CN111870616A - Composite nano particle for targeted inhibition of bone tumor and preparation method thereof - Google Patents
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Abstract
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.
Description
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 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%.
Example 1:
the composite nanoparticle for targeted inhibition of bone tumor comprises the following components in parts by mass:
3 parts of hyaluronic acid, 25 parts of tetrabutylammonium hydroxide aqueous solution, 3 parts of polyethylene glycol, 0.8 part of dimethylaminopyridine, 3 parts of nano-hydroxyapatite, 3 parts of azodicarbonyl diimidazole and 180 parts of anhydrous dimethyl sulfoxide; the concentration of the tetrabutylammonium hydroxide aqueous solution is 40%.
Example 2:
the composite nanoparticle for targeted inhibition of bone tumor comprises the following components in parts 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.
Example 3:
the composite nanoparticle for targeted inhibition of bone tumor comprises the following components in parts by mass:
5 parts of hyaluronic acid, 35 parts of tetrabutylammonium hydroxide aqueous solution, 5 parts of polyethylene glycol, 1.2 parts of dimethylaminopyridine, 5 parts of nano-hydroxyapatite, 5 parts of azodicarbonyl diimidazole and 240 parts of anhydrous dimethyl sulfoxide; the concentration of the tetrabutylammonium hydroxide aqueous solution is 40%.
The invention provides a preparation method of composite nanoparticles for targeted inhibition of bone tumor, which takes an example of an embodiment 2 as a specific component, and comprises the following steps as shown in figure 1:
0.4g of Hyaluronic Acid (HA) and 3mL of 40% tetrabutylammonium hydroxide aqueous solution (TBA) are uniformly mixed, stirred and dissolved, stirred at room temperature for 30min, and then filtered and lyophilized by using a sterile filter head with 450nm of Millex-HV to obtain an HA-TBA mixed product.
Step 200, preparation of PEG-HA Complex
The HA-TBA mixture obtained in the previous step and 0.4g of PEG8000 were dissolved in 10mL of anhydrous DMSO, 0.1g of Dimethylaminopyridine (DMAP) was added, and the mixture was reacted at 37 ℃ for 1d with stirring.
Step 300, preparing PEG-HA-HAP nano particles
0.4g of nano-hydroxyapatite and 0.4g of azodicarbonyl diimidazole (CDI) are added into 10mL of anhydrous DMSO, ultrasonic treatment is carried out for 20min in an ice bath, and the mixture is stirred at 40 ℃ for reaction for 4 h. And (3) adding the PEG-HA compound reaction liquid in the last step into the reaction liquid, continuously stirring and reacting for 24 hours, and closing the reaction device. The product solution was centrifuged, the supernatant discarded, and 10mL of anhydrous DMSO was added, dispersed using an ultrasonic disperser, repeated three times, and redispersed with 1mL of anhydrous DMSO. Filling the liquid, dialyzing with deionized water for 1d (the molecular weight of the dialysis bag is 3500), changing water every 1h, and freeze-drying to obtain the product PEG-HA-HAP nanoparticles.
Step 400, preparing the drug-loaded PEG-HA-HAP/Zol nano particles, namely the final product.
Taking 0.4g of PEG-HA-HAP nano particles and 0.2g of zoledronic acid sodium (ZOL), adding 2mL of deionized water for dispersion, stirring at a low speed for 2h, centrifuging, taking precipitates, and freeze-drying to obtain the drug-loaded PEG-HA-HAP/Zol nano particles.
The obtained PEG-HA-HAP/Zol nanoparticles carrying the medicine are respectively subjected to particle size, infrared spectrum, nuclear magnetic resonance hydrogen spectrum, dynamic light scattering, transmission electron microscope and in vitro release rate tests, and the results are respectively as follows:
(1) the results of the particle size are shown in the distribution curve of fig. 3.
(2) Infrared spectroscopy.
Taking a small amount of solid HAP, PEG, HA-TAB, PEG-HA-HAP nanoparticles and PEG-HA-HAP/Zol nanoparticle sample, mixing the sample with infrared dried potassium bromide powder, grinding, pressing into transparent sample potassium bromide tablet, and testing the appearance of fuchsin with a spectrum shown in figure 4.
(3) Hydrogen nuclear magnetic resonance spectroscopy.
Dissolving a small amount of solid HAP, PEG, HA-TAB, PEG-HA-HAP nanoparticles, Zol-loaded PEG-HA-HAP/Zol nanoparticles sample, and free Zol in heavy water, and determining nuclear magnetic resonance hydrogen spectrum. The content and the drug loading of each component are calculated according to a nuclear magnetic resonance hydrogen spectrum.
As shown in fig. 5, a: the zoledronic acid-loaded PEG-HA-HAP/Zol nanoparticles; b: zoledronic acid.
(4) Dynamic light scattering.
The prepared drug-loaded PEG-HA-HAP/Zol nano particles are placed in a cuvette, and the cuvette is placed in a dynamic light scattering particle size analyzer sample for testing, wherein each sample is tested for 3 times, and each time is 1 min. And (3) testing conditions are as follows: an argon ion laser with the wavelength of 658nm, the temperature of 25 +/-0.1 degrees and the dynamic light scattering angle of 90 degrees. Simultaneously, Zeta potential was measured, operating conditions: 11.4v/cm,13.0mA, 25 degrees C, sample solvent diluted with distilled water.
(5) And (4) a transmission electron microscope.
Dropping PEG-HA-HAP nanoparticles and PEG-HA-HAP/Zol nanoparticles on copper mesh coated with carbon support film, dyeing with 2% ursolic acid, naturally drying, and observing their forms under transmission electron microscope, as shown in FIG. 2.
(6) In vitro release rate.
Accurately weighing PEG-HA-HAP/Zol nanoparticles to be loaded, putting the PEG-HA-HAP/Zol nanoparticles into a dialysis bag (with the cut-off molecular weight of 8000- & ltSUB & gt 12000kDa), respectively putting the dialysis bag into HCl-PBS buffer solution with the Ph of 7.4 and 5.6, oscillating the solution in a dark place at 37 ℃ and 100rpm, respectively replacing the whole release medium with fresh medium at 0, 0.5, 1, 2, 4, 8, 12, 24 and 48h, measuring the release amount by adopting an ultraviolet-visible spectrophotometer, and calculating the release rate.
As shown in fig. 6, the upper curve in the figure is: a simple zoledronic acid aqueous solution release profile; the middle is as follows: nanoparticle drug release profile at PH 7.4. The following are: nanoparticle drug release profile at PH 5.4.
Nanoparticles in blood circulation: opsonin, integrin and the like in plasma can be adsorbed on the surfaces of the nanoparticles, so that the nanoparticles are easy to be identified by phagocytes and further phagocytized by a Mononuclear Phagocyte System (MPS) and a reticuloendothelial system (RES), and the bioavailability of the medicine is greatly reduced. For in vivo clearance of nanoparticles, the main approach at present is to modify the surface of nanoparticles, i.e. to adhere hydrophilic polymers to the particle surface by various methods, so as to minimize in vivo clearance. Polyethylene glycol (PEG) is a highly versatile polymer compound with excellent biocompatibility. Experiments prove that when PEG is grafted to the surface of the nanoparticle, the soft PEG chain segment can be highly modified and alternately overlapped to cover the surface of the nanoparticle to form compact conformation cloud, and the adsorption of protein in blood plasma is reduced through steric hindrance, so that the residence time of the PEG chain segment in blood is prolonged, and the effect of long circulation is achieved.
The cytotoxicity test results of osteosarcoma 143b treated by zoledronic acid-loaded PEG-HA-HAP/Zol nanoparticles are shown in FIGS. 7 to 10:
a: control group was untreated;
b: 1ug/ml nanoparticle;
c: 5ug/ml nanoparticles;
d: in vitro toxicity CCK curves for each group of cells.
143 b: human osteosarcoma cells; HSF: human fibroblasts; ASC: rat adipose-derived stem cells. HSF, ASC are controls.
After the nanoparticles are passively targeted to tumor tissues, the efficient uptake of giant cell tumor of bone can be realized through an active targeting mechanism, CD44 is one of characteristic surface markers of breast cancer stem cell expression, the application prospect of the CD44 as a tumor target is widely accepted by academia, and researches show that CD44 promotes invasive tumor metastasis and inhibits the activity of CD44 to induce invasive tumor apoptosis. Hyaluronic Acid (HA), commonly known as poliuric acid, is a glycosaminoglycan widely present in the body, and HAs certain effects on maintaining the structure of extracellular matrix and regulating intracellular activities, and is commonly used in cosmetic surgery and the like. Since CD44 is a receptor of hyaluronic acid, the hyaluronic acid can be used for preparing an amphiphilic block copolymer, so that giant cell tumor of the bone can be efficiently targeted. Can flexibly administer the medicine and effectively treat giant cell tumor of the bone.
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.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
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.
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| CN112675130A (en) * | 2020-12-29 | 2021-04-20 | 中国人民解放军总医院第三医学中心 | Application of hollow nano-particles in preparation of osteoporosis treatment drug |
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| CN112675130A (en) * | 2020-12-29 | 2021-04-20 | 中国人民解放军总医院第三医学中心 | Application of hollow nano-particles in preparation of osteoporosis treatment drug |
| CN112675130B (en) * | 2020-12-29 | 2023-04-25 | 中国人民解放军总医院第三医学中心 | Application of hollow nano particles in preparation of osteoporosis treatment medicine |
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