阅读说明：本技术 氧化石墨烯药物载体及其制备方法和应用 (Graphene oxide drug carrier and preparation method and application thereof ) 是由 冯晓黎 郭伟洪 陈志安 陈祈月 张雅晴 于 2020-04-03 设计创作，主要内容包括：本发明提供一种氧化石墨烯药物载体及其制备方法和应用,该氧化石墨烯药物载体的制备方法为：(1)在氧化石墨烯的分散液中加入聚乙二醇双胺,在活化剂的作用下进行酰胺缩合反应得到GO-PEG；(2)将GO-PEG溶于溶剂中配置成GO-PEG分散液,加入氧化海藻酸钠,在活化剂作用下进行席夫碱反应；反应结束后进行纯化、干燥,得到氧化石墨烯药物载体GO-PEG-OSA。将GO-PEG-OSA分散在溶剂中配置成GO-PEG-OSA分散液,加入药物溶液,搅拌10～20h,离心,得到纳米药物。本发明的药物载体和纳米药物对pH和光热敏感,具有“pH/温热”双重智能响应释放特性,具有优良的生物相容性、稳定性和水溶性,可高效快速携带药物进入病灶。(The invention provides a graphene oxide drug carrier and a preparation method and application thereof, wherein the preparation method of the graphene oxide drug carrier comprises the following steps: (1) adding polyethylene glycol diamine into the dispersion liquid of the graphene oxide, and carrying out amide condensation reaction under the action of an activating agent to obtain GO-PEG; (2) dissolving GO-PEG in a solvent to prepare GO-PEG dispersion, adding oxidized sodium alginate, and performing Schiff base reaction under the action of an activating agent; and after the reaction is finished, purifying and drying to obtain the graphene oxide drug carrier GO-PEG-OSA. Dispersing GO-PEG-OSA in a solvent to prepare GO-PEG-OSA dispersion, adding a medicinal solution, stirring for 10-20 h, and centrifuging to obtain the nano-medicament. The drug carrier and the nano-drug are sensitive to pH and photo-thermal, have the characteristics of pH/warm dual intelligent response and release, have excellent biocompatibility, stability and water solubility, and can efficiently and quickly carry the drug to enter a focus.)

1. A preparation method of a graphene oxide drug carrier is characterized by comprising the following steps: the method comprises the following steps:

(1) adding polyethylene glycol diamine into the dispersion liquid of the graphene oxide, and carrying out amide condensation reaction under the action of an activating agent to obtain GO-PEG;

(2) dissolving GO-PEG in a solvent to prepare GO-PEG dispersion, adding oxidized sodium alginate, and performing Schiff base reaction under the action of an activating agent; and after the reaction is finished, purifying and drying to obtain the graphene oxide drug carrier GO-PEG-OSA.

2. The method of claim 1, wherein: in the steps (1) and (2), the activating agent is a mixture of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

3. The method of claim 1, wherein: the temperature of the amide condensation reaction is 30-40 ℃.

4. The method of claim 1, wherein: the pH value of the dispersion liquid of the graphene oxide is 5-6.

5. The method of claim 1, wherein: the temperature of the Schiff base reaction is 10-30 ℃.

6. A graphene oxide drug carrier is characterized in that: the preparation method of the compound is characterized by comprising the following steps of 1-5.

7. Use of the graphene oxide drug carrier of claim 6 for the preparation of a drug for hyperthermia.

8. A method for preparing nano-drugs is characterized in that: the method comprises the following steps: dispersing the graphene oxide drug carrier of claim 6 in a solvent to prepare a graphene oxide drug carrier dispersion liquid, adding the drug, stirring for 10-20 h, and centrifuging to obtain the nano-drug.

9. The method of claim 8, wherein: the medicine is paclitaxel, adriamycin or platinum chemotherapeutic medicine.

10. A nano-drug characterized by: prepared by the preparation method of claim 8 or 9.

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to a graphene oxide drug carrier and a preparation method and application thereof.

Background

The traditional treatment scheme of malignant tumor mainly comprises surgical excision, chemotherapy and radiotherapy. Most patients are diagnosed at a locally intermediate or advanced stage, and chemotherapy is one of the main inevitable approaches. In addition to the side effects of chemotherapy on normal tissues of the patient's body, tumor resistance is a significant problem that cannot be ignored clinically. Once patients develop chemotherapy resistance, only chemotherapy drug replacement or dosage increase is needed, which undoubtedly aggravates the side effects of patients, reduces the compliance of treatment, and the curative effect is still uncertain. In order to overcome the drug resistance of the tumor, improve the tumor treatment effect and prolong the life cycle of patients, various schemes such as targeted therapy, photodynamic thermotherapy, immunotherapy and the like are clinically provided, but the use of the schemes is limited by the defects of few applicable groups, unstable curative effect and the like, and the comprehensive treatment scheme with low price, safety, reliability and high efficiency is difficult to realize clinically.

Paclitaxel (PTX) is a conventional chemotherapeutic drug for malignant tumors and is widely applied clinically, but because of the characteristics of poor water solubility and difficult oral absorption, paclitaxel is often limited to intravenous injection. Paclitaxel in blood circulation after intravenous injection lacks tumor targeting, so that not only is the focus difficult to reach effective controllable drug concentration, but also systemic toxic and side effects such as allergy, bone marrow suppression, neurotoxicity and the like are often caused. Moreover, due to the occurrence of tumor drug resistance in the long-term chemotherapy process, the dosage of the taxol can only be increased to achieve the expected curative effect, which further causes the toxic and side effects to be increased. This is a real problem that plagues clinical tumor treatment for a long time.

The nanotechnology-based drug delivery system provides opportunities for improving PTX antitumor efficacy, PTX-loaded nanomedicines have advantages over conventional drugs in terms of improved drug solubility and bioavailability, intelligent drug delivery is achieved by enhancing permeability and retention, and toxic side effects are reduced. For example, the novel preparation of albumin-bound paclitaxel takes nanoparticle albumin as a carrier, optimizes the water solubility of paclitaxel, improves the drug effect of tumor sites through an albumin receptor (Gp60) cell-penetrating pathway and a tumor extracellular matrix acidic Secreted Protein (SPARC) pathway, and is clinically used at present. Although the albumin-bound paclitaxel can increase the uptake of the drug in tumor cells, the endocytosed paclitaxel is re-pumped out by the drug-resistant glycoprotein (P-gpperotein) on the surface of the tumor cells, so that the current clinical nano-drugs still do not completely solve the problem of drug resistance of tumors to chemotherapeutic drugs. In order to maximize the antitumor benefit of paclitaxel, a new paclitaxel nano delivery scheme which has low cost, safety, high efficiency, low toxic and side effects, can aim at a target P-gp protein and overcomes tumor resistance is urgently needed in clinic.

P-gp protein is a member of ATP energy-dependent protein family, and ATP generation is dependent on mitochondrial redox respiratory chain to a great extent, so that damage to tumor cell mitochondria and blocking of ATP energy supply are potential means for inhibiting P-gp protein function. Research shows that photothermal therapy can generate a large amount of Reactive Oxygen Species (ROS) metabolites under the excitation of near infrared light, attack mitochondria and promote cancer cell apoptosis; meanwhile, the photothermal therapy can also heat local temperature, enhance the penetration and absorption of chemotherapeutic drugs in local cancer, and reverse the drug resistance to the chemotherapeutic drugs by inhibiting DNA damage repair. Therefore, chemotherapy based on a nano-drug delivery system and high-efficiency combination of near-infrared light (808nm wavelength) excitation and induction of photodynamic/photothermal can enhance intracellular concentration of paclitaxel, complete intelligent drug release under a warm condition, and even can be used as a promising strategy for reversing tumor drug resistance. However, most of the materials of the existing nano-drug delivery system only have the drug loading capability, but the materials do not have the treatment function, so that a plurality of drugs need to be loaded simultaneously to achieve the efficacy of synchronous heat and chemotherapy, the method has relatively high operation difficulty, and the synthesized nano-materials also lack biological stability. Therefore, the search for a material that can carry chemotherapeutic drugs and has the dual effect of 'light sensation/heat' is a breakthrough to solve the clinical problem.

Graphene Oxide (GO) is a biomaterial with considerable potential in recent years, and has been widely used in the biomedical field due to its unique lamellar structure, high drug loading and plasma membrane crossing ability, and high modifiability. Meanwhile, GO also has stronger photo-thermal conversion capability, especially in a near-infrared light wavelength region, generates stronger absorption and thermal conversion effects on light, and the generated heat can improve the temperature around a tumor focus, thereby indirectly or directly killing tumor cells. Therefore, theoretically, the nano-drug formed by combining GO and PTX is expected to improve the tumor treatment effect and the chemotherapy resistance in combination with the dual mode of thermo-chemotherapy. However, currently pure GO nanomaterials tend to aggregate in protein-rich or saline-rich environments (such as cell culture media and serum), and thus exhibit corresponding dose-dependent toxicity. To improve this biomaterial, the most effective approach is surface coating modification of GO by covalent or non-covalent conjugation. Polyethylene glycol diamine (PEG) is a commonly used modifying agent in biology due to its high solubility in organic solvents, minimal toxicity and protein resistance. Studies have found that PEG-modified GO (GO-PEG) nanoparticles can exhibit higher stability and better biocompatibility; the PTX @ GO-PEG nanoparticle loaded with PTX can generate better anti-tumor effect compared with the pure PTX.

In tumor organisms, the microenvironment of tumor foci is much different from that of normal tissues, and the pH value (pH 5.5) is often much lower than that of normal sites (pH 7.4). In order to realize intelligent accelerated administration of chemotherapeutic drugs in a tumor region, a drug release nano-drug triggered by low pH can be designed, and the material with the drug acidic release performance has good clinical application prospect. However, the PTX @ GO-PEG nanoparticles lack the ability to respond to external pH stimuli, and how to optimize the nano-drug and customize drug release to form precise treatment is another problem at present.

Disclosure of Invention

The invention aims to provide a graphene oxide drug carrier and a preparation method and application thereof.

The technical scheme adopted by the invention is as follows:

a preparation method of a graphene oxide drug carrier comprises the following steps:

(1) adding polyethylene glycol diamine into the dispersion liquid of the graphene oxide, and carrying out amide condensation reaction under the action of an activating agent to obtain GO-PEG;

(2) dissolving GO-PEG in a solvent to prepare GO-PEG dispersion, adding oxidized sodium alginate, and performing Schiff base reaction under the action of an activating agent; and after the reaction is finished, purifying and drying to obtain the graphene oxide drug carrier GO-PEG-OSA.

In the steps (1) and (2), the activating agent is a mixture of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

The mass ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) to the N-hydroxysuccinimide (NHS) is (0.5-1): (1-2).

The temperature of the amide condensation reaction is 30-40 ℃.

The time of the amide condensation reaction is 10-20 h. In some preferred embodiments, the time for the amide condensation reaction is 12 hours.

The pH value of the dispersion liquid of the graphene oxide is 5-6.

The mass ratio of the graphene oxide to the polyethylene glycol diamine is (0.01-0.05): (40-50).

The particle size of the graphene oxide is 50-200 nm.

The temperature of the Schiff base reaction is 10-30 ℃. In some preferred embodiments, the schiff base reaction is optionally carried out at room temperature.

The reaction time of the Schiff base is 10-20 h. In some preferred embodiments, the schiff base is reacted for a period of 12 hours.

The pH value of the GO-PEG dispersion liquid is 8-8.5.

The mass ratio of the GO-PEG to the oxidized sodium alginate is (0.01-0.02): (10-20).

The molecular weight of the GO-PEG-OSA is less than or equal to 3500.

The purification method is a dialysis method.

A graphene oxide drug carrier is obtained by the preparation method.

The graphene oxide drug carrier is applied to preparation of drugs for thermal therapy.

The medicine is an anti-tumor medicine.

A method for preparing nano-drugs comprises the following steps: dispersing the GO-PEG-OSA in a solvent to prepare GO-PEG-OSA dispersion, adding a medicine, stirring for 10-20 h, and centrifuging to obtain the nano medicine.

The medicine is an anti-tumor medicine.

The medicine is hydrophobic chemotherapy medicine such as Paclitaxel (PTX), adriamycin, platinum, etc.

A nanometer medicinal preparation is prepared by the above preparation method.

The method comprises the steps of firstly modifying the surface of graphene oxide GO with double-aminated polyethylene glycol PEG to form a GO-PEG nano composite, further mixing the GO-PEG nano composite with sodium alginate OSA under mild conditions, synthesizing a GO-PEG-OSA drug carrier through Schiff base reaction, and finally further synthesizing the drug-loaded nano drug by using a dialysis method.

OSA is a material obtained by oxidation treatment of natural polysaccharide Sodium Alginate (SA), retains excellent biodegradability, biocompatibility and water solubility of SA, and simultaneously shows pH-responsive properties due to the presence of aldehyde groups, and hydrophilic properties of OSA contribute to avoiding drug clearance of mononuclear phagocyte system after intravenous injection. Therefore, the introduction of OSA into drug carriers and the use of the present invention for the preparation of nano-drugs can help to achieve pH sensitive drug release effects and avoid drug clearance. The photo-thermal performance of GO is combined, and the pH/heat dual intelligent response release characteristics of a drug carrier and a nano drug can be endowed.

Compared with the prior art, the invention has the following beneficial effects:

(1) the drug carrier and the nano-drug are sensitive to pH and photo-heat and have the characteristic of pH/warm dual intelligent response release.

(2) The drug carrier and the nano-drug have excellent biocompatibility, stability and water solubility, and can carry the drug into the focus efficiently and quickly.

(3) The drug carrier can load the antitumor drug paclitaxel, and overcomes the defects of insufficient water solubility, unstable property and the like of the paclitaxel; meanwhile, the taxol derivative has good heat generating effect and active oxygen generating capacity under near-infrared irradiation, can reverse tumor resistance by combining photo-thermal/photodynamic/chemotherapy, completes the anti-tumor effect of the taxol with high efficiency and low toxicity, and generates a more efficient and safer anti-tumor effect than the single-drug taxol.

(4) The surface of the drug carrier of the invention has rich groups and high carrying capacity, and besides taxol, the drug carrier can also react with other drugs, nuclides, dyes and other molecules to load the drug carrier and carry out multi-functional derivatization such as multiple chemotherapy, photodynamic thermotherapy and the like.

Drawings

FIG. 1 is an infrared spectrum of GO-PEG-OSA;

FIG. 2 is a transmission electron micrograph of GO-PEG-OSA;

FIG. 3 is a graph of normal mucosal epithelial cytotoxicity of GO and GO-PEG-OSA in humans;

FIG. 4 is a pH/temperature response release curve for PTX @ GO-PEG, PTX @ GO-PEG-OSA;

FIG. 5 is a fluorescence plot of PTX @ GO-PEG-OSA uptake by gastric cancer cells, wherein a represents a fluorescence plot of FITC labeled PTX @ GO-PEG-OSA nano-drug, b represents a fluorescence plot of DAPI labeled gastric cancer cells, and c represents a fluorescence plot after superposition of a and b;

FIG. 6 is a graph of the chemotherapeutic effect of GO-PEG-OSA and PTX @ GO-PEG-OSA on gastric cancer cells;

FIG. 7 is a graph of the energy supply of GO-PEG-OSA and PTX @ GO-PEG-OSA to produce active oxygen, damage mitochondrial oxidation respiratory chain and inhibit P-gp drug-resistant protein under near infrared excitation;

FIG. 8 shows the anti-tumor effect of GO-PEG-OSA and PTX @ GO-PEG-OSA on the integrative heat treatment of gastric cancer cells under the near infrared excitation.

Detailed Description

The technical solution of the present invention will be further described with reference to the following examples.