CN108395537A - A kind of tetravalence platinum macromolecular prodrug PDA, tetravalence platinum macromolecular calcium phosphate nanoparticles and its application - Google Patents

Abstract

The invention discloses a tetravalent platinum macromolecular prodrug PDA, tetravalent platinum macromolecular calcium phosphate nanoparticles and applications thereof. First, the tetravalent platinum macromolecular prodrug PDA is synthesized, and then the tetravalent platinum macromolecular Molecular prodrug PDA, the nanoparticle obtained by modifying the surface with a hydrophilic block polymer. The tetravalent platinum macromolecule nano-calcium phosphate particles prepared by the present invention have good stability, high biocompatibility, good pH responsive release effect and good tumor cell killing effect. The disadvantages of low stability and easy clearance by the kidney have opened up a new way.

Description

A tetravalent platinum macromolecular prodrug PDA, tetravalent platinum macromolecular calcium phosphate nanoparticles and its application

technical field

The invention relates to tetravalent platinum macromolecular prodrug PDA, tetravalent platinum macromolecular calcium phosphate nanoparticles and applications thereof, belonging to the field of organic chemistry and nanometer material preparation.

Background technique

As an anticancer drug acting on DNA, cisplatin is currently widely used in the treatment of various cancers (such as ovarian cancer, cervical cancer, brain cancer, etc.). However, it has strong toxic side effects including nephrotoxicity and neurotoxicity, and is prone to drug resistance, which limits its application in antitumor drugs. In order to reduce the toxicity to normal cells and overcome the drug resistance of cancer cells, researchers have synthesized many chemically inert, non-toxic tetravalent platinum prodrugs, and studies have shown that tetravalent platinum molecules will be absorbed by cells after entering cancer cells. Internal substances such as glutathione and ascorbic acid are reduced to the form of divalent platinum to achieve anti-cancer effects.

In recent years, polymeric macromolecular drugs have attracted increasing attention from scientists. Yang Jun et al first synthesized two small tetravalent platinum molecules DHP and DSP derived from cisplatin, and further synthesized three tetravalent platinum macromolecular polymers P(DHP-DA)-PEG, P(DSP-PA ) and P(DSP-EDA). It has been verified in the literature that these three tetravalent platinum macromolecules can be taken up by tumor cells and that their cytotoxicity is generally higher than that of their corresponding parent small molecules. Jing Yabin's research group first synthesized a tetravalent platinum compound with a carboxyl group at the end, and then bonded with mPEG-b-PCL-b-PLL with an aminocation to self-assemble into a polymer micelle, and the research showed that The polymeric micelles are more cytotoxic than cisplatin and its corresponding parent polymer. However, since most macromolecular polymers are not biodegradable in vivo, their biosafety during circulation in vivo remains to be verified.

At the same time, calcium phosphate is a naturally occurring inorganic mineralization material. As we all know, calcium phosphate is an inorganic component of hard tissues such as teeth and bones, so it has the characteristics of non-toxicity, degradability and excellent biocompatibility, so nano-calcium phosphate has attracted more and more attention due to its excellent physical and chemical properties. more and more people's attention. Due to the excellent physical and chemical properties of nano-calcium phosphate, which allows it to maintain its own integrity during transport in the body, nano-calcium phosphate has more and more applications in the field of nano-drug carriers.

Based on this, the present invention first synthesizes a new tetravalent platinum macromolecular prodrug PDA, utilizes the numerous carboxyl groups on it to provide conditions for the mineralization of calcium phosphate, and then modifies the surface with polyethylene glycol to achieve the effect of long circulation , so as to prepare a new type of tetravalent platinum macromolecular calcium phosphate nanoparticles and study its drug release and anti-tumor effects.

Contents of the invention

In order to avoid the shortcomings of the prior art, the present invention aims to provide a tetravalent platinum macromolecular prodrug PDA, tetravalent platinum macromolecular calcium phosphate nanoparticles and applications thereof. The tetravalent platinum macromolecule nano-calcium phosphate particles prepared by the present invention have good stability, high biocompatibility, good pH responsive release effect and good tumor cell killing effect. The disadvantages of low stability and easy clearance by the kidney have opened up a new way.

The tetravalent platinum macromolecular prodrug PDA of the present invention, its structural formula is:

The molecular weight range of the tetravalent platinum macromolecular prodrug PDA is 8000-12000.

The tetravalent platinum macromolecular calcium phosphate nanoparticle of the present invention is a nanoparticle obtained by embedding the tetravalent platinum macromolecular prodrug PDA with calcium phosphate, and then modifying the surface with a hydrophilic block polymer.

The preparation method of tetravalent platinum macromolecular calcium phosphate nano-particles of the present invention comprises the following steps:

Step 1: Add cisplatin into water, raise the temperature to 60°C, then add hydrogen peroxide (30wt%) to oxidize, stir and react for 4 hours; after the reaction, let it stand for cooling, and freeze-dry at -50°C to obtain light yellow solid DHP, which is the axial band Small molecules of tetravalent platinum with hydroxyl groups;

The structural formula of DHP is as follows:

Step 2: Weigh DHP and pyromellitic dianhydride (PMDA) into dimethyl sulfoxide (DMSO), stir and react at 60°C for 2 hours; after the reaction, put the reaction solution in a dialysis bag (MWCO, 3500) dialyzed for 24 hours, freeze-dried at -50°C to obtain tetravalent platinum macromolecular prodrug PDA;

The structural formula of PDA is:

Step 3: Weigh the hydrophilic block polymer and PDA into distilled water to dissolve, adjust the pH to 9 with 0.1M NaOH solution, then add 0.1M Ca(NO ₃ ) ₂ solution dropwise to the reaction solution, and stir for 5 hours. Then, 0.1M Na ₂ HPO ₄ solution was added dropwise to the reaction liquid, stirred for 10 h, dialyzed for 2 days, and freeze-dried to obtain the target product.

In step 1, the ratio of H ₂ O ₂ to cisplatin in hydrogen peroxide is 159:1.

In step 2, the amount ratio of DHP and PMDA is 1:1.

In step 2, the molecular weight of PDA is 8000-12000.

In step 3, the hydrophilic block polymer is PEG _5k -PAA or PEG _5k -PAA-5-AF. When the hydrophilic block polymer is PEG _5k -PAA-5-AF, the tetravalent platinum calcium phosphate nanometer drug-loaded particle with fluorescence is prepared.

In step 3, the ratio of the amount of the hydrophilic block polymer to the carboxyl group on the PDA is 5-6:1; the ratio of the amount of Ca(NO ₃ ) ₂ to Na ₂ HPO ₄ is 1:2; The ratio of the carboxyl group on the PDA to the amount of Ca(NO ₃ ) ₂ is 1:2-3.

The particle size of the tetravalent platinum macromolecule calcium phosphate nanoparticle prepared by the invention is 50-100nm.

The synthetic route of PDA of the present invention is as follows:

Among them, PEG _5k -PAA is a hydrophilic polymer material synthesized by the inventor, and PEG _5k -PAA-5-AF is a polymer material with fluorescein obtained by bonding 5-AF and PEG _5k -PAA. The preparation process is as follows:

Synthesis of Hydrophilic Block Copolymer PEG _5k -PAA:

1. Mix and stir ethanethiol (24.85g, 400mmol) and 60mL distilled water, then slowly add 32g of NaOH solution (wherein NaOH 16g, 400mmol) with a mass concentration of 50% under ice-bath conditions, and then Add 20mL of acetone, stir for 30min, then add carbon disulfide (34.2g, 450mmol) and the solution becomes clear orange, and stir for 30min;

2. Slowly add 2-bromopropionic acid (62.73g, 410mmol) dropwise under ice-bath conditions, and then dropwise add 32g of NaOH solution with a mass concentration of 50% (NaOH 16g, 400mmol); remove after the exotherm stops ice bath, then add 60mL distilled water, and react at room temperature for 24h; Dichloromethane, then recrystallized with n-hexane, repeated 3 times to obtain light yellow crystal ETP;

3. Dissolve polyethylene glycol 5000 monomethyl ether (10g, 2mmol) in 60mL toluene, then add dicyclohexylcarbodiimide (DCC, 4.5386g, 22mmol) and 4-dimethylaminopyridine (DMAP, 25.9 mg, 0.212mmol), and the toluene solution (20mL) of ETP (4.2068g, 20mmol) was added dropwise with a constant pressure dropping funnel, and reacted for 12h; Precipitate in ether, repeat like this 3 times, centrifuge, drain to get ETP-PEG _5K ;

4. Dissolve ETP-PEG _5K (2.1616g, 0.4163mmol), acrylic acid (AA, 3g, 41.63mmol) and azobisisobutyronitrile (AIBN, 13.67mg, 0.08326mmol) in 30mL 1,4-dioxane In the ring, pass N ₂ for 30min under the condition of ice bath, move to 60°C oil bath for 24h reaction; after the reaction, part of 1,4-dioxane is removed by rotary evaporation, precipitated with anhydrous ether for 3 times, centrifuged, pumped The product PEG _5k -PAA was obtained by drying.

Synthesis of Hydrophilic Block Copolymer PEG _5k -PAA-5-AF Linked with Fluorescein

1. Dissolve 5-aminofluorescein (0.05g, 0.144mmol) in 20mL DMF, then add dicyclohexylcarbodiimide (DCC, 0.0594g, 0.288mmol) and 4-dimethylaminopyridine (DMAP, 35mg ,0.0288mmol);

2. Add the DMF solution (10mL) of PEG5k-PAA (0.1927g, 1.44mmol) dropwise with a constant pressure dropping funnel, and react for 12h; after the reaction, remove the insoluble matter DCU by filtration, and then precipitate in ice ether, repeat this process 3 times , centrifuged, and drained to obtain the product PEG _5k -PAA-5-AF.

The use of the tetravalent platinum macromolecular calcium phosphate nanoparticle of the present invention is the application in the preparation of anticancer drugs.

The beneficial effects of the present invention are reflected in:

1. The solution used in the process of preparing tetravalent platinum macromolecule nano-calcium phosphate particles in the present invention is a water system, which is safe, non-toxic and environmentally friendly;

2. The tetravalent platinum macromolecular prodrug PDA synthesized by the present invention has a large number of carboxyl groups, which is easier to bind with Ca ²⁺ and can increase the drug loading capacity of the system;

3. The tetravalent platinum macromolecule nano-calcium phosphate particles prepared by the present invention have good stability, high biocompatibility, good pH-responsive release effect and good tumor cell killing effect.

Description of drawings

Fig. 1 is the NMR spectrum of tetravalent platinum macromolecule prodrug PDA in deuterated DMSO;

Fig. 2 is the recorded GPC figure of PDA-Na in aqueous gel permeation chromatography;

Figure 3 is the NMR spectrum of the hydrophilic block copolymer PEG _5k -PAA in D ₂ O;

In Figure 4, a is the H NMR spectrum of the hydrophilic block copolymer PEG _5k -PAA-5-AF connected with fluorescein in D ₂ O, b is the H NMR spectrum of 5-AF in deuterated DMSO picture;

Among Fig. 5, a is the fluorescence curve of the measured PEG _5k- PAA-5-AF, b is the fluorescence curve of 5-AF, and c is the fluorescence curve of the tetravalent platinum macromolecular calcium phosphate particles with fluorescence;

Among Fig. 6, a, b are respectively the DLS number distribution figure and the DLS intensity distribution figure of the prepared three kinds of tetravalent platinum macromolecule nano-calcium phosphate particle sizes;

Fig. 7 is the transmission electron microscope picture of tetravalent platinum macromolecule nanometer calcium phosphate particle;

Curves a and b in Fig. 8 are respectively the graph of particle size variation with time in PBS (pH=7.4) and DMEM solution measured by DLS for tetravalent platinum macromolecular calcium phosphate particles;

Curve a in Fig. 9, b, c, d are the PBS solution of tetravalent platinum macromolecular calcium phosphate particle at pH=7.4, the PBS+5mM Vc solution of pH=7.4, the PBS solution of pH=5.0 and the PBS solution of pH=5.0 respectively The drug release curve measured by ICP-MS in the PBS+5mM Vc solution;

In Fig. 10, a and b are respectively calculated by XPSpeak software on the basis of the measured XPS data of divalent platinum and tetravalent platinum in the PBS solution of pH = 5.0 at different time points of tetravalent platinum macromolecular calcium phosphate particles. Relative content and the relative content of divalent platinum and tetravalent platinum at different time points in the PBS+5mM Vc solution of pH=5.0;

Figure 11 is the hemolysis value data calculated by ultraviolet test of tetravalent platinum macromolecular calcium phosphate particles with different concentrations;

Figure 12 is the cytotoxicity value of DHP, PDA and tetravalent platinum macromolecular nano calcium phosphate particles containing different Pt concentrations to MDA-MB-231;

Figure 13 is the cytotoxicity value of different concentrations of naked calcium phosphate nanoparticles that do not contain tetravalent platinum macromolecules to MDA-MB-231;

Fig. 14 is the graph that fluorescently labeled tetravalent platinum macromolecular nano calcium phosphate particles are measured by flow cytometry in MDA-MB-231 cells;

Figure 15 is the result of the quantitative uptake of platinum in DHP, PDA and tetravalent platinum macromolecule calcium phosphate nanoparticles by MDA-MB-231 cells.

Detailed ways

The technical solution of the present invention will be further analyzed and illustrated through specific examples below. The following examples are carried out on the premise of the technical solutions of the present invention, and detailed implementation methods and specific operation processes are provided, but the protection scope of the present invention is not limited to the following examples.

Embodiment 1: the synthesis of tetravalent platinum macromolecular prodrug PDA

1. Weigh 400mg (1.334mmol) of cisplatin and add it into 12mL of water. After heating up to 60°C, add 24mL of 30% (212mmol) hydrogen peroxide drop by drop. Phenomena changed, stirred and reacted for 4 hours; after the reaction was completed, the reaction solution was cooled to room temperature, left standing in an ice bath overnight to obtain a light yellow precipitate, filtered, washed with ice water, and freeze-dried to obtain a light yellow solid DHP;

2. Weigh DHP 150mg (0.45mmol) and PMDA 97.92mg (0.45mmol) into 15mL DMSO, stir and react at 60°C for 2h, then add excess ethanol to end capping; after the reaction, place the reaction solution in dialysis The bag (MWCO, 3500) was dialyzed for 24 hours, and freeze-dried to obtain tetravalent platinum macromolecule prodrug PDA.

Fig. 1 is the NMR spectrum of tetravalent platinum macromolecular prodrug PDA in deuterated DMSO, and the analyzed product is PDA.

Fig. 2 is the GPC graph measured in the aqueous phase gel permeation chromatography of the tetravalent platinum macromolecule prodrug PDA-Na. Specifically, the tetravalent platinum macromolecular prodrug PDA was adjusted to a pH of about 9 with 0.1M NaOH solution, and then tested by aqueous gel permeation chromatography, and the GPC aqueous phase flow rate was 1mL/min.

Table 1 is the corresponding data given by the tetravalent platinum macromolecular prodrug PDA-Na tested by aqueous gel permeation chromatography. It can be seen that the molecular weight of PDA-Na is about 9000, and the PDI is 2.993.

Table 1

compound Retention time/min MP mw mn PDI PDA-Na 25.436 8943 12752 4261 2.993

Example 2: Synthesis of Hydrophilic Block Copolymer PEG _5k -PAA

1. Mix and stir ethanethiol (24.85g, 400mmol) and 60mL distilled water, then slowly add 32g of NaOH solution (wherein NaOH 16g, 400mmol) with a mass concentration of 50% under ice-bath conditions, and then Add 20mL of acetone, stir for 30min, then add carbon disulfide (34.2g, 450mmol) and the solution becomes clear orange, and stir for 30min;

2. Slowly add 2-bromopropionic acid (62.73g, 410mmol) dropwise under ice-bath conditions, and then dropwise add 32g of NaOH solution with a mass concentration of 50% (NaOH 16g, 400mmol); remove after the exotherm stops ice bath, then add 60mL distilled water, and react at room temperature for 24h; Dichloromethane, then recrystallized with n-hexane, repeated 3 times to obtain light yellow crystal ETP;

3. Dissolve polyethylene glycol 5000 monomethyl ether (10g, 2mmol) in 60mL toluene, then add dicyclohexylcarbodiimide (DCC, 4.5386g, 22mmol) and 4-dimethylaminopyridine (DMAP, 25.9 mg, 0.212mmol), and the toluene solution (20mL) of ETP (4.2068g, 20mmol) was added dropwise with a constant pressure dropping funnel, and reacted for 12h; Precipitate in ether, repeat like this 3 times, centrifuge, drain to get ETP-PEG _5K ;

4. Dissolve ETP-PEG _5K (2.1616g, 0.4163mmol), acrylic acid (AA, 3g, 41.63mmol) and azobisisobutyronitrile (AIBN, 13.67mg, 0.08326mmol) in 30mL 1,4-dioxane In the ring, pass N ₂ for 30min under the condition of ice bath, move to 60°C oil bath for 24h reaction; after the reaction, part of 1,4-dioxane is removed by rotary evaporation, precipitated with anhydrous ether for 3 times, centrifuged, pumped The product PEG _5k -PAA was obtained by drying.

Fig. 3 is the H NMR spectrum of the hydrophilic block copolymer PEG _5k -PAA in D ₂ O. The analyzed product is PEG _5k -PAA, and the number of connected AA units is 87.

Embodiment 3: Synthesis of the hydrophilic block copolymer PEG _5k -PAA-5-AF connected with fluorescein

1. Dissolve 5-aminofluorescein (0.05g, 0.144mmol) in 20mL DMF, then add dicyclohexylcarbodiimide (DCC, 0.0594g, 0.288mmol) and 4-dimethylaminopyridine (DMAP, 35mg ,0.0288mmol);

2. Add the DMF solution (10mL) of PEG5k-PAA (0.1927g, 1.44mmol) dropwise with a constant pressure dropping funnel, and react for 12h; after the reaction, remove the insoluble matter DCU by filtration, and then precipitate in ice ether, repeat this way 3 times , centrifuged, and drained to obtain the product PEG _5k -PAA-5-AF.

In Figure 4, a is the H NMR spectrum of the hydrophilic block copolymer PEG _5k -PAA-5-AF connected with fluorescein in D ₂ O, b is the H NMR spectrum of 5-AF in deuterated DMSO picture.

In Fig. 5, a is the measured fluorescence curve of PEG _5k -PAA-5-AF, b is the fluorescence curve of 5-AF, and c is the fluorescence curve of tetravalent platinum macromolecule calcium phosphate nanoparticles with fluorescence. Based on the results of Figure 4 and Figure 5, it can be seen that 5-AF has been successfully bonded to PEG _5k -PAA and made it have a fluorescent effect.

Embodiment 4: Preparation of tetravalent platinum macromolecule nano calcium phosphate particles

Weigh 16.92mg PEG _5k -PAA (-COOH: 0.1220mmol), 6.25mg PDA (-COOH: 0.02272mmol) in a 25mL single-necked bottle, add 10mL distilled water to dissolve, adjust the pH to 9 with 0.1M NaOH solution; then add to the reaction 0.625 mL of 0.1M Ca(NO ₃ ) ₂ solution was slowly added dropwise into the solution, stirred for 5 hours, and then 1.250 mL of 0.1M Na ₂ HPO ₄ solution was slowly added dropwise, stirred for 10 hours, dialyzed for 2 days, and freeze-dried to obtain product.

When we added 1.692mg PEG _5k -PAA-5-AF before the reaction, we prepared tetravalent platinum macromolecule calcium phosphate nanoparticles with fluorescence. In addition, we changed the feeding amount of PDA into 5 mg and 3.13 mg, and prepared two other tetravalent platinum macromolecular calcium phosphate nanoparticles by using the same method as above.

In Fig. 6, a and b are respectively the DLS number distribution diagram and the DLS intensity distribution diagram of the prepared three kinds of tetravalent platinum macromolecule nano-calcium phosphate particles.

Fig. 7 is a transmission electron microscope image of the prepared tetravalent platinum macromolecular calcium phosphate particle size. It can be seen that the particle size is uniform and the particle size is about 40nm.

Curve a in Fig. 8, b is to select PDA charging amount to be that the tetravalent platinum macromolecule nano-calcium phosphate particle that makes 6.25mg is measured by DLS in PBS (pH=7.4) and DMEM solution particle size change figure with time , it can be seen that the particle stability is good.

Table 2 is the particle size data measured by DLS and the drug loading and drug loading efficiency data measured by ICP-MS for three tetravalent platinum macromolecular calcium phosphate nanoparticles.

Table 2

Example 5: Drug Release Experiment of Tetravalent Platinum Macromolecular Nano Calcium Phosphate Particles

We selected tetravalent platinum macromolecular nano calcium phosphate particles prepared with a PDA dosage of 6.25 mg to carry out corresponding release experiments. The specific implementation plan is as follows:

1. Measure 4 groups of 2mL 1.0mg/mL tetravalent platinum macromolecular calcium phosphate nanoparticle solutions and add them to the dialysis bag (boil the dialysis bag for 5 minutes before use, tie one end of the dialysis bag tightly with a rope and the other end with a clip), and prepare respectively PBS solution with pH=7.4, PBS solution with pH=5.0, PBS+5mM Vc solution with pH=7.4, PBS+5mM Vc solution with pH=5.0, add 15mL of the above buffer solution into the centrifuge tube at each time point, centrifuge The tube was placed in a constant temperature shaking box, and the temperature was set at 37°C. At different time points, the dialysis bag was taken out and put into fresh buffer solution. The time points were 0h, 2h, 4h, 12h, 24h, 48h, and 72h. The centrifuge tube was stored at -20°C, freeze-dried, and the content of Pt in the freeze-dried sample was determined by ICP-MS.

2. In addition, we measure 2 groups of 2mL3.0mg/mL tetravalent platinum macromolecular calcium phosphate nanoparticle solutions and add them to 10mL of PBS solution with pH=5.0 and 10mL of PBS+5mM Vc solution with pH=5.0, At different time points, 2 mL of sample solution was taken out, collected and freeze-dried, and the relative content of divalent platinum and tetravalent platinum was determined by XPS.

Curve a in Fig. 9, b, c, d are the PBS solution of tetravalent platinum macromolecular calcium phosphate particle at pH=7.4, the PBS+5mM Vc solution of pH=7.4, the PBS solution of pH=5.0 and the PBS solution of pH=5.0 respectively Utilize the drug release curve measured by ICP-MS in the PBS+5mM Vc solution of pH=7.4, as can be seen in the PBS+5mM Vc solution of pH=7.4 in the tetravalent platinum macromolecular calcium phosphate particle The drug is basically not released, and the drug is released faster in the PBS solution with pH=5.0, and the drug is released faster in the PBS+5mM Vc solution with pH=5.0.

In Fig. 10, a and b are the divalent platinum and tetravalent platinum of tetravalent platinum macromolecular calcium phosphate particles in PBS solution at pH=5.0 at different time points calculated by XPSpeak software on the basis of the measured XPS data respectively. and the relative content of divalent platinum and tetravalent platinum at different time points in the PBS+5mM Vc solution of pH=5.0, it can be seen that in the PBS solution of pH=5.0, the tetravalent platinum macromolecular calcium phosphate particles It is basically released in the form of tetravalent platinum, and the content of divalent platinum in the PBS+5mMVc solution of pH=5.0 gradually increases, and is basically completely converted into divalent platinum after 12 hours.

Embodiment 6: Hemolysis experiment of tetravalent platinum macromolecule nano calcium phosphate particles

1. Centrifuge 0.8mL of fresh mouse blood (2500rpm, 10min), pour off the supernatant, and then add 1mL of 0.01MPBS (pH=7.4) solution to dilute to obtain diluted blood;

2. Prepare three groups of 5mL 0.1mg/mL, 0.01mg/mL, and 0.001mg/mL tetravalent platinum macromolecular calcium phosphate nanoparticles in PBS (pH=7.4) respectively, and use three groups of 5mL PBS solutions (pH=7.4) 7.4) as a negative control, three groups of 5mL deionized water as a positive control;

3. Put the above 15 groups of solutions in a constant temperature shaking box, set the temperature at 37°C for 30 minutes, then add diluted blood and shake well, incubate at 37°C for 1 hour, measure the UV, and record the peak value at 540nm.

Figure 11 shows the hemolysis value data of 0.1mg/mL, 0.01mg/mL and 0.001mg/mL tetravalent platinum macromolecular calcium phosphate particles calculated by ultraviolet test, it can be seen that the concentration of the particles does not exceed 0.1mg/mL , its hemolysis value does not exceed 2%, and it has good compatibility with blood.

Example 7: Cytotoxicity Experiment of Tetravalent Platinum Macromolecular Nano Calcium Phosphate Particles

We selected tetravalent platinum small molecule DHP, tetravalent platinum macromolecular prodrug PDA and tetravalent platinum macromolecular calcium phosphate drug-loaded nanoparticles (Pt(IV)CPNP) to investigate their effects on MDA-MB-231 cells through MTT experiments. Toxicity, we also investigated the cytotoxicity of bare calcium phosphate nano drug-loaded particles to MDA-MB-231, the specific implementation scheme is as follows:

(1) Inoculate MDA-MB-231 cells into a 96-well plate, inoculate 1×10 ⁵ cells per well, then add 100 μL DDMEM complete medium to each well and place in a 37° C. incubator for 24 hours;

(2) Dilute PBS, DHP, PDA and Pt(IV)CPNP with DMEM complete medium to different concentrations to make solutions, take 100 μL of each group solution and add it to the well plate, set the parallel group as 3 groups, and then continue to place Cultivate in a 37°C incubator for 48 hours;

(3) At the end of the culture, the medium was aspirated and the same volume of DMEM complete medium was added. Add 25 μL MTT stock solution (5 mg mL ^-1 in PBS solution) to each well, add 25 μL PBS buffer solution to the blank well, and continue to incubate for 2 hours;

(4) Add 100 μL DMSO to each well and incubate for half an hour;

(5) Measure the absorbance (Abs) of the solution at 570 nm with a microplate reader. Cell viability was calculated using the following formula:

Among them, Abs(sample) is the absorbance value of the sample well; Abs(blank) is the absorbance value of the blank well; Abs(control) is the absorbance value of the PBS control well.

Figure 12 is the cytotoxicity value of DHP, PDA and tetravalent platinum macromolecular calcium phosphate particles containing different Pt concentrations to MDA-MB-231. It can be seen that compared with DHP and PDA at the same platinum concentration, tetravalent platinum Macromolecular nano calcium phosphate particles have stronger cytotoxicity and better killing effect.

Figure 13 shows the cytotoxicity values of different concentrations of naked calcium phosphate nanoparticles that do not contain tetravalent platinum macromolecules on MDA-MB-231. It can be seen that the cytotoxicity of naked nano calcium phosphate particles is low and the safety is good.

Embodiment 8: Cell qualitative uptake experiment of tetravalent platinum macromolecule calcium phosphate nanoparticle (Pt(IV)CPNP)

(1) Inoculate MDA-MB-231 cells in a 24-well plate, inoculate 1×10 ⁵ cells per well, add 100 μL DMEM complete medium, and then culture in a 37°C incubator for 24 hours;

(2) After the culture was over, remove the medium, and then replace it with a new DMEM complete medium containing 5-aminofluorescein-labeled Pt(IV)CPNP nanoparticles, and co-culture with the cells at 37°C for 2h, 4h, 6h;

(3) Remove the culture medium, wash twice with PBS buffer, use 0.25% trypsin (containing EDTA) to digest and collect the cells into the flow tube, use the flow cytometer to detect the green fluorescent signal in the cells, and the results are obtained by FlowJo_V10 software for analysis.

Figure 14 is a graph of fluorescence-labeled Pt(IV)CPNP measured by flow cytometry in MDA-MB-231 cells. It can be seen that Pt(IV)CPNP can be taken up by cells, and is basically taken up after 4h completely.

Example 9: Cell Quantitative Uptake Experiment of Tetravalent Platinum Macromolecular Nano Calcium Phosphate Particles (Pt(IV)CPNP)

(1) MDA-MB-231 cells were planted in 96-well plates at 5×10 ⁵ cells per well, using 100 μL DMEM complete medium, and cultured in a 37°C incubator for 24 hours;

(2) Remove the medium, replace it with a new DMEM complete medium, add DHP, PDA and Pt(IV)CPNP solution, so that the total concentration of platinum is 2, 4, 8 μg/mL, and mix with the cells at 37°C Co-cultivate for 3h;

(3) Remove the culture medium, wash three times with cold PBS, digest the cells with 0.25% trypsin, collect the cells into the PBS solution, put them in a clean beaker, add 500 μL concentrated nitric acid and evaporate it to dryness, then add 500 μL Wang water and make up to 5mL;

(4) Use ICP-MS to detect the content of platinum in cells.

Figure 15 is the quantitative uptake of platinum in DHP, PDA and Pt(IV) CPNP by MDA-MB-231 cells, from which it can be seen that the uptake of platinum in Pt(IV) CPNP by MDA-MB-231 cells is higher than DHP and PDA.

The above descriptions are only exemplary embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included in the protection of the present invention. within range.

Claims (10)

1. a kind of tetravalence platinum macromolecular prodrug PDA, it is characterised in that its structural formula is：

2. tetravalence platinum macromolecular prodrug PDA according to claim 1, it is characterised in that：

The molecular weight ranges of the tetravalence platinum macromolecular prodrug PDA are 8000~12000.

3. a kind of tetravalence platinum macromolecular calcium phosphate nanoparticles, it is characterised in that：It is with four described in calcium phosphate embedding claim 1 Valence platinum macromolecular prodrug PDA, surface carry out the nano particle of modification acquisition with hydrophilic block polymer again.

4. tetravalence platinum macromolecular calcium phosphate nanoparticles according to claim 3, it is characterised in that：

The grain size of the tetravalence platinum macromolecular calcium phosphate nanoparticles is 50~100nm.

5. a kind of preparation method of the tetravalence platinum macromolecular calcium phosphate nanoparticles described in claim 3, it is characterised in that including Following steps：

Step 1：Cis-platinum is added to the water, 60 DEG C is warming up to, hydrogen peroxide oxidation is then added, be stirred to react 4h；After reaction Cooling is stood, faint yellow solid DHP is lyophilized to obtain at -50 DEG C, for the axial tetravalence platinum small molecule for carrying hydroxyl；

Step 2：It weighs DHP and pyromellitic acid anhydride is added in dimethyl sulfoxide, 2h is stirred to react under the conditions of 60 DEG C；Reaction knot Shu Hou places reaction liquid into dialysis in bag filter and for 24 hours, is lyophilized at -50 DEG C, obtains tetravalence platinum macromolecular prodrug PDA；

Step 3：It weighs hydrophilic block polymer and PDA is added in distilled water and dissolves, pH to 9 is adjusted with 0.1M NaOH solutions, Then 0.1M Ca (NO are added dropwise into reaction solution₃)₂Solution is stirred to react 5h, 0.1M is then added dropwise into reaction solution again Na₂HPO₄Solution is stirred to react 10h, dialyses 2 days, and freeze-drying obtains target product.

6. preparation method according to claim 5, it is characterised in that：

In step 1, H in hydrogen peroxide₂O₂It is 159 with the ratio between the amount of substance of cis-platinum：1.

7. preparation method according to claim 5, it is characterised in that：

In step 2, the ratio between amount of substance of DHP and PMDA is 1：1.

8. preparation method according to claim 5, it is characterised in that：

In step 3, the hydrophilic block polymer is PEG_5k- PAA or PEG_5k-PAA-5-AF。

9. preparation method according to claim 5, it is characterised in that：

In step 3, the ratio between amount of substance of carboxyl is 5~6 on hydrophilic block polymer and PDA：1；Ca(NO₃)₂And Na₂HPO₄ The ratio between the amount of substance be 1：2；The upper carboxyls of PDA and Ca (NO₃)₂The ratio between the amount of substance be 1：2~3.

10. a kind of purposes of the tetravalence platinum macromolecular calcium phosphate nanoparticles described in claim 3, it is characterised in that：It is to make Application in standby anticancer drug.