阅读说明：本技术 一种PEG/MoS2量子点复合荧光纳米微球及其应用 (A kind of PEG/MoS2Quantum dot composite fluorescent nanosphere and its application ) 是由 董建 刘莉 董立峰 于 2019-07-11 设计创作，主要内容包括：本发明公开了一种PEG/MoS_2量子点(MoS_2-PEG)复合荧光纳米微球,其制备方法以及应用。所述纳米微球包括PEG基质以及嵌入PEG基质的MoS_2量子点。制备方法包括MoS_2量子点的制备和纳米微球MoS_2-PEG的制备。应用为以MoS_2-PEG为载体负载抗肿瘤药物。本发明提供的纳米微球利用肿瘤组织的EPR效应将药物被动地富集到肿瘤组织,提高疗效同时降低全身毒性；制备方法过程简单,条件温和,制备周期短,合成过程无有害物质产生。借助MoS_2-PEG纳米微球和抗肿瘤药物自身荧光实现同步示踪功能；该载药系统体外药物释放具有明显pH响应性,载药量大,在抗肿瘤药物传输领域可广泛应用。(The invention discloses a kind of PEG/MoS 2 Quantum dot (MoS 2 - PEG) composite fluorescence nanosphere, preparation method and application.The nanosphere includes PEG group matter and the MoS for being embedded in PEG group matter 2 Quantum dot.Preparation method includes MoS 2 The preparation of quantum dot and nanosphere MoS 2 The preparation of-PEG.Using for MoS 2 - PEG is carrier loaded anti-tumor drug.Drug is passively enriched to tumor tissues using the EPR effect of tumor tissues by nanosphere provided by the invention, and improve curative effect reduces general toxicity simultaneously；Preparation method process is simple, mild condition, short preparation period, and synthesis process unharmful substance generates.By MoS 2 - PEG nanosphere tracking function synchronous with the realization of anti-tumor drug autofluorescence；The drug-loading system vitro drug release has obvious pH responsiveness, and drugloading rate is big, can be widely applied in anti-tumor drug transmission field.)

1. a kind of PEG/MoS₂Quantum dot composite fluorescent nanosphere, which is characterized in that the nanosphere include PEG group matter with And multiple MoS of insertion PEG group matter₂Quantum dot.

2. a kind of PEG/MoS according to claim 1₂Quantum dot composite fluorescent nanosphere, which is characterized in that described PEG/MoS₂The average grain diameter of quantum dot composite fluorescent nanosphere is 123.5nm.

3. a kind of PEG/MoS according to claim 1₂Quantum dot composite fluorescent nanosphere, which is characterized in that described PEG/MoS₂The particle size range of quantum dot composite fluorescent nanosphere is 65-185nm.

4. a kind of PEG/MoS₂The preparation method of quantum dot composite fluorescent nanosphere, which is characterized in that the preparation method packet It includes:

Step 1: MoS₂The preparation of quantum dot: by Na₂MoO₄·2H₂O is dissolved in the water of certain volume, concentration 0.04mol/ L, after being ultrasonically treated 5min, it is 6.5 that solution, which is adjusted to pH value, with 0.1M HCl, and glutathione and two is then added into solution The water of times original volume, the mole of glutathione are Na₂MoO₄·2H₂4 times of O mole are ultrasonically treated 10min and are passed through Mixture is finally transferred in the stainless steel autoclave of teflon lining and reacts for 24 hours at 200 DEG C by argon gas；Natural cooling To room temperature, the speed centrifugation 5min of 9000rpm collects supernatant, and the bag filter for being 500~1000 with molecular cut off is in pure water Middle dialysis 12~48h, every 2~4h change a dialyzate；MoS is obtained after solution after dialysis is freeze-dried 3~4 days₂Quantum Point；

Step 2: MoS₂The preparation of-PEG nanosphere:

S1, by MoS made from step 1₂Quantum dot is scattered in solvent, and coupling agent is added, and ultrasonic treatment is stirred at room temperature, obtained To suspension；

S2, the suspension for obtaining S1 carry out first time dialysis；

S3, polyethylene glycol is added into the solution after dialysis, stirs, obtains suspension；

S4, the suspension for obtaining S3 carry out second and dialyse, and are then freeze-dried, obtain MoS₂- PEG nanosphere.

5. a kind of PEG/MoS according to claim 4₂The preparation method of quantum dot composite fluorescent nanosphere, feature exist In solvent is selected from PBS buffer solution, methanol and ethyl alcohol, MoS in the step 2 S1₂The dispersion of quantum dot in a solvent is dense Degree is 1~6mg/mL.

6. a kind of PEG/MoS according to claim 4₂The preparation method of quantum dot composite fluorescent nanosphere, feature exist In, coupling agent is glutaraldehyde in the step 2 S1, preferred concentration 2.5%, the molar ratio of glutaraldehyde and PEG be (1~ 1.5):1。

7. a kind of PEG/MoS according to claim 5₂The preparation method of quantum dot composite fluorescent nanosphere, feature exist In using double amino-terminated polyethylene glycol in the step 2 S3, average molecular weight range is 800~2500, with poly- second two The form of the PBS solution of alcohol is added, and Polyethylene glycol is 1~6mg/mL；

MoS in gained mixed liquor in the step 2 S3₂The mass ratio of quantum dot and PEG are (0.5~4): 1, stirring 8~for 24 hours.

8. a kind of PEG/MoS according to claim 4₂The preparation method of quantum dot composite fluorescent nanosphere, feature exist In, in the step 2 S1 be ultrasonically treated 5~30min, at room temperature stir 6~for 24 hours；

Dialysis uses molecular cut off to dialyse in pure water for 500~1000 bag filter for the first time in the step 2 S2 12~48h, every 2~4h change a dialyzate to remove free glutaraldehyde；

Molecular cut off is used to carry out second of dialysis 12 in pure water for 1000~3500 bag filter in the step 2 S4 ~48h, every 2~4h change a dialyzate to remove free PEG；

MoS is obtained after solution after dialysis is freeze-dried 3~4 days₂- PEG nanosphere.

9. a kind of PEG/MoS₂The application of quantum dot composite fluorescent nanosphere, which is characterized in that with MoS₂- PEG nanosphere is Carrier loaded anti-tumor drug.

10. a kind of PEG/MoS according to claim 9₂The application of quantum dot composite fluorescent nanosphere, which is characterized in that The anti-tumor drug includes but is not limited to adriamycin.

Technical field

The invention belongs to nano biological field of medicaments, are related to a kind of PEG/MoS₂Quantum dot composite fluorescent nanosphere and its Using.

Background technique

Cancer is to seriously threaten the malignant tumour of human life and health.The whole world increases 18,100,000 cancers newly within 2018 Example, for death toll up to 9,600,000, cancer has become global public health problem.And the China as populous nation, cancer Disease The dead quantity more occupy first place in the world.According to " Cancer in China report in 2018 " display, China is average true more than 10,000 people daily It examines as cancer, there are 7 people to be diagnosed as cancer per minute.How to cure cancer is the huge challenge that modern medicine faces.At present Clinically the treatment of tumour is still based on three kinds of operation, radiation and chemotherapy methods, and wherein chemotherapy is the most commonly used means.But It is traditional chemotherapy there are side effects serious, pharmacological dependence and the problems such as multidrug resistance (MDR), leads to its therapeutic effect not It is ideal.

In recent decades, with the development of nanotechnology, a variety of nano materials such as carbon nanotube, graphene oxide and fowler Alkene etc. has developed into conveying of the pharmaceutical carrier for anti-tumor drug.The water solubility and life of drug can be improved in these nano-carriers Object availability, and drug is passively enriched to tumor group using the high-permeability of tumor tissues and retention effect (EPR effect) It knits, and then improve curative effect to reduce general toxicity simultaneously.Nano-medicament carrier can also be conveyed drug by way of active transport To tumour cell, so that the outlet approach of Multidrug resistance tumour cell is blocked, thus efficiently against the Multidrug resistance of tumour.Separately Outside, there is the nano-carrier of stimulating responsive to be expected to control the release of drug on time, space and dosage for design and assembly, more It is easy to implement accurate treatment.PH is most common endogenous stimulus in current treatment of cancer and medicine controlled releasing.With normal tissue and Blood (pH 7.4) is compared, and acidic micro-environment (pH 6.0-7.0) caused by anaerobic sugar is degraded is the one of the kinds of tumors such as solid tumor A notable feature.PH value can further decrease (pH 3.0- in the acidic organelles such as endosome and lysosome in cancer cell 5.5), this is also of great significance to medicine controlled releasing in tumour.But phase so far, is treated with drug conveying and stimulating responsive The Intelligent fluorescent imaging research of pass is rarely reported.Most of nano-medicament carriers itself do not have tracking function, only by with Fluorescent marker or the compound of contrast agent are just able to achieve its positioning in cell.It would therefore be highly desirable to develop a kind of collection fluorescence imaging, The nano-medicament carrier that anti-tumor drug conveying and pH response medicine release function are integrated.Molybdenum disulfide quantum dot has Fluorescence is strong, stability is good, good water solubility, it is nontoxic the advantages that, be with a wide range of applications in field of biomedicine.Currently, MoS₂The research of quantum dot is concentrated mainly on bio-imaging, and its application in terms for the treatment of of cancer is still in early-stage development rank Section, there is not yet MoS₂It is one that quantum dot, which is applied to fluorescence imaging, anti-tumor drug conveying and pH response medicine release function, The report of the nano-medicament carrier of body.

Summary of the invention

It is an object of the invention to provide a kind of PEG/MoS in place of overcome the deficiencies in the prior art₂Quantum dot is compound glimmering Light nanosphere and application, with PEG/MoS₂Quantum dot composite fluorescent nanosphere is that carrier conveys anti-tumor drug, by it The fluorescence of itself realizes synchronous tracking function, and can realize that the pH response medicine in tumour cell discharges.The present invention provides such as Lower technology:

One of the objects of the present invention is to provide a kind of PEG/MoS₂Quantum dot composite fluorescent nanosphere；

The second object of the present invention is to provide a kind of PEG/MoS₂The preparation method of quantum dot composite fluorescent nanosphere；

The third object of the present invention is to provide a kind of PEG/MoS₂The application of quantum dot composite fluorescent nanosphere.

In order to achieve the above objectives, it is described that technical scheme is as follows:

The first aspect of the invention provides a kind of PEG/MoS₂Quantum dot composite fluorescent nanosphere, the nanometer are micro- Ball includes PEG group matter and the multiple MoS for being embedded in PEG group matter₂Quantum dot.

Preferably, the PEG/MoS₂The average grain diameter of quantum dot composite fluorescent nanosphere is 123.5nm.

Preferably, the PEG/MoS₂The particle size range of quantum dot composite fluorescent nanosphere is 65-185nm.

The second aspect of the invention provides a kind of PEG/MoS₂The preparation method of quantum dot composite fluorescent nanosphere, The preparation method includes:

Step 1: MoS₂The preparation of quantum dot: by Na₂MoO₄·2H₂O is dissolved in the water of certain volume, and concentration is Solution is adjusted to pH=6.5 with 0.1M HCl, gluathione is then added into solution by 0.04mol/L after being ultrasonically treated 5min Peptide (GSH) and the water for being twice in original volume, the mole of glutathione are Na₂MoO₄·2H₂4 times of O mole, ultrasonic treatment 10min is simultaneously passed through argon gas, is finally transferred to mixture in the stainless steel autoclave of teflon lining and reacts at 200 DEG C 24h.The speed centrifugation 5min of cooled to room temperature, 9000rpm collects supernatant, is 500~1000 with molecular cut off Bag filter is dialysed 12~48h in pure water, and every 2~4h changes a dialyzate.After solution after dialysis is freeze-dried 3~4 days Obtain MoS₂Quantum dot；

Step 2: MoS₂The preparation of-PEG nanosphere:

S1, by MoS₂Quantum dot is scattered in PBS buffer solution, and coupling agent is added, and ultrasonic treatment is stirred at room temperature, obtained Suspension；

S2, the suspension for obtaining S1 carry out first time dialysis；

S3, polyethylene glycol is added into the solution after dialysis, stirs, obtains suspension；

S4, the suspension for obtaining S3 carry out second and dialyse, and are then freeze-dried, obtain MoS₂- PEG nanosphere.

Wherein, the PBS buffer solution is as MoS₂The solvent of quantum dot, plays the effect of dispersion, and the solvent can be used Alcohols such as methanol, ethyl alcohol replace.

Preferably, MoS in the step 2 S1₂Dispersion concentration of the quantum dot in PBS buffer solution is 1~6mg/ mL；

Preferably, coupling agent is glutaraldehyde in the step 2 S1, and preferred concentration 2.5%, the molar ratio with PEG is (1~1.5): 1.

Preferably, in the step 2 S1 be ultrasonically treated 5~30min, at room temperature stir 6~for 24 hours.

Preferably, it is 500~1000 bag filter pure that dialysis, which uses molecular cut off, for the first time in the step 2 S2 12~48h of dialysis is carried out in water, every 2~4h changes a dialyzate to remove free glutaraldehyde.

Preferably, using double amino-terminated polyethylene glycol in the step 2 S3, average molecular weight range is 800~ 2500, it is added in the form of the PBS solution of polyethylene glycol, Polyethylene glycol is 1~6mg/mL；

Preferably, in the step 2 S3 gained mixed liquor in MoS₂The mass ratio of quantum dot and PEG are (0.5~4): 1, Stirring 8~for 24 hours.

Preferably, molecular cut off is used to carry out in pure water for 1000~3500 bag filter in the step 2 S4 Secondary dialysis 12~48h, every 2~4h change a dialyzate to remove free PEG；By the solution freeze-drying 3~4 after dialysis MoS is obtained after it₂- PEG nanosphere.

MoS₂The formation mechenism of-PEG nanosphere is analyzed: MoS₂Quantum dot surface during preparation is passivated through GSH, Therefore its surface is rich in amino.Using glutaraldehyde as coupling agent, the aldehyde radical at both ends respectively with the Amino End Group of PEG and MoS₂Quantum dot The amino on surface acts on forming schiff bases, realizes PEG in MoS in a manner of covalent bond₂The link of quantum dot surface.In addition, In Two MoS inevitably occur during reaction₂With PEG for " bridge " between quantum dot, by two Amino End Group of PEG respectively with two A MoS₂Amino covalence effect and the crosslinking of formation to a certain extent on quantum dot, to finally obtain a kind of MoS₂Quantum dot The special construction inlayed in PEG.

Using transmission electron microscope (TEM) to MoS₂The microscopic appearance and lattice of-PEG is characterized, MoS₂- PEG's Average grain diameter is 123.5nm, by multiple MoS₂Quantum dot be embedded in PEG group texture at；In general, the nanoparticle that size is several nanometers Son is easy to be discharged from kidney, and is easy to be retained by liver and spleen having a size of several hundred nanometers of particle.Correlative study shows The optimum size of EPR effect is improved in 100-200 nanometer range.From the perspective of partial size, the MoS of preparation₂- PEG nanometers EPR effect can be improved in the carrier that microballoon is used as drug conveying, and by drug-rich to tumor tissues, and then raising curative effect reduces simultaneously General toxicity.

MoS₂The ultraviolet and fluorescence spectrum of-PEG shows MoS₂- PEG has stable fluorescence within the scope of pH=4-12 Matter.MoS₂- PEG is interior at a wider pH range to have good fluorescent stability, this is most important to subsequent cell imaging.

In stability study under physiological environment, by MoS₂Quantum dot or MoS₂- PEG is scattered in water, PBS and thin respectively In born of the same parents' culture medium, observes it and stand the stability after different time.The result shows that MoS₂Stability of-the PEG under physiological environment It is substantially better than MoS₂Quantum dot.

Cell toxicity test shows MoS₂The cytotoxicity of-PEG is compared with MoS₂Quantum dot is substantially reduced, and works as MoS₂- PEG's is dense Without overt toxicity when degree is lower than 0.2mg/mL, when it is at concentrations up to 1.5mg/mL, cell survival rate is still up to 82%, it is ensured that Fluorescence imaging and the safety for carrying medicine.

The third aspect provided by the invention provides a kind of PEG/MoS₂The application of quantum dot composite fluorescent nanosphere, tool Body is with MoS₂- PEG is carrier, loads anti-tumor drug.

The anti-tumor drug includes but is not limited to adriamycin, with MoS₂- PEG is carrier loaded adriamycin (DOX), is formed Drug-loading system (MoS₂- PEG-DOX), wherein the load capacity of DOX is up to 71.6%.

Preparation process are as follows: take DOX to be dissolved in the concentration for being made into 1mg/mL in PBS solution (pH value be 7.4 or 9.0), then plus Enter MoS₂In-PEG solution (5mg/mL), it is protected from light stirring at room temperature for 24 hours；By MoS₂- PEG-DOX solution is placed in molecular cut off In the bag filter of Mw=3500, dialyse 48h in PBS solution, and every 4h replaces a PBS solution, is freeze-dried to obtain MoS₂-PEG- DOX solid.

MoS₂The vitro drug release of-PEG-DOX have apparent pH responsiveness, i.e., under acid condition (pH=5.0 and 6.0, simulate the microenvironment of cancer cell) DOX release rate and rate of release be apparently higher than pH=7.4 (simulation normal cell it is micro- Environment) when release rate.As it can be seen that MoS₂- PEG is expected to become a kind of pharmaceutical carrier of novel pH responsiveness.

Based on MoS₂- PEG nanosphere and the intrinsic fluorescence of DOX itself can be realized to synchronize and show while conveying drug Track function, the i.e. release of detection drug DOX and positioning carrier MoS₂-PEG。

The beneficial effects of the present invention are:

(1) a kind of new structural PEG/MoS is provided₂Quantum dot composite fluorescent nanosphere, average grain diameter are 123.5nm.Correlative study shows the optimum size for improving high-permeability and retention effect (EPR effect) in 100-200 nanometers of models In enclosing.From the perspective of partial size, the MoS of preparation₂- PEG is more suitable as the carrier of drug conveying, utilizes tumor tissues Drug is passively enriched to tumor tissues by EPR effect, and then is improved curative effect while being reduced general toxicity.

(2) preparation method technical process provided by the invention is simple, mild condition, short preparation period, and synthesis process is without having Evil substance generates.

(3) nanosphere MoS₂- PEG is interior at a wider pH range to have good fluorescent stability, under physiological environment Stability be substantially better than MoS₂Quantum dot, cytotoxicity are low, it is ensured that fluorescence imaging and the safety for carrying medicine.

(4) provided by the invention with MoS₂- PEG is carrier, the drug-loading system of anti-tumor drug is loaded, by MoS₂-PEG The fluorescence of nanosphere and anti-tumor drug itself realizes synchronous tracking function；And the drug-loading system vitro drug release has Apparent pH responsiveness, drugloading rate is big, can be widely applied in the transmission field of anti-tumor drug.

Detailed description of the invention

The Figure of description for constituting a part of the invention is used to provide further understanding of the present invention, and of the invention shows Examples and descriptions thereof are used to explain the present invention for meaning property, does not constitute improper limitations of the present invention.

Fig. 1 is nanosphere MoS provided by the invention₂The structural schematic diagram of-PEG；

Fig. 2 is nanosphere MoS provided by the invention₂The microscopic appearance and grain size distribution of-PEG；

Fig. 3 is MoS₂Quantum dot, PEG and MoS₂The XRD diagram of-PEG；

Fig. 4 is MoS₂Quantum dot, PEG and MoS₂The FT-IR of-PEG schemes；

Fig. 5 is MoS₂Quantum dot and MoS₂The xps energy spectrum figure of-PEG；

Fig. 6 is MoS₂The ultraviolet and fluorescence spectrum of-PEG；

Fig. 7 is MoS₂The fluorescence intensity curves figure of-PEG at different pH；

Fig. 8 is MoS₂Quantum dot and MoS₂- PEG the stability in aqueous solution, PBS solution and cell culture fluid respectively Schematic diagram；

Fig. 9 is MoS₂- PEG and MoS₂The cytotoxicity of quantum dot compares figure；

Figure 10 is MoS₂- PEG nanosphere carries medicine schematic diagram；

Figure 11 is MoS₂Hydrogen bond action between-PEG nanosphere and DOX；

Figure 12 is MoS₂The release in vitro behavior schematic diagram of-PEG-DOX at different pH；

Figure 13 is to use MoS₂The laser co-focusing photo of-PEG-DOX incubation U251 cell 2h, 8h and 16h.

Wherein, (a) is nanosphere MoS in Fig. 2₂The TEM photo of-PEG；It (b) is nanosphere MoS₂The partial size of-PEG point Butut；It (c) is single MoS₂The TEM photo of-PEG；It (d) is nanosphere MoS₂- PEG internal structure HRTEM photo；

(a) is MoS in Fig. 5₂The Mo 3d energy spectrum diagram of quantum dot；It (b) is MoS₂The S 2p energy spectrum diagram of quantum dot；(c) it is MoS₂The Mo 3d energy spectrum diagram of-PEG；It (d) is MoS₂The S 2p energy spectrum diagram of-PEG；

(a) is MoS in Figure 13₂- PEG is by 405nm laser excitation, the collecting signal within the scope of 450 ± 50nm；It (b) is DOX By 488nm laser excitation, the collecting signal within the scope of 595 ± 50nm；It (c) is MoS₂The blending image of-PEG and DOX.

Specific embodiment

It is noted that described further below be all exemplary, it is intended to provide further instruction to the present invention.Unless another It indicates, all technical and scientific terms used herein has usual with general technical staff of the technical field of the invention The identical meanings of understanding.

It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root According to exemplary embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singular Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet Include " when, indicate existing characteristics, step, operation, device, component and/or their combination.