CN113058027A - Preparation method of paclitaxel microparticles surface-modified by soybean agglutinin and tannic acid - Google Patents

Abstract

The invention discloses a preparation method of paclitaxel particles with surface modified by soybean agglutinin and tannic acid, which comprises the steps of crushing paclitaxel into powder, and coating a tannic acid layer on the surfaces of the paclitaxel particles by using an aqueous solution assembly technology; and then stably combining the soybean agglutinin of which the sugar structure end is modified by boric acid and polyvinyl alcohol with a tannin layer on the surface of the taxol particles in a hydrogen bond mode through a protein part of the soybean agglutinin, and finally dissociating the sugar structure end modified part of the soybean agglutinin under the condition of pH 3, so that the sugar structure end of the agglutinin is accurately pointed to the outside of the particles. After the structure of the soybean agglutinin is modified, the sugar structure end of the soybean agglutinin is crosslinked with the polyvinyl alcohol copolymer through boric acid, so that the tannin is only combined with the protein part of the soybean agglutinin, and the sugar structure end of the soybean agglutinin is accurately pointed to the outside of the particles, thereby ensuring the targeting property of the soybean agglutinin and improving the bioavailability.

Description

Preparation method of paclitaxel microparticles surface-modified by soybean agglutinin and tannic acid

Technical Field

The invention relates to a preparation method of a paclitaxel particle surface-modified by soybean agglutinin and tannic acid.

Background

Paclitaxel is the most excellent natural antitumor drug, and has been widely used for treating breast tumor, ovarian tumor, and partial head and neck tumor and lung tumor in clinic. Paclitaxel has attracted great interest to scientists due to its complex and novel chemical structure, unique biological mechanism of action, reliable antitumor activity and serious resource deficiencies. The method for preparing the membrane-coated paclitaxel nanoparticles by combining an aerosol atomization technology and a complex spontaneous membrane-forming coating technology to improve the solubility and stability of paclitaxel in water and further enhance the anti-tumor effect. A membrane-coated taxol nano-drug, its preparing process and use [ P ] Chinese patent CN201510014587.6, 2015-05-06 ] is disclosed. (Wangjianxin Hongtao, Zhuying, etc. a paclitaxel liposome and its preparation method [ P ] Chinese patent: CN202010934453.7, 2020-11-20.)

Lectins are a class of carbohydrate-binding proteins of non-immunological origin widely distributed in animals, plants and microorganisms, which contain in their structure at least one non-catalytic domain capable of reversibly binding to specific carbohydrate or glycoprotein, glycolipid surface carbohydrate groups without altering the carbohydrate structure. The lectin has three uses in the treatment of malignant tumor, namely 1) inducing apoptosis and autophagy of tumor cells, and can be directly used as a malignant tumor treatment medicament; 2) the treatment effect of the anti-tumor medicine is enhanced; 3) used as a carrier of antitumor drugs and used for developing tumor-targeted drugs.

The invention discloses a method for preparing matrine nanoparticles modified by wheat germ agglutinin, which comprises the following steps: the matrine nanoparticles modified by the wheat germ agglutinin are formed by connecting wheat germ agglutinin on matrine nanoparticles through chemical bonds, and the problems of low targeting property and poor using effect of the matrine nanoparticles modified by the wheat germ agglutinin are effectively solved. A preparation method and application of matrine nanoparticles modified by wheat germ agglutinin [ P ]. Chinese patent CN201510406710.9,2015-09-30 ] are disclosed before Lifeng and the like. The method improves the solubility of docetaxel, and the pea lectin modified docetaxel-loaded polyhydroxybutyrate nanoparticle improves the targeting property and the therapeutic index of the drug, and lays a foundation for developing a specific sugar-mediated tumor targeted drug delivery system. (Lifeng, Dongwei phoenix, Lifei, etc. carrying docetaxel polyhydroxybutyrate nanoparticle and preparation method, pea agglutinin modification method and application [ P ] Chinese patent: CN201310433512.2, 2013-12-18.)

Targeted drug delivery refers to the process by which a target agent selectively binds to a target cell to produce a pharmacological effect. Due to different administration routes, the target preparation can enter different positions of a human body, so that different targeting and action characteristics are generated. The main disadvantage of antitumor drugs is that the proliferation of normal cells is also severely inhibited while tumor cell growth is inhibited. The above reports demonstrate that lectins play a great role in targeted therapy, however there are many problems to be solved in order for a lectin particle delivery system to be a mature delivery system. Firstly, a degradable polymer which is easy to be phagocytized by epithelial cells is found as a particulate material; secondly, it is necessary to find an appropriate technique for modifying the surface of the degradable polymer particles with the lectin while ensuring the bioactivity of the lectin and the stability of the preparation. The tannic acid has multi-target and multi-effect anti-tumor activity, can act on multiple stages of generation and development of malignant tumors, and has small toxic and side effects, so that the tannic acid can be used as a chemosensitizer to overcome the drug resistance of some chemotherapeutic drugs to achieve the effect of adjuvant therapy; the end part of the sugar structure of the lectin is crosslinked with polyvinyl alcohol 1788 through boric acid, so that tannic acid is only bonded with the protein part of the soybean lectin, and finally the modified sugar structure end of the soybean lectin is dissociated, so that the sugar structure end of the soybean lectin is accurately pointed to the outside of the particle, and the targeting of the soybean lectin is ensured.

Disclosure of Invention

The invention aims to provide a preparation method of soybean agglutinin and tannin surface modified paclitaxel particles, which is based on the inhibition effect of tannin on tumor cells, the soybean agglutinin can specifically identify the tumor cells, a stable drug delivery system with stable structure of the soybean agglutinin and the tannin surface modified is constructed by utilizing the stable hydrogen bond network combination mode between the tannin and the soybean agglutinin, and the sugar structure end of the agglutinin is accurately pointed to the outside of the particles, thereby ensuring the targeting property of the soybean agglutinin, and the paclitaxel is targeted to the tumor cells through the mediation of the soybean agglutinin.

The technical scheme adopted by the invention is as follows:

a method for preparing paclitaxel microparticles with surface modified by soybean agglutinin and tannic acid comprises pulverizing paclitaxel into powder, and coating tannic acid layer on the surface of paclitaxel microparticles by aqueous solution assembly technique; and then stably combining the soybean agglutinin of which the sugar structure end is modified by boric acid and polyvinyl alcohol with a tannin layer on the surface of the taxol particles in a hydrogen bond mode through a protein part of the soybean agglutinin, and finally dissociating the sugar structure end modified part of the soybean agglutinin under the condition of pH 3, so that the sugar structure end of the agglutinin is accurately pointed to the outside of the particles.

Further, the tannic acid is derived from pomegranate rind, geranium wilfordii, grape rind or gallnut, and the mass concentration of the tannic acid aqueous solution is 0.05-0.2 g/ml;

incubating paclitaxel ground by a high-pressure homogenizer in a tannic acid aqueous solution for 8 hours at room temperature, and carrying out vacuum freeze drying for 30min to obtain paclitaxel particles with tannic acid surface modification;

in an aqueous solution with the pH value of 9.0, boric acid and polyvinyl alcohol 1788 are subjected to a crosslinking reaction on the sugar structure end of the soybean agglutinin to obtain the soybean agglutinin of which the sugar structure end is modified by the boric acid and the polyvinyl alcohol;

adjusting the pH of the aqueous suspension of the soybean agglutinin with the changed sugar structure end and the paclitaxel surface-modified with the tannic acid to 3.0 by using citric acid, dissociating the sugar structure end modified with the soybean agglutinin under an acidic condition, reacting for half an hour, placing the aqueous suspension of the soybean agglutinin and the paclitaxel surface-modified with the tannic acid in a low-temperature high-speed refrigerated centrifuge, centrifuging for 30min at 15000rpm, discarding the supernatant, washing the precipitate for 3 times by using distilled water, removing impurities such as boric acid and 1788 of polyvinyl alcohol, and freeze-drying to obtain the paclitaxel microparticles surface-modified with the soybean agglutinin and the tannic acid.

The more specific technical scheme is as follows:

a method for preparing surface modified paclitaxel microparticles with soybean agglutinin and tannic acid comprises the following steps:

1) modifying the tannin layer on the surface of paclitaxel: taking a tannic acid solution with the mass concentration of 0.05-0.2g/ml, putting paclitaxel crushed by a high-pressure homogenizer into the tannic acid solution, incubating for 8 hours at room temperature, and performing vacuum freeze drying to obtain tannic acid surface-modified paclitaxel particles; wherein the weight ratio of paclitaxel: the mass ratio of the tannic acid is 1:0.3 to 3; the tannic acid is derived from pericarpium Granati, herba Erodii seu Geranii, grape skin or Galla chinensis;

2) boric acid, soybean agglutinin, polyvinyl alcohol 1788 are dissolved in distilled water, and the ratio of soybean agglutinin: boric acid: the mass ratio of the polyvinyl alcohol 1788 is 1: 0.5-2: 0.5 to 2; adjusting pH of the solution to 9 with dipotassium hydrogen phosphate, heating and stirring at 60 deg.C for 50min to crosslink sugar structure end of soybean agglutinin with boric acid and polyvinyl alcohol 1788 to obtain soybean agglutinin with sugar structure end modified with boric acid and polyvinyl alcohol;

3) adding the taxol particles subjected to surface modification of the tannic acid obtained in the step 1) into the soybean lectin solution, obtained in the step 2), of which the sugar structure end is modified by boric acid and polyvinyl alcohol, wherein the taxol particles subjected to surface modification of the tannic acid: the mass ratio of the soybean agglutinin solution with the changed sugar structure end is 1:0.3-3, and the pH value of the mixed solution is adjusted to 3.0 by citric acid; after reacting for half an hour, placing the mixture in a low-temperature high-speed refrigerated centrifuge, centrifuging at 15000rpm for 30min, discarding the supernatant, removing impurities such as boric acid and polyvinyl alcohol 1788, washing the precipitate with distilled water for 3 times, and freeze drying to obtain the surface-modified paclitaxel microparticles of soybean agglutinin and tannic acid.

Compared with the prior art, the invention has the following beneficial effects: after the structure of the soybean agglutinin is modified, the sugar structure end of the soybean agglutinin is crosslinked with the polyvinyl alcohol copolymer through boric acid, so that the tannin is only combined with the protein part of the soybean agglutinin, and the sugar structure end of the soybean agglutinin is accurately pointed to the outside of the particles, thereby ensuring the targeting property of the soybean agglutinin and improving the bioavailability.

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

it is to be understood that these examples are preferred embodiments of the invention and are intended only for purposes of illustration. From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and technical scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

In addition, "g" in each example is a weight unit of "g"; "min" is the time unit "minutes"; 'mm' of³"volume unit" cubic millimeters "; "g/mL" is the concentration unit "grams per milliliter"; "mg/mL" is the concentration unit "milligrams per milliliter"; "rpm" is the speed of rotation "rpm"; "μ L" is volume unit "microliter"; "nm" is the length unit of "nanometer"; "μ g/ml" is the concentration unit "micrograms per milliliter"; "Cy5.5" is "a cyanine-type fluorescent dye, which is now widely used for detecting and labeling proteins or certain biomolecules. The cyanine dye is an unsaturated heterocyclic compound formed by connecting different heterocyclic nuclei through a methine chain, and has a structural general formula of M₂N-(CH＝CH)_n-NM₂According to the length of a methine chain between two nitrogen heterocycles, the dyes can be divided into cyanine dyes such as trimethine, pentamethine and heptamethine, which are respectively represented by Cy3, Cy5, Cy5.5 and Cy 7; balb/c is a hairless, nude, thymus-free, T cell immunodeficient inbred mouse. "human breast cancer cell MCF-7, human colon cancer cell HCT-116,Human non-small cell lung cancer a549 "is a distinct tumor cell line; Cy5.5-N-hydroxysuccinimide ester (CAS: 442912-55-2).

Example 1

1. A method for preparing surface-modified paclitaxel microparticles containing soybean agglutinin and tannic acid comprises:

(1) the method for preparing paclitaxel with tannin surface modification comprises incubating 10g of paclitaxel pulverized by high pressure homogenizer in 100ml of pomegranate bark tannin solution (0.1g/ml) at room temperature for 8 hr, and vacuum freeze drying for 30min to obtain paclitaxel microparticles with tannin surface modification.

(2) Soybean lectin with modified sugar structure: heating and stirring 12g of soybean agglutinin with 8g of boric acid and 8g of polyvinyl alcohol 1788 at 60 ℃ for 50min under the condition of pH 9.0 to crosslink sugar structure ends of the soybean agglutinin with the boric acid and the polyvinyl alcohol 1788, thereby obtaining the soybean agglutinin with the sugar structure ends occupied by the boric acid and the polyvinyl alcohol.

(3) Modified soybean lectin-modified tannic acid layer with modified sugar structure: adding 10g of the paclitaxel microparticles subjected to surface modification of pomegranate peel tannic acid in the step (1) into the soybean lectin solution in which 15g of sugar structure ends of the paclitaxel microparticles are occupied by boric acid and polyvinyl alcohol in the step (2), so that pomegranate peel tannic acid can only be combined with protein parts of the soybean lectin through stable hydrogen bonds, and the modified sugar structure ends of the soybean lectin point to the outside, thereby obtaining the soybean lectin with modified sugar structure ends and the paclitaxel microparticles subjected to surface modification of pomegranate peel tannic acid.

(4) Adjusting pH of suspension of (3) soybean agglutinin with changed sugar structure end and taxol particle surface modified with tannic acid with citric acid to 3.0, wherein the soybean agglutinin is dissociated by boric acid and sugar structure end modified with polyvinyl alcohol under acidic condition, after reacting for half an hour, placing the mixture in a low-temperature high-speed refrigerated centrifuge, centrifuging at 15000rpm for 30min, discarding supernatant, washing precipitate with distilled water for 3 times, removing impurities such as boric acid and polyvinyl alcohol 1788, and freeze drying to obtain the taxol particle surface modified with soybean agglutinin and tannic acid.

2. Inhibitory assay for tumor cells

Taking human breast cancer cells MCF-7 in logarithmic growth phase, human noduleIntestinal cancer cell HCT-116, human non-small cell lung cancer A549, 2.5g/L pancreatin digested plate (96-well plate, 5X 10)³One cell/well), incubating at 37 ℃ for 24h to allow the cells to adhere to the wall, setting PBS (phosphate buffer solution) wells as a control group, adding 5 mug/ml of paclitaxel into the drug treatment group 1, adding 5 mug/ml of the soybean agglutinin prepared in example 1 and tannic acid surface modified paclitaxel particles (100 mug/well) into the drug treatment group 2, setting five multiple wells per well, culturing for 48h, adding 30 μ L of thiazole blue (MTT, 5mg/mL) into each well, culturing for 4 hr, removing supernatant, adding 100 μ L of dimethyl sulfoxide into each well, shaking to dissolve the crystal, the absorbance at 490nm of each well was measured on a microplate reader, and the cell viability (absorbance value a490 nm/blank well absorbance value a490nm after drug treatment) x 100%, the in vitro toxicity results of the antitumor drugs on tumor cells are shown in table 1.

TABLE 1 in vitro toxicity of antitumor drugs to tumor cells

As can be seen from Table 1, the paclitaxel surface-modified with soybean lectin and tannic acid prepared in example 1 and Taxus chinensis have the same toxicological effects on tumor cells, and the paclitaxel surface-modified with soybean lectin and tannic acid has stronger effect on tumor cell proliferation.

4. Targeting property of soybean agglutinin and tannic acid surface modified paclitaxel microparticles in vivo

Balb/c female nude mice (5 weeks old) are inoculated with human breast cancer cells MCF-7 subcutaneously, and the tumor volume is taken as 100mm³30 mice bearing MCF-7 tumor of left and right human breast cancer cells are randomly divided into 3 groups, which are respectively: free sulfo-Cy5.5 (water-soluble cyanine dye Cy5.5) group, sulfo-Cy5.5-labeled paclitaxel group, sulfo-Cy5.5-labeled soybean lectin and tannic acid surface-modified paclitaxel microparticle group prepared in example 1.

sulfo-Cy5.5-N-hydroxysuccinimide ester reacts with paclitaxel to obtain sulfo-Cy5.5 labeled paclitaxel. And (3) reacting the sulfo-Cy5.5-N-hydroxysuccinimide ester with the soybean agglutinin and the tannin surface modified taxol microparticle to obtain the sulfo-Cy5.5 marked soybean agglutinin and tannin surface modified taxol microparticle. 0.1ml of sulfo-Cy5.5 fluorescent dye (1mg/ml), 1mg/ml of sulfo-Cy5.5 labeled paclitaxel (1mg/ml), 1mg/ml of sulfo-Cy5.5 labeled soybean agglutinin and 1mg/ml of tannic acid surface modified paclitaxel particles are respectively injected into tail veins, 0.5h, 2h, 4h, 12h and 24h are respectively administered, a chloral hydrate solution is injected into abdominal cavities, after nude mice are coma, a living body imager is adopted to analyze the distribution condition of the drug in the nude mice, after 24h, the experimental nude mice are euthanized, and the heart, liver, lung, kidney and tumor tissues of each group of nude mice are taken to observe fluorescence imaging.

TABLE 2 fluorescence intensity of free organs of tumor-bearing mice

At the time point of 4h administration, the paclitaxel microparticles with the surface modified by the soybean agglutinin and the tannic acid have obvious aggregation at the subcutaneous tumor part of the nude mice, while the paclitaxel microparticles without the surface modified by the soybean agglutinin and the tannic acid have no obvious aggregation at the tumor part of the nude mice, which is attributed to the active targeting effect of the soybean agglutinin recognition. As can be seen from table 2, at the time point of 24h, compared with the free fluorochrome group and the paclitaxel group without surface modification, the fluorescence signal in the tumor tissue after tail vein injection of the soybean lectin and tannin surface-modified paclitaxel microparticle group can last up to 24 h. The paclitaxel microparticles with the surface modified by the soybean agglutinin and the tannic acid can reduce the aggregation of normal tissue cells and reduce side effects while killing tumor cells. The result shows that the aggregation degree of the paclitaxel in the tumor tissue of a nude mouse can be prolonged by injecting the sulfo-Cy5.5 labeled soybean agglutinin and the tannin surface modified paclitaxel into the tail vein, and a strong fluorescence signal can be observed in the tumor tissue. In summary, the paclitaxel microparticles surface-modified by soybean agglutinin and tannic acid of the patent not only show significant anti-tumor activity, but also target tumor, and improve bioavailability.

The above description is only a preferred embodiment of the present invention and should not be taken as limiting the invention, and any modification, replacement, or improvement made on the principle of the present invention should be included in the protection scope of the present invention.

Claims (3)

1. A preparation method of paclitaxel particles surface-modified by soybean agglutinin and tannic acid is characterized in that: pulverizing paclitaxel into powder, and coating tannin layer on the surface of paclitaxel microparticle by aqueous solution assembly technology; and then stably combining the soybean agglutinin of which the sugar structure end is modified by boric acid and polyvinyl alcohol with a tannin layer on the surface of the taxol particles in a hydrogen bond mode through a protein part of the soybean agglutinin, and finally dissociating the sugar structure end modified part of the soybean agglutinin under the condition of pH 3, so that the sugar structure end of the agglutinin is accurately pointed to the outside of the particles.

2. The method of claim 1, wherein: the tannic acid is derived from pomegranate rind, geranium wilfordii, grape rind or gallnut, and the mass concentration of the tannic acid aqueous solution is 0.05-0.2 g/ml;

incubating paclitaxel ground by a high-pressure homogenizer in a tannic acid aqueous solution for 8 hours at room temperature, and carrying out vacuum freeze drying for 30min to obtain paclitaxel particles with tannic acid surface modification;

in an aqueous solution with the pH value of 9.0, boric acid and polyvinyl alcohol 1788 are subjected to a crosslinking reaction on the sugar structure end of the soybean agglutinin to obtain the soybean agglutinin of which the sugar structure end is modified by the boric acid and the polyvinyl alcohol;

adjusting the pH of the aqueous suspension of the soybean agglutinin with the changed sugar structure end and the paclitaxel surface-modified with the tannic acid to 3.0 by using citric acid, dissociating the sugar structure end modified with the soybean agglutinin under an acidic condition, reacting for half an hour, placing the aqueous suspension of the soybean agglutinin and the paclitaxel surface-modified with the tannic acid in a low-temperature high-speed refrigerated centrifuge, centrifuging for 30min at 15000rpm, discarding the supernatant, washing the precipitate for 3 times by using distilled water, removing impurities such as boric acid and 1788 of polyvinyl alcohol, and freeze-drying to obtain the paclitaxel microparticles surface-modified with the soybean agglutinin and the tannic acid.

3. The method of claim 1, wherein: the method comprises the following steps:

1) modifying the tannin layer on the surface of paclitaxel: taking a tannic acid solution with the mass concentration of 0.05-0.2g/ml, putting paclitaxel crushed by a high-pressure homogenizer into the tannic acid solution, incubating for 8 hours at room temperature, and performing vacuum freeze drying to obtain tannic acid surface-modified paclitaxel particles; wherein the weight ratio of paclitaxel: the mass ratio of the tannic acid is 1:0.3 to 3; the tannic acid is derived from pericarpium Granati, herba Erodii seu Geranii, grape skin or Galla chinensis;

2) boric acid, soybean agglutinin, polyvinyl alcohol 1788 are dissolved in distilled water, and the ratio of soybean agglutinin: boric acid: the mass ratio of the polyvinyl alcohol 1788 is 1: 0.5-2: 0.5 to 2; adjusting pH of the solution to 9 with dipotassium hydrogen phosphate, heating and stirring at 60 deg.C for 50min to crosslink sugar structure end of soybean agglutinin with boric acid and polyvinyl alcohol 1788 to obtain soybean agglutinin with sugar structure end modified with boric acid and polyvinyl alcohol;

3) adding the taxol particles subjected to surface modification of the tannic acid obtained in the step 1) into the soybean lectin solution, obtained in the step 2), of which the sugar structure end is modified by boric acid and polyvinyl alcohol, wherein the taxol particles subjected to surface modification of the tannic acid: the mass ratio of the soybean agglutinin solution with the changed sugar structure end is 1:0.3-3, and the pH value of the mixed solution is adjusted to 3.0 by citric acid; after reacting for half an hour, placing the mixture in a low-temperature high-speed refrigerated centrifuge, centrifuging at 15000rpm for 30min, discarding the supernatant, removing impurities such as boric acid and polyvinyl alcohol 1788, washing the precipitate with distilled water for 3 times, and freeze drying to obtain the surface-modified paclitaxel microparticles of soybean agglutinin and tannic acid.