CN109745338B

CN109745338B - Preparation method and application of Pt (IV) polymer prodrug micelle encapsulating vorinostat and having reduction response

Info

Publication number: CN109745338B
Application number: CN201910042080.XA
Authority: CN
Inventors: 王燕铭; 沈杰; 杨硕; 余宙; 李静; 王雅洁
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2019-01-17
Filing date: 2019-01-17
Publication date: 2021-03-30
Anticipated expiration: 2039-01-17
Also published as: CN109745338A

Abstract

A Pt (IV) polymer prodrug micelle which has reduction response performance and can entrap Vorinostat and an application thereof are disclosed, cis-trans-diammoniumdichlorodisuccinate and cystamine dihydrochloride are used as raw materials, 1-hydroxy-7-azobenzotriazole and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride are used as condensing agents, and the Pt (IV) polymer prodrug micelle with stable physicochemical properties can be obtained through controllable microwave polymerization and PEG chemical technology, wherein the chemical structure is shown in a formula (1). The micelle not only can regulate and control the self drug-loading capacity, but also can release drugs aiming at the response of a tumor microenvironment, and has good inhibition effect on human cervical cancer cells and human ovarian cancer cells. Meanwhile, the micelle entrapped vorinostat can achieve a better synergistic tumor treatment effect at a dosage far lower than the normal administration dosage, and has a good application prospect.

Description

Preparation method and application of Pt (IV) polymer prodrug micelle encapsulating vorinostat and having reduction response

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a preparation method and application of Pt (IV) polymer prodrug micelle which is used for entrapping vorinostat and has reduction response for treating tumors.

Background

Platinum drugs represented by cisplatin have become the first choice for treating cancer by virtue of the advantages of wide antitumor spectrum, high anticancer activity, no cross-resistance with other anticancer drugs, suitability for combined medication and the like. However, the solubility is poor, the clinical administration is difficult, selectivity is not available, the drug is easy to combine with plasma protein, and the like, so that the clinical application of the drug is limited, and therefore, the effective structural modification and modification of cisplatin is always one of the focused works in the medical field. At present, pt (iv) drugs in clinical research have made great progress in improving the solubility of platinum drug molecules and improving the metabolic stability, but still face the problems of systemic non-targeted distribution, rapid in vitro excretion, and poor treatment effect when used alone. In the aspect of treating tumors by combining platinum drugs, a small-molecule drug synergistic treatment strategy is mainly adopted clinically at present, but the method also has many problems, such as lack of targeting and selectivity, high systemic toxicity, incapability of simultaneously reaching tumor tissues by multi-component drugs, complex pharmacokinetics caused by different administration modes, and the like. The physiological characteristics of the tumor microenvironment are utilized to design the nano drug-loading system with the environment response drug release performance, and micromolecule drugs with different mechanisms are delivered to the tumor part at the same time and released in a grading way, so that the problems can be effectively solved, and the effect of tumor treatment is greatly improved.

The traditional nano drug delivery system generally adopts two modes to cooperatively deliver multi-component drug molecules, one mode is to embed two or more drug molecules in a polymer core for transportation and delivery in a physical packaging mode, the other mode is to embed other components of drugs in a physical packaging mode by covalently coupling one drug molecule on a polymer skeleton, and although a great deal of research work has been reported in the two aspects, the two modes still have the defects of complex preparation process, poor reproducibility, non-uniform physicochemical properties, unstable drug release and the like, and have great limitation on medical application.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a preparation method and application of a functional nano delivery system, namely a Pt (IV) polymer prodrug micelle which is used for entrapping vorinostat and has reduction response and is used for synergistically treating tumors.

The technical scheme of the invention is as follows:

description of the symbols:

SAHA is an abbreviation for vorinostat;

mPEG_5K-P(DSP-AED)_nabbreviation for pt (iv) polymeric prodrug micelle with reduction response;

Bz-P(DSP-AED)_nabbreviation for benzylamine hydrochloride-terminated pt (iv) polymeric prodrug micelle with reduction response;

mPEG_5K-P(DSP-AED)_n@ SAHA is an abbreviation for SAHA-entrapped Pt (IV) polymeric prodrug micelles with a reduction response.

The invention firstly provides a preparation method of Pt (IV) polymer prodrug micelle with reduction response, and the micelle is named mPEG_5K-P(DSP-AED)_nWherein mPEG_5KRepresents PEG chain segment with the number average molecular weight of 5000, and n represents polymerization degree; the method takes cis-trans-Diammoniodidichloroplatinum (DSP) and cystamine dihydrochloride as raw materials, 1-hydroxy-7-azobenzotriazol (HOAT) and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (ECDI & HCl) as a condensing agent, and Pt (IV) polymer prodrug micelle which has different drug loading capacity, reduction response and drug release performance and stable physicochemical properties can be obtained through controllable microwave polymerization and PEG chemical processes, and the steps are as follows:

1) preparing cis-trans-Diamminedichloroplatinum (DSP) from Cisplatin (CDDP);

putting CDDP in distilled water, dropwise adding 30wt% of hydrogen peroxide under stirring, reacting overnight at 70 +/-5 ℃ in a dark place under the protection of argon, removing water by rotary evaporation, refrigerating overnight until solids are fully separated out, washing with ice water, ethanol and diethyl ether, and drying to obtain bright yellow solids; dissolving the bright yellow solid and succinic anhydride in anhydrous N, N-Dimethylformamide (DMF), reacting at 70 +/-5 ℃ in a dark place under the protection of argon for 24 hours, removing the DMF by rotary evaporation, adding anhydrous acetone for dissolving, dropwise adding the anhydrous acetone into anhydrous ether to separate out a precipitate, filtering and drying, adding the anhydrous acetone into the precipitate again, fully performing ultrasonic treatment, filtering, washing the anhydrous acetone and the anhydrous ether, and drying to obtain a white solid.

2) Rapid preparation of hydrophobic segment P (DSP-AED) from DSP and cystamine dihydrochloride by microwave technique_n；

Adding DSP, cystamine dihydrochloride, EDCI HCl and HOAT into a microwave tube, dissolving anhydrous DMF, freezing liquid nitrogen, pumping air by an oil pump, repeating the operation for three times, adding N, N-Diisopropylethylamine (DIEA), reacting on a microwave synthesizer (Biotage Initiator 2.5, Sweden) at 50 +/-0.5 ℃, supplementing the anhydrous DMF solution of DSP after the reaction is finished, continuing to react and seal the end under the same condition, dialyzing in dimethyl sulfoxide (DMSO) for 36h by using a dialysis bag with the molecular weight cutoff of 3.5KDa, dialyzing in water for 24h, and freeze-drying to obtain yellow powder, namely a hydrophobic chain segment P (DSP-AED)_nCalculating the molecular weight by nuclear magnetic hydrogen spectroscopy, and obtaining the molecular weight distribution by GPC;

3)P(DSP-AED)_npreparation of mPEG after PEG (polyethylene glycol)_5K-P(DSP-AED)_nMicelles;

p (DSP-AED)_n、mPEG_5K-NH₂Adding EDCI HCl and 4-Dimethylaminopyridine (DMAP) into a three-neck flask, injecting anhydrous DMF under the protection of argon, stirring and reacting for 48 hours at normal temperature, transferring the reaction solution into a dialysis bag with the molecular weight cutoff of 7.0KDa after the reaction is finished, dialyzing for 48 hours with distilled water, and freeze-drying to obtain fluffy yellow solid; dissolving fluffy yellow solid in dimethyl sulfoxide (DMSO), adding into ultrapure water dropwise at 40 + -0.5 deg.C under stirring, transferring into dialysis bag with molecular weight cutoff of 3.5KDa, dialyzing with distilled water for 24 hr, and lyophilizing to obtain mPEG_5K-P(DSP-AED)_nMicelles;

the chemical structure of the Pt (IV) polymer prodrug micelle with reduction response is shown as a formula (1), n is 21-13

The dosage ratio of the CDDP, the distilled water and the 30wt% hydrogen peroxide in the step 1) is 10.0 mmol: 80mL of: 17.1 mL;

the ratio of the bright yellow solid, succinic anhydride, anhydrous DMF, anhydrous acetone, anhydrous ether and the added anhydrous acetone is 2.0 g: 2.4 g: 30mL of: 20mL of: 200mL of: 15 mL.

Step 2) the dosage ratio of the solutions of DSP, cystamine dihydrochloride, EDCI & HCl, HOAT, anhydrous DMF, DIEA, supplemented DSP and anhydrous DMF is 0.94 mmol: 0.94 mmol: 3.76 mmol: 2.07 mmol: 12mL of: 2.07 mmol: 0.38 mmol: 2mL, the reaction time on a microwave synthesizer is 5-30min, and the end capping time for adding the DSP is 10 min.

The method for calculating the molecular weight of the hydrophobic chain segment P (DSP-AED) n through the nuclear magnetic hydrogen spectrum in the step 2) specifically comprises the following steps:

hydrophobic chain segment P (DSP-AED)_nThe end amino group reacts with excessive benzylamine hydrochloride to form an end cap, and methylene hydrogen atoms (-CH) in cystamine are utilized₂S-) nuclear magnetic integrated area and blocked benzylidene hydrogen atom (PhCH) in benzyl₂-) ratio of nuclear magnetic integrated area to obtain P (DSP-AED)_nThe polymerization degree n of (2) is further calculated to obtain P (DSP-AED)_nThe formula is as follows:

P(DSP-AED)_nmolecular weight M_n＝650×(n+1)+534 (2)

Wherein: area (g) is-CH₂Integrated area of S- (cystamine); area (a) is PhCH₂Integrated area of- (benzylamine).

Step 3) the P (DSP-AED)_n、mPEG_5K-NH₂EDCI & HCl, DMAP, anhydrous DMF, fluffy solid, DMSO, ultrapure water in a ratio of 0.02 mmol: 0.08 mmol: 0.08 mmol: 0.08 mmol: 10mL：25mg：1mL：25mL。

The invention further provides a preparation method of Pt (IV) polymer prodrug micelle with reduction response and encapsulated Voritodrine, which is characterized in that on the basis of preparing Pt (IV) polymer prodrug with reduction response, Pt (IV) polymer prodrug and Voritodrine (SAHA) are used for preparing Pt (IV) polymer prodrug micelle mPEG5K-P (DSP-AED) n @ SAHA with reduction response and encapsulated SAHA by an ultrafiltration method, and the preparation method comprises the following steps:

mPEG prepared by the above method_5K-P(DSP-AED)_nDissolving SAHA in DMSO at 30 + -0.5 deg.C, adding into ultrapure water dropwise under stirring, stirring at room temperature for about 5 hr, transferring the solution into ultrafiltration tube with cut-off molecular weight of 10KDa, and centrifuging for ultrafiltration to obtain mPEG_5K-P(DSP-AED)_n@ SAHA micelles. The mPEG_5K-P(DSP-AED)_nThe dosage ratio of SAHA to DMSO to ultrapure water is 10 mg: 0.4mg or 0.1 mg: 500. mu.L: 10mL, stirring speed 1000r/min, ultrafiltration and centrifugation 5000r/min 15 min.

The method for calculating the encapsulation capacity (LC) and the Encapsulation Efficiency (EE) of SAHA is as follows:

mPEG_5K-P(DSP-AED)_nfreeze-drying the @ SAHA micelle solution to determine the concentration, collecting filtrate, freeze-drying, and determining the content of the non-entrapped SAHA by using high performance liquid chromatography, thereby calculating the entrapment amount and the entrapment rate of the SAHA, wherein the calculation formula is as follows:

wherein: w_SAHAIs the total mass of the SAHA; w_freeSAHAMass of the unencapsulated SAHA; w_loadedSAHAIs the quality of the SAHA encapsulated; w_polymerIs mPEG_5K-P(DSP-AED)_nThe quality of (c).

The Pt (IV) polymer prodrug micelle with reduction response and loaded vorinostat (SAHA) provided by the invention can be applied to preparation of antitumor drugs.

The invention has the advantages and beneficial effects that:

the method for preparing the Pt (IV) polymer prodrug micelle with reduction response is quick, simple, convenient, green and environment-friendly, and has the potential of batch production. The Pt (IV) polymer prodrug micelle can be used as a medicine, has the capability of regulating and controlling self medicine carrying, releases the medicine aiming at the response of a tumor microenvironment, and has the advantage of good inhibition effect on human cervical carcinoma cells and human ovarian carcinoma cells, and meanwhile, the micelle can be used as a nano medicine carrier to entrap SAHA, can realize a better synergistic treatment effect on tumors under the condition of far lower than normal administration dosage, and has good application prospect. The invention provides application of a Pt (IV) polymer prodrug with reduction response and a Pt (IV) polymer prodrug with reduction response encapsulating SAHA in preparation of antitumor drugs.

Drawings

FIG. 1 shows infrared spectra of (a) CDDP, (b) DHP, and (c) DSP.

FIG. 2 is a DSP nuclear magnetic diagram¹H NMR。

FIG. 3 shows Bz-P (DSP-AED)₂₁Nuclear magnetic map¹H NMR。

FIG. 4 shows Bz-P (DSP-AED)₁₆Nuclear magnetic map¹H NMR。

FIG. 5 shows Bz-P (DSP-AED)₁₃Nuclear magnetic map¹H NMR。

FIG. 6 shows mPEG_5K-P(DSP-AED)₂₁Nuclear magnetic map¹H NMR。

FIG. 7 shows mPEG in example 4_5K-P(DSP-AED)₂₁Particle size distribution of micelles and electron microscopy.

FIG. 8 shows mPEG in example 4_5K-P(DSP-AED)₂₁、mPEG_5K-P(DSP-AED)₁₆And mPEG_5K-P(DSP-AED)₁₃Stability of micelles at (A)4 ℃ and (B)37 ℃.

FIG. 9 shows mPEG in example 5_5K-P(DSP-AED)₂₁Cell viability of micelles on different cells.

FIG. 10 shows an example8 middle mPEG_5K-P(DSP-AED)₂₁The release behaviour of @ SAHA (0.88%) in a reducing environment.

FIG. 11 shows mPEG (A) in example 8_5K-P(DSP-AED)₂₁@ SAHA (0.88%) and (B) mPEG_5K-P(DSP-AED)₂₁@ SAHA (0.15%) was shown to have a synergistic therapeutic effect in HeLa cells.

FIG. 12 shows mPEG in example 8_5K-P(DSP-AED)₂₁@ SAHA (0.88%) synergized with treatment of apoptosis on HeLa cells.

FIG. 13 shows mPEG in example 8_5K-P(DSP-AED)₂₁@ SAHA (0.88%) was used in a western blot assay with concurrent therapy on HeLa cells.

Detailed Description

The invention is further elucidated with reference to the drawing. It should be understood that the examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.

Example 1:

a preparation method of a Pt (IV) polymer prodrug micelle with reduction response is characterized in that cis-trans-diammine dichlorodisuccinic platinum (DSP) and cystamine dihydrochloride are used as raw materials, 1-hydroxy-7-azobenzotriazol (HOAT) and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (ECDI & HCl) are used as condensing agents, and the Pt (IV) polymer prodrug micelle with stable physicochemical properties can be obtained through controllable microwave polymerization and PEG chemical technology, and comprises the following steps:

1) cis-trans-Diamminedichloroplatinum (DSP) was prepared from Cisplatin (CDDP).

3.0g (10.0mmol) of CDDP is mixed and rotated in 80mL of distilled water, 17.1mL (150mmol) of 30wt% hydrogen peroxide is added into the solution under stirring, the temperature is 70 +/-5 ℃, argon is protected, and the reaction is carried out overnight in a dark place. After the reaction is finished, the reaction solution is subjected to rotary evaporation to remove water to about 15mL, refrigerated overnight to precipitate a solid, filtered, fully rinsed with ice water (20mL), ethanol (20mL) and diethyl ether (40mL), and dried to obtain 2.7g of a bright yellow solid with the yield of 81.0%. The bright yellow solid 2.0g (6.0mmol) and succinic anhydride 2.4g (24.0mmol) were dissolved in 30mL of anhydrous DMF at 70 + -5 deg.C under protection of argon and protected from light for 24 h. After the reaction is finished, performing rotary evaporation to remove DMF, adding 20mL of anhydrous acetone to dissolve, dropwise adding 200mL of anhydrous ether under vigorous stirring to generate a light yellow solid precipitate, filtering, washing with anhydrous ether (50mL), ultrasonically dispersing the solid in anhydrous acetone (15mL) for 15min, filtering, washing with anhydrous acetone (10mL) and anhydrous ether (20mL), and drying to obtain 1.55g of white solid with the yield of 48.3%.

2) Rapid preparation of hydrophobic segment P (DSP-AED) from DSP and cystamine dihydrochloride by microwave technique_n。

Putting 500mg (0.94mmol) of DSP and 210.8mg (0.94mmol) of cystamine dihydrochloride, 3.76mmol of EDCI-HCl 720.8mg and 2.07mmol of HOAT 281.5mg in a microwave tube, dissolving 12mL of anhydrous DMF, freezing liquid nitrogen, pumping oil, repeating the operation for three times, adding 342 μ L (2.07mmol) of N, N-Diisopropylethylamine (DIEA) at 50 +/-0.5 ℃ in a microwave synthesizer, reacting for 30min, adding 2mL of DSP 200mg (0.38mmol) solution dissolved in anhydrous DMF after the reaction is finished, continuing the reaction in the microwave synthesizer for 10min under the same condition, transferring to DMSO with the molecular weight cutoff of 3.5KDa for dialysis for 36h, dialyzing for 24h with distilled water, and freeze-drying to obtain yellow solid powder, namely hydrophobic chain segment P (DSP-AED)_nThe degree of polymerization was calculated as 21 by nuclear magnetic hydrogen spectroscopy, and the molecular weight was further calculated (see table 1).

P(DSP-AED)_nThe formula for calculating the molecular weight is as follows:

P(DSP-AED)_nmolecular weight M_n＝650×(n+1)+534 (2)

3)P(DSP-AED)₂₁Preparation of Pt (IV) polymer prodrug mPEG with reduction response after PEGylation_5K-P(DSP-AED)₂₁Micelles;

weighing P (DSP-AED)₂₁283mg(0.02mmol)、mPEG_5K-NH₂Adding 400mg (0.08mmol), EDCI & HCl 15.3mg (0.08mmol) and DMAP 9.8mg (0.08mmol) into a 25mL three-neck flask, dissolving with 10mL of anhydrous DMF, reacting at normal temperature for 48h under the protection of argon, transferring the reaction system into a dialysis bag with molecular weight cutoff of 7.0KDa after the reaction is finished, dialyzing with distilled water for 48h, and freeze-drying to obtain light yellow fluffy solid mPEG_5K-P(DSP-AED)₂₁. Weighing mPEG_5K-P(DSP-AED)₂₁Preparing a 25mg/mLDMSO solution, and mixing the organic phase and the aqueous phase according to the volume ratio of 1: stirring at 40 + -0.5 deg.C and 1000r/min, adding dropwise into ultrapure water, stirring for 10min, transferring into dialysis bag with cut-off molecular weight of 3.5KDa, dialyzing with distilled water for 24 hr, and lyophilizing to obtain mPEG_5K-P(DSP-AED)₂₁Micelles.

Example 2:

the preparation method of Pt (IV) polymer prodrug micelle with reduction response comprises the following steps:

1) same as example 1, step 1;

Putting 500mg (0.94mmol) of DSP and 210.8mg (0.94mmol) of cystamine dihydrochloride, 3.76mmol of EDCI-HCl 720.8mg and 2.07mmol of HOAT 281.5mg in a microwave tube, dissolving 12mL of anhydrous DMF, freezing liquid nitrogen, pumping oil, repeating the operation for three times, adding 342 muL (2.07mmol) of N, N-Diisopropylethylamine (DIEA), reacting in a microwave synthesizer at 50 +/-0.5 ℃ for 10min, adding 2mL of DSP 200mg (0.38mmol) solution dissolved in anhydrous DMF after the reaction is finished, continuing the reaction in the microwave synthesizer for 10min to terminate the end under the same condition, transferring to DMSO with the molecular weight cutoff of 3.5KDa for dialysis for 36h, dialyzing with distilled water for 24h, and freeze-drying to obtain yellow solid powder, namely hydrophobic chain segment P (DSP-AED)_nThe degree of polymerization was calculated as 16 by nuclear magnetic hydrogen spectroscopy, and the molecular weight was further calculated (see table 1).

3)P(DSP-AED)₁₆Preparation of Pt (IV) polymer prodrug mPEG with reduction response after PEGylation_5K-P(DSP-AED)₁₆Micelles;

weighing P (DSP-AED)₁₆218mg(0.02mmol)、mPEG_5K-NH₂Adding 400mg (0.08mmol), EDCI & HCl 15.3mg (0.08mmol) and DMAP 9.8mg (0.08mmol) into a 25mL three-neck flask, dissolving with 10mL of anhydrous DMF, reacting at normal temperature for 48h under the protection of argon, transferring the reaction system into a dialysis bag with molecular weight cutoff of 7.0KDa after the reaction is finished, dialyzing with distilled water for 48h, and freeze-drying to obtain light yellow fluffy solid mPEG_5K-P(DSP-AED)₁₆. Weighing mPEG_5K-P(DSP-AED)₁₆Preparing a 25mg/mL DMSO solution, and mixing according to the volume ratio of an organic phase to an aqueous phase of 1: stirring at 40 + -0.5 deg.C and 1000r/min, adding dropwise into ultrapure water, stirring for 10min, transferring into dialysis bag with cut-off molecular weight of 3.5KDa, dialyzing with distilled water for 24 hr, and lyophilizing to obtain mPEG_5K-P(DSP-AED)₁₆Micelles.

Example 3:

1) same as example 1, step 1;

Putting 500mg (0.94mmol) of DSP and 210.8mg (0.94mmol) of cystamine dihydrochloride, 3.76mmol of EDCI-HCl 720.8mg and 2.07mmol of HOAT 281.5mg in a microwave tube, dissolving 12mL of anhydrous DMF, freezing liquid nitrogen, pumping oil, repeating the operation for three times, adding 342 muL (2.07mmol) of N, N-Diisopropylethylamine (DIEA), reacting in a microwave synthesizer at 50 +/-0.5 ℃ for 5min, adding 2mL of DSP 200mg (0.38mmol) solution dissolved in anhydrous DMF after the reaction is finished, continuing the reaction in the microwave synthesizer for 10min to terminate the end under the same condition, transferring to DMSO with the molecular weight cutoff of 3.5KDa for dialysis for 36h, dialyzing with distilled water for 24h, and freeze-drying to obtain yellow solid powder, namely hydrophobic chain segment P (DSP-AED)_nThe degree of polymerization was calculated as 13 by nuclear magnetic hydrogen spectroscopy, and the molecular weight was further calculated (see table 1).

3)P(DSP-AED)₁₃Is prepared by PEGPt (IV) polymer prodrug mPEG with reduction response_5K-P(DSP-AED)₁₃Micelles;

weighing P (DSP-AED)₁₃180mg(0.02mmol)、mPEG_5K-NH₂Adding 400mg (0.08mmol), EDCI & HCl 15.3mg (0.08mmol) and DMAP 9.8mg (0.08mmol) into a 25mL three-neck flask, dissolving with 10mL of anhydrous DMF, reacting at normal temperature for 48h under the protection of argon, transferring the reaction system into a dialysis bag with molecular weight cutoff of 7.0KDa after the reaction is finished, dialyzing with distilled water for 48h, and freeze-drying to obtain light yellow fluffy solid mPEG_5K-P(DSP-AED)₁₃. Weighing mPEG_5K-P(DSP-AED)₁₃Preparing a 25mg/mL DMSO solution, and mixing according to the volume ratio of an organic phase to an aqueous phase of 1: stirring at 40 + -0.5 deg.C and 1000r/min, adding dropwise into ultrapure water, stirring for 10min, transferring into dialysis bag with cut-off molecular weight of 3.5KDa, dialyzing with distilled water for 24 hr, and lyophilizing to obtain mPEG_5K-P(DSP-AED)₁₃Micelles.

And (4) analyzing results: as can be seen from the IR spectrum of FIG. 1, 3516cm appeared in (b)^-1(O-H) and 559cm^-1The characteristic peak of (Pt-O) shows that the DHP structure is correct and disappears along with the characteristic peak of DHP; (c) appear 1708cm^-1(-COOH) and 1662cm^-1(PtOC ═ O-) new characteristic peaks, and fig. 2 further illustrates that the DSP structure is correct. To determine the molecular weight of the polymer, P (DSP-AED)_nThe both ends were reacted with benzylamine hydrochloride, and the polymerization degree was 21, 16, 13, further calculated from fig. 3, 4, 5, respectively, and the molecular weight was calculated, and the molecular weight distribution of the polymer was obtained by GPC (DMSO is mobile phase) (see table 1).

Table 1, examples 1 to 3 different polymerization times P (DSP-AED)_nMolecular weight of (2) and distribution thereof

[a] Calculating the molecular weight through nuclear magnetic hydrogen spectrum; [b] molecular weight distribution by GPC.

Example 4:

mPEG with reducing response_5K-P(DSP-AED)_nAnd (5) characterization of the micelle.

(1) mPEG determination using Malvern particle sizer_5K-P(DSP-AED)_nThe Critical Micelle Concentration (CMC) of (a) is specifically:

mPEG prepared according to examples 1-3_5K-P(DSP-AED)₂₁、mPEG_5K-P(DSP-AED)₁₆And mPEG_5K-P(DSP-AED)₁₃Respectively preparing water solutions with mass concentrations of 0.1, 0.25, 0.5, 0.75, 1, 2.5, 5, 7.5, 10, 25, 50, 75, 100 and 250 mu g/mL, detecting the scattering signal intensity of the water solutions, making a scatter diagram fitting curve for the scattering intensity under each concentration, and searching a mutation point to obtain the critical micelle concentration (see table 2).

mPEG in Table 2 and example 4_5K-P(DSP-AED_nCharacterization of physicochemical Properties of micelles

[a] Calculating the molecular weight through nuclear magnetic hydrogen spectrum; [b] the Pt content was determined by ICP-MS, as a percentage of the mass of cisplatin corresponding to Pt (IV) contained in the polymer to the mass of the polymer.

(2) mPEG determination using Malvern particle sizer_5K-P(DSP-AED)_nThe particle size and the potential distribution of (a) are as follows:

mPEG prepared according to examples 1-3_5K-P(DSP-AED)₂₁、mPEG_5K-P(DSP-AED)₁₆And mPEG_5K-P(DSP-AED)₁₃The micelles were prepared as 0.5mg/mL aqueous solutions, and placed in a test dish, and the particle size and potential distribution were measured at 25 deg.C (see Table 2, see FIG. 7).

(3) Measurement of mPEG Using ICP-MS_5K-P(DSP-AED)_nThe content of the cisplatin in the composition is as follows:

mPEG prepared as in examples 1-3 was weighed separately_5K-P(DSP-AED)₂₁、mPEG_5K-P(DSP-AED)₁₆And mPEG_5K-P(DSP-AED)₁₃5.0mg, adding 2mL of concentrated nitric acid, and heating to 100 +/-5 DEG CEvaporating nitric acid to dryness, diluting with ultrapure water to appropriate concentration, measuring Pt content, and further calculating mPEG_5K-P(DSP-AED)₂₁、mPEG_5K-P(DSP-AED)₁₆And mPEG_5K-P(DSP-AED)₁₃The content of cisplatin in the composition (see Table 2).

(4) Observation of mPEG with Transmission Electron microscope (TME)_5K-P(DSP-AED)_nMicelle morphology and size, in mPEG_5K-P(DSP-AED)₂₁Micelles are exemplified (see fig. 7).

(5) mPEG determination using Malvern particle sizer_5K-P(DSP-AED)_nThe stability of the micelle is specifically as follows: mPEG prepared as in examples 1-3 was mixed with PBS (pH 7.4)_5K-P(DSP-AED)₂₁、mPEG_5K-P(DSP-AED)₁₆And mPEG_5K-P(DSP-AED)₁₃The micelles were prepared to 0.5mg/mL respectively, and the particle size distribution was measured at 10min, 30min, 1h, 2h, 4h, 6h, 8h, 12h, and 24h in a 37 ℃ environment (see FIG. 8).

And (4) analyzing results: mPEG_5K-P(DSP-AED)₂₁、mPEG_5K-P(DSP-AED)₁₆And mPEG_5K-P(DSP-AED)₁₃Micelles can form nano-micelles well, but mPEG can be found_5K-P(DSP-AED)₂₁The micelle has lower critical micelle concentration, which shows that the micelle forming capability is optimal and the stability is better at 37 ℃, so that the Pt (IV) polymer prodrug micelle becomes a subsequent research object.

Example 5: mPEG with reducing response_5K-P(DSP-AED)₂₁Use of micelles

mPEG with reducing response_5K-P(DSP-AED)₂₁Micellar cytotoxicity assay:

according to the growth conditions of the cells, PC3, A2780, PANC-1, HeLa, MCF-7, A549 and H8 cells are respectively inoculated in a 96-well plate with the density of 5000 cells/well, the outermost ring is supplemented with PBS due to the edge hole effect, the mixture is kept stand for five minutes and is put into a cell incubator containing 5 percent of carbon dioxide and the temperature of 37 ℃ for 24 hours. DSP and mPEG were treated with PBS_5K-P(DSP-AED)₂₁The micelles were prepared as solutions of different solubilities, and added to 6 well plates in an amount of 10. mu.L/well, and then placedCulturing in carbon dioxide cell culture box for 48 hr. The MTT solution was added to a 96-well plate in an amount of 10. mu.L per well in a dark environment, and then placed in an incubator for further incubation for 4 hours. The supernatant in the well plate was aspirated, a certain amount of DMSO was added, and the resulting crystals were completely dissolved by gentle shaking. The absorbance of each well was measured and recorded at a wavelength of 570nm using an enzyme linked immunosorbent instrument. The cell inhibition ratios of PC3, A2780, PANC-1, HeLa, MCF-7, A549 and H8 cells were calculated from the obtained absorbances.

And (4) analyzing results: from FIG. 9, it can be found that DSP and mPEG_5K-P(DSP-AED)₂₁The cell inhibition rate of the micelle on PC3, PANC-1 and MCF-7 cells is poor; DSP has better cell killing effect on A549 cells, but the cytotoxicity of micelles is lower; DSP and mPEG_5K-P(DSP-AED)₂₁The micelle has good cell inhibition effect on HeLa and A2780, the cell inhibition effect of the micelle is slightly lower than that of DSP, but the cell killing effect of DSP and the cell killing effect of the micelle on normal cells H8 are obviously different. These results indicate that the application potential of the micelle in killing a2780 and HeLa cells, especially, the cytostatic rate of the micelle on H8 cells is significantly lower than that on HeLa cells, which indicates that the micelle can well kill tumor cells, but has low toxicity to normal cells, probably because there is a higher reducing substance in HeLa cells, the micelle releases cisplatin with better reduction responsiveness, thereby killing tumor cells.

Example 6: preparation of mPEG according to different prescription amounts by ultrafiltration_5K-P(DSP-AED)₂₁@ SAHA micelle

Using mPEG prepared in example 1_5K-P(DSP-AED)₂₁And SAHA is prepared into Pt (IV) polymer prodrug mPEG with SAHA entrapment and reduction response by an ultrafiltration method_5K-P(DSP-AED)₂₁@ SAHA micelles, the specific experimental procedure was as follows:

weighing mPEG_5K-P(DSP-AED)₂₁Dissolving 10.0mg in 500 mu LDMSO, adding 40 mu L of 10mg/mL SAHA DMSO solution, uniformly mixing, dropwise adding 10mL ultrapure water at the temperature of 30 +/-0.5 ℃, rotating at the speed of 1000r/min, after dropwise adding, continuously stirring at room temperature for about 5h, transferring to a retention tankRepeating the operation for 4 times in an ultrafiltration tube with the molecular weight of 10KDa and at 5000r/min for 15min to obtain mPEG_5K-P(DSP-AED)₂₁@ SAHA micelles.

1mL of the micelle solution is taken for freeze-drying, the concentration of the micelle solution is determined, and then filtrate is collected for freeze-drying, and the content of the non-entrapped SAHA is determined by using high performance liquid chromatography, so that the entrapment amount (LC) and the Entrapment Efficiency (EE) of the SAHA are calculated (see table 3).

Wherein: w_SAHAIs the total mass of the SAHA; w_freeSAHAMass of the unencapsulated SAHA; w_loadedSAHAIs the quality of the SAHA encapsulated; w_polymerIs mPEG_5K-P(DSP-AED)₂₁The quality of (c).

Example 7: preparation of mPEG according to different prescription amounts by ultrafiltration_5K-P(DSP-AED)₂₁@ SAHA micelle

Using mPEG prepared in example 1_5K-P(DSP-AED)₂₁And SAHA is prepared into Pt (IV) polymer prodrug mPEG with SAHA entrapment and reduction response by an ultrafiltration method_5K-P(DSP-AED)_n@ SAHA micelles, the specific experimental procedure was as follows:

weighing mPEG_5K-P(DSP-AED)₂₁Dissolving 10.0mg in 500 mu LDMSO, adding 10 mu L of 10mg/mL SAHA DMSO solution, uniformly mixing, dropwise adding 10mL ultrapure water, controlling the temperature to be 30 +/-0.5 ℃, rotating speed to be 1000r/min, after dropwise adding, continuously stirring at room temperature for about 5h, transferring to an ultrafiltration tube with molecular weight cutoff of 10KDa, carrying out 5000r/min, carrying out 15min, repeating the operation for 4 times, and obtaining mPEG_5K-P(DSP-AED)₂₁@ SAHA micelles.

1mL of the micelle solution is taken for freeze-drying, the concentration of the micelle solution is determined, then the filtrate is collected for freeze-drying, and the content of the SAHA which is not loaded is determined by high performance liquid chromatography, so that the loading amount (LC) and the Encapsulation Efficiency (EE) of the SAHA are calculated (see table 3).

And (4) analyzing results: obtained mPEG by PEGylation_5K-P(DSP-AED)_nThe nuclear magnetization is shown in FIG. 6 with the polymerization degree of 21 as an example, and the structure is confirmed to be correct. The calculated SAHA Loading (LC) and Encapsulation Efficiency (EE) by detecting the content of the unencapsulated SAHA by high performance liquid chromatography are shown in table 3.

TABLE 3 mPEG in examples 5-6_5k-P(DSP-AED)₂₁Characterization of physicochemical Properties of the @ SAHA micelles

[a] Theoretical drug loading; [b] actual encapsulation capacity and encapsulation efficiency.

Example 8: characterization and co-treatment of SAHA-entrapped Pt (IV) polymeric prodrug micelles

Characterization of SAHA-entrapped pt (iv) polymeric prodrug micelles:

mPEG determination using Malvern particle sizer_5K-P(DSP-AED)₂₁The particle size and potential distribution of the @ SAHA micelle are specifically: mPEG prepared in examples 6-7_5K-P(DSP-AED)₂₁@ SAHA (0.88%) micelles and mPEG_5K-P(DSP-AED)₂₁@ SAHA (0.15%) micelles were prepared as a 0.5mg/mL solution and placed in a test dish and the particle size and potential distribution were measured at 25 deg.C (see Table 3).

The drug release performance of the Pt (IV) polymer prodrug micelle encapsulating the SAHA is as follows:

with mPEG_5K-P(DSP-AED)₂₁@ SAHA (0.88%) micelle as an example:

1mL of mPEG_5K-P(DSP-AED)₂₁@ 0.88% micelle solution 3mg/mL was loaded into a dialysis bag with a molecular weight cut-off of 1.0KDa, placed in 40mL PBS (containing 10mM or 0.1mM Dithiothreitol (DTT)) at pH 7.4 or pH 5.5, and placed in a shaker at 260r/min at 37 deg.C, and 3mL of the dialysate was taken as a test sample at 1.5h, 2.5h, 4h, 7h, 9h, 24h, 32h, 48h, and 72h, respectively, while an equal amount of fresh buffer was supplemented. 1mL of the test sample is subjected to nitrolysis by concentrated nitric acid, diluted to a proper concentration by ultrapure water, and the Pt content is measured by ICP-MS(ii) a After 2mL of the test sample was lyophilized, the SAHA content was measured by high performance liquid chromatography, and the cumulative drug release curve was plotted (see FIG. 10).

And (4) analyzing results: mPEG_5K-P(DSP-AED)₂₁The content and speed of drug release of the @ SAHA (0.88%) micelle in 10mM environment are obviously higher than those in 0.1mM environment, and the neutral environment is more favorable for SAHA release at the same DTT concentration, probably because SAHA is a weak acid and is greatly dissociated in neutral environment and acidic environment, which indicates that mPEG_5K-P(DSP-AED)₂₁The @ SAHA micelle has a reduction-responsive ability to release platinum-based and SAHA drugs.

Evaluation of SAHA-entrapped pt (iv) polymeric prodrug micellar co-therapy:

according to the growth condition of the cells, the HeLa cells are inoculated in a 96-well plate with the density of 5000 cells/well, the outermost ring is supplemented with PBS due to the edge-hole effect, the mixture is kept stand for five minutes and is placed into a cell culture box containing 5 percent of carbon dioxide and at 37 ℃ for 24 hours. The micelles were made into solutions of different solubilities with PBS, and added to 6 parallel wells at 10. mu.L per well, and then placed in a carbon dioxide cell incubator for 48 h. The MTT solution was added to a 96-well plate in an amount of 10. mu.L per well in a dark environment, and then placed in an incubator for further incubation for 4 hours. The supernatant in the well plate was aspirated, a certain amount of DMSO was added, and the resulting crystals were completely dissolved by gentle shaking. The absorbance of each well was measured and recorded at a wavelength of 570nm using an enzyme linked immunosorbent instrument. The inhibition rate of HeLa cells was calculated from the obtained absorbance. Evaluation was carried out by means of the synergy index CI (combination index), according to the King equation, as follows:

in the formula E_AAnd E_BRespectively representing the inhibition rate of single drugs A and B on cells, E_A+BRepresents the inhibition rate of the two drugs on cells when used together, when CI<0.85 indicates two-drug antagonism; when CI is more than or equal to 0.85 and less than or equal to 1.15, the two medicines are added; when CI is present>A score of 1.15 indicates a synergistic effect (FIG. 11).

And (4) analyzing results:

mPEG_5K-P(DSP-AED)₂₁micelles, SAHA and mPEG_5K-P(DSP-AED)₂₁The proliferation inhibition effect of the @ SAHA (0.88%) micelle on HeLa is gradually reduced along with the increase of concentration, the cell survival rate is in a dose-dependent relationship, and mPEG_5K-P(DSP-AED)₂₁The survival rate of the cells of the @ SAHA (0.88%) micelle group is lower than that of mPEG_5K-P(DSP-AED)₂₁The survival rate of the cells of the micelle group and the SAHA group shows that the two drugs have synergistic or additive effect. FIG. 11 shows that Pt concentrations ranging from 10 μ M to 40 μ M show synergistic effects at 0.88% SAHA loading, especially mPEG at 20 μ M Pt concentration_5K-P(DSP-AED)₂₁The cell survival rate under the action of micelle is about 90 percent, the cell survival rate under the action of SAHA is about 70 percent, and mPEG_5K-P(DSP-AED)₂₁The survival rate of the cells under the action of the @ SAHA (0.88%) micelle is reduced to about 35%, and the synergistic effect is obvious. When the SAHA loading amount is 0.15%, the Pt concentration range is 20-40 mu M, and the synergistic effect is also shown, and the result shows that the low-dose SAHA can enhance the synergistic anti-cancer effect of Pt.

P(DSP-AED)₂₁@ SAHA (0.88%) micelle apoptosis assay:

the degree of apoptosis of the cells was determined by annexin V-FITC/PI double staining. The specific method comprises the following steps: at 2X 10⁵The cell density of/mL is inoculated in a 6-well plate, and after the cells are attached to the wall, single SAHA and mPEG are added_5K-P(DSP-AED)₂₁Empty micelles and drug-loaded micelles mPEG with SAHA encapsulation capacity of 0.88 percent_5K-P(DSP-AED)₂₁@ SAHA (0.88%) was incubated with the cells for 48 h. The cells of each group were collected after digestion with pancreatin and the cell concentration was adjusted to about 1X 10⁶one/mL. 200 μ L of the cell suspension was put into a 5mLEP tube, 10 μ L of Annexin V-FITC and 20 μ L of 20 μ g/mL PI solution were added, mixed well and incubated for 15 minutes at room temperature in the dark. Add 800. mu.L PBS to the EP tube and analyze with BD flow cytometer.

And (4) analyzing results:

mPEG_5K-P(DSP-AED)₂₁micelles, SAHA and mPEG_5K-P(DSP-AED)₂₁The @ SAHA (0.88%) micelles all led to apoptosis of the cells (FIG. 1)2). mPEG was shown by quantitative analysis_5K-P(DSP-AED)₂₁The total apoptosis rate of the micelle is 13.00%; the total apoptosis rate of SAHA is 10.64%; mPEG_5K-P(DSP-AED)₂₁@ SAHA (0.88%) micelle-mediated therapy mainly caused late apoptosis to be 48.2% with a total apoptosis rate of 56.71%. The experimental results show that mPEG_5K-P(DSP-AED)₂₁The @ SAHA (0.88%) micelle can cause cell death through an apoptosis pathway, the apoptosis effect of cells after synergistic treatment is obviously superior to that after single medicine, and the treatment effect is 1+1 > 2.

mPEG_5K-P(DSP-AED)₂₁@ SAHA (0.88%) micellar western blot experiment:

acetylation of histone H3 and expression of apoptosis-related protein clear caspase3 were detected by Western Blot method. The specific method comprises the following steps: at 2X 10⁵The cells were seeded in 6-well plates at a density of one mL/mL, and after about 24 hours, SAHA and mPEG alone were added_5K-P(DSP-AED)₂₁Empty micelles and drug-loaded micelles mPEG with SAHA encapsulation capacity of 0.88 percent_5K-P(DSP-AED)₂₁@ SAHA (0.88%) was incubated with the cells. After 48h, the cells were harvested and lysed with a RIPA buffer containing 1% protease inhibitor, incubated on ice for 30min, 13000r/min, centrifuged at 4 ℃ for 15min, and the supernatant protein solution was retained. The protein separation adopts an SDS-PAGE system, each lane is loaded with 30-60 mu g of sample, the sample is electrically transferred to a nitrocellulose membrane (purchased from GE Healthcare), after being sealed for 1h by 5% of skim milk, specific antibodies (purchased from Abcam) of different proteins are added for incubation for 2h at room temperature, secondary antibody marked by HRP is added for incubation for 1h at room temperature, and finally, a Kodak X-ray chemiluminescence detector is used for color development. Tubulin was used as an internal reference for each experiment.

And (4) analyzing results: from FIG. 13, it was found that mPEG_5K-P(DSP-AED)₂₁The clear-caspase 3 band for the @ SAHA (0.88%) micelle group was significantly thicker than the SAHA group and mPEG_5K-P(DSP-AED)₂₁The result of the band of micelle group shows that the expression of apoptosis protein caused by synergistic treatment is stronger than that of apoptosis protein after single medicine application and is consistent with the apoptosis experimental result, which indicates that mPEG_5K-P(DSP-AED)₂₁The @ SAHA (0.88%) micelle has a good synergistic treatment effect; mPEG_5K-P(DSP-AED)₂₁There was no significant difference in Ac-H3 bands between the @ SAHA (0.88%) micelle group and the SAHA group, indicating that the combination of platinum in the drug-loaded micelle and SAHA did not achieve the effect of synergistically inhibiting tumor cell proliferation by enhancing the acetylation degree of histone H3. The lateral evidence shows that the mechanism of synergistic cancer treatment of HDACI and platinum drugs is that HDACI increases the availability of DNA by enhancing histone acetylation, thereby providing an opportunity for complexing platinum metal compounds and DNA, enhancing the treatment effect of platinum drugs and further synergistically inhibiting the proliferation of tumor cells.

Claims

1. Preparation method of Pt (IV) polymer prodrug micelle with reduction response, wherein micelle is named mPEG_5K-P(DSP-AED)_nWherein mPEG_5KRepresents PEG chain segment with the number average molecular weight of 5000, and n represents polymerization degree; the method is characterized in that: the Pt (IV) polymer prodrug micelle with different drug-loading rates and reduction response release performance can be obtained by using cis-trans-diammine dichlorodisuccinic acid platinum (DSP) and cystamine dihydrochloride as raw materials and 1-hydroxy-7-azobenzotriazol (HOAT) and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (ECDI & HCl) as condensing agents through controllable microwave polymerization and PEG chemical processes, and the steps are as follows:

1) preparing cis-trans-Diamminedichloroplatinum (DSP) from Cisplatin (CDDP);

putting CDDP in distilled water, dropwise adding 30wt% of hydrogen peroxide under stirring, reacting overnight at 70 +/-5 ℃ in a dark place under the protection of argon, removing water by rotary evaporation, refrigerating overnight until solids are fully separated out, washing with ice water, ethanol and diethyl ether, and drying to obtain bright yellow solids; dissolving bright yellow solid and succinic anhydride in anhydrous N, N-Dimethylformamide (DMF), reacting at 70 +/-5 ℃ in a dark place under the protection of argon for 24 hours, removing the DMF by rotary evaporation, adding anhydrous acetone for dissolving, dropwise adding the anhydrous acetone into anhydrous ether to separate out a precipitate, filtering and drying, adding the anhydrous acetone into the precipitate again, fully performing ultrasonic treatment, filtering, washing the anhydrous acetone and the anhydrous ether, and drying to obtain a white solid;

2) rapid preparation of DSP and cystamine dihydrochloride by microwave technologyHydrophobic chain segment P (DSP-AED)_n；

Adding DSP, cystamine dihydrochloride, EDCI HCl and HOAT into a microwave tube, dissolving anhydrous DMF, freezing liquid nitrogen, pumping air by an oil pump, repeating the operation for three times, adding N, N-Diisopropylethylamine (DIEA), reacting at 50 +/-0.5 ℃ in a Biotage Initiator 2.5 and Sweden, adding the anhydrous DMF solution of DSP after the reaction is finished, continuing to react and seal the end under the same condition, dialyzing in dimethyl sulfoxide (DMSO) for 36h by using a dialysis bag with the molecular weight cutoff of 3.5KDa, continuously dialyzing in water for 24h, and freeze-drying to obtain yellow powder, namely a hydrophobic chain segment P (DSP-AED)_nCalculating the molecular weight by nuclear magnetic hydrogen spectroscopy, and obtaining the molecular weight distribution by GPC;

3）P(DSP-AED)_npreparation of Pt (IV) polymer prodrug micelle mPEG with reduction response after PEGylation_5K-P(DSP-AED)_n；

P (DSP-AED)_n、mPEG_5K-NH₂Adding EDCI HCl and 4-Dimethylaminopyridine (DMAP) into a three-neck flask, injecting anhydrous DMF under the protection of argon, stirring and reacting for 48 hours at normal temperature, transferring the reaction solution into a dialysis bag with the molecular weight cutoff of 7.0KDa after the reaction is finished, dialyzing for 48 hours with distilled water, and freeze-drying to obtain fluffy yellow solid; dissolving fluffy yellow solid in dimethyl sulfoxide (DMSO), adding dropwise into ultrapure water at 40 + -0.5 deg.C under stirring, transferring into dialysis bag with molecular weight cutoff of 3.5KDa, dialyzing with distilled water for 24 hr, and lyophilizing to obtain Pt (IV) polymer prodrug micelle mPEG_5K-P(DSP-AED)_n。

2. The method for preparing Pt (IV) polymer prodrug micelle with reduction response according to claim 1, wherein in the step 1) of preparing the DSP: the dosage ratio of CDDP, distilled water and 30wt% hydrogen peroxide is 10.0 mmol: 80mL of: 17.1 mL;

3. The method for preparing Pt (IV) polymer prodrug micelle with reduction response according to claim 1, wherein the hydrophobic segment P (DSP-AED) in the step 2)_nThe preparation process comprises the following steps: the dosage ratio of the solutions of the DSP, the cystamine dihydrochloride, the EDCI & HCl, the HOAT, the anhydrous DMF, the DIEA, the supplemented DSP and the anhydrous DMF is 0.94 mmol: 0.94 mmol: 3.76 mmol: 2.07 mmol: 12mL of: 2.07 mmol: 0.38 mmol: 2mL, the reaction time on a microwave synthesizer is 5-30min, and the end capping time for adding the DSP is 10 min.

4. The method of preparing Pt (IV) polymer prodrug micelle with reduction response according to claim 1, wherein the hydrophobic segment P (DSP-AED)_nThe molecular weight calculation method of (2) is as follows:

hydrophobic chain segment P (DSP-AED)_nThe end amino group reacts with excessive benzylamine hydrochloride to form end capping, and methylene hydrogen atom (-C) in cystamine is utilizedH ₂S-) nuclear magnetic integrated area and blocked benzylidene hydrogen atom (PhC) in benzylH ₂-) ratio of nuclear magnetic integrated area to obtain P (DSP-AED)_nThe polymerization degree n of (2) is further calculated to obtain P (DSP-AED)_nThe formula is as follows:

polymerization degree:

(1)

P(DSP-AED)_nmolecular weight:

(2)

wherein:Area(g) is-CH ₂Integrated area of S- (cystamine);Area(a) Is PhCH ₂Integrated area of- (benzylamine).

5. The preparation method of Pt (IV) polymer prodrug micelle with reduction response according to claim 1, wherein the preparation method is characterized in thatIn the pegylation process described in step 3): the P (DSP-AED)_n、mPEG_5K-NH₂EDCI & HCl, DMAP, anhydrous DMF, fluffy yellow solid, DMSO, ultrapure water with the dosage ratio of 0.02 mmol: 0.08 mmol: 0.08 mmol: 0.08 mmol: 10mL of: 25 mg: 1mL of: 25 mL.

6. Preparation method of Pt (IV) polymer prodrug micelle coated with vorinostat (SAHA) and having reduction response, wherein the micelle is named mPEG_5K-P(DSP-AED)_n@ SAHA, characterized by: mPEG prepared according to claim 1_5K-P(DSP-AED)_nAnd vorinostat (SAHA) in a ratio and prepared by ultrafiltration, the steps of:

mixing mPEG_5K-P(DSP-AED)_nDissolving SAHA in DMSO at 30 + -0.5 deg.C, adding into ultrapure water dropwise under stirring, stirring at room temperature for 5 hr, transferring the solution into ultrafiltration tube with cut-off molecular weight of 10KDa, and centrifuging for ultrafiltration to obtain mPEG_5K-P(DSP-AED)_n@ SAHA micelles.

7. The preparation method of Pt (IV) polymer prodrug micelle carrying vorinostat (SAHA) with reduction response according to claim 6, wherein the mPEG is used as the active ingredient_5K-P(DSP-AED)_nThe dosage ratio of SAHA to DMSO to ultrapure water is 10 mg: 0.4mg or 0.1 mg: 500. mu.L: 10mL, stirring speed 1000r/min, ultrafiltration and centrifugation 5000r/min 15 min.

8. The preparation method of Pt (IV) polymer prodrug micelle carrying vorinostat (SAHA) with reduction response according to claim 6, wherein the mPEG is used as the active ingredient_5K-P(DSP-AED)_nThe SAHA loading in the @ SAHA micelle was 0.88% or 0.15%.

9. The preparation method of vorinostat (SAHA) -encapsulated pt (iv) polymeric prodrug micelle having reduction response according to any one of claims 6 to 8, wherein: the method for calculating the encapsulation capacity (LC) and the Encapsulation Efficiency (EE) of SAHA is as follows:

wherein:W _SAHAis the total mass of the SAHA;W _freeSAHAmass of the unencapsulated SAHA;W _loadedSAHAis the quality of the SAHA encapsulated;W _polymeris mPEG_5K-P(DSP-AED)_nThe quality of (c).

10. Use of Pt (IV) polymer prodrug micelle with reduction response prepared by the method of claim 1 or Pt (IV) polymer prodrug micelle with reduction response entrapped vorinostat (SAHA) prepared by the method of claim 6 in preparation of antitumor drugs.