CN114984237B - 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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CN114984237B
CN114984237B CN202210467569.3A CN202210467569A CN114984237B CN 114984237 B CN114984237 B CN 114984237B CN 202210467569 A CN202210467569 A CN 202210467569A CN 114984237 B CN114984237 B CN 114984237B
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tanshinone iia
modifier
solution
estrogen receptor
carrier
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CN114984237A (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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    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
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    • A—HUMAN NECESSITIES
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    • A61P35/00—Antineoplastic agents
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D307/92—Naphthofurans; Hydrogenated naphthofurans
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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 functions similar to those of estrogen, and the traditional Chinese medicine salvia miltiorrhiza contains various active ingredients of phytoestrogens, 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 diseases, 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, the present invention provides a method 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 an anti-tumor drug Dox, the carrier shows better tumor targeting and anti-tumor effects at 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.
Drawings
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 the estrogen receptor beta protein (A: beta estradiol; B: tanIIA; 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 changes of the tumor-bearing mouse model over the course of treatment (days 2-12).
Fig. 8 is a picture of isolated tumors in 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:
1. modification of tanshinone IIA as active component of phytoestrogen Chinese medicine
(1) Tanshinone IIA (4.5g, 15.3mmol) was dissolved in acetic acid (360 mL), glycine ethyl ester hydrochloride (4.25g, 30.6 mmol) 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 x 5). The mixture was filtered and chromatographed on silica gel (eluent: petroleum ether/ethyl acetate = 60/40) to give intermediate 1.
(2) Intermediate 1 (2.0 g, 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, 70% yield) as a red solid.
(3) Intermediate 2 (1.3g, 3.4 mmol) was added to DMF (10 mL) 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, 10.2mmol). Stir at room temperature for 1h. The disappearance of the intermediate was checked by LCMS. After the reaction is completed. The mixture was extracted with ethyl acetate (300ml × 3) and the organics were washed with aqueous ammonia (50ml × 2). The organic phase was concentrated to give intermediate 3 (1.5 g).
(4) To a solution of intermediate 3 (1.5 g) in DCM (10 mL) was slowly added TFA (10 mL). The reaction was carried out at room temperature for 1 hour. LCMS monitored completion of reaction. The tanshinone IIA modification (265mg, 0.477mmol, 13.7%) was obtained by the reversed phase preparation method of Pre-HPLC under acidic conditions. The modification is in the form of trifluoroacetate.
2. The preparation of the target nanoparticle 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 for 3 times by using PBS buffer solution, and drying the precipitate for 12h in vacuum at 40 ℃ to obtain Dox-MSN nanoparticles.
(2) Weighing a proper amount of Dox-MSN, 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, 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:
1. modification of tanshinone IIA as active component of phytoestrogen 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-azobenzotriazole) -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.
2. The preparation of the target nanoparticle 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 tanshinone IIA modifier and triethylamine (2 eq) by mass, and reacting. 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:
1. modification of tanshinone IIA as active component of phytoestrogen Chinese medicine
(1) Tanshinone IIA is dissolved in acetic acid. And then adding glycine ethyl ester hydrochloride and a paraformaldehyde solution, uniformly stirring, 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, stirring uniformly, 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.
2. The preparation of the target nanoparticle 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 (6 eq) 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:
1. molecular docking study
Ligand preparation: the structure of tanshinone IIA comes from a 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: estrogen receptor alpha (PDB: 1 QKT) and estrogen receptor beta (PDB: 3 OLL) crystal structures were downloaded from Protein databases (Protein Data Bank, PDB database, http:// www. Rcsb. Org.) and stored in mol2 format. Introducing the protein structure into Discovery Studio software, dehydrating, selecting the binding site of the original ligand, 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 docking with Estrogen receptor alpha protein
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.
2. Preparation of Dox-MSN and Tan-Dox-MSN preparations
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 by using the PBS buffer solution, vacuum-drying the precipitate at 40 ℃ for 12h to obtain Dox-MSN nanoparticles, and combining all the supernate and washing solution for calculating the drug loading. FIG. 3 TEM image of Tan-Dox-MSN prepared in example 1.
3. Cell uptake study
Collecting MCF-7 cells in log-extended period, and measuring density at 5 × 10 5 One well, inoculated in 6-well plates, 2mL per well, placed in an incubator, 5% CO 2 And cultured at 37 ℃ for 24h. 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 culturing for 1, 2, and 4 hours. After the completion of the culture, the cells were washed three times with PBS, digested with 1mL of 0.25% pancreatin +0.02% EDTA, digested with two times of the complete medium, centrifuged, dispersed with PBS, and analyzed by flow cytometry, and the excitation wavelength of Dox was set to 470nm and the emission wavelength was set to 580nAnd m is selected. Quantitative analysis was performed on the uptake of different preparations by MCF-7 cells.
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.
4. In vivo tumor targeting study
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 1mg/kg; the dose of Dox in each group was 2mg/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.
5. Study of antitumor Activity
The antitumor effect of the nano-structure lipid preparation for improving the active targeting property of the adriamycin tumor and protecting the kidney in vivo is investigated by constructing a BALB/c nude mouse breast cancer model of tumor-bearing MCF-7 cells, administering different medicines, recording the volume, the weight and the like of the tumor, and the antitumor effect of the medicine 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 is diluted to 1X 10 by MEM medium 8 Add equal volume of matrigel per mL. Wiping the axilla of a female BALB/c nude mouse with alcohol cotton, inoculating 100 μ L of cell suspension containing matrigel under the axilla of the forelimb of the nude mouse by using a precooled 1mL syringe, and taking care to uniformly mix the cell suspension during inoculation and keeping the temperature at 0-4 ℃ to prevent the matrigel from solidifying. Observing the growth condition of the tumor every day, and measuring the size of the tumor by using a vernier caliper until the tumor volume reaches 100mm 3 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 the fourth pharmaceutical factory of Shijiazhuang.
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.
Preparing a blank mesoporous silicon nanoparticle (Tan-MSN) modified by tanshinone IIA: weighing a proper amount of MSN-COOH, ultrasonically dispersing in 10mL 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 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 mesoporous silicon nanoparticle group (Tan-Dox-MSN) loaded with doxorubicin hydrochloride: 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, 12), tumor volume V = ab was measured by measuring tumor major axis (a) and minor axis (b) with a vernier caliper 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 a Tan-Dox-MSN group and the ratio of a 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 in tumor parts, 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 (9)

1. A tanshinone IIA modifier having a structure represented by the following formula (I):
Figure FDA0003922073680000011
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 FDA0003922073680000012
(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 80 ℃, 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 targeted molecule covalently connected with the carrier, wherein the targeted molecule is the tanshinone IIA modifier or the salt thereof according to claim 1, and the carrier is mesoporous silicon nanoparticles.
8. The method for preparing the estrogen receptor targeted delivery system according to claim 7, wherein the method comprises dispersing the 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.
9. Use of the tanshinone IIA modifier according to claim 1 or a salt thereof, or the estrogen receptor targeted delivery system according to claim 7 in preparation of a medicament for resisting an estrogen receptor over-expression tumor.
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