阅读说明：本技术 一种靶向抑制骨肿瘤的复合纳米粒子及制备方法 (Composite nano particle for targeted inhibition of bone tumor and preparation method thereof ) 是由 吴宏伟 钟午 许彦 黄钢 黄少兵 于 2020-05-15 设计创作，主要内容包括：本发明实施例公开了一种靶向抑制骨肿瘤的复合纳米粒子,按照重量份数包括如下组分：透明质酸3～5份、四丁基氢氧化铵水溶液25～35份、聚乙二醇3～5份、二甲氨基吡啶0.8～1.2份、纳米羟基磷灰石3～5份、氮氮羰基二咪唑3～5份、无水二甲基亚砜180～240份,其制备方法包括依次制备HA-TBA混合产物、PEG-HA复合物、PEG-HA-HAP纳米粒子和载唑来膦酸PEG-HA-HAP/Zol纳米粒子；本发明将纳米羟基磷灰石制成了靶向制剂发挥骨肿瘤抑制作用,既可局部给药抑制肿瘤和骨破坏,也可全身给药靶向聚集在骨肿瘤部位,促进骨修复,是一种潜在的既能抗肿瘤,又能修复骨质的新型纳米靶向制剂。(The embodiment of the invention discloses a composite nanoparticle for targeted inhibition of bone tumor, which comprises the following components in parts by weight: 3-5 parts of hyaluronic acid, 25-35 parts of tetrabutylammonium hydroxide aqueous solution, 3-5 parts of polyethylene glycol, 0.8-1.2 parts of dimethylaminopyridine, 3-5 parts of nano-hydroxyapatite, 3-5 parts of azodicarbonyl diimidazole and 180-240 parts of anhydrous dimethyl sulfoxide, wherein the preparation method comprises the steps of sequentially preparing an HA-TBA mixed product, a PEG-HA compound, PEG-HA-HAP nanoparticles and zoledronic acid-loaded PEG-HA-HAP/Zol nanoparticles; the nano hydroxyapatite is prepared into a targeting preparation to play a role in inhibiting bone tumor, can be locally administered to inhibit tumor and bone destruction, can be systemically administered to target and gather at the bone tumor part to promote bone repair, and is a potential novel nano targeting preparation which can resist tumor and repair bone.)

1. The composite nanoparticle for targeted inhibition of bone tumor is characterized by comprising the following components in parts by weight:

3-5 parts of hyaluronic acid, 25-35 parts of tetrabutylammonium hydroxide aqueous solution, 3-5 parts of polyethylene glycol, 0.8-1.2 parts of dimethylaminopyridine, 3-5 parts of nano-hydroxyapatite, 3-5 parts of azodicarbonyl diimidazole and 180-240 parts of anhydrous dimethyl sulfoxide.

2. The composite nanoparticle for targeted inhibition of bone tumor according to claim 1, comprising the following components in parts by weight:

4 parts of hyaluronic acid, 30 parts of tetrabutylammonium hydroxide aqueous solution, 4 parts of polyethylene glycol, 1 part of dimethylaminopyridine, 4 parts of nano-hydroxyapatite, 4 parts of azodicarbonyl diimidazole and 210 parts of anhydrous dimethyl sulfoxide.

3. The composite nanoparticle for targeted inhibition of bone tumor according to claim 2, wherein the concentration of the tetrabutylammonium hydroxide aqueous solution is 40%.

4. A preparation method of composite nanoparticles for targeted inhibition of bone tumor is characterized by comprising the following steps:

step 100, preparing an HA-TBA mixed product, uniformly mixing 0.4g of hyaluronic acid and 3mL of 40% tetrabutylammonium hydroxide aqueous solution, stirring and dissolving, filtering the stirred and dissolved mixed solution by using a sterile filter head with the wavelength of 450nm, and freeze-drying to obtain the HA-TBA mixed product;

step 200, preparing a PEG-HA compound, dissolving the HA-TBA mixed product and 0.4g of polyethylene glycol 8000 in 10mL of anhydrous dimethyl sulfoxide, adding 0.1g of dimethylaminopyridine, and stirring for reaction for 1 day to obtain the PEG-HA compound;

step 300, preparing PEG-HA-HAP nanoparticles, adding 0.4g of nano-hydroxyapatite and 0.4g of azodicarbonyl diimidazole into 10mL of anhydrous dimethyl sulfoxide for primary reaction, then adding a PEG-HA compound, stirring for a second time for reaction for 24 hours, stopping the reaction, sequentially centrifuging the product solution, removing supernatant, performing dialysis treatment after ultrasonic dispersion, and finally performing freeze-drying to obtain the PEG-HA-HAP nanoparticles;

step 400, preparing drug-loaded PEG-HA-HAP/Zol nanoparticles, namely taking 0.4g of PEG-HA-HAP nanoparticles and 0.2g of sodium zoledronate, dispersing and stirring, centrifuging, taking precipitate, and performing freeze-drying treatment to obtain the final product, namely the drug-loaded PEG-HA-HAP/Zol nanoparticles, namely the composite nanoparticles capable of inhibiting the bone tumor in a targeted manner.

5. The method for preparing the composite nanoparticle for targeted inhibition of bone tumor according to claim 4, wherein the stirring and dissolution in the step 100 is performed at room temperature for 30 minutes.

6. The method for preparing the composite nanoparticle for targeted inhibition of bone tumor according to claim 4, wherein the stirring reaction for 1 day in the step 200 is performed at a constant temperature of 37 ℃.

7. The method for preparing the composite nanoparticle for targeted inhibition of bone tumor according to claim 4, wherein in the step 300, the preliminary reaction is performed by performing ultrasonic treatment in ice bath for 20min, and then stirring at 40 ℃ for reaction for 4 h.

8. The method for preparing the composite nanoparticle for targeted inhibition of bone tumor according to claim 4, wherein in the step 300, 10mL of anhydrous dimethyl sulfoxide is added again after removing the supernatant, and ultrasonic dispersion is performed three times, and re-dispersion is performed with 1mL of anhydrous dimethyl sulfoxide.

9. The preparation method of the composite nanoparticle for targeted inhibition of bone tumor according to claim 8, wherein the dialysis treatment method comprises: and filling the redispersed liquid into a dialysis bag with the molecular weight of 3500, dialyzing with deionized water for 1 day, changing water every 1h, and freeze-drying to obtain the product PEG-HA-HAP/Zol nano particles.

10. The method for preparing the composite nanoparticle for targeted inhibition of bone tumor according to claim 4, wherein the step 400 of dispersing and stirring comprises: dispersing 2mL of deionized water and stirring at a low speed for 2 h.

Technical Field

The embodiment of the invention relates to the technical field of nanoparticle preparation, in particular to a composite nanoparticle for targeted inhibition of bone tumor and a preparation method thereof.

Background

Clinical treatment currently presents a number of challenges for primary and metastatic bone tumors, and osteosarcomas and bone metastases are often treated with chemotherapy or targeted drugs to kill tumor cells and mitigate the tumorous bone destruction. The medicines are required to be targeted to tumor parts as much as possible, so that the tumor killing effect is increased, and the side effects of the whole body are reduced no matter the medicines are used for systemic treatment or local treatment.

Zoledronic acid is a common medicine for inhibiting osteoclast, and has obvious therapeutic effect on osteolytic bone destruction caused by giant cell tumor of bone, osteoporosis and malignant bone tumor. However, the systemic administration of zoledronic acid can generate obvious toxic and side effects such as jaw necrosis, ectopic ossification, fever, nausea, vomiting of esophagus burn and other adverse reactions, and intravenous injection can be quickly cleared by the kidney due to poor water solubility, poor intestinal absorption and low biological benefit, so how to solve the problem of targeted administration of zoledronic acid is the current one.

The nano-hydroxyapatite is one of the main components of normal bone tissues, the structure of the nano-hydroxyapatite can be combined with a high molecular substance, and the medicament is loaded into the nano-particles, so that the slow release of the medicament can be realized. However, the simple in vivo injection of nano-hydroxyapatite has a physical injury.

Hyaluronic Acid (HA) and polyglycolic acid (PEG) are two very widely used materials in the field of drug sustained release. Hyaluronic acid is an endogenous high molecular polymer, has good biocompatibility, and can be widely combined with CD44 on the surfaces of cells, particularly tumor cells. Therefore, the nano-particle can have the capability of actively targeting and combining with tumor cells by selecting the nano-particle to be combined with the nano-hydroxyapatite. In addition, the nano hydroxyapatite composite targeting nano particles modified by hyaluronic acid and polyethylene glycol also have the function of repairing bone destruction.

The nano targeting preparation can utilize the characteristics of abundant blood vessels, wider vascular wall gaps and poor structural integrity in solid tumor tissues, so that nano particles with the particle size of 50-500nm can penetrate through the capillary walls of bone tumors to realize the enrichment of the nano targeting preparation in the tumor tissues, and the nano targeting preparation is a passive targeting behavior.

Therefore, based on the two abilities of active targeting and passive targeting of the nano particles to bone tumor tissues, the nano particle is expected to become a novel nano targeting preparation, so that the nano particle can not only target bone tumor focuses to kill tumor cells, but also can generate bone repair effect on areas damaged by bones, and can meet the requirements of timed, positioned and quantitative release of zoledronic acid.

Disclosure of Invention

Therefore, the embodiment of the invention provides a composite nanoparticle for targeted inhibition of bone tumor and a preparation method thereof, so as to solve the problems in the prior art.

In order to achieve the above object, an embodiment of the present invention provides the following:

in a first aspect of the embodiments of the present invention, there is provided a composite nanoparticle for targeted inhibition of bone tumor, comprising the following components in parts by weight:

3-5 parts of hyaluronic acid, 25-35 parts of tetrabutylammonium hydroxide aqueous solution, 3-5 parts of polyethylene glycol, 0.8-1.2 parts of dimethylaminopyridine, 3-5 parts of nano-hydroxyapatite, 3-5 parts of azodicarbonyl diimidazole and 180-240 parts of anhydrous dimethyl sulfoxide.

As a preferable scheme of the invention, the paint comprises the following components in percentage by mass:

hyaluronic acid HA4 parts, tetrabutylammonium hydroxide aqueous solution 30 parts, polyethylene glycol 4 parts, dimethylaminopyridine 1 part, nano-hydroxyapatite 4 parts, azodicarbonyl diimidazole 4 parts and anhydrous dimethyl sulfoxide 210 parts.

In a preferred embodiment of the present invention, the concentration of the tetrabutylammonium hydroxide aqueous solution is 40%.

In a second aspect of the embodiments of the present invention, there is provided a method for preparing a composite nanoparticle for targeted inhibition of bone tumor, comprising the following steps:

step 100, preparing an HA-TBA mixed product, uniformly mixing 0.4g of hyaluronic acid and 3mL of 40% tetrabutylammonium hydroxide aqueous solution, stirring and dissolving, filtering the stirred and dissolved mixed solution by using a sterile filter head with the wavelength of 450nm, and freeze-drying to obtain the HA-TBA mixed product;

step 200, preparing a PEG-HA compound, dissolving the HA-TBA mixed product and 0.4g of polyethylene glycol 8000 in 10mL of anhydrous dimethyl sulfoxide, adding 0.1g of dimethylaminopyridine, and stirring for reaction for 1 day to obtain the PEG-HA compound;

step 300, preparing PEG-HA-HAP nanoparticles, adding 0.4g of nano-hydroxyapatite and 0.4g of azodicarbonyl diimidazole into 10mL of anhydrous dimethyl sulfoxide for primary reaction, then adding a PEG-HA compound, stirring for a second time for reaction for 24 hours, stopping the reaction, sequentially centrifuging the product solution, removing supernatant, performing dialysis treatment after ultrasonic dispersion, and finally performing freeze-drying to obtain the PEG-HA-HAP nanoparticles;

step 400, preparing drug-loaded PEG-HA-HAP/Zol nanoparticles, namely taking 0.4g of PEG-HA-HAP nanoparticles and 0.2g of sodium zoledronate, dispersing and stirring, centrifuging, taking precipitate, and performing freeze-drying treatment to obtain the final product, namely the drug-loaded PEG-HA-HAP/Zol nanoparticles, namely the composite nanoparticles capable of inhibiting the bone tumor in a targeted manner.

In a preferred embodiment of the present invention, the stirring and dissolving in step 100 is performed at room temperature for 30 minutes.

In a preferred embodiment of the present invention, in the step 200, the stirring reaction is performed for 1 day in a constant temperature environment of 37 ℃.

In a preferred embodiment of the present invention, in step 300, the preliminary reaction is performed by performing ultrasonic treatment in an ice bath for 20min, and stirring at 40 ℃ for 4 h.

In a preferred embodiment of the present invention, in step 300, after removing the supernatant, 10mL of anhydrous dimethylsulfoxide is added again, ultrasonically dispersed three times, and redispersed with 1mL of anhydrous dimethylsulfoxide.

As a preferable aspect of the present invention, a method of dialysis treatment includes: filling the redispersed liquid into a dialysis bag with the molecular weight of 3500, dialyzing with deionized water for 1 day, changing water every 1h, and freeze-drying to obtain the product PEG-HA-HAP/Zol nano particles.

In a preferred embodiment of the present invention, in the step 400, the dispersion stirring includes: dispersing 2mL of deionized water and stirring at a low speed for 2 h.

The embodiment of the invention has the following advantages:

the nano hydroxyapatite is prepared into the targeting preparation to play a role in filling bones, so that the defect that the traditional medicine-carrying artificial bone system is difficult to flexibly administer is overcome, and the nano targeting preparation can resist tumors and repair bones.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

FIG. 1 is a schematic flow chart of a production process in an embodiment of the present invention;

fig. 2 is a transmission electron microscope image of nano hydroxyapatite particles in an embodiment of the present invention;

FIG. 3 is a particle size distribution curve of PEG-HA-HAP/Zol nanoparticles carrying drug in accordance with an embodiment of the present invention;

FIG. 4 is a graph of infrared spectra of nanoparticles of different compositions in accordance with embodiments of the present invention;

FIG. 5 is a nuclear magnetic spectrum of nanoparticles in accordance with an embodiment of the present invention;

FIG. 6 is a release curve of zoledronic acid loaded PEG-HA-HAP/Zol nanoparticles in accordance with an embodiment of the present invention;

FIG. 7 is a schematic diagram of the cytotoxicity experiment of osteosarcoma 143b treated with zoledronic acid-loaded PEG-HA-HAP/Zol nanoparticles in the embodiment of the present invention.

FIG. 8 shows the result of apoptosis of osteosarcoma 143b cells treated with the nanoparticles of the present invention and other nanoparticles.

FIG. 9 shows the results of apoptosis-related proteins of osteosarcoma 143b cells treated with the nanoparticles of the present invention and other nanoparticles.

FIG. 10 is a graph of the distribution of nanoparticles of the present invention at various time points after intragastric and caudal vein injection into mice.

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides a composite nanoparticle for targeted inhibition of bone tumor, which comprises the following components in parts by weight:

3-5 parts of hyaluronic acid, 25-35 parts of tetrabutylammonium hydroxide aqueous solution, 3-5 parts of polyethylene glycol, 0.8-1.2 parts of dimethylaminopyridine, 3-5 parts of nano-hydroxyapatite, 3-5 parts of azodicarbonyl diimidazole and 180-240 parts of anhydrous dimethyl sulfoxide; the concentration of the tetrabutylammonium hydroxide aqueous solution is 40%.