CN104174035B - Preparation method for the chitosan molecule beacon nano-complex that the targeting octreotide of pulmonary cancer diagnosis is modified - Google Patents
Preparation method for the chitosan molecule beacon nano-complex that the targeting octreotide of pulmonary cancer diagnosis is modified Download PDFInfo
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Abstract
Chitosan (CS) molecular beacon (MB) nano-complex that targeting somatostatin analogue octreotide (OCT) for pulmonary cancer diagnosis of the present invention is modified, by synthesizing chitosan molecule beacon nano-complex with self-assembly method, then chitosan miR 155 (microRNA 155) the molecular beacon nano-complex modified with glutaraldehyde as cross linker synthesis targeting octreotide by chemical reaction, the chitosan molecule beacon nano-complex that this targeting octreotide is modified can be according to the generation of fluorescence intensity dynamic monitoring pulmonary carcinoma, evolution, can be applicable to lung carcinoma cell and pulmonary carcinoma mice with tumor living animal imaging, there is targeting strong, transfection efficiency is high, specificity is high, good stability, the features such as background is low, new approaches are provided by the early diagnosis for pulmonary carcinoma, new method and new technique.
Description
Technical field
The present invention relates to biomedicine technical field, especially relate to a kind of targeting octreotide for pulmonary cancer diagnosis and repair
The preparation method of the chitosan molecule beacon nano-complex of decorations.
Background technology
Pulmonary carcinoma is the malignant tumor of serious threat human health, and its M & M rises year by year, early diagnosis
And early treatment is the key improving patient's prognosis, but patients with lung cancer is at pathological changes the most often non-evident sympton, goes to a doctor
Time be mostly middle and advanced stage, lose the best opportunity for the treatment of.Therefore " origin " of occurring from pulmonary carcinoma sets about, and seeks
Efficiently, the new method of special early diagnosis, it will to the prevention of pulmonary carcinoma, diagnose and treat generation far-reaching influence.
Tumor stem cell theory is thought and be there is the tumor cell that a group quantity is few in tumor tissue cell, is referred to as swollen
Tumor stem cell, all of tumor cell all is broken up to come by tumor stem cell.Microrna (miRNA, microRNA)
Playing a significant role in the generation evolution of tumor, the miRNAs that detection NSCLC stem cell is relevant is pulmonary carcinoma morning
One Critical policies of phase diagnosis.Research shows that the miRNAs of common different tumor type expression characteristicses expresses
Spectrum, this will be for the tissue-derived of tumor and diagnosis offer foundation, and distinctive miRNA express spectra can be as tumor
Early diagnosis molecular marker, it is possible to judge the different prognosis of tumor, predicted treatment effect.Multinomial research shows
MiR-155 is high expressed in NSCLC, detection lung tissue in miR-155 expression can distinguish patients with lung cancer with
Non-lung cancer patient, and detect the miR-155 expression in tumor tissues and can predict the tumor recurrence and not of patient
Good prognostic risk, in pulmonary carcinoma stem cell, miR-155 expresses higher than common lung carcinoma cell.If able to directly carry glimmering
Stimulative substance or chemotherapeutics target lung carcinoma cell even pulmonary carcinoma stem cell, will be expected to be finally reached early diagnosis and radical cure
The purpose of pulmonary carcinoma.Molecular beacon (molecular beacon, MB) is based on FRET (fluorescence resonance energy transfer)
A kind of high sensitivity of (fluorescence resonance energy transfer, FRET) principle design and height
Specific detection instrument, a kind of double labelling oligonucleotide probe in loop-stem structure, the nucleic acid sequence at two ends
Row complementary pairing.During uncombined object chain, it is positioned at the fluorophor of its end and quenching group immediately adjacent to, fluorescence
It is quenched；When molecular beacon is combined with object chain, the space conformation of molecular beacon changes, and fluorophor is remote
From quencher, fluorescence signal recovers, and therefore molecular beacon is that a kind of specific detection DNA and RNA target are to sequence
Hair clip type nucleic probe, visual research in nucleic acid-nucleic acid and living cells can be used for.
Common molecular beacon itself is easily degraded by nuclease or core associated proteins, and existence and stability is poor, cellular uptake is tired
The shortcomings such as difficulty.And with lock nucleic acid (1ocked nucleic acid, LNA) modified base, due to geometry and three-dimensional
Matter changes, and it is energy stable existence in the environment of higher temperature, and the ability of nuclease-resistant degraded is obviously enhanced, and has
The hybridization affinity powerful with DNA/RNA.Utilize molecular beacon it is crucial that how to obtain preferable transport vehicle,
Both can effectively protect against being degraded, and efficiently molecular beacon can be delivered again and play targets identification into host cell
The function of nucleotide sequence.Chitosan (chitosan, CS) nanometer is natural cationic polysaccharide, external and internal reality
Testing the safety and efficacy all confirming chitosan as transfer vector, its main feature has: 1. histocompatibility
Good, immunogenicity is low, toxicity is little；2. lotus positive electricity, easy and DNA molecular self assembly is combined into nano-complex, shape
Become stable nucleocapsid structure, and effect protected to DNA；3. manufacturing process is simple, gentle, to DNA, antibody
Little Deng the impact of biomacromolecule physicochemical property；4. the active amine groups that chitosan nano surface is intensive is easy to macromole
Modify and couple.
At present, polypeptide receptoroid has become the focus of research as the molecular target of diagnosis of malignant tumor and treatment, its
Middle somatostatin receptor (SSTR) is exactly Typical Representative therein, and many tumors and metastasis tissue thereof have SSTR
Expression, compared with normal tissue substantially increases, mostly to express SSTR2, such as nonsmall-cell lung cancer, minicell
Pulmonary carcinoma, endocrine pancreas cancer, pituitary tumor, lymphoma, meningioma and colon, rectum, mammary gland and prostatitis adenoncus
Tumor etc..The SSTR analog (SSA) of synthetic is alternative after modifying at present strengthens and extends its biology
Activity, long half time, powerful, as octreotide (octreotide, OCT), Lanreotide (lanreotide),
Vapreotide (vapreotide) etc..SSA is possible not only to specific binding with the SSTR of expression of tumor tissue in vivo,
And can also be combined by the SSTR on tumor vascular endothelial cell, and then reach the mesh of dual-target tumor tissues
's.
In the prior art, study the active targeting group representing octreotide (OCT) as drug delivery system,
Antitumor drug can be delivered to tumor locus；Also have been reported that octreotide radioisotope labeling thing is ratified by FDA
Diagnostic reagent as tumor.But the complex preparation methoies such as molecular beacon of the prior art or octreotide are complicated,
Process conditions are harsh, and these complex are only used in diagnosing, late period, and this can make patients with lung cancer miss
Golden hour, affect the treatment of patients with lung cancer, simultaneously these complex for diagnosing targeting,
At least on the one hand poor effect in the aspect such as specificity or stability, is therefore badly in need of a kind of preparation method easy and can
Targeting is strong, specificity is high, the reagent of the diagnosing of the New methods in working of good stability, especially in acquisition
In early days can be with the excellent reagent of diagnosing.
Summary of the invention
It is an object of the invention to provide a kind of chitosan molecule beacon for the targeting octreotide modification of pulmonary cancer diagnosis to receive
The preparation method of rice complex, solve in prior art for the medicine of pulmonary cancer diagnosis or the preparation method of reagent is complicated,
Process conditions are harsh and its targeting, specificity or stability are the best and in can only diagnosing, the problem of advanced lung cancer.
The present invention solves technical problem and be the technical scheme is that a kind of targeting Austria for pulmonary cancer diagnosis of offer is bent
The preparation method of the chitosan molecule beacon nano-complex that peptide is modified, comprises the steps:
A, by chitosan nano and molecular beacon according to the ratio of mass ratio 7:1～10:1 pH6.0-7.0's
PBS mixes, and whirlpool concussion 40s～120s, at room temperature stand 40min～120min, obtain shell
Glycan molecule beacon nano-complex；
B, in chitosan molecule beacon nano-complex drip 0.5%～2.0% glutaraldehyde solution, described penta 2
Aldehyde solution is 900:1～1300:1 with the mass ratio of described chitosan molecule beacon nano-complex, by first
Secondary stirring mixing carries out reacting 1h～2h, and mixing speed is 100rpm～300rpm for the first time, passes through trace after reaction
Dialysis cartridge dialysis 0.5h～2h for the first time removes unconjugated glutaraldehyde；Then according to octreotide divides with described chitosan
The ratio that mass ratio is 120:1～80:1 of sub-beacon nano-complex adds octreotide, carries out second time and stirs
Hybrid reaction 3h～6h, mixing speed is 100rpm～300rpm for the second time, by microdialysis post second after reaction
Secondary dialysis 11h～15h removes unconjugated octreotide, obtains the shell for the targeting octreotide of pulmonary cancer diagnosis is modified and gathers
Glycan molecule beacon nano-complex.
In the preparation process in accordance with the present invention, in step, the particle diameter≤300nm of described chitosan nano.
In the preparation process in accordance with the present invention, in step, described Nano chitosan and the quality of described molecular beacon
Ratio is 7:1.
In the preparation process in accordance with the present invention, in step, the pH of described PBS is 6.0.
In the preparation process in accordance with the present invention, in step, the whirlpool concussion time is 60s.
In the preparation process in accordance with the present invention, in stepb, the concentration of described glutaraldehyde solution is 1.0%, described penta
Dialdehyde solution is 1000:1 with the mass ratio of described chitosan molecule beacon nano-complex.
In the preparation process in accordance with the present invention, in stepb, described octreotide and described chitosan molecule beacon nanometer
The mass ratio of complex is 100:1.
In the preparation process in accordance with the present invention, in stepb, the stirring hybrid reaction time is 1.5h for the first time, first
Secondary mixing speed is 150rpm.
In the preparation process in accordance with the present invention, in stepb, the second time stirring hybrid reaction time is 4h, for the first time
Mixing speed is 150rpm.
In the preparation process in accordance with the present invention, in stepb, dialysis time is 1h for the first time；Dialysis time for the second time
For 12h.
Implement the chitosan molecule beacon nano-complex of the targeting octreotide modification for pulmonary cancer diagnosis of the present invention
Preparation method, has the advantages that the target polypeptide octreotide (OCT) for pulmonary cancer diagnosis of the present invention is repaiied
Chitosan (CS) molecular beacon (MB) nano-complex of decorations, by believing by self-assembly method synthesis chitosan molecule
Mark nano-complex, the chitosan then modified with glutaraldehyde as cross linker synthesis targeting octreotide by chemical reaction
MiR-155 (microRNA-155) molecular beacon nano-complex, this preparation method is simple and easy to do and facilitates industrialization
Producing, the chitosan molecule beacon nano-complex that obtained targeting octreotide is modified can move according to fluorescence intensity
State the monitoring generation of pulmonary carcinoma, evolution, can be applicable to lung carcinoma cell and pulmonary carcinoma mice with tumor living animal imaging, tool
There are the features such as targeting is strong, transfection efficiency is high, specificity is high, good stability, background are low, by the diagnosis for pulmonary carcinoma
There is provided New methods in working and new technique, in particular for the early diagnosis of pulmonary carcinoma；Additionally research at present is about carefully
Born of the same parents and living animal imaging aspect, mainly study in terms of albumen, and this research to be the gene utilizing high expressed enter
Row imaging, both at home and abroad report almost without, belong to pioneering.
Accompanying drawing explanation
Fig. 1 is liquid phase molecule hybrid experiment checking the design susceptiveness of molecular beacon, specificity experiments in embodiment 8
Result figure；
Fig. 2 is that in embodiment 9, different quality divides than the gel blocking of the chitosan molecule beacon nano-complex of synthesis
Analysis figure；
Fig. 3 is that in embodiment 10, different quality is tested than the envelop rate of the chitosan molecule beacon nano-complex of synthesis
Result figure；
Fig. 4 is that in embodiment 11, different quality is more real than the fluorescence shielding of the chitosan molecule beacon nano-complex of synthesis
Test result figure；
Fig. 5 is the zeta potentiometric analysis figure of CS and CS-MB in embodiment 12；
Fig. 6 is the DNase I experimental analysis figure of CS and CS-MB in embodiment 12；
Fig. 7 is that embodiment 13 uses laser co-focusing commercialization siPORT lipofectamine detection pulmonary carcinoma thin
The expression experimental result of miR-155 in born of the same parents, pulmonary carcinoma stem cell (LCSL) and negative control vero, CT-26 cell
Figure, CS-RSMB group is negative control group；
Fig. 8 is that embodiment 14 uses laser co-focusing chitosan molecule beacon nano-complex (CS-MB) to detect lung
The expression experimental result picture of miR-155 in cancerous cell and pulmonary carcinoma stem cell LCSL, CS-RSMB group is negative control group；
Fig. 9 is that embodiment 15 uses laser co-focusing immunofluorescence technique detection lung carcinoma cell and transplanted tumor cancerous lung tissue
The expression experimental result picture of middle SSTR2；
Figure 10 is the chitosan molecule beacon nano-complex that embodiment 16 uses laser co-focusing octreotide to modify
(CS-MB-OCT-FITC) the expression experimental result picture of miR-155, CS-RSMB-OCT-FITC in detection lung carcinoma cell
Group is negative control group；
Figure 11 is in embodiment 17 animal level, miR-155 in bioluminescence imaging technology detection subcutaneous transplantation tumor tissue
The experimental result picture of expression, CS-RSMB is negative control group；
Figure 12 is the experimental result picture that in embodiment 17, frozen section laser co-focusing is observed, and CS-RSMB is negative
Matched group；
Figure 13 be in embodiment 18 animal level tail vein injection CS-MB and CS-MB-OCT nano-complex and then
Detection molecules beacon nano-complex to the experimental result picture of the identification of lung carcinoma cell, diagnostic effect in lung tissue,
CS-RSMB is negative control group；
Figure 14 is to observe, with frozen section laser co-focusing, the experimental result picture carrying out verifying in embodiment 18,
CS-RSMB is negative control group.
Detailed description of the invention
Below in conjunction with the accompanying drawings and embodiment, the chitosan modified the present invention for the targeting octreotide of pulmonary cancer diagnosis divides
The implementing of preparation method of sub-beacon nano-complex is described further:
The present invention relates to one be combined with molecular beacon by chitosan nano, targeting octreotide is modified, by identifying lung
In cancerous cell or pulmonary carcinoma stem cell, the expression of micro-RNA identifies cancerous cell, is realized tumor by fluorescence imaging
The identification function of cell even tumor stem cell, reaches the purpose of pulmonary cancer diagnosis.The one provided for this present invention is used
In the preparation method of the chitosan molecule beacon nano-complex that the targeting octreotide of pulmonary cancer diagnosis is modified, it is specifically real
Under executing such as, but present disclosure is not limited solely to following example.
Embodiment 1-7 is the chitosan molecule beacon nano-complex of the targeting octreotide modification for pulmonary cancer diagnosis
Preparation.As long as wherein chitosan nano be Nano grade all within protection scope of the present invention, be preferably≤300nm,
More preferably≤200nm, chitosan molecule beacon nano-complex prepared by such chitosan nano be more beneficial for into
One step prepares the chitosan molecule beacon nano-complex that the targeting octreotide that stability is high, targeting is strong is modified.
Embodiment 1:
Chitosan nano is mixed according to the ratio of mass ratio 7:1 with molecular beacon in the PBS of pH6.0
Close, and whirlpool concussion 60s, at room temperature stand 60min, obtain chitosan molecule beacon nano-complex；At shell
Glycan molecule beacon nano-complex drips the glutaraldehyde solution of 1.0%, glutaraldehyde solution and chitosan molecule beacon
The mass ratio of nano-complex is 1000:1, carries out reacting 1.5h by stirring mixing for the first time, for the first time stirring
Speed is 150rpm, removes unconjugated glutaraldehyde by microdialysis post dialysis 1h for the first time after reaction；Then
Add octreotide according to the ratio that mass ratio is 100:1 of octreotide with chitosan molecule beacon nano-complex, enter
Row second time stirring hybrid reaction 4h, mixing speed is 150rpm for the second time, by microdialysis post second after reaction
Secondary dialysis 12h removes unconjugated octreotide, obtains the chitosan that the targeting octreotide of particle diameter 45nm～55nm is modified
Molecular beacon nano-complex.
Embodiment 2: difference from Example 1 is, chitosan nano and molecular beacon are 10:1 according to mass ratio,
Finally give the chitosan molecule beacon nano-complex that the targeting octreotide of particle diameter 40nm～55nm is modified.
Embodiment 3: difference from Example 1 is, chitosan nano and molecular beacon are 8:1 according to mass ratio,
Finally give the chitosan molecule beacon nano-complex that the targeting octreotide of particle diameter 45nm～60nm is modified.
Embodiment 4: difference from Example 1 is, chitosan nano and molecular beacon are 9:1 according to mass ratio,
Finally give the chitosan molecule beacon nano-complex that the targeting octreotide of particle diameter 40nm～60nm is modified.
Embodiment 5:
Chitosan nano is mixed according to the ratio of mass ratio 9:1 with molecular beacon in the PBS of pH6.5
Close, and whirlpool concussion 40s, at room temperature stand 100min, obtain chitosan molecule beacon nano-complex；?
Dripping the glutaraldehyde solution of 0.5% in chitosan molecule beacon nano-complex, glutaraldehyde solution is believed with chitosan molecule
The mass ratio of mark nano-complex is 1300:1, carries out reacting 2h by stirring mixing for the first time, for the first time stirring
Speed is 100rpm, removes unconjugated glutaraldehyde by microdialysis post dialysis 1.5h for the first time after reaction；So
After add according to the ratio that mass ratio is 80:1 of octreotide and described chitosan molecule beacon nano-complex difficult to understand bent
Peptide, carries out second time and stirs hybrid reaction 3h, and mixing speed is 100rpm for the second time, passes through microdialysis after reaction
Post second time dialysis 11h removes unconjugated octreotide, obtains the targeting octreotide modification of particle diameter 30nm～50nm
Chitosan molecule beacon nano-complex.
Embodiment 6:
Chitosan nano is mixed according to the ratio of mass ratio 8:1 with molecular beacon in the PBS of pH7.0
Close, and whirlpool concussion 120s, at room temperature stand 120min, obtain chitosan molecule beacon nano-complex；?
Dripping the glutaraldehyde solution of 2.0% in chitosan molecule beacon nano-complex, glutaraldehyde solution is believed with chitosan molecule
The mass ratio of mark nano-complex is 900:1, carries out reacting 1h by stirring mixing for the first time, stirring speed for the first time
Degree is 300rpm, removes unconjugated glutaraldehyde by microdialysis post dialysis 2h for the first time after reaction；Then press
The ratio that mass ratio is 120:1 according to octreotide with described chitosan molecule beacon nano-complex adds octreotide,
Carrying out second time and stir hybrid reaction 5h, mixing speed is 250rpm for the second time, by microdialysis post the after reaction
Secondary dialysis 15h removes unconjugated octreotide, and the shell of the targeting octreotide modification obtaining particle diameter 45nm～65nm gathers
Glycan molecule beacon nano-complex.
Embodiment 7:
Chitosan nano is mixed according to the ratio of mass ratio 10:1 with molecular beacon in the PBS of pH6.0
Close, and whirlpool concussion 120s, at room temperature stand 40min, obtain chitosan molecule beacon nano-complex；?
Dripping the glutaraldehyde solution of 1.5% in chitosan molecule beacon nano-complex, glutaraldehyde solution is believed with chitosan molecule
The mass ratio of mark nano-complex is 1200:1, carries out reacting 1.8h by stirring mixing for the first time, stirs for the first time
Mixing speed is 200rpm, removes unconjugated glutaraldehyde by microdialysis post dialysis 0.5h for the first time after reaction；
Then according to octreotide adds difficult to understand with the ratio that mass ratio is 110:1 of described chitosan molecule beacon nano-complex
Bent peptide, carries out second time and stirs hybrid reaction 6h, and mixing speed is 200rpm for the second time, saturating by trace after reaction
Analysis post second time dialysis 14h removes unconjugated octreotide, and the targeting octreotide obtaining particle diameter 50nm～70nm is modified
Chitosan molecule beacon nano-complex.
Embodiment 8-18 is that the chitosan molecule beacon nano-complex of targeting octreotide modification is for diagnosing
Related experiment.
Embodiment 8: liquid phase molecule hybrid experiment
The correctness of liquid phase molecule hybrid experiment checking design molecular beacon sequences and specificity.
MiR-155 sequential design according to people also synthesizes the miR-155 molecular beacon that lock nucleic acid is modified, and designs simultaneously
The random molecular beacon sequence (random sequence, RS-MB) not being combined with any target gene is as negative right
According to.
Wherein miR-155MB:
5’-Cy5-CCAGCG-ACC+CCT+ATCA+CGAT+TAGCATTAA-CGCTGG-BHQ3-3’；
RS MB:
5’-Cy5-CCAGCG-AC+GCCA+ATG+ACC+TTA+AGCATTAA-CGCTGG-BHQ2-3’。
+ N represents that base is modified by locking nucleic acid structure, and Cy5 represents fluorophor, and BHQ2 represents quenching group, line
Part represents the stem mechanism of molecular beacon；By Shanghai, bio-engineering corporation synthesizes.
Preparing hybrid buffer, wherein containing 10mM KCl, 5mM MgCl2With 10mM Tris-HCl.By same concentration
Molecular beacon be sufficiently mixed in hybridization buffer with targeting sequence, join in 96 hole blackboards, every pore volume
250 μ l, the final concentration of 100nmol/L of molecular beacon, return to zero with hybridization buffer, and Varioskan Flash is many
Function microplate reader detects the fluorescence intensity in every hole, arranges excitation wavelength 649nm, launches wavelength 670nm, slit 5nm,
Fluorescence intensity is often organized in detection.From figure 1 it appears that when only molecular beacon exists when, fluorescence signal is non-
The lowest；When targeting sequence exists when, molecular beacon loop-stem structure is opened, and produces strong fluorescence signal, about
For 50 times of independent molecular beacon group, difference has statistical significance；When the situation with the presence of not complementary series gene
Under, molecular beacon still keeps loop-stem structure, shows specificity, susceptiveness that molecular beacon synthesizes.
Embodiment 9: gel retardation assay
By chitosan nano and miR-155MB in PBS buffer solution (pH6.0), by different mass ratio (WCS:
WmiR-155MB) mix, WmiR-155MBFixed mass is 2 μ g and rapid vortex oscillation 60s, and room temperature places 60min,
Utilize gel electrophoresis analysis.From figure 2 it can be seen that along with the increase of chitosan nano quality, miR-155MB
Amount gradually decrease, when mass ratio is 7:1 and 10:1, chitosan is substantially completely combined with miR-155MB.
Embodiment 10: entrapment efficiency determination
By chitosan nano and miR-155 molecular beacon in PBS buffer solution, by different mass ratio (WCS:
WmiR-155MB) mix, cumulative volume is 300 μ l, the final concentration of 200nM of miR-155MB, and whirlpool shakes rapidly
Swinging 60s, room temperature places 60min, and on high speed centrifuge, 20000rpm is centrifuged 60 minutes, takes supernatant in ultraviolet
The concentration of miR-155MB, and computational envelope rate is detected on spectrophotometer.Computing formula is: envelop rate EE%=
(WAlways-WTrip)/WAlways× 100%.From figure 3, it can be seen that along with the increase of chitosan mass, envelop rate gradually strengthens,
Reach maximum when mass ratio is 7:1 and 10:1, about about 95%, this is more consistent with gel retardation assay result.
Embodiment 11: fluorescence screening experiment
MiR-155Cy5probes:5 ,-Cy5-is synthesized by Shanghai Sheng Gong company
TTAATGCTAATCGTGATAGGGGT-3 ', with same method by the chitosan nano of different quality ratio and
MiR-155-Cy5 (2 μ g) fluorescent probe carries out self assembly, microplate reader fluorescence intensity.Can also from Fig. 4
Going out, along with the increase of chitosan nano quality, fluorescence intensity gradually weakens, glimmering when mass ratio is 7:1 and 10:1
Light intensity is minimum, shows that chitosan can wrap up miR-155-Cy5 probe, is combined formation nucleocapsid structure with DNA,
DNA can be effectively protected and exempt from interfered by outside, and more consistent with gel retardation assay, entrapment efficiency determination result.
Embodiment 12: chitosan molecule beacon nano-complex analysis of physical and chemical property
Chitosan molecule beacon nano-complex is prepared according to the ratio that mass ratio is 7:1, by shell by same method
Polysaccharide nanometer and chitosan molecule beacon nano-complex drop on copper sheet respectively, static 10 minutes of room temperature, 2% phosphorus tungsten
Acid dyeing 1 minute, by transmission electron microscope detection chitosan and chitosan molecule beacon nano-complex, uses nanoparticle
Degree analyser detects chitosan nano and chitosan molecule beacon nanometer particle size and zeta current potential (Malvern
3000HS, UK) (TEM, Philips TECNAI10), and divided by DNase I experimental verification synthesis chitosan
The stability of sub-beacon nano-complex, operates according to DNase I test kit (the green skies), the end of molecular beacon
Concentration is 100nM.In reactant liquor, 37 DEG C after 10 minutes, add EDTA, 65 DEG C, within 10 minutes, terminate reaction,
Microplate reader measures each hole fluorescence intensity.
Transmission electron microscope shows that chitosan and chitosan molecule beacon nano-complex particle diameter are less, is evenly distributed, greatly
Little respectively may be about 20nm, 30nm, more consistent with nano-particle size analysis instrument result.As it is shown in figure 5, Zeta potential
Show that chitosan nano and complex are positive charge, because cell membrane is electronegative, the beneficially combination of cell and phagocytosis；
CS-MB nano-complex electric charge decreases and may counteract the positive charge of part chitosan nano after being combined with DNA
Relevant.As shown in Figure 6, DNase I experiment show chitosan molecule beacon nano-complex under the effect of enzyme in
Existing low-down fluorescence signal, shows that CS-MB nano-complex stability is splendid, it is possible to the degraded of opposing nuclease.
Embodiment 13: siPORT lipofectamine (Ambion, USA) the transfection molecular beacon inspection of commercialization
Survey nonsmall-cell lung cancer (NSCLC) cell (A549, SPC-A1) and pulmonary carcinoma stem cell (LCSC) miR-155
Express
Cell is seeded in Tissue Culture Dish (laser confocal microscope is special), fills with PBS after cell attachment
Divide rinsing, 3min × 3 time, fully remove fragment and dead cell；SiPORT transfection reagent operates to specifications,
Diluting (1:20 dilution) by OPTI MEM I culture medium, the siPORT transfection reagent that each culture dish takes 5 μ l is dilute
Release, incubated at room 10min；With the miR-155 molecular beacon of OPTI MEM I culture medium preparation 400nM with random
Acid molecules beacon；Take the siPORT transfection reagent after equal-volume dilution and the miR-155 molecular beacon after dilution
With random sequence molecular beacon (negative control), miR-155 molecular beacon and the final concentration of random sequence molecular beacon
For 200nM, each culture dish all adds the miR-155 molecular beacon after the 200 above-mentioned dilutions of μ l and random sequence molecule
Beacon；120min is hatched in cell culture incubator；PBS rinses 3min × 3 time；Each culture dish adds 300 μ l's
Hoechst33342 dyeing liquor redyes 20min；PBS rinses 3min × 3 time, after adding appropriate PBS in each culture dish
Detect under laser confocal microscope and take pictures, as shown in Figure 7.Split with 1ml RIPA lysate after having taken photo
Solve cell, take 100ul and join in 96 orifice plates, often the multiple hole of 6, cell of group, microplate reader fluorescence intensity.With
Carcinoma of prostate (PC-3) cell of expression people miR-155 is as positive control, simultaneously not express people miR-155
African green monkey kidney cell vero cell, mouse junction cancer CT-26 cell as negative control, with CD133, CD338
As the surface marker of pulmonary carcinoma stem cell, flow cytometer sorts the double of CD133 and CD338 from A549 cell
Positive cell is pulmonary carcinoma stem cell (CD133+CD338+), use the method detection NSCLC cell of qRT-PCR simultaneously
And pulmonary carcinoma stem cell (CD133+CD338+) expression of interior miR-155.Test result indicate that and expressing miR-155
Lung carcinoma cell and pulmonary carcinoma stem cell in it can be seen that red fluorescence, and in stem cell, fluorescence signal is higher, location
In cytoplasm, and not expressing the vero cell of miR-155, CT-26 cell is not detected by red fluorescence, and use
The fluorescence signal intensity that microplate reader detects is consistent with qRT-PCR result trend, to sum up shows design synthesis
MiR-155 molecular beacon can be good at detecting the cancerous cell of high expressed miR-155.
Embodiment 14: chitosan molecule beacon nano-complex (CS-MB) detection NSCLC cell and LCSC cell
The expression of miR-155
By the CS-MB nano-complex of preceding method synthesis, fully rinse with PBS after same cell attachment,
3min × 3 time, add the CS-MB nano-complex of OPTI MEM I culture medium dilution in each Tissue Culture Dish,
Final concentration of 200nM.Hatch 120min, PBS for 37 DEG C and rinse 3min × 3 time；Each culture dish adds 300 μ l
Hoechst33342 dyeing liquor redyes 20min；PBS rinses 3min × 3 time, adds appropriate PBS in each culture dish
After detect under laser confocal microscope and take pictures, as shown in Figure 8, strong with microplate reader detection fluorescence after having taken photo
Degree.To express the PC-3 cell of miR-155 as positive control, the greenest not express the Africa of people miR-155
Monkey-kidney cells vero cell, mouse junction cancer CT-26 cell are as negative control.Test result indicate that and expressing
It can be seen that stronger red fluorescence in the lung carcinoma cell of miR-155 and pulmonary carcinoma stem cell, and fluorescence in stem cell
Signal is higher, is positioned at cytoplasm, and do not express the vero cell of miR-155, CT-26 cell is not detected by red
Color fluorescence, experimental result is with siPORT liposome transfection result.And the fluorescence signal intensity detected by microplate reader with
QRT-PCR result trend is consistent, and compares with the siPORT lipofectamine of commercialization, fluorescence signal intensity
Higher.
Embodiment 15: cellular immunofluorescence technology for detection SSTR2 is in NSCLC cell and in-house expression
Cell climbing sheet, density is about 70-80%；After drawing culture medium, fix 15min with 1 milliliter of 4% paraformaldehyde；
PBS rinses, 3 × 3min；5%BSA room temperature closes 20min；. add an anti-SSTR2,1:200 of PBS dilution
(Sata Cruz), in 4 DEG C of refrigerator overnight incubation；PBS is as negative control.PBS rinses, 3 × 3min；Add
Enter 1:200 dilution after two resist, hatch 30 minutes in 37 DEG C；(this step and later step notice that lucifuge processes)
PBS rinses, 3 × 3min；The DAPI dyeing 5min of dropping 50ul；PBS rinses, 3 × 3min；Anti-cancellation is sealed
Tablet mounting.Set up subcutaneous transplantation tumor model, make frozen section, SSTR2 in same method detection tumor tissues
Expression, as shown in Figure 9, it is seen that SSTR2 expresses in cell membrane and cytoplasm in endoglin expression, tissue,
Therefore can carry out targeting with octreotide, be not detected by the expression of SSTR2 at stem cell levels.
Embodiment 16: chitosan molecule beacon nano-complex (CS-MB-OCT) that targeting octreotide (OCT) is modified
And the chitosan molecule beacon nano-complex that the octreotide (OCT) of FITC modification is modified
(CS-MB-OCT-FITC) expression of miR-155 in detection lung carcinoma cell
Prepare chitosan molecule beacon complex (200nM) and the octreotide of FITC modification that targeting octreotide is modified
Chitosan molecule beacon nano-complex (200nM) modified, turns by same method and molecular beacon concentration respectively
Dye (200nM) NSCLC cell, in lung carcinoma cell cytoplasm it is seen that higher red fluorescence is believed after 1 hour
Number, green fluorescence, at the periphery of cell, shows that octreotide and somatostatin receptor are combined, and then causes more
Many nano-complexes enter intracellular, cause the miR-155 in miR-155 molecular beacon and cytoplasm to combine.
As shown in Figure 10, test result indicate that in the lung carcinoma cell expressing miR-155 it can be seen that stronger red fluorescence,
It is positioned at cytoplasm, and the time shortens to 1 hour, and it is thin not express the vero cell of miR-155, CT-26
Born of the same parents are not detected by red fluorescence, and experimental result is with siPORT liposome transfection result.And with microplate reader detect glimmering
Light signal strength is consistent with qRT-PCR result trend, and poly-with the siPORT lipofectamine of commercialization and shell
Glycan molecule beacon nano-complex is compared, and shows higher fluorescence signal intensity.
Fluorescence Intensity Assays is carried out: three kinds of i.e. commercialization siPORT liposomees of material by embodiment 13,14,16
The chitosan molecule beacon nanometer that transfection reagent, chitosan molecule beacon nano-complex and targeting octreotide are modified is multiple
Compound is compared, and fluorescence signal intensity gradually strengthens, and the nano material incubation time that targeting is modified is shorter, is 1
Hour.By same method, after three kinds of materials transfection lung carcinoma cells, the transfection efficiency of flow cytometry analysis cell,
Again show that the chitosan molecule beacon nano-complex that targeting octreotide is modified is demonstrated by higher transfection efficiency.Combine
On show that the chitosan molecule beacon nano-complex that targeting octreotide is modified not only show higher fluorescence intensity,
And transfection efficiency is higher, and the fluorescence signal intensity detected by microplate reader is consistent with qRT-PCR result trend,
Show to reach EARLY RECOGNITION lung carcinoma cell or lung by the miR-155 of molecular beacon identification cancerous cell high expressed
The purpose of cancer stem cell.
Embodiment 17: in living animal imaging technique detection subcutaneous transplantation tumor tissue, miR-155 expresses
Set up subcutaneous transplantation tumor animal model, first by three kinds of nano-complex (i.e. CS-RSMB, CS-miR155MB
And CS-miR155MB-OCT) be injected directly into tumor tissues respectively in carry out preliminary identification (concentration 2 μMs, volume
100 μ l), as shown in figure 11, test result indicate that and CS-miR155MB group can be seen strong fluorescence signal,
And more consistent with the size distribution of tumor, and CS-RSMB group (negative control group) has not seen red fluorescent,
Owing to there is minimal amount of stem cell in cancerous lung tissue, show that molecular beacon possibly even and can swell with tumor cell
MiR-155 in tumor stem cell combines, thus causes the generation of fluorescence signal.Subcutaneous transplantation tumor is taken out simultaneously,
Frozen section is observed, as shown in figure 12, it appeared that CS-miR155MB-OCT group shows higher fluorescence signal,
Fluorescence signal is combined (picture is as a example by A549 cell) from the miR-155 in tumor cell, shows octreotide
The chitosan molecule beacon nano-complex modified can be by reaching more with the combination of the miR-155 in tumor cell
The purpose of good ground tumor cell；Show can be by identifying cancerous cell or cancer stem cell high expressed simultaneously
MiR-155, reaches to identify lung carcinoma cell or the purpose of pulmonary carcinoma stem cell.
Embodiment 18: animal horizontal tail vein injection chitosan molecule beacon nano-complex (CS-MB) and song difficult to understand
Peptide modify chitosan molecule beacon nano-complex (CS-MB-OCT) nano combined analyte detection its to lung carcinoma cell
Identification, diagnostic effect
Setting up lung transplantation tumor animal model by tail vein injection A549 lung carcinoma cell, tail vein injection CS-MB is (dense
Spend 2 μMs, volume 300ul) or the CS-MB-OCT of same molecular beacon concentration, living imaging inspection is to swollen in lung
The identification situation of tumor, as shown in figure 13, it can be seen that produce bright fluorescence signal in pulmonary after 1 hour, and right
Fluorescence signal is had no according to group.Lung tissue being taken out, frozen section is observed simultaneously, as shown in figure 14, it appeared that
CS-MB-OCT nano-complex shows higher fluorescence signal, more cancer in simultaneously can also be observed that lung tissue
Tuberosity is formed, and fluorescence signal, from the cancerous node in lung tissue, shows that CS-MB-OCT can be by with intracellular
The combination of miR-155 reaches the purpose of preferably tumor cell, and show can be by identifying cancerous cell or cancer
The miR-155 of stem cell high expressed, reaches to identify lung carcinoma cell or the purpose of pulmonary carcinoma stem cell.
It should be noted that embodiment 1-7 prepares the side of the chitosan molecule beacon nano-complex that octreotide is modified
Method is applicable not only to somatostatin analogue octreotide, is equally applicable to somatostatin analogue Austria song that FITC modifies
Peptide.
It should be noted that the concentration unit occurred in an embodiment " mM " expression " mmol/L "；" nM " represents
“nmol/L”；" μM " represents " μm ol/L ".
It should be appreciated that for those of ordinary skills, can be improved according to the above description or be become
Change, within all these improvement or conversion all should belong to the protection domain of claims of the present invention.
Claims (10)
1. the preparation side for the chitosan molecule beacon nano-complex of the targeting octreotide modification of pulmonary cancer diagnosis
Method, it is characterised in that comprise the steps:
A, by chitosan nano and molecular beacon according to the ratio of mass ratio 7:1～10:1 pH6.0-7.0's
PBS mixes, and whirlpool concussion 40s～120s, at room temperature stand 40min～120min, obtain shell
Glycan molecule beacon nano-complex；
B, in chitosan molecule beacon nano-complex drip 0.5%～2.0% glutaraldehyde solution, described penta 2
Aldehyde solution is 900:1～1300:1 with the mass ratio of described chitosan molecule beacon nano-complex, by first
Secondary stirring mixing carries out reacting 1h～2h, and mixing speed is 100rpm～300rpm for the first time, by micro-after reaction
Amount dialysis cartridge dialysis 0.5h～2h for the first time removes unconjugated glutaraldehyde；Then according to octreotide and described chitosan
The mass ratio of molecular beacon nano-complex is that the ratio of 120:1～80:1 adds octreotide, carries out second time and stirs
Mixing hybrid reaction 3h～6h, mixing speed is 100rpm～300rpm for the second time, by microdialysis post after reaction
Second time dialysis 11h～15h removes unconjugated octreotide, obtains the targeting octreotide modification for pulmonary cancer diagnosis
Chitosan molecule beacon nano-complex.
Preparation method the most according to claim 1, it is characterised in that in step, described chitosan nano
Particle diameter≤the 300nm of rice.
Preparation method the most according to claim 1, it is characterised in that in step, described nanoshell
Polysaccharide is 7:1 with the mass ratio of described molecular beacon.
Preparation method the most according to claim 1, it is characterised in that in step, described PBS delays
The pH rushing liquid is 6.0.
Preparation method the most according to claim 1, it is characterised in that in step, during whirlpool concussion
Between be 60s.
Preparation method the most according to claim 1, it is characterised in that in stepb, described glutaraldehyde
The concentration of solution is 1.0%, and described glutaraldehyde solution with the mass ratio of described chitosan molecule beacon nano-complex is
1000:1.
Preparation method the most according to claim 1, it is characterised in that in stepb, described octreotide
It is 100:1 with the mass ratio of described chitosan molecule beacon nano-complex.
Preparation method the most according to claim 1, it is characterised in that in stepb, for the first time stirring
The hybrid reaction time is 1.5h, and mixing speed is 150rpm for the first time.
Preparation method the most according to claim 1, it is characterised in that in stepb, second time stirring
The hybrid reaction time is 4h, and mixing speed is 150rpm for the first time.
Preparation method the most according to claim 1, it is characterised in that in stepb, for the first time dialysis
Time is 1h；Dialysis time is 12h for the second time.
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| 以壳聚糖为载体固定化青霉素酰化酶的研究;宋建彬等;《化工进展》;20041231;第23卷(第2期);第181页右栏第2-4段，第182页左栏第2-5段，第183页右栏结论部分 * |
| 壳聚糖纳米包裹miR-155分子信标实时成像肺癌细胞的实验研究;安江宏;《中国博士学位论文全文数据库医药卫生科技辑》;20090315(第3期);第57页第3-7段，第58页最后2段，第64页1-9段，第68页最后1段，第69页图23 * |
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