CN104353082A - Functional nano material drug delivery system for identifying, capturing and restraining circulating tumor cells - Google Patents
Functional nano material drug delivery system for identifying, capturing and restraining circulating tumor cells Download PDFInfo
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- CN104353082A CN104353082A CN201410639414.9A CN201410639414A CN104353082A CN 104353082 A CN104353082 A CN 104353082A CN 201410639414 A CN201410639414 A CN 201410639414A CN 104353082 A CN104353082 A CN 104353082A
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Abstract
The invention belongs to the field of application of a nano material coating technology in identifying, capturing and activity regulating of circulating tumor cells. For pre-warning and preventing of cancer metastasis, particularly, the invention relates to a functional nano material drug delivery system for identifying, capturing and restraining circulating tumor cells. The functional nano material drug delivery system consists of a central nano material carrier, a surface targeted antibody or an aptamer, and a drug for resisting cancers or preventing the cancer metastasis. The functional nano material drug delivery system disclosed by the invention can be used for in-vitro simulation or specific identification and capturing research of trace circulating tumor cells of a blood sample of a clinical patient, and also can be used for regulating the activities of the captured circulating tumor cells, so that the application prospect in pre-warning and preventing of the cancer metastasis is expanded.
Description
Technical field
the invention belongs to nano material coating technique in circulating tumor cell identification, catch the application with activity regulation, especially for early warning and the prevention aspect of cancer metastasis, particularly relate to a kind of specific recognition, catch and suppress the function nano material drug carrier system of circulating tumor cell.
Background technology
over more than 50 year, the mortality rate of multiple major disease (as heart disease) have dropped > 60%, but cancer mortality only reduces about 5%, and wherein the overwhelming majority dies from shifting again of Post operation tumor.Traditional " anticarcinogen " mainly for those malignant proliferations tumor cell but not be in the circulating tumor cell of resting state, therefore not only optionally can not kill circulating tumor cell, and broken the immune system of human body, balance between " self premunition of disease-human body " is tilted towards unfavorable aspect, even causes mutagenesis, carcinogenic.Therefore, the slow stupid route that what the research and development of cancer therapy drug were walked is " anticancer again after cancer metastasis ", produces little effect.The application approach of current nanotechnology in cancer also mainly lays particular emphasis on and the nano material of the chemicals of postoperative metastasis and monoclonal antibody body bag quilt is combined, and this mode is due to its toxicity and non-specific problem, the cancer metastasis process caused by circulating tumor cell effectively can not be intervened.Therefore research and development can specific recognition and catch trace circular tumor cell in blood and lower the functionalized nano material drug carrier system of its activity, effectively can prevent the neoplasm metastasis because bringing out after circulating tumor cell activation, and then the hidden danger that elimination Post operation tumor shifts and recurs.
goal of the invention
the object of the invention is to the non-specific problem avoiding Conventional nano technology for detection He catch circulating tumor cell, and then provide a kind of specific recognition, catch and suppress the function nano material drug carrier system of circulating tumor cell, such nano material drug-loading system, because can catching specifically and suppressing caught circulating tumor cell, is expected to the early warning and the prevention aspect that are applied to cancer metastasis.
A kind of function nano material drug carrier system of the present invention, described function nano material drug carrier system is made up of center nano material carrier, surperficial targeting antibodies or aptamers, medicine that is anticancer or prophylaxis of cancer transfer.
Described center nano material carrier is PAMAM dendrimers, gold nano grain AuNPs, nano silicon particles SiNPs, magnetic nanoparticle MNPs or graphene oxide graphene oxide.
Described surface targets can specific recognition and forming in conjunction with the targeting antibodies of circulating tumor cell surface biomarkers or aptamers by two or more to targeting antibodies or aptamers, and described biomarker is EpCAM, Sialyl Lewis X, HER2, EGFR, ALDH1 or CD44.
The medicine of described anticancer or prophylaxis of cancer transfer comprises ursolic acid and derivant, mifepristone and derivant thereof, captopril and derivant, emodin and derivant thereof.
Described center nano material carrier and surperficial targeting antibodies or aptamers adopt covalent attachment to carry out, and described covalent attachment is 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate/N-hydroxysuccinimide method, avidin/biotin method or N-hydroxysuccinimide/Maleimide chemistry method.
Described function nano material drug carrier system by the medicine of described anticancer or prophylaxis of cancer transfer and center nano material carrier are carried out chemical coupling, Electrostatic Absorption, physically trapping mode prepare.
Described center nano material carrier surface has reactive functionality, and described reactive functionality is-OH ,-NH
2,-COOH or-SH.
Described center nano material carrier adopts intermediate connector to be connected with surperficial targeting antibodies or aptamers, and described intermediate connector is carboxylated Polyethylene Glycol, the Polyethylene Glycol of sulfhydrylation, hydrazide-polyethylene glycol-dithiol or OPSS-PEG-NHS.
A kind of specific recognition of the present invention, catch and suppress the function nano material drug carrier system of circulating tumor cell, the medicine shifted by center nano material carrier, surperficial targeting antibodies or aptamers and anticancer or prophylaxis of cancer forms, and wherein core props up be configured to by having the abundant nano material of multivalence Complex effect, excellent dimensions effect, height load capacity and surface functional group; Periphery is then by can specific recognition and forming in conjunction with two or more targeting antibodies of circulating tumor cell (CTCs) surface biomarkers or aptamers; Medicine that is anticancer or prophylaxis of cancer transfer is then that the efficient small molecular weight chemical medicine of low toxicity shifted by anticancer or prophylaxis of cancer is formed.The nano material drug-loading system that the present invention obtains, not only Stability Analysis of Structures, and the good characteristic of nano material and targeting antibodies or aptamers itself can be kept simultaneously, also can be used for the external specific recognition to micro-CTCs in simulation and clinical patient blood sample and catch research, and can be used for regulating and controlling caught CTCs activity, thus open up application prospect in the early warning and prevention of neoplasm metastasis.
The present invention is described in detail as follows:
Nano material used in the present invention should have the load capacity of multivalence Complex effect, good dimensional effect and biological stability and height, can adopt but be not limited to following material: tree (dendrimers), gold nano grain (AuNPs), nano silicon particles (SiNPs), magnetic nanoparticle (MNPs), graphene oxide (graphene oxide).Preferentially for of the present invention be polyamidoamine dendritic macromole (PAMAM dendrimers), except having the total characteristic of common nano material (outside regular meticulous chondritic, hydrophobic cavity characteristics, the load that can be hydrophobic drug and inorganic probe molecule provides place, reach the object such as the controlled release of medicine and the targeted delivery of slow release and inorganic probe) outward, its surface also has a large amount of controllable function group (amino), chemical modification can be carried out to it, multivalence complexation targeting antibodies, part and drug molecule etc., thus build biological composite (bioconjugate) safely and effectively.
Targeting antibodies used in the present invention is selected to express at tumor cell especially circulating tumor cell apparent height for those, and on normal cell especially hemocyte and leukocyte low expression or the biomarker of not expressing, can adopt but be not limited to following label: EpCAM, Sialyl Lewis X, HER2, EGFR, ALDH1, CD44.Preferentially for of the present invention be the antibody of EpCAM and Sialyl Lewis X for epithelium circulating tumor cell high expressed, wherein EpCAM is also called CD326, TACSTDl, C017-lA, GA733-2 and KSA etc., belong to single pass transmembrane I type glycoprotein, molecular mass is 30 ~ 40 kD, be made up of 3 parts, i.e. ectodomain, single pass transmembrane domain and intracellular domain.Ectodomain starts with a signal sequence, thereafter and then an epithelium growth factor sample repetitive sequence, mankind's Elityran sequence and one lack the domain of cysteine, epithelium growth factor sample repetitive sequence and mankind's Elityran sequence form a chondritic, are responsible for the homologous cell adhesion properties of EpCAM molecule.Intracellular domain is one and has 26 amino acid whose short chains, containing 2 a-actinine (a-actinin) binding sites, can be combined with actin, thus and cytoskeleton interaction.Under physiological conditions, EpCAM is expressed in normal epithelial all except squamous epithelial cancer to some extent, and multidigit is in intercellular tight junction.The expression of obvious EpCAM is lacked in the cell of connective tissue and hematopoietic origin, cerebral tissue and vascular endothelial cell.Under pathologic condition, EpCAM is almost expressed in all adenocarcinoma, comprises Colon and rectum adenocarcinoma, adenocarcinoma of stomach, breast carcinoma, ovarian cancer, adenocarcinoma of lung, carcinoma of prostate, cancer of pancreas and hepatocarcinoma and retinoblastoma.Therefore, the index of EpCAM Chang Zuowei cancer earlier detection and diagnosis.Another one antibody is for antigen Sialy Lewis X (Slex).Slex is also called CD15s; it is the isomers of tumor markers single sialic acid gangliosides fat CA19-9; also be that adhesion molecule selects element (E-selectin; one of i.e. CD62) aglucon determined of the comparison that identifies, be combined into by galactose (Gal), glucose (Glc), sialic acid (NeuAc), N-acetyl group (NAc), fucose (Fuc) 5 molecules.Cell adhesion molecule, as mediated cell and the mutual identification of cell or cell and substrate and the molecular basis of effect, plays an important role in tumor invasion, transfer process.The high expressed of carbohydrate antigen, reflects the change of tumor cell membrane carbohydrate antigen sugar chain.The sugar chain heterogeneity produced by chemical modification effect is changed and distribution, some tumor antigen may be covered, reduce tumor cell immunogenicity, increase the net negative charge of molecule, weaken the effect of lymphocyte and macrophage combination and killing tumor cell, impel tumor cell generation immune evasion.The repulsive force mutual due to cell surface increases, and absorption and binding ability reduce, and thus make tumor cell be easy to shift from tissue loss.Adhesion molecule plays dual function in the Infiltration and metastasis process of tumor cell, and tumor cell must first be lowered by cell adhesion molecule on the one hand, comes off from primary tumor; Need again the rise by extracellular matrix receptor or endothelial cell ligand on the other hand, adhere to extracellular matrix or vascular endothelial cell and move, could metastasis be formed.It is the important symbol that neoplasm metastasis is inclined to that CD15s high expressed and stove dedifferente.Therefore, CD15s is also as one of the target spot of CTCs research.
The medicine of formation function nano material drug carrier system used in the present invention, be often referred to the chemicals of the anticancer of those high-efficiency low-toxicities or prophylaxis of cancer transfer, can adopt but be not limited to following compounds: ursolic acid and derivant, mifepristone and derivant thereof, captopril and derivant, emodin and derivant thereof.Preferential recommendation is used for mifepristone of the present invention and derivant thereof.Because they can suppress the adhesion of circulating tumor cell to tunica intima and the invasion and attack to peripheral tissues in blood, therefore can play a significant role in the prevention of cancer metastasis.
The covalent attachment of center used in the present invention nano material and surperficial targeting antibodies or aptamers, can adopt but be not limited to following method: 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC)/N-hydroxysuccinimide (NHS) method, Avidin (avidin)/biotin (biotin) method and N-hydroxysuccinimide (NHS)/maleimide (maleimide) chemical method.Preferential for EDC/NHS catalysis method of the present invention, double antibody (anti-EpCAM, anti-Slex) not only can be made covalently bound on PAMAM dendrimers surface successively, also can reduce the physical absorption (anti-fouling effect) of antibody at material surface, reduce the toxicity of nano material itself, increase the stability of nano material-antibody complex in biotic environment.
The preparation of function nano material drug carrier system used in the present invention, can adopt but be not limited to following method and realize: by chemicals covalent coupling nano-material surface, by chemicals Electrostatic Absorption at nano-material surface, chemicals is embedded in nano material cavity.Preferential recommendation is used for physically trapping method of the present invention, is embedded in the 3D cavity structure of PAMAM dendrimers by chemicals mifepristone and derivant thereof in certain environment.The method had both avoided chemicals and antibody is connected to the surperficial space steric effect produced of PAMAM dendrimers jointly, farthest make use of again the space structure of PAMAM dendrimers, improves drug load.
Center used in the present invention nano material, surface should have reactive functionality, and what be convenient to targeting antibodies is covalently bound, can select but be not limited to following groups :-OH ,-NH
2,-COOH ,-SH.
When nano material used in the present invention is connected with targeting antibodies, reduce the intermediate of space steric effect, can adopt but be not limited to following material: the Polyethylene Glycol (PEG-SH) of carboxylated Polyethylene Glycol, sulfhydrylation, hydrazide-polyethylene glycol-dithiol, orthopyridyl-disulfide-polyethyleneglycol-N-hydroxy-succ inimide (OPSS-PEG-NHS).
Center used in the present invention nano material and surperficial targeting antibodies, in order to realize maximum multivalence Complex effect, the consumption of targeting antibodies is many more than nano material itself, specifically how much will determine according to the exposed reactive functionality of nano-material surface.
Extracorporeal circulation tumor models used in the present invention should choose that those researchs are concentrated, JEG-3 high in postoperative metastasis and recurrence degree, can adopt but be not limited to following JEG-3: breast carcinoma cell strain, Prostatic cancer cell lines, colon cancer cell line, hepatoma cell strain, cervical cancer cell lines, stomach cancer cell line, lung cancer cell line.Preferential recommendation selects human colon cancer cell strain HT29 as extracorporeal circulation tumor models.Because of colon cancer cell line HT29 transfer and recurrence degree moderate, risk is less; And biomarker EpCAM and Slex expresses at this cell surface stability height, therefore to choose HT29 be external CTCs model, will contribute to carrying out smoothly of this research.
The external blood sample containing circulating tumor cell used in the present invention takes from that those researchs are concentrated, middle and terminal cancer patient high in postoperative metastasis and recurrence degree, can choose and be not limited to following cancer patient: colon cancer patient, breast cancer patients, hepatocarcinoma patient, prostate cancer patient, Patients with Cervical Cancer, Patients with Gastric Cancer, lung cancer patient.
The physicochemical property characterization method of function nano material drug carrier system used in the present invention, can adopt but be not limited to following method: nuclear magnetic resoance spectrum (
1h NMR), infrared spectrum (FTIR), uv-vis spectra (UV-Vis), fluorescence spectrum or collection of illustrative plates, laser particle size (DLS)/Zeta potential, field emission scanning electron microscope (FSEM) figure, atomic force microscope (AFM) figure, projection Electronic Speculum figure, electrophoretogram.
The external CTCs of function nano material drug carrier system used in the present invention catches the means of research, can take but be not limited to following method: according to CTCs content (1/10 in patient blood
3-10
6), first to adding a certain amount of target tumor in a large amount of interference cells (normal cell that such as leukocyte, erythrocyte, biomarker are not expressed or other tumor cell) or add a certain amount of target tumor in whole blood, after add quantitative nano material-bis-/many targeting antibodies or aptamers complex and carry out and catch research.Preferential recommendation use in the present invention by investigate separately the dual anti-complex of PAMAM dendriemrs-to suspend and adherent HT29 cell identification and catch effect, or investigate under a large amount of interference cell (leukaemia HL-60 or erythrocyte RBCs) exists, situation is caught to a small amount of HT29 cell, adopt fluorescence to take pictures and flow cytometry analysis assess dual anti-complex external in simulate blood sample to the specificity capture effect of CTCs model.
Function nano material drug carrier system used in the present invention catches research means to CTCs's in clinical patient blood sample, can adopt but be not limited to this method: the blood sample obtaining tumour patient after postoperative or chemotherapy, after treated acquisition buffy coat, add nano material-bis-/many targeting antibodies or aptamers complex is hatched altogether or directly add nano material-bis-/many targeting antibodies or aptamers complex in whole blood carries out and catch research.Preferential recommendation uses in the present invention by obtaining the postoperative colon cancer blood sample of tumour hospital, add the quantitative dual anti-complex of PAMAM dendrimers-to carry out catching experiment, after blood post-processed, streaming quantitative analysis its catch CTCs number, laser co-focusing is analyzed it and is caught situation.
Function nano material drug carrier system used in the present invention is to the activity regulation research means of catching CTCs, can adopt but be not limited to following assessment measure: with the cytotoxicity of MTT, XTT, LDH method test nano material-bis-/many targeting antibodies complex, the distribution situation of cell cycle is affected, by the apoptosis situation of the staining assessment cells such as Annexin V-FITC/PI, TUNEL, Annexin V-PE/7-AAD, DAPI, AO/EB, Caspase 3/8/9 with the test such as PI staining, BrdU incorporation methods complex.Adopt mtt assay, the dual anti-complex of morphology dyeing observational method, flow cytometry analysis cell cycle and apoptosis method further investigation PAMAM dendrimers-to the inhibiting mechanism of catching CTCs activity in preferential recommendation the present invention.
Beneficial effect of the present invention:
(1) the invention provides a kind of specific recognition, catch and suppress the function nano material complex drug-loading system of circulating tumor cell, this drug-loading system can identify, catches and suppress the circulating tumor cell in blood specifically, thus is expected to play a significant role in the early warning of cancer metastasis and prevention area;
(2) the invention provides the technical information two or more antibody or aptamers being coupled to same nano-material surface, this preparation method is simple, is easy to operation, and reaction condition is gentle, low to the requirement of equipment, and has the prospect of industrialized implementation.
Accompanying drawing explanation
Fig. 1 is the invention schematic diagram of the function nano material drug carrier system identifying, catch and suppress circulating tumor cell.I, nano material PAMAM dendrimers carries out carboxylated modification in surface; Ii, the covalently bound two kinds of antibody (aEpCAM and aSlex) in dendrimers surface of modification; Iii-v, prepared dendrimers-double antibody drug-loading system under existing with or without interference cell to the specific recognition of target colon cancer cell line HT29 with catch; Vi, prepared dendrimers-double antibody drug-loading system carries out activity inhibition to the cancerous cell of catching.
Fig. 2 is the corresponding derivant (CC G6) of G6 PAMAM dendrimers of embodiment 1,2 gained and the hydrogen nuclear magnetic spectrum of antibody complex (G6-5aEpCAM-5aSlex).
Fig. 3 is the corresponding derivant (CC G6) of G6 PAMAM dendrimers of embodiment 1,2 gained and the infared spectrum of antibody complex (G6-5aEpCAM-5aSlex).
Fig. 4 is the corresponding derivant (CC G6) of G6 PAMAM dendrimers of embodiment 1,2 gained and the ultraviolet absorpting spectrum of antibody complex (G6-5aEpCAM-5aSlex).
Fig. 5 is the scanning electron microscope (SEM) photograph of the dual anti-complex G6-5aEpCAM-5aSlex of embodiment 1,2 gained.
The external identification to adherent HT29 cell of fluorescent labeling dual anti-complex PE-5aEpCAM-G6-3aSlex-FITC that Fig. 6 obtains for embodiment 3 and combined with fluorescent collection of illustrative plates.
The external identification to non-adherent HT29 cell of fluorescent labeling dual anti-complex PE-5aEpCAM-G6-3aSlex-FITC that Fig. 7 obtains for embodiment 3 and catch fluorescence pattern.
The capture rate flow cytometer showed of fluorescent labeling dual anti-complex PE-5aEpCAM-G6-3aSlex-FITC to micro-target HT29 cell under a large amount of RBCs interference that Fig. 8 obtains for embodiment 3.
The fluorescent labeling dual anti-complex PE-5aEpCAM-G6-3aSlex-FITC that Fig. 9 obtains for embodiment 4 catches fluorescence pattern to micro-CTCs in postoperative colon cancer patient blood sample.
The fluorescent labeling dual anti-complex PE-5aEpCAM-G6-3aSlex-FITC that Figure 10 obtains for embodiment 4 catches streaming collection of illustrative plates to micro-CTCs in postoperative colon cancer patient blood sample.
Figure 11 is that the dual anti-complex G6-5aEpCAM-3aSlex of the variable concentrations of embodiment 5 gained affects the period profile of HT29 cell.* represent under equal conditions with the comparing of blank group,
p<0.01.
Figure 12 is that the dual anti-complex G6-5aEpCAM-3aSlex of the variable concentrations of embodiment 5 gained is to the Apoptosis of HT29 cell.* represent with * * under equal conditions with the comparing of blank group,
#with
##comparison under expression equal conditions between same sample variable concentrations; When for * or
#time,
p<0.05; When for * * or
##time,
p<0.01.
Detailed description of the invention
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.In addition should be understood that those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after the content of having read the present invention's instruction.
Embodiment 1: the preparation of the polyamidoamine dendritic macromole complex of double antibody bag quilt
With or without fluorescent-labeled antibody bag quilt polyamidoamine dendritic macromole complex by by antibody and polyamidoamine dendritic macromole covalently bound and obtain, thus for the external specific recognition to CTCs, catch and activity regulation, concrete preparation process is as shown in Figure 1.
(1) the complete carboxylated modification of polyamidoamine dendritic macromole:
What get dissolve with methanol is amino the 6th PAMAM type dendritic macromole (G6 PAMAM dendrimers) containing 60 mg ends, after rotary evaporation falls methanol solution, complete with 2 ml DMSO solubilize, after add 246 mg butanedioic anhydrides, under room temperature, the strong magnetic agitation of lucifuge reacts 24 h, be 8 by product molecular cut off subsequently, 000 ~ 14, the cellulose dialysis film of 000 is dialysed in 500 ml × 3 24 h in ultra-pure water, after Economical Purification, last lyophilization obtains the completely Carboxylation product C C G6 of dendrimer surface amino groups (G6-(COOH)
256),
(2) partial acetylation of polyamidoamine dendritic macromole is modified:
By G6-(COOH)
256be dissolved in the phosphate buffered solution of pH 7.4, make its concentration be 0.276 μ g/ml, get G6-(COOH) that 8 ml concentration are 0.276 μ g/ml
256, add EDC 302 ng and NHS 181.5 ng, room temperature lucifuge stir-activating 1 h, thus obtain the acetylizad intermediate product of dendrimer surface portion;
(3) double antibody (aEpCAM) of polyamidoamine dendritic macromole and (aSlex) modify
Get the intermediate product solution after activation 1 ml, add the aSlex solution that aEpCAM solution that 118.6 μ l concentration are 50 μ g/ml and 7.12 μ l concentration are 500 μ g/ml, under room temperature, lucifuge stirs and spends the night, then through dialysis purification, lyophilization obtains the dual anti-complex of G6-5aEpCAM-3aSlex of dendrimer surface coupled antibody aEpCAM and aSlex;
(4) double antibody (aEpCAM) of polyamidoamine dendritic macromole and (aSlex) modify
Get the intermediate product solution after activation 1 ml, add the aSlex solution that aEpCAM solution that 118.6 μ l concentration are 50 μ g/ml and 11.86 μ l concentration are 500 μ g/ml, under room temperature, lucifuge stirs and spends the night, then through dialysis purification, lyophilization obtains the dual anti-complex of G6-5aEpCAM-5aSlex of dendrimer surface coupled antibody aEpCAM and aSlex.
(5) double antibody (aEpCAM and aSlex) of fluorescently-labeled polyamidoamine dendritic macromole is modified
Get the product C C G6 of the above-mentioned activation of 1 ml, under first adding the aSlex of 3.56 μ l and the two anti-igg-FITC solution room temperatures of 3.56 μ l, lucifuge reacts 12 h, after add 15 μ l aEpCAM-PE solution continue reaction 12 h, then through product dialysis purification 24 h, last lyophilization obtains the PE-5aEpCAM-G6-3aSlex-FITC complex of double fluorescence labeling.
(6) double antibody (aEpCAM and aSlex) of fluorescently-labeled polyamidoamine dendritic macromole is modified
Get the product C C G6 of the above-mentioned activation of 1 ml, under first adding the aSlex of 5.93 μ l and the two anti-igg-FITC solution room temperatures of 5.93 μ l, lucifuge reacts 12 h, after add 15 μ l aEpCAM-PE solution continue reaction 12 h, then through product dialysis purification 24 h, last lyophilization obtains the PE-5aEpCAM-G6-5aSlex-FITC complex of double fluorescence labeling.
Embodiment 2: the sign of the polyamidoamine dendritic macromole complex of double antibody bag quilt
(1) get freshly prepd a certain amount of CC G6, G6-5aEpCAM-5aSlex material, use the D of 700 μ l respectively
2o dissolves fully and is placed in nuclear magnetic tube, and rear use 400 MHz NMR spectrometer with superconducting magnet is done
1h NMR analyzes.(2) freshly prepd a certain amount of CC G6, G6-5aEpCAM-5aSlex material is got, dissolve fully with PBS (pH 7.4), through ultrasonic Treatment 5 min, after the disposable water solublity metre filter of 0.22 μm, measure respective water solublity particle diameter and Potential Distributing in DLS/Zeta potential instrument.(3) get freshly prepd a certain amount of CC G6, G6-5aEpCAM-5aSlex material, with appropriate KBr powder mixing after tabletting, in radar stealthy materials, test its FTIR collection of illustrative plates respectively.
(4) freshly prepd a certain amount of CC G6, G6-5aEpCAM-5aSlex material is got, after being dissolved be mixed with finite concentration with PBS (pH 7.4), and with PBS (pH 7.4) for benchmark, on Q5000 ultramicrospectrophotometer, measure the ultraviolet absorpting spectrum of these materials at wavelength 210-600 nm respectively.
(5) freshly prepd G6-5aEpCAM-5aSlex complex is got, first conducting resinl is bonded on specimen holder, draw the 0.1%(W/W of ultrasonic disperse with capillary tube afterwards) this complex solution, dropwise drip on conducting resinl, put into scanning electron microscope after air-dry and observe.
Characterization result analysis is known, CC G6's and G6-5aEpCAM-5aSlex
1h NMR profile variation (as shown in Figure 2) demonstrates the difference that antibody connects the front and back structure of matter, and the especially appearance at ester peak, describes the existence of antibody on G6 PAMAM dendrimers surface.In FTIR collection of illustrative plates (as shown in Figure 3), when after antibody aEpCAM and aSlex in CC G6 connection, though remain on 1680-1630 cm
-1the C=O stretching vibration at place, but at 1420-1400 cm
-1the amido link characteristic absorption peak that the C-N at place is flexible strengthens, and the difference of visible material surface group also reflects the successful coupling of double antibody at nano-material surface.Because of CC G6, this does not have uv absorption in 220 nm places, and antibody aEpCAM and aSlex all has feature ultraviolet absorption value at 220 nm places, therefore selects the absworption peak of 220 nm as judging whether antibody is connected to the index in nano material.UV collection of illustrative plates display (as shown in Figure 4), though be the G6-5aEpCAM-5aSlex complex of low concentration, still has characteristic absorption peak at 220 nm places, and visible nano-material surface successfully wraps by antibody.What physicochemical property prepared dual anti-complex G6-5aEpCAM-5aSlex has, its monodispersed aqueous solution particle is carried out particle diameter and potential determination shows (see table 1), when after the covalently bound double antibody of nano material CC G6, though electric negative characteristic does not have significant change, but its water solublity particle diameter sharply increases, the existence of visible antibody can cause certain aggregation tendency.Scanning electron microscope collection of illustrative plates (as shown in Figure 5) also further determined that pattern and the diameter of prepared dual anti-complex, and this complex surface almost spherical, horizontal diameter is at about 100 nm.
Table 1 is water solublity diameter and the Zeta electric potential of derivant (CC G6) that G6 PAMAM dendrimers is corresponding and antibody complex (G6-5aEpCAM-5aSlex).
Embodiment 3: the external identification to CTCs model (HT29 cell) of polyamidoamine dendritic macromole complex of double antibody bag quilt and catch analysis (1) and will the HT29 cell of exponential phase be in 10
5the density of/ml is seeded on the special ware of laser co-focusing of 35 mm, after cell attachment, suspend with the PBS liquid containing 1% BSA and close 30 min, after cyclic washing, with concentration be the fluorescently-labeled dual anti-complex (PE-5aEpCAM-G6-3aSlex-FITC) of 20 μ g/ml at 37 DEG C, 5% CO
2incubator in Dual culture 1 h.Finally, wash away unconjugated complex or antibody, after fixing with fixative, the PBS solution lucifuge added containing nuclear staining agent DAPI dyes 15 min.After dyeing, cyclic washing, finally use serum-free is without the covering of phenol red culture medium and in laser confocal microscope DAPI λ
ex405 nm, λ
em425-475 nm, FITC λ
ex488 nm, λ
em500-535 nm, PE λ
ex550 nm, λ
emphotographic analysis under 570-610 nm pattern.
(2) get the HT29 cell being in exponential phase, after trypsinization, often HT29 cell concentration 10 got by pipe
6individual, first with Hoechst 33258 dyeing liquor 15 min that dye, labelling is carried out to nucleus, close 30 min removing non-specific binding with the PBS fluid-tight of 1%BSA again with after PBS washing.Finally, control tube and sample cell add the PBS of 1 ml or concentration is respectively that the fluorescently-labeled complex PE-5aEpCAM-G6-3aSlex-FITC of 20 μ g/ml educates 1 h altogether in 37 DEG C of water-bath lucifuges.After the unconjugated complex of washing removing, suspend with the PBS of 100-500 μ l, and in fluorescence microscope DAPI λ
ex365 nm, λ
em445/50 nm, λ
ex470/40 nm, λ
emunder 525/50 nm pattern, situation is caught in qualitative analysis.
(3) get the HT29 cell being in exponential phase, after trypsinization, often HT29 cell concentration 10 got by pipe
3individual, add 10 respectively
6, 10
8individual RBCs simulates clinical patient blood sample (i.e. 1 CTCs/ 10
3-10
6hemocyte), after PBS confining liquid process cell mixture 30 min of 1% BSA, often manage that directly to add 1 ml concentration be that the fluorescently-labeled dual anti-complex PE-5aEpCAM-G6-3aSlex-FITC of 20 μ g/ml educates 1 h altogether in 37 DEG C of water-baths.Cell mixture adds two kinds of Isotype antibody IgG-PE and IgG-FITC as negative control simultaneously.After the unconjugated complex of washing removing, the cell PBS of 500 μ l suspends, after the cell number of catching in flow cytometer 488 nm laser place's quantitative analysis complex.
Preparation-obtained fluorescently-labeled double antibody complex PE-5aEpCAM-G6-3aSlex-FITC is used for the external identification to CTCs model (HT29 cell) and catches experiment (as shown in Figure 1).Under not having interference cell to exist, for adherent HT29 cell, laser co-focusing atlas analysis (as shown in Figure 6), the surface of cell membrane that identifies by dual anti-complex and combine can demonstrate the yellow-green fluorescence (bright spot display) of superposition, and not combined cell, only have nucleus to demonstrate blue-fluorescence (Lycoperdon polymorphum Vitt display).For the HT29 cell suspended, fluorescence microscope collection of illustrative plates (as shown in Figure 7) also demonstrates similar results, and dual anti-complex can identify and catch HT29 cell in 1 h internal specific ground, and captured HT29 cell surface has Two Colour Fluorescence (bright spot display).Under interference erythrocyte exists in a large number, along with interference mean constant of red blood cell is from 10
6be increased to 10
8the cell number that then dual anti-complex is caught from 1000 target HT29 cells is reduced to 25 (as shown in Figure 8) from 39, increasing of visible disturbance cell number, can cause the increase of space and contact resistance, can affect complex to the identification of target cell and capture effect further.Even if but at nearly Clinical CT Cs content (i.e. 1CTCs/10
3~ 10
6hematopoietic cell) under, the PAMAM dendrimers of the double antibody bag quilt prepared by us still can identify highly sensitive from a large amount of erythrocyte, with high specificity and capture CTCs, and this also illustrates the advantage place of double antibody complex.
Embodiment 4: the polyamidoamine dendritic macromole complex of double antibody bag quilt is to the identification of CTCs in clinical patient blood sample and catch analysis
Get 1 ml colon cancer patient blood sample, after 1%BSA sealing treatment, directly adding 1 ml concentration is the fluorescently-labeled dual anti-complex PE-5aEpCAM-G6-3aSlex-FITC of 20 μ g/ml, after educating 1 h altogether in 37 DEG C of water-baths, and the unconjugated complex of PBS washing removing; Then use the erythrocyte cracked liquid of 8-10 ml in 37 DEG C of water-bath cracking 5-10 min, until erythrocyte cracks completely, 1500 rpm centrifugalize go out remaining cell; Finally educate 30 min labelling leukocyte altogether with the anti-CD45 of APC labelling, after background colour is removed in washing, on flow cytometer, analyze CTCs number that dual anti-complex catches or with after the further labeled cell core of DAPI, identify under laser co-focusing the CTCs that catches by dual anti-complex.
Obtain postoperative colon cancer patient blood sample, the PAMAM dendrimers of the dual anti-bag quilt further prepared by confirmation catches the high specific of CTCs, sets forth its clinical value.Laser confocal microscope collection of illustrative plates shows (as shown in Figure 9), this dual anti-complex can capture the CTCs of extremely trace, its surface demonstrates specific yellow-green fluorescence (bright spot display), in order to difference by the leukocyte of anti-CD45 institute labelling (film surface red fluorescence, shown in bright spot).Streaming quantitative analysis is known (as shown in Figure 10), and for Isotype control group, dual anti-complex can capture CTCs, and the CTCs captured is presented at PE
+fITC
+aPC
-region.Statistics shows, this dual anti-complex PE-5aEpCAM-G6-3aSlex-FITC prepared can from 8 patient 1 ml blood average capture to 11 ± 3 CTCs.
Embodiment 5: the activity regulation of polyamidoamine dendritic macromole complex to the HT29 cell of catching of double antibody bag quilt is analyzed
(1) get the HT29 cell being in growth logarithmic (log) phase, after trypsinization, be mixed with cell suspension with culture medium piping and druming, after cell counting, getting density is 8 × 10
3~ 1 × 10
4the cell suspension inoculation of individual/ml is in 96 orifice plates, and every hole 100 μ l, is placed on 37 DEG C, the CO of 5%
224 h are cultivated in incubator.Then on aseptic operating platform, first use 0.22 μm of membrane filtration dual anti-complex mother solution G6-5aEpCAM-3aSlex, the culture medium that rear removal is old, every hole adds the complex (0 of the variable concentrations of 100 μ l culture medium dilutions respectively, 1.25,2.5,5,10,20 μ g/ml) Dual culture.Blank group, solvent control are separately set, often organize 6 multiple holes.After acting on 48 h, remove the culture medium containing complex, in every hole, add 100 μ l containing MTT(500 μ g/ml) serum-free without phenol red medium.After continuing to hatch 4 h, take out 96 orifice plates and stop cultivating.Remove the supernatant in each hole gently, every hole adds DMSO 150 μ l, and shaking table vibrates 10 min, and after bluish violet crystallization is all dissolved, in microplate reader, 570 nm wavelength places measure the absorbance value (A in each hole
570 nm).Survival rate can be obtained by formula: survival rate (%)=(experimental group A
570 nm-solvent control group A
570 nm)/(blank group A
570 nm-solvent control group A
570 nm) × 100%.(2) choose the human colon cancer cell HT29 of exponential phase after trypsinization, adjustment cell density is 5 × 10
5individual/ml, is inoculated in 6 orifice plates with 1.5 ml/ holes, is placed in 37 DEG C, 5 % CO
2incubator in cultivate 24 h; With 0.22 μM of above-mentioned complex mother solution of membrane filtration, prepare a series of Concentraton gradient (0,10,20 μ g/ml), CC G6 is used as negative control, without complex processed group as blank group.Old culture medium is abandoned in suction, after PBS washed cell, adds complex corresponding to 2 ml in every hole, continues to hatch 48 h in incubator.After Dual culture, with PBS washed cell three times, removing complex, digestion collecting cell moves in centrifuge tube, centrifugal 6 min of 200 g, after supernatant discarded, with the PBS washed cell twice of pre-cooling.Careful Aspirate supernatant, the PBS of about 200 μ l is stayed in centrifuge tube, avoid siphoning away cell, afterwards often pipe to add 70% ethanol mixing of 1 ml pre-cooling fixing ,-20 DEG C are spent the night, centrifugal 6 min of 200 g, discard ethanol, clean once with the PBS of pre-cooling, draw supernatant, the PBS of residual about 50 μ about l, attack is with cell dispersion.By the propidium iodide stain liquid prepared (dye solution 500 μ l, propidium iodide stain liquid (20X) 25 μ l, RNase A(50X) 10 μ l) often pipe 500 μ l join in corresponding blank pipe and sample cell, room temperature lucifuge dyes 30 min.Cell cycle distribution situation is detected at excitation channel 488 nm place with flow cytometer.
(3) take the logarithm the HT29 cell of trophophase, trypsinization adjustment cell concentration is 5 × 10
5individual/ml, is inoculated on six well culture plates with 1.5ml/hole, puts 37 DEG C, 5%CO
2incubator in cultivate.After cell attachment, remove old culture medium, negative hole adds fresh culture medium, and sample well then adds above-mentioned complex and the CC G6 (0,10 of variable concentrations, 20 μ g/ml), after acting on 48 h, trypsinization, centrifugal collecting cell, wash three times with PBS, remain the PBS re-suspended cell of 50 μ l.Two dye liquor, i.e. 500 μ l Binding Buffer+5 μ l Annexin V-FITC+5 μ l PI are prepared in proportion by sample size.Getting the above-mentioned dyeing mixed liquor of 500 μ l joins in negative control pipe and sample cell one by one, and room temperature lucifuge reacts 15 min.Two positive compensating pipes of list and dead cell+5 μ l Annexin V-FITC or dead cell+5 μ l PI are set, regulation voltage, detect apoptosis situation with flow cytometer at excitation channel 488 nm place.
After prepared dual anti-complex specificity captures targeted cancerous cells, can its activity that regulate and control caught cancerous cell further be also a bright spot of the present invention.Cell proliferation experiment shows (see table 2), and for CC G6, dual anti-complex G6-5aEpCAM-3aSlex demonstrates the ability of stronger antiproliferative effect, and in concentration dependent trend.Complex can change the distribution of HT29 cell in each period of cell cycle further, for blank group, cell after complex effect its obviously increase at the cell percentage composition of S phase, and decline (as shown in figure 11) at the content of G0/G1 phase, this dual anti-complex visible cell division capable of blocking in the S phase, thus is avoided entering the G2/M phase prematurely.Streaming quantitative analysis cell apoptosis situation is known (as shown in figure 12), and complex not only can change cellular morphology, also slightly can enter apoptosis in early days by inducing cell, but apoptosis degree is no more than 20%.Unitary analysis is known, the PAMAM dendrimers complex of this double antibody bag quilt can produce activity inhibition to the cancerous cell of catching to a certain extent, but does not produce serious cytotoxicity.
Table 2 is mtt assay CC G6 and the G6-5aEpCAM-3aSlex that analyzes embodiment 5 gained with concentration increase (5,10,20 μ g/ml) to the suppression ratio on HT29 cell-unit mole.
##under expression equal conditions compared with CC G6,
p<0.01.
Embodiment 6: the preparation of the magnetic nano-balls of antibody bag quilt
First carboxylated magnetic nanoparticle (MNPs-COOH) is prepared.Get end for amino, core be Fe
2o
3nano-particle, add succinic anhydride and react 2 h in dimethyl formamide (DMF) solution, through ethanol cyclic washing and ultra-pure water dialysis after, namely obtain water miscible MNPs-COOH.
Secondly, Anti-EpCAM antibody is covalently attached to MNPs-COOH surface.Get 5 mg MNPs-COOH, add 50 mM EDC and 50 mM NHS, in PBS (pH 6.8) solution of 1 ml 0.01 M, activate 30 min.MNPs-COOH after activation, after Magnetic Isolation, dissolves with the PBS (pH 7.2) of 1 ml 0.01 M, and rear and 50 μ g anti-EpCAM antibody Keep agitation react 4 h.After PBS (pH 7.2) cyclic washing, namely obtain the magnetic nano-balls of antibody bag quilt.
Embodiment 7: the preparation of the gold nanosphere of aptamers bag quilt
First gold nano grain (AuNPs) is prepared.Get 100 ml 1mM gold chloride (HAuCl4) vlil, after add 10 ml 38.8 mM sodium citrates and to reflux 20 min, namely prepare AuNPs.
Secondly, the DNA aptamers chain of sulfhydrylation is covalently attached to AuNPs surface.Such as get the aptamers of 9 μ l 1 mM, the acetum and 1.5 μ l 10 mM trichloroethyl phosphate (TCEP) that add 1 μ l 500 mM activate 1 h.The AuNPs added afterwards prepared by 3 ml reacts 16 h.The NaCl of the Tris acetic acid and 300 μ l 1M that finally add 30 μ l 500 ml reacts 24 h again.The aptamers of non-complexation, then through the centrifugal 15 min removings of 14000 rpm, namely prepares the gold nanosphere of aptamers bag quilt.
Claims (8)
1. a function nano material drug carrier system, is characterized in that: described function nano material drug carrier system is made up of center nano material carrier, surperficial targeting antibodies or aptamers, medicine that is anticancer or prophylaxis of cancer transfer.
2. function nano material drug carrier system according to claim 1, is characterized in that: described center nano material carrier is PAMAM dendrimers, gold nano grain AuNPs, nano silicon particles SiNPs, magnetic nanoparticle MNPs or graphene oxide graphene oxide.
3. function nano material drug carrier system according to claim 1, it is characterized in that: described surface targets can specific recognition and forming in conjunction with the targeting antibodies of circulating tumor cell surface biomarkers or aptamers by two or more to targeting antibodies or aptamers, and described biomarker is EpCAM, Sialyl Lewis X, HER2, EGFR, ALDH1 or CD44.
4. function nano material drug carrier system according to claim 1, is characterized in that: the medicine of described anticancer or prophylaxis of cancer transfer comprises ursolic acid and derivant, mifepristone and derivant thereof, captopril and derivant, emodin and derivant thereof.
5. function nano material drug carrier system according to claim 1, it is characterized in that: described center nano material carrier and surperficial targeting antibodies or aptamers adopt covalent attachment to carry out, and described covalent attachment is 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate/N-hydroxysuccinimide method, avidin/biotin method or N-hydroxysuccinimide/Maleimide chemistry method.
6. function nano material drug carrier system according to claim 1, is characterized in that: described function nano material drug carrier system by the medicine of described anticancer or prophylaxis of cancer transfer and center nano material carrier are carried out chemical coupling, Electrostatic Absorption, physically trapping mode prepare.
7. function nano material drug carrier system according to claim 2, is characterized in that: described center nano material carrier surface has reactive functionality, and described reactive functionality is-OH ,-NH
2,-COOH or-SH.
8. function nano material drug carrier system according to claim 5, it is characterized in that: described center nano material carrier adopts intermediate connector to be connected with surperficial targeting antibodies or aptamers, described intermediate connector is carboxylated Polyethylene Glycol, the Polyethylene Glycol of sulfhydrylation, hydrazide-polyethylene glycol-dithiol or OPSS-PEG-NHS.
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