antagonism antigen vaccine inoculation.
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 character and the severity of illness to be treated.The prescription for the treatment of 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, other factors that medication and doctor are known.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 48 hours western blottings from the Her-2/neu albumen of isopyknic SKBR3 cell lysate after the Her-2/neusiRNA transfection.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 72 hours western blottings from the Her-2/neu albumen of isopyknic SKBR3 cell lysate after the Her-2/neusiRNA transfection.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 the 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 the resistance to chemotherapeutics, plays a crucial role; Sliwkowski, 2001).In about 1/3rd people's breast cancer or ovarian cancer case, Her-2/neu is overexpression, and its overexpression and the prognosis relevant (Berchuck etc., 1990) that differs from.
Having made many trials comes the Her-2/neu in the anticancer to express as potential methods for the treatment of.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 the 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 effectively urgees angiogenesis factor (Kumar﹠amp; Yarmand-Bagheri, 2001), its level significantly reduces after the Her-2/neu expression silencing.The downward modulation of the Her-2/neu of reverse transcription siRNA has improved the level of thrombospondin-1, thrombospondin-the 1st, strong angiogenesis inhibitor (Izumi etc., 2002).Vitro data has proved the also remarkable HLAI class surface expression of going up in the tumour of transferring person (Choudhury etc., 2004) of HER2siRNA treatment.
A. tactful; Reticent
Her2/NeucDNA 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 makes up
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 (MW7416.25 of K-salt), antisense sequences r (UUCAAGAUCUCUGUGAGGC) d (TT) 3ThSS (MW7409.57 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 the K-salt of annealing 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 gsiRNA.
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 synthesize 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 annealing from Qiagen-Xeragon Inc.Specification is:
DNA target sequence AAGCCTCACAGAGATCTTGAA.
3 '-sulfydryl on adopted siRNA r (GCCUCACAGAGAUCUUGAA) 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 the 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) divide several parts of addings, and fastVibration.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 mean particle size of particle 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 approximate 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 20mMtris from the siRNA/ nano-Au composite that 148 μ gsiRNA 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. double-stranded siRNA liquid storage (or 12.8 μ l nano-Au composites) that 3.3 μ l are suitable 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 fully mix by inhaling up and down.
3. in each hole (except the nano-Au composite hole) adds 6 μ lRNAiFect and moves 5 times and fully mix 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 to 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 Signalling Technology 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 gsi RNA/ 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.
The siRNA-Au-Glc nano particle is to the 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 to cell count, 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 cell count has been shown real response to the siRNA-nano particle.
Reference
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WO02/32404 enjoys the PCT application of GB-A-0313259.4 right of priority.
Claims (12)
1. reduce the in vitro method of target gene, this method comprises makes the cell that contains this gene contact with the nano particle that comprises the Au core, the core of wherein said nano particle have 0.5 and 10nm between mean diameter, and wherein said core and a plurality of part are covalently bound, this part comprises (i), and at least one contains the part of carbohydrate group and (ii) at least one RNA part, wherein said carbohydrate group is selected from polysaccharide, oligosaccharides and monose, described RNA part comprises the siRNA molecule, and wherein in that to lack under the situation of transfection agents the described cell of contact and wherein said nano particle covalently bound by mercaptan linking group and described RNA part.
2. the process of claim 1 wherein that described siRNA part comprises 3 ' overhang of 2 ribonucleotides.
3. the process of claim 1 wherein that described RNA part comprises first of RNA molecule justice or antisense strand and covalently bound with the nano particle core by its 5 ' and/or 3 ' end are arranged.
4. the method for claim 3, wherein said RNA part comprise with first chain of RNA molecule complementary and with second chain of the RNA molecule of first chain annealing of RNA molecule.
5. the method for claim 4, wherein said the 2nd RNA chain is covalently bound with the nano particle core by its 5 ' and/or 3 ' end.
6. the process of claim 1 wherein RNA part target Her2 gene order.
7. the process of claim 1 wherein that described RNA part is strand or double-stranded.
8. the process of claim 1 wherein that described RNA part has:
Ribonucleotide between 10 and 40,
Ribonucleotide between 17 and 30,
Ribonucleotide between 19 and 25, or
Ribonucleotide between 21 and 23.
9. the process of claim 1 wherein that this method causes the instantaneous rejecting of described target gene.
10. the method for claim 9, wherein this method is used the nano particle with at least two kinds of different RNA parts, described part is conjugated on the identical nano particle or is present in the composition of at least two kinds of dissimilar nano particles, with at least two kinds of expression of gene in the downward modulation approach.
11. the method for claim 10, wherein said approach are signal pathway, route of metastasis or pathways metabolism in pathways of inflammation, antiviral pathway, the cancer.
12. the process of claim 1 wherein described RNA part based on the conserved domain of gene family to reduce a plurality of members' of described gene family expression.
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| GB60/573,805 | 2004-05-24 | ||
| US0411537.4 | 2004-05-24 | ||
| PCT/GB2005/002058 WO2005116226A2 (en) | 2004-05-24 | 2005-05-24 | Nanoparticles comprising rna ligands |
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| CN201210359631.3A Division CN102872463B (en) | 2004-05-24 | 2005-05-24 | Nanoparticles comprising RNA ligands |
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| CN101180400A CN101180400A (en) | 2008-05-14 |
| CN101180400B true CN101180400B (en) | 2013-07-10 |
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| CN2005800240377A Expired - Fee Related CN101180400B (en) | 2004-05-24 | 2005-05-24 | Nanoparticles comprising RNA ligands |
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| ES (2) | ES2655069T3 (en) |
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| WO2008073856A2 (en) * | 2006-12-08 | 2008-06-19 | Massachusetts Institute Of Technology | Delivery of nanoparticles and/or agents to cells |
| AU2009316286B2 (en) * | 2008-11-24 | 2016-05-26 | Northwestern University | Polyvalent RNA-nanoparticle compositions |
| US20100233270A1 (en) | 2009-01-08 | 2010-09-16 | Northwestern University | Delivery of Oligonucleotide-Functionalized Nanoparticles |
| KR20170072367A (en) * | 2009-04-15 | 2017-06-26 | 노오쓰웨스턴 유니버시티 | Delivery of oligonucleotide-functionalized nanoparticles |
| CN102631687A (en) * | 2012-05-07 | 2012-08-15 | 西安电子科技大学 | Multifunctional magnetic nano-carrier for targeted delivery of microRNA, preparation method and application thereof |
| CA2873274C (en) * | 2012-06-08 | 2021-06-01 | Ethris Gmbh | Pulmonary delivery of messenger rna |
| EP2865759B1 (en) * | 2012-06-20 | 2017-05-17 | NapaJen Pharma, Inc. | Nucleic acid complex and nucleic acid-polysaccharide complex |
| US10894963B2 (en) | 2013-07-25 | 2021-01-19 | Exicure, Inc. | Spherical nucleic acid-based constructs as immunostimulatory agents for prophylactic and therapeutic use |
| WO2015126502A2 (en) | 2013-12-03 | 2015-08-27 | Northwestern University | Liposomal particles, methods of making same and uses thereof |
| CN106535876B (en) | 2014-06-04 | 2020-09-11 | 埃克西奎雷股份有限公司 | Multivalent delivery of immunomodulators through liposomal spherical nucleic acids for prophylactic or therapeutic applications |
| JP2017537619A (en) | 2014-11-21 | 2017-12-21 | ノースウェスタン ユニバーシティ | Sequence-specific intracellular uptake of spherical nucleic acid nanoparticle complexes |
| CN111110846B (en) * | 2018-10-30 | 2021-07-23 | 国家纳米科学中心 | Metal-nucleic acid nano-particle and preparation method and application thereof |
| CN113413467A (en) * | 2021-07-01 | 2021-09-21 | 清华大学 | Carrier-free mRNA delivery method |
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| CA2262018C (en) * | 1996-07-29 | 2007-10-02 | Nanosphere Llc | Nanoparticles having oligonucleotides attached thereto and uses therefor |
| GB0025414D0 (en) * | 2000-10-16 | 2000-11-29 | Consejo Superior Investigacion | Nanoparticles |
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2004
- 2004-05-24 GB GBGB0411537.4A patent/GB0411537D0/en not_active Ceased
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Non-Patent Citations (2)
| Title |
|---|
| Choudhury A. et al..Small interfering RNA (siRNA) inhibits the expression of theHer2/Neu gene, upregulates HLA class I and inducesapoptosis of Her2/Neu positive tumor cell lines.Int. J. Cancer108 1.2004,108(1),71-77. |
| Choudhury A. et al..Small interfering RNA (siRNA) inhibits the expression of theHer2/Neu gene, upregulates HLA class I and inducesapoptosis of Her2/Neu positive tumor cell lines.Int. J. Cancer108 1.2004,108(1),71-77. * |
Also Published As
| Publication number | Publication date |
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| ES2655069T3 (en) | 2018-02-16 |
| ES2533238T3 (en) | 2015-04-08 |
| CN102872463B (en) | 2015-04-08 |
| CN102872463A (en) | 2013-01-16 |
| GB0411537D0 (en) | 2004-06-23 |
| CN101180400A (en) | 2008-05-14 |
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