阅读说明：本技术 一种基于透明质酸修饰氟化石墨烯的靶向药物载体的制备方法 (Preparation method of targeted drug carrier based on hyaluronic acid modified fluorinated graphene ) 是由 周旸 徐城锋 黄芊蔚 胡锦华 王秉 万军民 于 2019-10-05 设计创作，主要内容包括：本发明涉及医用材料领域,公开了一种基于透明质酸修饰氟化石墨烯的靶向药物载体的制备方法,本发明先以鳞片石墨为原料制备得到氧化石墨烯,然后通过溶液法以二乙氨基三氟化硫为氟化剂制得部分氟化石墨烯,其表面所拥有的大量含氧的亲水官能团,有助于其通过氢键对药物进行负载和提高生物相容性。随后通过超声波处理得到部分氟化石墨烯水溶液。在氟化石墨烯上接上PEI,后利用透明质酸对其修饰,得到靶向药物载体。(The invention relates to the field of medical materials, and discloses a preparation method of a targeted drug carrier based on hyaluronic acid modified fluorinated graphene. And then obtaining a partially fluorinated graphene aqueous solution through ultrasonic treatment. PEI is connected to the fluorinated graphene, and then the fluorinated graphene is modified by hyaluronic acid to obtain the targeted drug carrier.)

1. A preparation method of a targeted drug carrier based on hyaluronic acid modified fluorinated graphene is characterized by comprising the following steps of in terms of mg, g and mL:

1) cooling the container to 0-5 ℃ in an ice water bath, and adding 20-25mL of concentrated sulfuric acid; then 0.5-2g of flake graphite powder and 0.2-1g of sodium nitrate are added, and ultrasonic treatment is carried out for 20-30 min;

2) stirring for reaction, keeping the reaction temperature below 10 ℃, then weighing 2-4g of potassium permanganate, adding in batches within 25-30min, and reacting for 1.5-2.5 h;

3) heating to 35-40 deg.C, and reacting for 25-30 min;

4) measuring 70-120mL of water, cooling to 0-5 ℃ through an ice water bath, and adding; then adjusting the temperature to 90-95 ℃, and continuously stirring for reaction for 25-30 min;

5) adding 50-70mL of water to stop reaction, and adding 20-30mL of 25-35% H ₂O ₂Continuously stirring the solution for reaction for 15-20 min;

6) adding 30-50mL of HCl solution with volume concentration of 8-12%, and standing for 4-5 days;

7) removing the supernatant of the solution, taking the lower-layer viscous liquid, centrifuging until the viscous liquid is neutral, and pouring, freezing and drying the viscous liquid for 24-48 hours to obtain a graphene oxide sheet;

8) grinding graphene oxide sheets into graphene oxide powder;

9) adding 0.5-1.5g of graphene oxide powder and 20-40mL of dichloromethane or 1, 2-dichloroethane into another container, and stirring for 10-14h to uniformly disperse the graphene oxide powder;

10) dropwise adding 0.5-1mL of diethylaminosulfur trifluoride, then carrying out ultrasonic treatment for 4-6h at room temperature, and stirring for reaction for 24-72 h;

11) adding 15-30mL of methanol to terminate the reaction, filtering to obtain a partially fluorinated graphene product, fully washing with ethanol and deionized water, and drying at 50-70 ℃;

12) weighing 25-30mg of partial fluorinated graphene, dispersing in 45-60mL of water, and performing ultrasonic treatment to obtain a partial fluorinated graphene solution;

13) adding 1-1.5g of sodium hydroxide and 1.2-1.5g of sodium hypochlorite into a part of fluorinated graphene solution, and carrying out ultrasonic treatment for 3-4 h;

14) centrifuging, removing supernatant, adding water to obtain uniform dispersion, adding dilute hydrochloric acid until pH is neutral, dialyzing in water for 48-72 hr, and freeze drying;

15) weighing 20-30mg of fluorinated graphene, dispersing in 5-10mL of DMSO, adding 10-15mL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, activating, stirring and reacting for 3-5h to obtain an activated fluorinated graphene solution;

16) dissolving 35-40mg of PEI in 15-25mL of DMSO (dimethyl sulfoxide), dropwise adding the PEI solution into the activated graphene fluoride solution under stirring, performing ultrasonic stirring reaction for 24-36h at room temperature, dialyzing to remove unreacted impurities, and performing freeze drying to obtain fluorinated graphene-PEI;

17) weighing 0.1-0.12g of hyaluronic acid, dissolving in 5-10mL of water, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, activating, stirring and reacting for 3-5h to obtain an activated hyaluronic acid solution;

18) weighing 20-30mg of fluorinated graphene-PEI, dispersing in 5-15mL of water, dropwise adding the obtained fluorinated graphene-PEI solution into the activated hyaluronic acid solution under stirring, reacting for 24-36h under stirring at room temperature, and dialyzing to remove unreacted impurities;

19) dialyzing the solution obtained in the step 18) by using a sodium bicarbonate solution with the pH value of 7.8-8.5, replacing water every 3-4h, transferring the solution into the water after 48-60h, and continuously dialyzing for 24-36 h;

20) and removing water in the solution by adopting a rotary evaporation method, and drying to obtain the target drug carrier based on the hyaluronic acid modified fluorinated graphene.

2. The method as claimed in claim 1, wherein the stirring speed in step 2) is 400-800rpm and the stirring time is 20-40 min.

3. The method of claim 1, wherein in the step 6), the supernatant is removed every 1 to 2 days during the standing, and an equal amount of deionized water is added.

4. The method according to claim 1, wherein in step 7), the centrifugation process is specifically: centrifuging at 4000rpm, 6000rpm, 8000rpm, 10000rpm and 12000rpm for 10min respectively by using a high-speed centrifuge, removing supernatant in the centrifuge tube after each centrifugation and adding equal amount of deionized water.

5. The method according to claim 1, wherein each of the reactions in step 9), step 10), and step 11) is carried out at room temperature.

6. The preparation method according to claim 1, wherein in step 12), the ultrasonic treatment is specifically: and performing ultrasonic treatment for 2.5-3h by using an ultrasonic cell crusher under the power of 600-650W to obtain the uniformly dispersed nano-sized partially fluorinated graphene solution.

7. The method according to claim 1, wherein in step 13), the sonication conditions are: carrying out ultrasonic treatment for 3-4h at the power of 550-600W.

8. The method of claim 1, wherein the dialysis bag used in the steps 14) and 19) has a size of 8000-14000D.

9. The method of claim 1, wherein in step 20), the rotary evaporation is performed at a speed of 2 to 3rpm/s and at a temperature of 35 to 45 ℃.

Technical Field

The invention relates to the field of medical materials, in particular to a preparation method of a targeted drug carrier based on hyaluronic acid modified fluorinated graphene.

Background

Since the first successful preparation by the Geim task group in 2010, the fluorinated graphene is taken as a novel derivative of graphene, and is widely concerned, and similar to the fluorinated graphene, the fluorinated graphene still has good mechanical properties, and fluorine atoms are introduced on the basis of the fluorinated graphene, so that a plurality of unique and excellent properties are obtained. It has excellent electronic, optical and mechanical performance, interface energy reducing effect, high heat stability and high antioxidant performance. This makes the main research focus of fluorinated graphene in the fields of optics, electronics, theoretical calculation, etc.

The fluorinated graphene not only inherits the excellent performance of the graphene material, but also has the remarkable advantages (such as stable paramagnetism, stable photoluminescence and special medical effect of fluorine) which are not possessed by other carbon materials, and has the potential value of serving as a carrier of a novel tumor drug. But the application of the fluorinated graphene in the biological field is severely limited by poor biocompatibility caused by hydrophobicity, harsh preparation conditions, complicated synthesis steps and other factors. Therefore, it is of great significance to explore how to apply fluorinated graphene in the biological field.

Disclosure of Invention

In order to solve the technical problems, the invention provides a preparation method of a targeted drug carrier based on hyaluronic acid modified fluorinated graphene, which comprises the steps of firstly, taking crystalline flake graphite as a raw material, preparing graphene oxide by improving a hummers method and a series of post-treatments, and then, converting partial oxygen-containing groups of the graphene oxide into fluorine-containing groups by taking diethylaminosulfur trifluoride as a fluorinating agent through a solution method, so as to prepare the hydrophilic partially fluorinated graphene which can be stably dispersed in water. The surface of the graphene oxide sheet layer has a large number of oxygen-containing hydrophilic functional groups, which helps to load the drug through hydrogen bonds and improve biocompatibility. And dispersing the obtained partially fluorinated graphene in water, and performing ultrasonic treatment to obtain a uniformly dispersed nano-sized partially fluorinated graphene aqueous solution. And adding sodium hydroxide and sodium hypochlorite into the solution to convert-OH into-COOH, thereby obtaining the partially fluorinated graphene-COOH. Dispersing fluorinated graphene-COOH powder in DMSO, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to activate-COOH, adding PEI to prepare fluorinated graphene-PEI, and dropwise adding fluorinated graphene-PEI into hyaluronic acid activated by 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to obtain a final product.

The specific technical scheme of the invention is as follows: a preparation method of a targeted drug carrier based on hyaluronic acid modified fluorinated graphene comprises the following steps:

1) cooling the container to 0-5 ℃ in an ice water bath, and adding 20-25mL of concentrated sulfuric acid; then 0.5-2g of flake graphite powder and 0.2-1g of sodium nitrate are added, and ultrasonic treatment is carried out for 20-30 min.

2) Stirring for reaction, keeping the reaction temperature below 10 ℃, then weighing 2-4g of potassium permanganate, adding in batches within 25-30min, and reacting for 1.5-2.5 h.

The invention adds potassium permanganate in batches to ensure that the reaction is mild, is convenient to control the temperature of a reaction system, and prevents the excessive violent reaction and the generation of unnecessary impurities. At the same time, the graphite-sulfuric acid first-order intercalation material can be formed by sulfuric acid intercalation under the oxidation action of the graphite-sulfuric acid first-order intercalation material.

3) Heating to 35-40 deg.C, and reacting for 25-30 min.

4) Measuring 70-120mL of water, cooling to 0-5 ℃ through an ice water bath, and adding; then adjusting the temperature to 90-95 ℃, and continuously stirring and reacting for 25-30 min.

The graphite oxide prepared in the step 3) is reacted with water, and simultaneously the water enters the interlayer to replace acid in the graphite oxide so as to obtain the graphene oxide through stripping.

5) Adding 50-70mL of water to stop reaction, and adding 20-30mL of 25-35% H ₂O ₂And continuously stirring the solution for reaction for 15-20 min.

6) Adding 30-50mL of HCl solution with volume concentration of 8-12%, and standing for 4-5 days.

In the invention, the product is kept stand for 4-5 days in the step 6), so that impurities such as acid and the like in the lower-layer precipitate are separated and permeated into the supernatant, thereby reducing the centrifugation time.

7) And removing the supernatant of the solution, taking the lower-layer viscous liquid, centrifuging until the viscous liquid is neutral, and pouring, freezing and drying the viscous liquid for 24-48 hours to obtain the graphene oxide sheet.

8) The graphene oxide sheets are ground into graphene oxide powder.

9) And adding 0.5-1.5g of graphene oxide powder and 20-40mL of dichloromethane or 1, 2-dichloroethane into another container, and stirring for 10-14h to uniformly disperse the graphene oxide powder.

In the step 9), dichloromethane or 1, 2-dichloroethane is used as a solvent, so that the graphene oxide powder can be uniformly dispersed in the solvent.

10) Dropwise adding 0.5-1mL of diethylaminosulfur trifluoride, then carrying out ultrasonic treatment for 4-6h at room temperature, and stirring for reaction for 24-72 h.

The method takes diethylaminosulfur trifluoride as a fluorinating agent, has strong fluorination effect, and can convert oxygen-containing functional groups in graphene oxide into fluorine-containing functional groups at normal temperature and normal pressure. Hydrophilic partially fluorinated graphene is prepared and can be stably dispersed in water. The surface of the graphene oxide sheet layer has a large number of oxygen-containing hydrophilic functional groups, which helps to load the drug through hydrogen bonds and improve biocompatibility.

11) Adding 15-30mL of methanol to terminate the reaction, filtering to obtain a partially fluorinated graphene product, fully washing with ethanol and deionized water, and drying at 50-70 ℃.

12) Weighing 25-30mg of partial fluorinated graphene, dispersing in 45-60mL of water, and performing ultrasonic treatment to obtain a partial fluorinated graphene solution.

According to the invention, the nano-sized partially fluorinated graphene solution uniformly dispersed in water is obtained by using the ultrasonic cell crusher, so that convenience is brought to subsequent modification of hyaluronic acid.

13) And adding 1-1.5g of sodium hydroxide and 1.2-1.5g of sodium hypochlorite into the partial graphene fluoride solution, and performing ultrasonic treatment for 3-4 h.

According to the method, sodium hydroxide and sodium hypochlorite are added to convert-OH in the partially fluorinated graphene into-COOH, so that the partially fluorinated graphene-COOH is obtained.

14) Centrifuging, removing supernatant, adding water to obtain uniform dispersion, adding dilute hydrochloric acid until pH is neutral, dialyzing in water for 48-72 hr, and freeze drying.

15) Weighing 20-30mg of fluorinated graphene, dispersing in 5-10ml of DMSO, adding 10-15ml of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, activating and stirring for reaction for 3-5h to obtain an activated fluorinated graphene solution.

16) Dissolving 35-40mg of PEI in 15-25ml of DMSO, dropwise adding the PEI solution into the activated graphene fluoride solution under stirring, performing ultrasonic stirring reaction for 24-36h at room temperature, dialyzing to remove unreacted impurities, and freeze-drying to obtain the fluorinated graphene-PEI.

17) 0.1-0.12g of hyaluronic acid is weighed and dissolved in 5-10ml of water, and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added for activation and stirring reaction for 3-5h to obtain activated hyaluronic acid solution.

18) Weighing 20-30mg of fluorinated graphene-PEI, dispersing in 5-15ml of water, dropwise adding the obtained fluorinated graphene-PEI solution into the activated hyaluronic acid solution under stirring, reacting for 24-36h under stirring at room temperature, and dialyzing to remove unreacted impurities.

According to the invention, PEI and fluorinated graphene are firstly combined, and then hyaluronic acid activated by 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is added to obtain the fluorinated graphene-PEI-HA with a targeting function.

19) Dialyzing the solution obtained in the step 18) by using a sodium bicarbonate solution with the pH value of 7.8-8.5, replacing water every 3-4h, transferring the solution into the water after 48-60h, and continuously dialyzing for 24-36 h.

20) And removing water in the solution by adopting a rotary evaporation method, and drying to obtain the target drug carrier based on the hyaluronic acid modified fluorinated graphene.

Preferably, in the step 2), the stirring speed is 400-800rpm, and the stirring time is 20-40 min.

Preferably, in step 6), during the standing process, every 1-2d of the supernatant is removed, and an equal amount of deionized water is added.

Preferably, in step 7), the centrifugation process is specifically: centrifuging at 4000rpm, 6000rpm, 8000rpm, 10000rpm and 12000rpm for 10min respectively by using a high-speed centrifuge, removing supernatant in the centrifuge tube after each centrifugation and adding equal amount of deionized water.

Preferably, in step 9), step 10), and step 11), each reaction is carried out at room temperature.

Preferably, in step 12), the ultrasonic treatment is specifically: and performing ultrasonic treatment for 2.5-3h by using an ultrasonic cell crusher under the power of 600-650W to obtain the uniformly dispersed nano-sized partially fluorinated graphene solution.

Preferably, in step 13), the ultrasonic conditions are as follows: carrying out ultrasonic treatment for 3-4h at the power of 550-600W.

Preferably, the dialysis bag used in the steps 14) and 19) has a specification of 8000- + 14000D.

Preferably, in step 20), the rotation speed of the rotary evaporation is 2-3rpm/s, and the temperature is 35-45 ℃.

Compared with the prior art, the invention has the beneficial effects that: according to the method, firstly, crystalline flake graphite is used as a raw material, graphene oxide is prepared by improving a hummers method and a series of post-treatments, then diethylaminosulfur trifluoride is used as a fluorinating agent by a solution method, partial oxygen-containing groups of the graphene oxide are converted into fluorine-containing groups, and the hydrophilic partially fluorinated graphene which can be stably dispersed in water is prepared. The surface of the graphene oxide sheet layer has a large number of oxygen-containing hydrophilic functional groups, which helps to load the drug through hydrogen bonds and improve biocompatibility. And dispersing the obtained partially fluorinated graphene in water, and performing ultrasonic treatment to obtain a uniformly dispersed nano-sized partially fluorinated graphene aqueous solution. And adding sodium hydroxide and sodium hypochlorite into the solution to convert-OH into-COOH, thereby obtaining the partially fluorinated graphene-COOH. Dispersing fluorinated graphene-COOH powder in DMSO, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to activate-COOH, adding PEI to prepare fluorinated graphene-PEI, and dropwise adding fluorinated graphene-PEI into hyaluronic acid activated by 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to obtain a final product.

Drawings

Fig. 1 is an infrared detection spectrum of fluorinated graphene (which is not modified by hyaluronic acid and is respectively labeled as fluorinated graphene 1-3) prepared in the processes of examples 1-3.

Detailed Description

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