CN114984237A - Tanshinone IIA modifier and preparation method and application thereof - Google Patents

Tanshinone IIA modifier and preparation method and application thereof Download PDF

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CN114984237A
CN114984237A CN202210467569.3A CN202210467569A CN114984237A CN 114984237 A CN114984237 A CN 114984237A CN 202210467569 A CN202210467569 A CN 202210467569A CN 114984237 A CN114984237 A CN 114984237A
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tanshinone iia
solution
estrogen receptor
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carrier
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CN114984237B (en
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刘志东
张瀛
祁东利
党文利
皮佳鑫
郭盼
邓秀平
邢斌
于长祥
李佳玮
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Tianjin University of Traditional Chinese Medicine
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Abstract

The invention discloses a tanshinone IIA modifier, a preparation method and application thereof. The tanshinone IIA modifier with the terminal amino group is successfully prepared through the steps of Mannich addition reaction, ester hydrolysis reaction, condensation reaction and the like. The affinity of tanshinone IIA and estrogen receptor is utilized to construct a nano delivery system with active targeting. After the anti-tumor drug Dox is loaded, the anti-tumor drug Dox shows better tumor targeting and anti-tumor effects on the cellular level and the water level in animals, has higher safety, and is expected to become a targeted treatment mode of diseases with estrogen receptor overexpression characteristic represented by breast cancer.

Description

Tanshinone IIA modifier and preparation method and application thereof
Technical Field
The invention relates to the field of medicines, in particular to a tanshinone IIA modifier, a preparation method and application thereof, and especially relates to preparation of mesoporous silicon nanoparticles targeting estrogen receptors and application thereof in treating breast cancer.
Background
Breast cancer has become one of the most serious diseases threatening female health and causing female death in today's world, and it is predicted that the total number of breast cancer patients will continue to increase in the next decade, and more than 70% of breast cancer is estrogen-dependent. Nano medicine provides new opportunities for the diagnosis and treatment of breast cancer, and based on estrogen-dependent breast cancer, the overexpression of estrogen receptors is one of the diagnostic bases. The well designed nanometer preparation targeting the estrogen receptor has great significance for diagnosing and treating estrogen-dependent breast cancer.
The phytoestrogen has a structure and a function similar to those of estrogen, and the traditional Chinese medicine salvia miltiorrhiza contains a plurality of active ingredients of phytoestrogen, wherein tanshinone IIA (Tan IIA) is reported to be used as the active ingredient for treating cardiovascular diseases, has the functions of expanding blood vessels, reducing blood pressure and resisting thrombus, and has obvious curative effects on treating coronary heart disease, angina pectoris and tachycardia.
The information in this background is only for the purpose of illustrating the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art that is known to a person skilled in the art.
Disclosure of Invention
In order to solve at least part of technical problems in the prior art, the invention provides a tanshinone IIA modifier which is used as a targeting molecule for preparing a drug delivery system, can specifically target a drug delivery part, and greatly improves the active targeting property of drug delivery. Specifically, the present invention includes the following.
In a first aspect of the present invention, there is provided a tanshinone IIA-modifying compound having a structure represented by the following formula (I):
Figure BDA0003625065510000021
wherein n is an integer in the range of 2 to 10, preferably 3 to 8, more preferably 3 to 5.
Preferably, the salt is an organic acid salt, examples of which include, but are not limited to, trifluoroacetic acid, methanesulfonic acid, and toluenesulfonic acid.
In a second aspect of the present invention, there is provided a process for preparing a tanshinone IIA-modifying compound or a salt thereof, comprising the steps of:
(1) adding amino-PEGn-amino with a protecting group at one end into an organic solution of a compound with a structure shown in a formula (II) to react with a catalyst, then extracting, washing with ammonia water, and concentrating to obtain an intermediate 3;
Figure BDA0003625065510000022
(2) and adding organic acid into the intermediate 3 organic solution to react to obtain the tanshinone IIA modifier.
Alternatively, the preparation method of the present invention may further comprise (3) adding glycine ethyl ester hydrochloride to the acetic acid solution of tanshinone IIA to react with an aqueous aldehyde solution, examples of which include an aqueous formaldehyde solution, or an aqueous paraformaldehyde solution such as an aqueous trioxymethylene solution, etc., to obtain intermediate 1;
Figure BDA0003625065510000031
dissolving the intermediate 1 in a hydrochloric acid solution, stirring at high temperature, concentrating and freeze-drying to obtain an intermediate 2;
Figure BDA0003625065510000032
according to the preparation method of the present invention, preferably, the amino protecting group is at least one of a Cbz protecting group, a Boc protecting group, a PMB protecting group, and a Trt protecting group.
According to the preparation method of the present invention, preferably, the organic solution in the step (3) is at least one solution of dimethylacetamide, tetrahydrofuran and dichloromethane.
According to the preparation method of the invention, preferably, the catalyst of the step (3) comprises 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate and N, N-diisopropylethylamine.
According to the preparation method of the present invention, preferably, the organic acid of step (4) includes at least one of trifluoroacetic acid, methanesulfonic acid and toluenesulfonic acid.
In a third aspect of the present invention, an estrogen receptor targeted delivery system is provided, which comprises a carrier and a targeting molecule covalently linked to the carrier, wherein the targeting molecule is a tanshinone iia modification compound or a salt thereof as described in the first aspect.
According to the estrogen receptor targeted delivery system, the carrier is preferably a nanoparticle, such as a silicon mesoporous silica particle.
The fourth aspect of the present invention provides a method for preparing an estrogen receptor targeting delivery system, which comprises dispersing a carrier in an aqueous solution, activating carboxyl groups on the surface of the carrier, adding tanshinone IIA modifier or salt thereof and triethylamine to react, and obtaining the delivery system. The carrier may be a drug-loaded carrier or an empty drug-unloaded carrier.
In the fifth aspect of the invention, the tanshinone IIA modifier or salt thereof, or the application of the estrogen receptor targeting delivery system in preparing the medicine for resisting the estrogen receptor over-expression tumor is provided.
The tanshinone IIA modifier with the terminal amino group is successfully prepared through the steps of Mannich addition reaction, ester hydrolysis reaction, condensation reaction and the like. Further, the modifier is grafted to a carrier to construct a nanoparticle-level targeted delivery carrier, and after the carrier is loaded with the antitumor drug Dox, the carrier shows better tumor targeting and antitumor effect on the cellular level and the water level in an animal body, and has higher safety. Tan-MSN is used as a nano-platform-based traditional Chinese medicine active ingredient mediated administration route, and is expected to become a targeted treatment mode for diseases with estrogen receptor overexpression characteristic represented by breast cancer.
The invention utilizes the affinity of Tan IIA and estrogen receptor to construct a nano delivery system with active targeting. In a preferred embodiment, the mesoporous silicon nanoparticles are used as a carrier, and have the advantages of good biocompatibility, high effective component loading, easy surface modification and the like. Therefore, tanshinone IIA is modified on the mesoporous silicon nanoparticles, and the mesoporous silicon nanoparticles with estrogen receptor targeting property are prepared by utilizing the affinity effect of the tanshinone IIA with estrogen receptors, thereby playing an important role in diagnosis and treatment of breast cancer.
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FIG. 1 molecular docking of different ligands to estrogen receptor alpha protein (A: beta estradiol; B: TanIIA; C: Tan-COOH).
FIG. 2 molecular docking of different ligands to estrogen receptor beta protein (A: beta estradiol; B: Tan IIA; C: Tan-COOH).
FIG. 3 TEM image of Tan-Dox-MSN prepared in example 1.
FIG. 4 results of quantitative analysis of nanoparticle uptake by MCF-7 cells incubated for various periods of time. Note (×: P < 0.001, ×: P < 0.01, mean ± SD, n ═ 3).
FIG. 5 shows the fluorescence distribution of indocyanine green in tumor-bearing nude mice at different time points.
FIG. 6 shows the fluorescence intensity of doxorubicin hydrochloride in tumors and organs at 12 h.
FIG. 7 is a graph of the relative tumor volume change of the tumor-bearing mouse model over the course of treatment (days 2-12).
Fig. 8 is a picture of isolated tumors of each group of mice after 12 days. A: physiological saline; b: dox injection; c: a Dox solution; d: Dox-MSN; e: MSN-Tan; f: Tan-Dox-MSN.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is percent by weight.
Example 1
The embodiment is a preparation method of mesoporous silicon nanoparticles for improving active targeting of tumors, which comprises the following steps:
modification of tanshinone IIA as active component of phytoestrogen Chinese medicine
(1) Tanshinone IIA (4.5g, 15.3mmol) was dissolved in acetic acid (360mL), glycine ethyl ester hydrochloride (4.25g, 30.6mmol) and formaldehyde (3mL, 37% aqueous solution) were added, and stirring was carried out at 65 ℃ for 12 hours. LCMS monitor reaction. After completion of the reaction, the mixture was cooled to 25 ℃ and concentrated under vacuum. The mixture was washed with clean water (200mL 5). The mixture was filtered and chromatographed on silica gel (eluent: petroleum ether/ethyl acetate 60/40) to give intermediate 1.
(2) Intermediate 1(2.0g, 4.88mmol) was added to a solution of 6N hydrochloric acid (20 mL). The solution was stirred at 80 ℃ for 4 hours. LCMS showed reaction completion. Then concentrated in vacuo and lyophilized directly to yield intermediate 2(1.3g, 3.4mmol, yield 70%) as a red solid.
(3) Intermediate 2(1.3g, 3.4mmol) was added to DMF (10mL) followed by a solution of t-Boc-N-Amido-PEGn-amine (N ═ 3, 1.2g, 4.1mmol), HATU (1.56g, 4.1.1mmol) and DIEA (1.3g,10.2 mmol). Stir at room temperature for 1 h. The disappearance of the intermediate was checked by LCMS. After the reaction is completed. The mixture was extracted with ethyl acetate (300mL x 3) and the organics were washed with aqueous ammonia (50mL x 2). The organic phase was concentrated to give intermediate 3(1.5 g).
(4) To intermediate 3(1.5g) in DCM (10mL) was added TFA (10mL) slowly. The reaction was carried out at room temperature for 1 hour. LCMS monitored completion of reaction. Through a reversed-phase preparation method by Pre-HPLC under acidic conditions, tanshinone IIA modification (265mg, 0.477mmol, 13.7%) was obtained. The modification is in the form of trifluoroacetate.
Secondly, the preparation of the target nano-particle modified by phytoestrogen tanshinone IIA comprises the following steps:
(1) weighing a proper amount of MSN-COOH, dispersing in PBS buffer solution containing Dox, stirring (keeping out of the sun) for a certain time at room temperature, centrifuging (10000rpm, 25 ℃,10 min), washing the precipitate with PBS buffer solution for 3 times, and vacuum drying the precipitate at 40 ℃ for 12h to obtain Dox-MSN nanoparticles.
(2) Weighing a proper amount of Dox-MSN, ultrasonically dispersing the Dox-MSN in 10mL of MES buffer solution (pH 6.0), adding a proper amount of EDC/NHS, uniformly mixing, oscillating for 1h at room temperature to activate carboxyl, centrifugally separating (10000rpm, 25 ℃,10 min), taking precipitate, re-dispersing the precipitate in 10mL of phosphate buffer solution with pH 7.4, adding a proper amount of tanshinone IIA modifier and triethylamine, reacting for 3h, centrifugally separating (10000rpm, 25 ℃,10 min), washing the precipitate for 3 times by using PBS buffer solution, and obtaining the tanshinone IIA modified mesoporous silicon nanoparticles (Tan-Dox-MSN).
As can be seen from fig. 3, the mesoporous silicon nanoparticles prepared in this example have a small and uniform particle size.
Example 2
The embodiment is a preparation method of mesoporous silicon nanoparticles for improving active targeting of tumors, which comprises the following steps:
modification of tanshinone IIA as active ingredient of phytoestrogen traditional Chinese medicine
(1) Dissolving tanshinone IIA in acetic acid, adding glycine ethyl ester hydrochloride and formaldehyde water solution, stirring uniformly, reacting for a proper time, and purifying by a silica gel column to obtain an intermediate 1.
(2) The intermediate 1 was dissolved in hydrochloric acid solution. The solution was stirred at high temperature, concentrated and lyophilized to give intermediate 2.
(3) Dissolving the intermediate 2 in dimethylacetamide, adding amino-PEGn-Cbz amino protecting group (N ═ 4), 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate and N, N-diisopropylethylamine, stirring uniformly, extracting with ethyl acetate, washing with ammonia water, and concentrating to obtain an intermediate 3.
(4) And dissolving the intermediate 3 in DMSO, adding trifluoroacetic acid, reacting completely, and obtaining the tanshinone IIA modifier by preparative chromatography. The modification is in the form of trifluoroacetate.
Secondly, the preparation of the target nano-particle modified by phytoestrogen tanshinone IIA comprises the following steps:
(1) carboxyl functionalized mesoporous silicon (MSN-COOH) is used as a delivery carrier, and indocyanine green (ICG) is loaded by an adsorption method.
(2) Dispersing the carrier loaded with the effective components in an aqueous solution, activating carboxyl by adopting 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS), centrifuging, adding 0.065 times of mass of tanshinone IIA modifier and triethylamine (2eq) to react. The multifunctional mesoporous silicon nano-particle of the targeting estrogen receptor is obtained.
Through detection, the particle size of the mesoporous silicon nanoparticle targeting the estrogen receptor of the embodiment is similar to that of the embodiment 1.
Example 3
The embodiment is a preparation method of a multifunctional mesoporous silicon nanoparticle for improving active targeting of tumors, which comprises the following steps:
modification of tanshinone IIA as active component of phytoestrogen Chinese medicine
(1) Tanshinone IIA is dissolved in acetic acid. Then adding glycine ethyl ester hydrochloride and paraformaldehyde solution, stirring uniformly, reacting for a proper time, and purifying by a silica gel column to obtain an intermediate 1.
(2) Dissolving the intermediate 1 in hydrochloric acid solution. The solution was stirred at high temperature, concentrated and lyophilized to give intermediate 2.
(3) Dissolving the intermediate 2 in dimethylacetamide, adding amino-PEGn-PMB amino protecting group (N ═ 6), 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate and N, N-diisopropylethylamine, uniformly stirring, extracting with ethyl acetate, washing with ammonia water, and concentrating to obtain an intermediate 3.
(4) And dissolving the intermediate 3 in chloroform, adding trifluoroacetic acid, and reacting completely to obtain the tanshinone IIA modifier by preparative chromatography. The modification is in the form of trifluoroacetate.
Secondly, the preparation of the target nano-particle modified by phytoestrogen tanshinone IIA comprises the following steps:
(1) carboxyl functionalized mesoporous silicon is used as a delivery carrier, and coumarin-6 is loaded by an adsorption method.
(2) Dispersing the carrier loaded with the effective components in an aqueous solution, activating carboxyl by adopting 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, centrifuging, adding 0.2 time of tanshinone IIA modifier and triethylamine (6eq) by mass, and reacting. The mesoporous silicon nano-particle of the targeting estrogen receptor is obtained.
Through detection, the particle size of the mesoporous silicon nanoparticle targeting the estrogen receptor of the embodiment is similar to that of the embodiment 1.
Test example
In order to determine whether the modification group affects the binding activity of the drug, the inventors performed molecular docking studies, cell uptake tests, tumor targeting studies, and anti-tumor activity studies on the mesoporous silicon nanoparticles targeting the estrogen receptor of example 1, with the following methods and results:
first, molecular docking study
Ligand preparation: the structure of tanshinone IIA is from the traditional Chinese medicine database (http:// tcm. cmu. edu. tw), the structures of the original ligand beta-estradiol and the modifier are drawn by using ChemDraw software, the structures are stored in a mol2 format, and all ligands are introduced into Discovery Studio software. After hydrogenation and energy minimization operations are carried out on the ligand, the ligand is in butt joint with a receptor.
Receptor preparation: the estrogen receptor alpha (PDB: 1QKT) and estrogen receptor beta (PDB: 3OLL) crystal structures were downloaded from Protein databases (Protein Data Bank, PDB database, http:// www.rcsb.org) and stored in mol2 format. And (3) introducing the protein structure into Discovery Studio software, dehydrating, selecting an original ligand binding site, deleting the original ligand, and docking with a preselected ligand.
In the butt joint process, a CDOCKER mode is adopted, the radius of the selected cavity is 10, and other parameters are system default values.
As can be seen from tables 1 and 2, the tanshinone iia and its modified substance, the estrogen receptor α and the estrogen receptor β, the-cdoccker INTERACTION assay, are positive values, and the lower the ENERGY, the better the stability, which indicates that the tanshinone iia and its modified substance are more stably combined with the estrogen receptor α and the estrogen receptor β.
TABLE 1 results of Pre-selected ligand to Estrogen receptor alpha protein docking
Figure BDA0003625065510000091
TABLE 2 results of Pre-selected ligand docking with Estrogen receptor beta protein
Figure BDA0003625065510000092
As can be seen in FIG. 1-A, the pro-ligand β estradiol forms hydrogen bond interactions with the amino acid residues ARG394, GLU353 in the estrogen receptor α and hydrophobic interactions with LEU346, LEU384, ALA350, MET388, LEU387, LEU 391. ARG394, GLU353, LEU346, LEU384, ALA350, MET388, LEU387, LEU391 are therefore considered to be key amino acids for binding of estrogen receptor alpha to ligands.
As can be seen from FIG. 1-B, tanshinone IIA forms hydrogen-bond interactions with the amino acid residues GLU353, MET388 of estrogen receptor alpha and hydrophobic interactions with LEU349, LEU346, ALA350, MET421, LEU525, HIS524, MET343, LEU384, LEU387, LEU391, PHE 404.
As shown in FIG. 1-C, the tanshinone IIA terminal carboxyl modifier forms hydrogen bond interaction with the amino acid residue THR347 in the estrogen receptor alpha, forms hydrophobic interaction with ALA350, LYS529, MET388, PHE404 and LEU384, forms Pi-sulfurr interaction with MET421 and forms Pi-Sigma interaction with LEU 525.
As can be seen from FIG. 2-A, the pro-ligand β estradiol forms hydrogen bond interactions with the amino acid residues HIS475, GLU305 of the estrogen receptor β and hydrophobic interactions with LEU298, LEU476, ILE373, MET336, ILE376, MET340, ALA302, LEU339, LEU343, PHE 356. HIS475, GLU305, LEU298, LEU476, ILE373, MET336, ILE376, MET340, ALA302, LEU339, LEU343, PHE356 are therefore considered to be key amino acids for binding of estrogen receptor beta to ligands.
As can be seen from FIG. 2-B, Tan IIA forms hydrogen bond interactions with the amino acid residues GLU305, MET340 of the estrogen receptor β and hydrophobic interactions with LEU301, LEU339, ALA302, LEU298, LEU476, MET295, ILE373, HIS475, LEU343, PHE 356.
As can be seen from fig. 2-C, intermediate 2 forms hydrogen bond interactions with the amino acid residues MET340, LEU298, GLU305 of the estrogen receptor β, hydrophobic interactions with PHE356, LEU343, LAL302, LEU339, HIS475, MET295, LEU476, ILE373, ILE376 and Pi-sulfur interactions with MET 336.
Preparation of di, Dox-MSN and Tan-Dox-MSN preparation
Weighing a proper amount of MSN-COOH, dispersing in doxorubicin hydrochloride PBS buffer solution with a certain concentration, stirring (keeping out of the sun) for a certain time at room temperature, centrifuging (10000rpm, 25 ℃,10 min), washing the precipitate for 3 times with the PBS buffer solution, vacuum-drying the precipitate at 40 ℃ for 12h to obtain Dox-MSN nanoparticles, and combining all the supernatants and the washing solution for calculating the drug loading. FIG. 3 TEM image of Tan-Dox-MSN prepared in example 1.
Third, study of cellular uptake
Collecting MCF-7 cells in log-extended period, and measuring density at 5 × 10 5 One well/well, inoculated in 6-well plates, 2mL per well, placed in incubator, 5% CO 2 And cultured at 37 ℃ for 24 h. The original culture medium was aspirated, and 2mL of a culture medium containing Dox-MSN and Tan-Dox-MSN (Dox concentration: 5. mu.g/mL) was added thereto, followed by culture for 1, 2, and 4 hours. After the end of the culture, the cells were washed three times with PBS, digested with 1mL of 0.25% pancreatin + 0.02% EDTA, digested with twice the total medium, centrifuged, dispersed with PBS, and analyzed by flow cytometry, with Dox excitation wavelength set at 470nm and emission wavelength set at 580 nm. The uptake of the different preparations by MCF-7 cells was quantified.
As can be seen from FIG. 4, the uptake of MCF-7 cells in the prepared modified group (Tan-Dox-MSN) was higher than that in the unmodified group (Dox-MSN) in 1, 2 and 4 hours, and the targeting effect was better.
Fourth, research of tumor targeting in vivo
Corresponding nanoparticles (ICG-MSN, Tan-ICG-MSN) were prepared by substituting Dox with indocyanine green (ICG) using the procedure of example 1.
Tumor-bearing nude mice were randomly divided into 7 groups of 3 mice each. The specific grouping is as follows: a normal saline group, an ICG solution group, an ICG-MSN group, a Tan-ICG-MSN group, a Dox solution group, a Dox-MSN group, and a Tan-Dox-MSN group. The dosage of the medicament is calculated according to the weight after each group is respectively marked, 200 mu L of solution or preparation is injected into the tail vein of each group of mice, and the physiological saline group is injected with the physiological saline with the same volume. Wherein the dosage of ICG of each group is 1 mg/kg; the dose of Dox in each group was 2 mg/kg.
The physiological saline group, the ICG solution group, the ICG-MSN group and the Tan-ICG-MSN group are subjected to in-vivo imaging photographing at 1h, 4h, 12h and 24h after injection, and the fluorescence distribution condition in the mice of each group is observed. Mice in the Dox solution group, the Dox-MSN group and the Tan-Dox-MSN group are sacrificed 12h after administration, dissected, and respectively taken heart, liver, spleen, lung, kidney and tumor tissues, placed in a living body imaging system of small animals for observation, and the distribution conditions of the carrier and the drug in each tissue organ and tumor part are determined.
As can be seen from FIGS. 5 and 6, the fluorescent intensity of Tan-ICG-MSN at the tumor sites of mice at 12h and 24h is obvious, and good targeting property is shown, and the fluorescent intensity of the tumor sites of the Tan-Dox-MSN group is higher than that of the Dox-MSN group, which indicates that the Tan-Dox-MSN has good tumor targeting effect.
Fifth, research on antitumor Activity
The antitumor effect of the nano-structure lipid preparation for improving the active targeting property and the kidney protection of the adriamycin tumor in vivo is investigated by constructing a BALB/c nude mouse breast cancer model of tumor-bearing MCF-7 cells, administering different medicaments, recording the volume and weight of the tumor and the like, and the antitumor effect of the medicament is verified by investigating the cell proliferation and apoptosis conditions of the tumor part.
Taking MCF-7 cells of breast cancer in an exponential growth phase for subculture operation, after the cell density reaches a certain number, resuspending the cells by using MEM (minimum essential factor) culture medium, centrifuging the cells, washing the cells for 3 times by using PBS (phosphate buffer solution) to remove serum, diluting the cells by using MEM culture medium to adjust the cell concentration, and counting the cells by using a counting plate. The MCF-7 cell suspension was diluted to 1X 10 in MEM 8 Add equal volume of matrigel per mL. Wiping the oxter of a female BALB/c nude mouse with alcohol cotton, inoculating 100 mu L of cell suspension containing matrigel under the oxter of the forelimb of the nude mouse by a precooled 1mL syringe, and during the inoculation, taking care of the uniform mixing of the cell suspension and keeping the temperature at 0-4 ℃ to prevent the matrigel from coagulating. Observing the growth condition of the tumor every day, and measuring the size of the tumor by using a vernier caliper until the volume of the tumor reaches 100mm 3 On the left and right (a: tumor width, b: tumor length, tumor volume, V ═ ab: 2 and/2) carrying out random grouping.
Tumor-bearing mice were randomly divided into 6 groups. The specific grouping is as follows:
physiological saline group
Positive drug adriamycin hydrochloride injection (DOX injection)
Doxorubicin hydrochloride solution group (DOX solution)
Mesoporous silicon nanoparticle group loaded with doxorubicin hydrochloride (Dox-MSN)
Tanshinone IIA modified blank mesoporous silicon nanoparticle group (Tan-MSN)
Tanshinone IIA modified mesoporous silicon nanoparticle group (Tan-Dox-MSN) loaded with doxorubicin hydrochloride
The preparation method comprises the following steps:
physiological saline group: purchased from Shijiazhuang, fourth pharmaceutical factory.
Positive drug doxorubicin hydrochloride injection (DOX injection): purchased from Shanxi Pude pharmaceutical Co.
Preparation of doxorubicin hydrochloride solution (solution D): dissolving doxorubicin hydrochloride in physiological saline according to the mass ratio of 1:1000 to prepare a salvianolic acid A solution.
Preparing a blank mesoporous silicon nanoparticle (Tan-MSN) modified by tanshinone IIA: weighing a proper amount of MSN-COOH, ultrasonically dispersing in 10mL of MES buffer solution (pH 6.0), adding a proper amount of EDC/NHS, uniformly mixing, oscillating for 1h at room temperature to activate carboxyl, centrifugally separating (10000rpm, 25 ℃,10 min), taking precipitate, re-dispersing in 10mL of phosphate buffer solution with pH 7.4, adding a proper amount of tanshinone IIA modifier and triethylamine, reacting for 3h, centrifugally separating (10000rpm, 25 ℃,10 min), washing the precipitate for 3 times by using PBS buffer solution to obtain tanshinone IIA modified mesoporous silicon nanoparticles (Tan-MSN), and combining all supernate and washing solution for calculating the dosage loss.
Tanshinone IIA modified doxorubicin hydrochloride-loaded mesoporous silicon nanoparticle group (Tan-Dox-MSN): the sample prepared in example 1.
The dosage of the medicament is calculated according to the weight after each group is respectively marked, about 200 mu L of solution or preparation is injected into the tail vein of each group of mice, and the normal saline with the same volume is injected into the control group. The injection is given 1 time every 3 days for a total of 5 times.
As shown in FIG. 7, after administration ( days 0, 3, 6, 9, and 12), the vernier caliper was usedMeasuring the tumor major (a) and minor (b) diameters, and the tumor volume V ═ ab 2 /2. Relative tumor volume-time curves were plotted using the time of administration as the abscissa and the ratio of the tumor volume of each group at different times to the average tumor volume of the control group 12 days after administration as the ordinate (relative tumor volume).
The tumor-bearing mice are sacrificed the next day after the 5 th administration, the subcutaneous transplanted tumors and the cell membranes are completely stripped to form tumor tissues, as shown in figure 8, the relative tumor volume of the Tan-Dox-MSN group and the ratio of the DOX solution group and the Dox-MSN group are both remarkably reduced (p is less than 0.01), which shows that the nano-structure lipid preparation can improve the effective accumulation of the medicament at the tumor part, improve the bioavailability of the medicament and improve the in-vivo anti-tumor effect of the medicament.
Experiments prove that the targeting property of the multifunctional mesoporous silicon nanoparticles targeting estrogen receptors prepared in the embodiments 2 and 3 in cell uptake experiments and in vivo tumor targeting is similar to that in the embodiment 1.
While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Many modifications and variations may be made to the exemplary embodiments of the present description without departing from the scope or spirit of the present invention. The scope of the claims is to be accorded the broadest interpretation so as to encompass all modifications and equivalent structures and functions.

Claims (10)

1. A tanshinone IIA modifier having a structure represented by the following formula (I):
Figure FDA0003625065500000011
wherein n is an integer in the range of 2 to 10.
2. The method for preparing tanshinone IIA-modifying compounds or salts thereof according to claim 1, comprising the steps of:
(1) adding amino-PEGn-amino with a protecting group at one end into an organic solution of a compound with a structure shown in a formula (II) to react with a catalyst, then extracting, washing with ammonia water, and concentrating to obtain an intermediate 3;
Figure FDA0003625065500000012
(2) and adding organic acid into the organic solution of the intermediate 3 to react to obtain the tanshinone IIA modifier.
3. The preparation method according to claim 2, further comprising (3) a step of adding glycine ethyl ester hydrochloride and an aqueous aldehyde solution to the tanshinone IIA acetic acid solution to react to obtain an intermediate 1, dissolving the intermediate 1 in a hydrochloric acid solution, stirring at high temperature, concentrating and lyophilizing to obtain the compound having the structure shown in formula (II).
4. The method according to claim 2, wherein the organic solution in the step (1) is at least one solution of dimethylacetamide, tetrahydrofuran and dichloromethane.
5. The method according to claim 2, wherein the catalyst of step (1) comprises 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethyluronium hexafluorophosphate and N, N-diisopropylethylamine.
6. The method according to claim 2, wherein the organic acid of step (2) comprises at least one of trifluoroacetic acid, methanesulfonic acid and toluenesulfonic acid.
7. An estrogen receptor targeted delivery system, which is characterized by comprising a carrier and a targeting molecule covalently linked with the carrier, wherein the targeting molecule is the tanshinone IIA modifier or the salt thereof according to claim 1.
8. The estrogen receptor-targeted delivery system of claim 7, wherein the carrier is a nanoparticle.
9. The method for preparing the estrogen receptor targeted delivery system according to claim 7 or 8, which comprises dispersing a carrier in an aqueous solution, activating carboxyl on the surface of the carrier, adding tanshinone IIA modifier or salt thereof and triethylamine to react to obtain the delivery system.
10. Use of the tanshinone IIA modification compound or salt thereof according to claim 1 or the estrogen receptor targeting delivery system according to claim 7 or 8 in the preparation of a medicament for resisting an estrogen receptor over-expression tumor.
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