CN104293921A - Magnetic nano composite for treating lung cancer and preparation method thereof - Google Patents
Magnetic nano composite for treating lung cancer and preparation method thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
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- A61K49/0017—Fluorescence in vivo
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- A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
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- A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
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- A—HUMAN NECESSITIES
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- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
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Abstract
The invention discloses a magnetic nano composite for treating lung cancer. The magnetic nano composite for treating lung cancer is characterized by comprising a magnetic nano microsphere, a molecular beacon which is adsorbed on the magnetic nano microsphere and a microRNA specificity inhibitor (comprising antisense nucleic acid); the sequence of the molecular beacon is 5'-Aleax488-CCAGCG-gaaacc+Cagc+Aga+Caa+Tgtagct-CGCTGG-BHQ1-3', wherein CCAGCG is a 5' end stem-shaped sequence, CGCTGG is a 3' end stem-shaped sequence, Aleax488 is a luminophore, BHQ1 is a quenching group and gaaacc+Cagc+Aga+Caa+Tgtagct is a cyclic sequence; the capitalized basic group in the cyclic sequence undergoes modification treatment of locked nucleic acid; and the magnetic nano microsphere comprises magnetic nano particles and polylactic acid-glycolic acid copolymers which are coated on the surfaces of the magnetic nano particles. Compared with the prior art, the magnetic nano composite for treating lung cancer has the advantages of resisting the degradation of nuclease in living cells, specifically suppressing the expression of miR-221 in the lung cancer, further suppressing the multiplication and migration of the lung cancer cells and realizing the treatment of the lung cancer.
Description
Technical field
The present invention relates to a kind of for suppressing the magnetic nanocomposites of lung cancer expression of nucleic acid, particularly a kind of magnetic nanocomposites for lung cancer therapy and preparation method thereof.
Background technology
The detection method of Microrna (microRNA, miRNA) is a lot.Northern hybridization detects the easy and reliable method of the one of the expression level of miRNA in histocyte, can be Northern-blot through the method for the molecular size classics of detected through gel electrophoresis miRNA, but need total serum IgE amount more.Biochip technology and real-time fluorescence quantitative PCR are applied to systematic study and the detection by quantitative of miRNA, but step is loaded down with trivial details, and cost is higher, are vulnerable to the restriction of Reverse Transcription Efficiency.The methods such as fluorescence in situ hybridization (FISH) are the methods of relative simplicity, but miRNAs in situ detection system is relatively less, and chief reason is low copy and segment is less.Although can significantly improve miRNA/cDNA hybridization complex by taking the probe of base modification to melt chain temperature, it is too low that miRNA/cDNA hybridization complex melts chain temperature, is difficult to tolerate the processes such as complicated rinsing.What is more important, traditional miRNA detection method is difficult to the specific detection realizing miRNA in intact cell.Molecular beacon can be guaranteed to react not by the interference of its factor such as precursor and contaminating genomic DNA, also can accurately distinguish the miRNA of sequence very high homology; The quantitative linearity scope of ultra-wide and the detection sensitivity of height, copy several ten thousand copies to from several, quantitative linearity range spans 7 orders of magnitude; Sample consumption is few, only needs the total serum IgE of 1 ~ 10ng; Applied widely, the RNA of total serum IgE, cell lysate and purifying can be used for the detection by quantitative of miRNA, is particularly useful for the detection of active somatic cell miRNA.
The molecular beacon of expressing for detection by quantitative miR-221 is had in prior art.And the miRNA found recently may become the biological novel targets of oncotherapy.Different tumour has different miRNA expression patterns, by the analysis of miRNA express spectra, by contributing to diagnosing tumour clinically, by stages and the estimation of prognosis.The expression of microRNA is relevant to the existence of the adenocarcinoma of lung comprising I phase tumour.Research finds, people miR-221 (hsa-miR-221) high expression level is relevant with prognosis mala; Hsa-miR-221 high expression level is the factor of prognosis mala.MiR-221 can be used as a therapy target of lung cancer.It is low that molecular beacon has background signal, highly sensitive, specific recognition is strong, simple to operate, need not can detect in real time with unreacted probe separates, can be used for the advantages such as in-vivo analysis.At biochemical analysis, have a wide range of applications in biomedical research and clinical diagnosis value.Filter out lung cancer miRNA express spectra by gene chip and its function has been identified at present, selection of the present invention detects to the miR-221 that lung cancer occurs and prognosis is relevant, but when common molecular beacon is used for the detection analysis of mRNA in viable cell, because intracellular nucleic acid enzyme is to the degraded of molecular beacon skeleton and destruction, often cause the generation of false positive signal.
Summary of the invention
The present invention is to solve above-mentioned deficiency, provides a kind of magnetic nanocomposites for lung cancer therapy and preparation method thereof.
Above-mentioned purpose of the present invention is realized by following technical scheme: a kind of magnetic nanocomposites for lung cancer therapy, is characterized in that: comprise magnetic Nano microsphere and be adsorbed on the molecular beacon on magnetic Nano microsphere, and its sequence of described molecular beacon is
5 '-Aleax488-CCAGCG-gaaacc+Cagc+Aga+Caa+Tgtagct-CGCTGG-BHQ1-3 ', wherein CCAGCG is 5 ' end bulbous sequence, CGCTGG is 3 ' end bulbous sequence, luminophore is Alexa488, extracting the group that goes out is BHQ1, gaaacc+Cagc+Aga+Caa+Tgtagct is ring-shaped sequence, is wherein carry out the base after locking nucleic acid moditied processing with capitalizing the base that represents in ring-shaped sequence; Described magnetic Nano microsphere comprises magnetic nanoparticle and is wrapping by the Poly(D,L-lactide-co-glycolide on magnetic nanoparticle surface.
Sequence information according to ripe microRNA designs molecular beacon, the specific design of molecular beacon is carried out according to the principle of design of website (www.molecular-beacons.org) Middle molecule beacon, and cycling probe principles of structural design is: length is between 15 ~ 30 bases; Can complementary pairing with target miRNA.Bulbous sequential structure principle of design: the length of probe sequence is 5 ~ 7 bases; GC content one between 70 ~ 80%; Melt chain temperature and want 7 ~ 10 DEG C than detected temperatures; G base can not connect luminophore.Luminophore and extraction are gone out the selection of group: luminophore can be extracted accordingly the group that goes out extract and go out, two groups have a high signal to background ratio (more than 2.0).Be the Degradation resisting nucleic acid in living cell enzyme according to above principle the present invention, the present invention carries out lock nucleic acid to the number of base in the molecular beacon designed and modifies.Lock nucleic acid (Locked nucleic acid, LNA) be a kind of novel nucleotide derivative, 2 '-O of β-D-RIBOSE in its structure, 4 '-C position forms the condensation structure of this rigidity of Oxymethylene bridge of annular by shrink effect, change geometry and the tridimensional character of Nucleotide, reduce the snappiness of ribose structure, thus considerably improve its stability, the antienzyme that this conformation not only significantly improves nucleic acid cuts ability, and improves cross compatibility.The base number that LNA modifies should more than 6, otherwise slower in conjunction with speed with miRNA.4 bases in people miR-221 molecular beacon have been carried out LNA modification by the present invention, and namely the capitalization in above-mentioned molecular beacon ring-shaped sequence has done LNA modification.
In order to improve sensitivity and the stability of molecular beacon detection by quantitative further, magnetic Nano microsphere is adopted to carry out Isolation and purification molecular beacon of the present invention.The principle that magnetic Nano microsphere extracts DNA is: the macromolecular material (as Poly(D,L-lactide-co-glycolide) being rich in positive charge chemical group at magnetic nanoparticle pan coating, is prepared into positively charged magnetic Nano microsphere.Magnetic Nano microsphere is joined in DNA extraction liquid, and in the damping fluid of slant acidity (PH6.5), the DNA in solution can be adsorbed on positively charged Nano microsphere.Adopt magnetic Nano microsphere as the carrier of molecular beacon, extract corresponding DNA, the much impurity affecting pcr amplification can be removed, improve the stability of pcr amplification.In addition, a certain amount of magnetic Nano microsphere can adsorb a certain amount of DNA, contributes to the template DNA obtaining relative constant, improves the accuracy that fluorescent molecular bacon probe quantitative detects.
Magnetic nanoparticle (MNPs) is formed centered by magneticsubstance, can wrap by the nucleocapsid structure of biomolecules, not only possesses good magnetic conductance tropism, also there is good biocompatibility, can be combined with several functions molecule, be used widely in viable cell and biological tissue imaging.But not modified nano particle has metal toxicity to viable cell, therefore finishing to be carried out to nano particle.
Further, described magnetic nanoparticle is Fe
3o
4magnetic nanoparticle.
Fe
3o
4magnetic nanoparticle is convenient to nucleus magnetic resonance (MRI) video picture, and molecular beacon is convenient to fluorescent microscope video picture, and this nanotechnology has dual image displaying function.
Further, described magnetic nanocomposites is the magnetic nanocomposites of surface through polyoxyethylene glycol process.
Nano particle after polyoxyethylene glycol (PEG) modification has good long circulating stability, can escape the system of engulfing and engulf, and can improve biocompatibility in body and external.
The preparation method of a kind of magnetic nanocomposites for lung cancer therapy of the present invention, is characterized in that: comprise the following steps:
A. design needs the molecular beacon of synthesis: determine that people miR-221 sequence is 5 '-AGCTACATTGTCTGCTGGGTTTC-3 ', the molecular beacon ring-shaped sequence of designer miR-221 is GAAACCCAGCAGACAATGTAGCT, luminophore is Alexa488, and extracting the group that goes out is BHQ1; The base of carrying out LNA modification is needed in selected 4 ring-shaped sequences, then design its sequence of molecular beacon and be defined as 5 '-Aleax488-CCAGCG-gaaacc+Cagc+Aga+Caa+Tgtagct-CGCTGG-BHQ1-3 ', be wherein namely defined as by the base that capitalization represents the base will carrying out lock nucleic acid moditied processing in ring-shaped sequence;
Protocols in Molecular Biology (shearing enzyme, polysaccharase etc. as used DNA) can being adopted to synthesize according to the above-mentioned molecular beacon sequences determined, chemosynthesis mode also can be adopted on DNA synthesizer to synthesize;
B. magnetic Nano Fe is prepared
3o
4particle: the KOH solution of 20mL is poured in flask and stirs, then the KNO solution of 20mL is added wherein, after fully stirring, the FeSO4 solution of 10mL is added, until reaction soln is blackish green, and produces some flocculent substances;
Move into ageing 4h in the water-bath of 90 DEG C after the above-mentioned reaction soln of abundant stirring, obtain black Fe
3o
4gelating soln.Above-mentioned gelating soln is placed in magnetic field, carries out Fe
3o
4magnetic-particle is separated with solution; Washed with de-ionized water is adopted to be separated the Fe obtained
3o
4magnetic-particle; By cleaned Fe
3o
4particle vacuum-drying goes out the Fe of black
3o
4particle powder.
C. magnetic Nano microsphere is prepared: the Fe upper step finally obtained with polyoxyethylene glycol
3o
4particle carries out surface treatment, then by the Fe after process
3o
4granular composite, in 100mL dehydrated alcohol, forms magnetic fluid; Magnetic fluid to be poured in there-necked flask and to add sodium lauryl sulphate 0.3g, 10mL vinylbenzene, stirring 1h; Then at N
2add and Fe under environment
3o
4the mass ratio of particle is the Poly(D,L-lactide-co-glycolide of 1: 1 and the Vinylstyrene of 1mL, heating in water bath to 60 DEG C reaction 1h; Be warming up to 75 DEG C, under alkaline condition (controlling to pH=10 by 0.1mol/L NaOH solution), carry out suspension polymerization 10h; And then ageing at 90 DEG C, use ethanol and water washing after ageing, finally carry out Magneto separate, isolate magnetic Nano microsphere;
D. adopt absorption method that the magnetic Nano microsphere of preparation and the molecular beacon of synthesis, miR-221 specific inhibitor are formed magnetic nanocomposites.
The using method of magnetic nanocomposites of the present invention: the miR-221 molecular beacon preparing 250nmol/L in 100 μ L PBS, adds 3 μ l liposomes, in 37 DEG C of cell culture incubators, jointly hatches 20min; Making the section of human body lung carcinoma cell is directly added in lung carcinoma cell alive by the PBS containing molecular beacon and liposome, and hatch 60min in 37 DEG C of cell culture incubators, then after redying 10min with the DAPI of 5mg/L, apply the expression of confocal laser scanning microscope lung carcinoma cell miR-221 alive.
Visible stronger green fluorescence in focusing results visible A549, SPC-A1 lung carcinoma cell altogether, mainly be positioned at endochylema, a small amount of punctate fluorescence signal that karyon is faint as seen, background is very low, and after adding miR-221 specific inhibitor, in cell, green florescent signal obviously weakens.The result that the miR-221 of molecular beacons detection expresses is verified with Real-time PCR.Focusing results prompting cancerous lung tissue all can see the green fluorescence that intensity does not wait altogether, and be mainly seen in tumour cell endochylema, also show the fluorescent signal of a small amount of point-like in core, background is very low, and after adding miR-221 specific inhibitor, in tissue, fluorescent signal obviously weakens.By the detected result of biological cells and tissues miR-221 expression level, feasibility and the practicality of the molecular beacon that the present invention designs is described.Mixture DNase I Protection and cell protein hatch biologically stable and the release effects of experimental observation mixture, and magnetic nanocomposites real time imagery A549 and SPC-A1 lung carcinoma cell, observe imaging results with laser co-focusing.Under living imaging system visible intravenous injection miR-221 specific antagonists magnetic nanocomposites after lung's fluorescence intensity do not add miR-221 antagonist group fluorescence intensity and obviously weaken, this can further demonstrate that magnetic nanocomposites is used for the treatment of feasibility and the practicality of lung cancer.
What in above-mentioned magnetic nanocomposites preparation method, b step adopted is that sol-gel method prepares magnetic Nano Fe
3o
4particle, its advantage can ensure strictly to control stoichiometric ratio, easily realizes High Purity; Technique is simple, and reaction time is short, and temperature of reaction is low; Particle size is little, is evenly distributed, good dispersity.In addition to the Fe that b step generates
3o
4the Fe that particle powder can generate with tem observation
3o
4the pattern of nano particle; The structure of its crystal is measured with electron diffraction (ED) and XRD; Its magnetic is measured with VSM; Measure qualified after, then carry out step c.Finally isolate magnetic Nano microsphere to step c, available x-ray instrument observes Fe in magnetic-particle
3o
4whether the crystalline form of magnetic core is complete, and rough calculation Fe
3o
4particle diameter. use transmission electron microscope observing Fe
3o
4the structure of magnetic core and magnetic-particle and pattern, form with the functional group of determination of infrared spectroscopy magnetic-particle, measures the magnetic saturation intensity of magnetic-particle with vibrating sample magnetometer." absorption method " described in Step d refers to that oligonucleotide probe or target molecule are connected by secondary key (such as ionic linkage) with solid-phase matrix surface and fix, or with non covalent bond effect by few core former times acid probe or target molecule directly or constant potential be adsorbed onto the surface of solid-phase matrix, or pass through electrostatic interaction by the phosphate radical negative ion in probe of the present invention with the decorative layer of solid-phase matrix surface band positive charge and fix.
The present invention's advantage is compared with prior art: magnetic nanocomposites of the present invention can resist the Degradation of nucleic acid in living cell enzyme, can suppress the expression of miR-221 in lung cancer, thus realizes the treatment to lung cancer.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail:
For a magnetic nanocomposites for lung cancer therapy, comprise magnetic Nano microsphere and be adsorbed on the molecular beacon on magnetic Nano microsphere, its sequence of described molecular beacon is
5 '-Aleax488-CCAGCG-gaaacc+Cagc+Aga+Caa+Tgtagct-CGCTGG-BHQ1-3 ', wherein CCAGCG is 5 ' end bulbous sequence, CGCTGG is 3 ' end bulbous sequence, luminophore is Alexa488, extracting the group that goes out is BHQ1, gaaacc+Cagc+Aga+Caa+Tgtagct is ring-shaped sequence, is wherein carry out the base after locking nucleic acid moditied processing with capitalizing the base that represents in ring-shaped sequence; Described magnetic Nano microsphere comprises magnetic nanoparticle and is wrapping by the Poly(D,L-lactide-co-glycolide on magnetic nanoparticle surface.
Sequence information according to ripe microRNA designs molecular beacon, the specific design of molecular beacon is carried out according to the principle of design of website (www.molecular-beacons.org) Middle molecule beacon, and cycling probe principles of structural design is: length is between 15 ~ 30 bases; Can complementary pairing with target miRNA.Bulbous sequential structure principle of design: the length of probe sequence is 5 ~ 7 bases; GC content one between 70 ~ 80%; Melt chain temperature and want 7 ~ 10 DEG C than detected temperatures; G base can not connect luminophore.Luminophore and extraction are gone out the selection of group: luminophore can be extracted accordingly the group that goes out extract and go out, two groups have a high signal to background ratio (more than 2.0).Be the Degradation resisting nucleic acid in living cell enzyme according to above principle the present invention, the present invention carries out lock nucleic acid to the number of base in the molecular beacon designed and modifies.Lock nucleic acid (Locked nucleic acid, LNA) be a kind of novel nucleotide derivative, 2 '-O of β-D-RIBOSE in its structure, 4 '-C position forms the condensation structure of this rigidity of Oxymethylene bridge of annular by shrink effect, change geometry and the tridimensional character of Nucleotide, reduce the snappiness of ribose structure, thus considerably improve its stability, the antienzyme that this conformation not only significantly improves nucleic acid cuts ability, and improves cross compatibility.The base number that LNA modifies should more than 6, otherwise slower in conjunction with speed with miRNA.4 bases in people miR-221 molecular beacon have been carried out LNA modification by the present invention, and namely the capitalization in above-mentioned molecular beacon ring-shaped sequence has done LNA modification.
In order to improve sensitivity and the stability of molecular beacon detection by quantitative further, magnetic Nano microsphere is adopted to carry out Isolation and purification molecular beacon of the present invention.The principle that magnetic Nano microsphere extracts DNA is: the macromolecular material (as Poly(D,L-lactide-co-glycolide) being rich in positive charge chemical group at magnetic nanoparticle pan coating, is prepared into positively charged magnetic Nano microsphere.Magnetic Nano microsphere is joined in DNA extraction liquid, and in the damping fluid of slant acidity (PH6.5), the DNA in solution can be adsorbed on positively charged Nano microsphere.Adopt magnetic Nano microsphere as the carrier of molecular beacon, extract corresponding DNA, the much impurity affecting pcr amplification can be removed, improve the stability of pcr amplification.In addition, a certain amount of magnetic Nano microsphere can adsorb a certain amount of DNA, contributes to the template DNA obtaining relative constant, improves the accuracy that fluorescent molecular bacon probe quantitative detects.
Magnetic nanoparticle (MNPs) is formed centered by magneticsubstance, can wrap by the nucleocapsid structure of biomolecules, not only possesses good magnetic conductance tropism, also there is good biocompatibility, can be combined with several functions molecule, be used widely in viable cell and biological tissue imaging.But not modified nano particle has metal toxicity to viable cell, therefore finishing to be carried out to nano particle.
Further, described magnetic nanoparticle is Fe
3o
4magnetic nanoparticle.
Fe
3o
4magnetic nanoparticle is convenient to nucleus magnetic resonance (MRI) video picture, and molecular beacon is convenient to fluorescent microscope video picture, and this nanotechnology has dual image displaying function.
Further, described magnetic nanocomposites is the magnetic nanocomposites of surface through polyoxyethylene glycol process.
Nano particle after polyoxyethylene glycol (PEG) modification has good long circulating stability, can escape the system of engulfing and engulf, and can improve biocompatibility in body and external.
For a preparation method for the magnetic nanocomposites of lung cancer therapy, comprise the following steps:
A. design needs the molecular beacon of synthesis: determine that people miR-221 sequence is 5 '-AGCTACATTGTCTGCTGGGTTTC-3 ', the molecular beacon ring-shaped sequence of designer miR-221 is GAAACCCAGCAGACAATGTAGCT, luminophore is Alexa488, and extracting the group that goes out is BHQ1; The base of carrying out LNA modification is needed in selected 4 ring-shaped sequences, then design its sequence of molecular beacon and be defined as 5 '-Aleax488-CCAGCG-gaaacc+Cagc+Aga+Caa+Tgtagct-CGCTGG-BHQ1-3 ', be wherein namely defined as by the base that capitalization represents the base will carrying out lock nucleic acid moditied processing in ring-shaped sequence;
Protocols in Molecular Biology (shearing enzyme, polysaccharase etc. as used DNA) can being adopted to synthesize according to the above-mentioned molecular beacon sequences determined, chemosynthesis mode also can be adopted on DNA synthesizer to synthesize; ;
B. magnetic Nano Fe is prepared
3o
4particle: the KOH solution of 20mL is poured in flask and stirs, then the KNO solution of 20mL is added wherein, after fully stirring, the FeSO4 solution of 10mL is added, until reaction soln is blackish green, and produces some flocculent substances;
Move into ageing 4h in the water-bath of 90 DEG C after the above-mentioned reaction soln of abundant stirring, obtain black Fe
3o
4gelating soln.Above-mentioned gelating soln is placed in magnetic field, carries out Fe
3o
4magnetic-particle is separated with solution; Washed with de-ionized water is adopted to be separated the Fe obtained
3o
4magnetic-particle; By cleaned Fe
3o
4particle vacuum-drying goes out the Fe of black
3o
4particle powder.
C. magnetic Nano microsphere is prepared: the Fe upper step finally obtained with polyoxyethylene glycol
3o
4particle carries out surface treatment, then by the Fe after process
3o
4granular composite, in 100mL dehydrated alcohol, forms magnetic fluid; Magnetic fluid to be poured in there-necked flask and to add sodium lauryl sulphate 0.3g, 10mL vinylbenzene, stirring 1h; Then at N
2add and Fe under environment
3o
4the mass ratio of particle is the Poly(D,L-lactide-co-glycolide of 1: 1 and the Vinylstyrene of 1mL, heating in water bath to 60 DEG C reaction 1h; Be warming up to 75 DEG C, under alkaline condition (controlling to pH=10 by 0.1mol/L NaOH solution), carry out suspension polymerization 10h; And then ageing at 90 DEG C, use ethanol and water washing after ageing, finally carry out Magneto separate, isolate magnetic Nano microsphere;
D. adopt absorption method that the magnetic Nano microsphere of preparation and the molecular beacon of synthesis, miR-221 specific inhibitor are formed magnetic nanocomposites.
The using method of magnetic nanocomposites of the present invention: the miR-221 molecular beacon preparing 250nmol/L in 100 μ L PBS, adds 3 μ l liposomes, in 37 DEG C of cell culture incubators, jointly hatches 20min; Making the section of human body lung carcinoma cell is directly added in lung carcinoma cell alive by the PBS containing molecular beacon and liposome, and hatch 60min in 37 DEG C of cell culture incubators, then after redying 10min with the DAPI of 5mg/L, apply the expression of confocal laser scanning microscope lung carcinoma cell miR-221 alive.
Visible stronger green fluorescence in focusing results visible A549, SPC-A1 lung carcinoma cell altogether, be mainly positioned at endochylema, a small amount of punctate fluorescence signal that karyon is faint as seen, background is very low.The result that the miR-221 of molecular beacons detection expresses is verified with Real-time PCR.Focusing results prompting cancerous lung tissue all can see the green fluorescence that intensity does not wait altogether, and be mainly seen in tumour cell endochylema, also show the fluorescent signal of a small amount of point-like in core, background is very low.By the detected result of biological cells and tissues miR-221 expression level, feasibility and the practicality of the molecular beacon that the present invention designs is described.Mixture DNase I Protection and cell protein hatch biologically stable and the release effects of experimental observation mixture, and magnetic nanocomposites real time imagery A549 and SPC-A1 lung carcinoma cell, observe imaging results with laser co-focusing.This can further demonstrate that feasibility and the practicality of miR-221 molecular beacon.
What in above-mentioned magnetic nanocomposites preparation method, b step adopted is that sol-gel method prepares magnetic Nano Fe
3o
4particle, its advantage can ensure strictly to control stoichiometric ratio, easily realizes High Purity; Technique is simple, and reaction time is short, and temperature of reaction is low; Particle size is little, is evenly distributed, good dispersity.In addition to the Fe that b step generates
3o
4the Fe that particle powder can generate with tem observation
3o
4the pattern of nano particle; The structure of its crystal is measured with electron diffraction (ED) and XRD; Its magnetic is measured with VSM; Measure qualified after, then carry out step c.Finally isolate magnetic Nano microsphere to step c, available x-ray instrument observes Fe in magnetic-particle
3o
4whether the crystalline form of magnetic core is complete, and rough calculation Fe
3o
4particle diameter. use transmission electron microscope observing Fe
3o
4the structure of magnetic core and magnetic-particle and pattern, form with the functional group of determination of infrared spectroscopy magnetic-particle, measures the magnetic saturation intensity of magnetic-particle with vibrating sample magnetometer." absorption method " described in Step d refers to that oligonucleotide probe or target molecule are connected by secondary key (such as ionic linkage) with solid-phase matrix surface and fix, or with non covalent bond effect by few core former times acid probe or target molecule directly or constant potential be adsorbed onto the surface of solid-phase matrix, or pass through electrostatic interaction by the phosphate radical negative ion in probe of the present invention with the decorative layer of solid-phase matrix surface band positive charge and fix.
The foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every utilize specification sheets of the present invention to do equivalent structure or the conversion of equivalent flow process, or be directly or indirectly used in other relevant technical fields, be all in like manner included in scope of patent protection of the present invention.
Claims (4)
1. the magnetic nanocomposites for lung cancer therapy, it is characterized in that: comprise magnetic Nano microsphere and be adsorbed on the molecular beacon on magnetic Nano microsphere, its sequence of described molecular beacon is 5 '-Aleax488-CCAGCG-gaaacc+Cagc+Aga+Caa+Tgtagct-CGCTGG-BHQ1-3 ', wherein CCAGCG is 5 ' end bulbous sequence, CGCTGG is 3 ' end bulbous sequence, luminophore is Aleax488, extracting the group that goes out is BHQ1, gaaacc+Cagc+Aga+Caa+Tgtagct is ring-shaped sequence, wherein carry out the base after locking nucleic acid moditied processing with capitalizing the base that represents in ring-shaped sequence, described magnetic Nano microsphere comprises magnetic nanoparticle and is wrapping by the Poly(D,L-lactide-co-glycolide on magnetic nanoparticle surface.
2. a kind of magnetic nanocomposites for lung cancer therapy according to claim 1, is characterized in that: described magnetic nanoparticle is Fe
3o
4magnetic nanoparticle.
3. a kind of magnetic nanocomposites for lung cancer therapy according to claim 1, is characterized in that: described magnetic nanocomposites is the magnetic nanocomposites of surface through polyoxyethylene glycol process.
4. for a preparation method for the magnetic nanocomposites of lung cancer therapy, it is characterized in that: comprise the following steps:
A. design needs the molecular beacon of synthesis: determine that people miR-221 sequence is 5 '-AGCTACATTGTCTGCTGGGTTTC-3 ', the molecular beacon ring-shaped sequence of designer miR-221 is GAAACCCAGCAGACAATGTAGCT, luminophore is Alexa488, and extracting the group that goes out is BHQ1; The base of carrying out LNA modification is needed in selected 4 ring-shaped sequences, then design its sequence of molecular beacon and be defined as 5 '-Aleax488-CCAGCG-gaaacc+Cagc+Aga+Caa+Tgtagct-CGCTGG-BHQ1-3 ', be wherein namely defined as by the base that capitalization represents the base will carrying out lock nucleic acid moditied processing in ring-shaped sequence;
Protocols in Molecular Biology (shearing enzyme, polysaccharase etc. as used DNA) can being adopted to synthesize according to the above-mentioned molecular beacon sequences determined, chemosynthesis mode also can be adopted on DNA synthesizer to synthesize;
B. magnetic Nano Fe is prepared
3o
4particle: the KOH solution of 20mL is poured in flask and stirs, then the KNO solution of 20mL is added wherein, after fully stirring, the FeSO4 solution of 10mL is added, until reaction soln is blackish green, and produces some flocculent substances;
Move into ageing 4h in the water-bath of 90 DEG C after the above-mentioned reaction soln of abundant stirring, obtain black Fe
3o
4gelating soln.Above-mentioned gelating soln is placed in magnetic field, carries out Fe
3o
4magnetic-particle is separated with solution; Washed with de-ionized water is adopted to be separated the Fe obtained
3o
4magnetic-particle; By cleaned Fe
3o
4particle vacuum-drying goes out the Fe of black
3o
4particle powder.
C. magnetic Nano microsphere is prepared: the Fe upper step finally obtained with polyoxyethylene glycol
3o
4particle carries out surface treatment, then by the Fe after process
3o
4granular composite, in 100mL dehydrated alcohol, forms magnetic fluid; Magnetic fluid to be poured in there-necked flask and to add sodium lauryl sulphate 0.3g, 10mL vinylbenzene, stirring 1h; Then at N
2add and Fe under environment
3o
4the mass ratio of particle is the Poly(D,L-lactide-co-glycolide of 1: 1 and the Vinylstyrene of 1mL, heating in water bath to 60 DEG C reaction 1h; Be warming up to 75 DEG C, carry out suspension polymerization 10h in the basic conditions; And then ageing at 90 DEG C, use ethanol and water washing after ageing, finally carry out Magneto separate, isolate magnetic Nano microsphere;
D. adopt absorption method that the magnetic Nano microsphere of preparation and the molecular beacon of synthesis, miR-221 specific inhibitor are formed magnetic nanocomposites.
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