阅读说明：本技术 叶酸功能化的链霉亲和素修饰的磁性纳米颗粒、其制备方法和应用 (Magnetic nanoparticle, preparation method and the application of the Streptavidin modification of folic acid functionalization ) 是由 杨延莲 郑望舒 李平 刘长亮 王琛 于 2018-05-17 设计创作，主要内容包括：本发明提供了一种叶酸功能化的链霉亲和素修饰的磁性纳米颗粒,所述纳米颗粒为使用亲水性羧基化超顺磁性纳米颗粒以链霉亲和素修饰、进而结合生物素标记的偶联聚乙二醇的叶酸分子制得。还提供了该纳米颗粒的制备方法和应用。本发明所述的纳米颗粒成本低,生物相容性好,为在临床血液样品中的应用提供可能。另外,本发明不同于传统的靶向上皮细胞粘附分子的循环肿瘤细胞分离技术,叶酸功能化磁性纳米颗粒可捕获上皮细胞粘附分子表达受限、恶性程度较高的肿瘤细胞,提供了一种富集、分离和检测循环肿瘤的快速、准确和低成本的检测技术,为临床肿瘤转移患者的预后判断、疗效监测、转移复发监测、早期检测等提供了有效方法。(The present invention provides a kind of magnetic nanoparticle of the Streptavidin of folic acid functionalization modification, the nano particle is to combine the folate molecule of the conjugated polyethylene glycol of biotin labeling to be made with Streptavidin modification, in turn using hydrophily carboxylated superparamagnetic nano particle.Additionally provide the preparation method and application of the nano particle.Nano particle of the present invention is at low cost, good biocompatibility, provides possibility for the application in clinical blood sample.In addition, the present invention is different from the circulating tumor cell isolation technics of traditional targeting epithelial cell adhesion molecule, folic acid functional magnetic nano particle can capture epithelial cell adhesion molecule and express limited, the higher tumour cell of grade malignancy, a kind of quick, accurate and inexpensive detection technique for being enriched with, separating and detect circulating tumor is provided, Index for diagnosis, curative effect monitoring, transfer and relapse monitoring, the early detection etc. for shifting patient for clinical tumor provide effective ways.)

1. the magnetic nanoparticle that a kind of Streptavidin of folic acid functionalization is modified, which is characterized in that the nano particle is Using hydrophily carboxylated superparamagnetic nano particle with Streptavidin modification and then in conjunction with the poly- second of coupling of biotin labeling The folate molecule of glycol is made.

2. the preparation method of nano particle according to claim 1, which is characterized in that the described method comprises the following steps:

(1) preparation of carboxylated magnetic nanoparticle: by FeCl₃·6H₂After O is dissolved in ethylene glycol formation clear solution, it is added anhydrous Sodium acetate and anhydrous sodium acrylate, heating are vigorously stirred, and form ultrasound after suspension, and carboxyl is made in the suspension after heating ultrasound Change magnetic nanoparticle；

(2) preparation of the magnetic nanoparticle of Streptavidin modification: carboxylated magnetic nanoparticle made from step (1) is turned It moves to and forms dispersion in buffer solution；It is right in above-mentioned magnetic nanoparticle dispersion that EDC and sulfo-NHS are dissolved in The carboxylated magnetic nanoparticle is activated；It is affine that magnetic nanoparticle after washing the activation is scattered in strepto- again The magnetic nanoparticle of Streptavidin modification is made in plain solution oscillating reactions；

(3) preparation of the magnetic nanoparticle of the Streptavidin modification of folic acid functionalization: strepto- made from step (2) is affine The magnetic nanoparticle of element modification is scattered in bovine serum albumen solution and the folic acid of the conjugated polyethylene glycol of biotin labeling point The magnetic nanoparticle of the Streptavidin modification of folic acid functionalization is made in sub common incubation.

3. according to the method described in claim 2, it is characterized in that, in the step (1), the FeCl₃·6H₂The ethylene glycol of O Solution concentration is 0.05M~0.5M, preferably 0.1M~0.2M, most preferably 0.125M.

4. according to the method in claim 2 or 3, which is characterized in that in the step (1), the heating temperature be 150~ 250 DEG C, preferably 180 DEG C~220 DEG C, most preferably 200 DEG C；The heating time be 4~10 hours, preferably 6-9 hours, Most preferably 8 hours.

5. method according to any one of claim 2 to 4, which is characterized in that in the step (2):

The buffer solution is selected from one or more of: MES buffer solution, PBS buffer solution；Preferably MES buffer solution； And/or

The solvent of the solution of streptavidin is PBS.

6. the method according to any one of claim 2 to 5, which is characterized in that in the step (3), the incubation temperature Degree is 4 DEG C~37 DEG C, preferably 4 DEG C -25 DEG C, most preferably 4 DEG C；The incubation time be 1 hour~16 hours, preferably 4 - 12 hours, most preferably 10 hours hour.

7. nano particle according to claim 1 or the nanometer prepared according to any one of claim 2 to 6 the method Application of the particle in drug and/or medical product of the preparation for circulating tumor cell capture.

8. application according to claim 7, which is characterized in that the tumour cell is that the tumour of folate receptor-positive is thin Born of the same parents；Preferably, the tumour cell is selected from one or more of: ovarian cancer tumor cell, Cervical Tumor cell, three feminine genders Breast cancer tumor cells, colon cancer tumours cell, non-small cell lung cancer tumour cell.

9. a kind of nano material for being used to capture circulating tumor cell based on folacin receptor, which is characterized in that the material packet Contain: according to claim 1 or according to nano particle prepared by any one of claim 2 to 6 the method；With And carrier or auxiliary material in need for capturing the circulating tumor cell.

10. a kind of kit for circulating tumor cell capture, which is characterized in that the kit includes according to claim Nano particle described in 1 or the nano particle prepared according to any one of claim 2 to 6 the method.

Technical field

The invention belongs to the liquid Biopsy fields based on functionalized nano material, and in particular to a kind of folic acid functionalization Streptavidin modification magnetic nanoparticle, and its preparation method and application.

Background technique

Although the circulating tumor cell capture technique based on targeting epithelial cell adhesion molecule achieves certain success, A possibility that being lost in the presence of the circulating tumor cell as caused by epithelial-mesenchymal conversion process, therefore it is viscous to need to establish epithelial cell Attached molecule supplemental markers library.Folacin receptor is a kind of glycosyl-phosphatidyl inositol receptor, and extensive overexpression is in oophoroma, uterine neck In the tumour cells such as cancer, triple negative breast cancer, colon cancer, non-small cell lung cancer, and its expression quantity level and cancer development degree Correlation, therefore be expected to as the target separated for circulating tumor cell.

At present the folacin receptor targeted therapy scheme applied to cancer and inflammation disease be based primarily upon to folacin receptor antibody, The research and development of folic acid conjugates and antifol, however its as effective circulating tumor cell marker in correlative study and Still sufficiently do not paid attention to and developed in clinical application.The commercialization lung cancer uniquely through CFDA approval existing now recycles Tumour cell detection -- folate receptor-positive circulating tumor cell detection kit (Shanghai Ge Nuo Biotechnology Co., Ltd) base In more complicated round pcr, by effectively combining the ratiometric conversion of number of probes and circulating tumor cell number of CTC final Obtain circulating tumor cell testing result.This technology can only carry out circulating tumor cell simply and preliminary technology, and nothing The intact form that method retains cell realizes further molecular biology characterization and analysis.

Summary of the invention

Therefore, the purpose of the present invention is to overcome the defects in the prior art, provides a kind of strepto- parent of folic acid functionalization The magnetic nanoparticle modified with element, and its preparation method and application.

Before illustrating the content of present invention, it is as follows to define term used herein:

Term " EDC " refers to: 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride.

Term " sulfo-NHS " refers to: N- hydroxy thiosuccinimide.

Term " PBS " refers to: phosphate buffered saline solution.

Term " MNP " refers to: magnetic nanoparticle.

Term " DiO " refers to: 3- octadecyl -2- [3- (3- octadecyl -2 (3H)-benzoxazoles -2- subunit) -1- third Alkene -1- base] benzoxazoles perchlorate.

Term " MES " refers to: 2-morpholine ethane sulfonic acid.

To achieve the above object, the first aspect of the present invention provides a kind of Streptavidin modification of folic acid functionalization Magnetic nanoparticle, the nano particle be using hydrophily carboxylated superparamagnetic nano particle with Streptavidin modification, And then the folate molecule of the conjugated polyethylene glycol of biotin labeling is combined to be made.

The second aspect of the present invention provides the preparation method of nano particle described in first aspect, which can be with The following steps are included:

(1) preparation of carboxylated magnetic nanoparticle: by FeCl₃·6H₂After O is dissolved in ethylene glycol formation clear solution, it is added Anhydrous sodium acetate and anhydrous sodium acrylate, heating are vigorously stirred, and form ultrasound after suspension, and the suspension after heating ultrasound is made Carboxylated magnetic nanoparticle；

(2) preparation of the magnetic nanoparticle of Streptavidin modification: by carboxylated magnetic Nano made from step (1) Grain, which is transferred in buffer solution, forms dispersion；EDC and sulfo-NHS are dissolved in above-mentioned magnetic nanoparticle dispersion In the carboxylated magnetic nanoparticle is activated；Magnetic nanoparticle after washing the activation is scattered in strepto- again The magnetic nanoparticle of Streptavidin modification is made in avidin solution oscillating reactions；

(3) preparation of the magnetic nanoparticle of the Streptavidin modification of folic acid functionalization: by strepto- made from step (2) The magnetic nanoparticle of Avidin modification is scattered in bovine serum albumen solution and the leaf of the conjugated polyethylene glycol of biotin labeling Acid molecule is incubated for jointly, and the magnetic nanoparticle of the Streptavidin modification of folic acid functionalization is made.

Preparation method according to a second aspect of the present invention, wherein in the step (1), the FeCl₃·6H₂The second two of O Alcoholic solution concentration is 0.05M~0.5M, preferably 0.1M~0.2M, most preferably 0.125M.

Preparation method according to a second aspect of the present invention, wherein in the step (1), the heating temperature be 150~ 250 DEG C, preferably 180 DEG C~220 DEG C, most preferably 200 DEG C；The heating time is 4~10 hours, and preferably 6~9 is small When, most preferably 8 hours.Preparation method according to a second aspect of the present invention, wherein in the step (2):

The buffer solution is selected from one or more of: MES buffer solution, PBS buffer solution；Preferably MES buffering Solution；And/or

The solvent of the solution of streptavidin is PBS.

Preparation method according to a second aspect of the present invention, wherein in the step (3), the incubation temperature is 4 DEG C~37 DEG C, preferably 4 DEG C~-25 DEG C, most preferably 4 DEG C；The incubation time is 1 hour~16 hours, and preferably 4 hours~12 is small When, most preferably 10 hours.

The third aspect of the present invention provides nano particle described in first aspect or according to method described in second aspect Application of the nano particle of preparation in drug and/or medical product of the preparation for circulating tumor cell capture.

Application according to a third aspect of the present invention, wherein the tumour cell is the tumour cell of folate receptor-positive；It is excellent Selection of land, the tumour cell are selected from one or more of: ovarian cancer tumor cell, Cervical Tumor cell, three negative breasts Cancerous swelling oncocyte, colon cancer tumours cell, non-small cell lung cancer tumour cell.

The fourth aspect of the present invention provides a kind of nanometer material for being used to capture circulating tumor cell based on folacin receptor Material, the material includes: according to first aspect or according to nano particle prepared by second aspect the method；With And carrier or auxiliary material in need for capturing the circulating tumor cell.

The fifth aspect of the present invention provides a kind of kit for circulating tumor cell capture, and the kit includes The nano particle according to first aspect or the nano particle prepared according to second aspect the method.

The present invention relates to the applications that folic acid functional magnetic nano particle captures circulating tumor cell specific recognition, originally The purpose of invention is to provide a kind of functionalized nano material and its in the capture of circulating tumor cell and the application of context of detection.Institute Hydrophily carboxylated superparamagnetic nano particle can be prepared in the technology of stating by the solvothermal method of optimization, and passes through EDC/ Sulfo-NHS engagement means Streptavidin modified outcome, and then combine the folic acid of the conjugated polyethylene glycol of biotin labeling Molecule (biotin-PEG-FA) realizes specific function, final to realize efficiently catching to the tumour cell of folate receptor-positive It obtains.The method of the present invention is simple, at low cost, and folic acid functional magnetic nano particle is with higher to circulating tumor cell sensitive Property and weaker non-specific adsorption；Enrich the catching method of circulating tumor cell now.

The application is exempted from for realizing the high magnetic to folate receptor-positive, the higher circulating tumor cell of grade malignancy Epidemic disease capture, improves sensitivity and purity.

The present invention provides a kind of method for preparing the functionalized nano material for efficient capture circulating tumor cell, described Method includes:

1) hydrophily carboxylated superparamagnetic nano particle is prepared by the solvothermal method optimized, and passed through EDC/sulfo-NHS engagement means Streptavidin modified outcome, and then combine the conjugated polyethylene glycol of biotin labeling Folate molecule (biotin-PEG-FA) realizes specific function.

2) it is incubated for jointly by the circulating tumor cell in folic acid functional magnetic nano particle and blood and is adding magnetic outside The process for carrying out Magnetic Isolation off field is realized and is captured to the high sensitivity and high specific of circulating tumor cell.

Specifically, the high magnetic of folate receptor-positive, the higher circulating tumor cell of grade malignancy is exempted from above-mentioned realization Epidemic disease catching method, comprising the following steps:

1) by FeCl₃·6H₂After O is dissolved in ethylene glycol formation clear solution, anhydrous sodium acetate and anhydrous sodium acrylate is added, Heating is vigorously stirred, and is formed ultrasound after suspension, is shifted and be sealed in four polyvinyl fluoride stainless steel cauldrons, slow from room temperature It is warming up to reaction temperature and continuous heating.

2) by magnetic nanoparticle in being transferred to buffer solution；EDC and sulfo-NHS are dissolved in above-mentioned magnetic Nano In particle dispersion system, violent oscillating reactions at room temperature.

3) magnetic nanoparticle of Streptavidin modification is scattered in bovine serum albumen solution the simultaneously idol of biotin labeling The folate molecule of connection polyethylene glycol is incubated for jointly.

4) tumour cell for taking culture is digested and is scattered in buffer solution to be unicellular, with DIO pre-dyed cytoplasma membrane.It takes In Tumor dispersal through sufficiently dyeing to the bovine serum albumen solution through folic acid functional magnetic nano particle.Tumour cell It is vibrated with magnetic nanoparticle after being incubated for altogether, it is isolated under the action of externally-applied magnetic field to be swollen by what magnetic nanoparticle captured Oncocyte, and it is observed, analyzed and is taken pictures using fluorescence microscope.

Preferably, tumour cell is lung carcinoma cell, breast cancer cell, cervical cancer cell, ovarian cancer cell, colon cancer cell Deng.

The magnetic nanoparticle of the Streptavidin modification of folic acid functionalization of the invention can have but be not limited to following The utility model has the advantages that

It is of the present invention that folic acid function used in the new method of specific recognition is carried out to tumor cell surface folacin receptor Magnetic nanoparticle can be changed, and the cost of material is low, good biocompatibility, provides possibility for the application in clinical blood sample.Separately Outside, the present invention is different from the circulating tumor cell isolation technics of traditional targeting epithelial cell adhesion molecule, folic acid functionalization magnetic Property nano particle can capture that epithelial cell adhesion molecule expression is limited, the higher tumour cell of grade malignancy, provide a kind of richness Collection, separation and quick, the accurate and inexpensive detection technique for detecting circulating tumor, the prognosis for shifting patient for clinical tumor are sentenced Disconnected, curative effect monitoring, transfer and relapse monitoring, early detection etc. provide effective ways.

Detailed description of the invention

Hereinafter, carrying out the embodiment that the present invention will be described in detail in conjunction with attached drawing, in which:

Fig. 1 shows carboxylated Fe₃O₄The transmission electron microscope micro-image of nano particle；Wherein Fig. 1 a shows unit length For the transmission electron microscope micro-image under 1 micron；It is the transmission electron microscope micro-image under 0.5 micron that Fig. 1 b, which shows unit length,.

Fig. 2 shows carboxylated Fe₃O₄The particle diameter distribution of nano particle；Wherein Fig. 2 a shows transmission electron microscope micro-image Statistical result, Fig. 2 b show dynamic light scattering statistical result.

Fig. 3 shows carboxylated Fe₃O₄Nano particle magnetic lag curve.

Fig. 4 shows carboxylated Fe₃O₄Nano particle ftir analysis；Wherein a is unmodified Fe₃O₄ Nano particle infrared absorption curve；B is carboxylated Fe₃O₄Nano particle infrared absorption curve；C is Streptavidin modification Fe₃O₄Nano particle infrared absorption curve；D is the Fe of folic acid functionalization₃O₄Nano particle infrared absorption curve.

Fig. 5 shows the variation that dynamic light scattering measures different classes of magnetic nanoparticle hydrated diameter in pure water；Its Middle a is unmodified Fe₃O₄The distribution of nano particle hydrated diameter in pure water；B is carboxylated Fe₃O₄Nano particle is in pure water The distribution of hydrated diameter；C is the Fe of Streptavidin modification₃O₄The distribution of nano particle hydrated diameter in pure water；D is folic acid The Fe of functionalization₃O₄The distribution of nano particle hydrated diameter in pure water.

Fig. 6 shows folic acid functional magnetic nano particle and captures to obtain the fluorescence microscope images of Hela cell；Wherein a is Optical imagery；B is the DIO pre-dyed living cells image of green fluorescence label；C is the superimposed image of a and b.

Specific embodiment

Present invention will be further explained by specific examples below, it should be understood, however, that, these embodiments are only It is used, is but should not be understood as present invention is limited in any form for specifically describing in more detail.

This part carries out general description to the material and test method that arrive used in present invention test.Although being It realizes many materials used in the object of the invention and operating method is it is known in the art that still the present invention still uses up herein It may detailed description.It will be apparent to those skilled in the art that within a context, if not specified, material therefor of the present invention and behaviour It is well known in the art as method.

Unless specifically stated otherwise, Cell line Hela used in following embodiment is purchased from the Chinese Academy of Medical Sciences.

Unless specifically stated otherwise, the solvent of aqueous solution used is sterile ultra-pure water solution in following embodiment.

Unless specifically stated otherwise, reagent used in following embodiment is analytical reagents.

Unless specifically stated otherwise, PBS solution used in following embodiment is 1 × PBS solution.

Reagent and instrument used in the following embodiment are as follows:

Reagent:

FeCl₃·6H₂O, ethylene glycol, anhydrous sodium acetate, anhydrous sodium acrylate, ethyl alcohol have purchased from Chinese medicines group chemical reagent Limit company；

MES buffer solution, EDC, sulfo-NHS, the solution of streptavidin of PBS, PBS, bovine serum albumen solution, RPMI-1640 culture medium is purchased from Thermo Fisher Scientific Inc.；

The coupling polyethylene glycol folate molecule of biotin labeling is purchased from upper sea PengShuo Biotechnology Co., Ltd；

Human acute myeloid leukaemia HL-60 cell, it is purchased from the Chinese Academy of Medical Sciences.

Instrument:

200KV lanthanum hexaboride transmission electron microscope is purchased from FEI Co., the U.S., model Tecnai G2 20S-TWIN；

Nano particle size and Zeta potential analyzer are purchased from Malvern Instr Ltd., Britain, model Zetasizer Nano ZS；

Analysis of Physical instrument (PPMS) magnetometer is purchased from Quantum Design, Inc., the U.S., model PPMS-9；

Fourier Transform Infrared Spectrometer is purchased from U.S. Perkin Elmer InstrumentsCo.Ltd., model Spectrum One；

Flow cytometer is purchased from U.S. BD Medical Devices Co., Ltd., model Accuri C6；

Fluorescence microscope is purchased from Olympus Co., Ltd, model IX75.