CN116966200A - Vanadium-based nano-drug capable of reversing cisplatin resistance through multiple targets and preparation method thereof - Google Patents

Abstract

The invention discloses a vanadium-based nano-drug capable of reversing cisplatin resistance by multiple targets and a preparation method thereof. The preparation method of the medicine comprises the following steps: (1) preparation of mesoporous silica; (2) vanadium ion doping; (3) surface amino modification; (4) loading a Pt (IV) prodrug; (5) PDGF modification is carried out on the surface of the nanoparticle after drug loading, and the nanoparticle is prepared. The method can stably and efficiently prepare the vanadium-based nano-drug with excellent performance, and the vanadium-based nano-drug prepared by the method can reverse cisplatin resistance at multiple targets, thereby providing a new idea for constructing a lung cancer accurate treatment scheme for reversing cisplatin resistance at multiple targets.

Description

A vanadium-based nanomedicine capable of reversing cisplatin resistance at multiple targets and its preparation method

Technical field

The invention relates to the technical field of biomedical materials, and in particular to a vanadium-based nanomedicine that can reverse cisplatin resistance at multiple targets and a preparation method thereof.

Background technique

According to reports, the mortality rate of lung cancer ranks first, and it is urgent to achieve efficient and precise treatment of lung cancer. Lung cancer patients are usually treated with cisplatin as first-line chemotherapy. However, during long-term cisplatin treatment, patients are prone to acquire drug resistance, seriously reducing the efficacy of lung cancer. Multi-target reversal of cisplatin resistance has always been the research frontier in the field of lung cancer. A large amount of research is devoted to developing treatment options for reversing cisplatin resistance. For example, Chinese scholars use cisplatin in combination with small molecule drugs to change the uptake mode of cisplatin or activate the expression of functional proteins to achieve high accumulation of cisplatin. In addition, the researchers developed a kinetically inert Pt(IV) prodrug that easily reacts with glutathione (GSH), reduces GSH levels, weakens the inactivation effect of GSH on platinum drugs, and helps reverse cisplatin ( CDDP) resistance.

However, current research has found that simple DNA repair inhibitory drugs may achieve certain synergistic enhancement effects, but tumor cells will inevitably develop new drug resistance to DNA repair inhibitory drugs, and will also evolve Novel cisplatin resistance mechanisms. Therefore, starting from the mechanism of cisplatin resistance, it is urgent and challenging to develop a new multi-target precision treatment regimen for reversing cisplatin resistance in lung cancer.

Contents of the invention

In order to solve the above-mentioned deficiencies in the existing technology, the purpose of the present invention is to provide a vanadium-based nanomedicine that can reverse cisplatin resistance at multiple targets and a preparation method thereof.

The technical solutions of the present invention to solve the above technical problems are as follows:

A method for preparing a vanadium-based nanomedicine (pLi-VMSN-Pt) that can reverse cisplatin resistance at multiple targets, including the following steps:

(1) Disperse the mesoporous silica and the precursor containing vanadium ions in water, stir evenly, add ammonia water, and continue stirring to obtain solution A;

(2) Subject the solution A obtained in step (1) to a hydrothermal reaction to prepare vanadium-doped mesoporous silica;

(3) Take the vanadium-doped mesoporous silica prepared in step (2) and disperse it in 3-aminopropyltriethoxysilane and ammonia solution to prepare vanadium-doped mesoporous silica with amino groups on the surface. silicon oxide;

(4) Mix the Pt(IV) prodrug with the vanadium-doped mesoporous silica with amino groups on the surface prepared in step (3) and avoid light and stir to obtain nanoparticles loaded with Pt(IV) prodrug;

(5) Mix the hydrogenated soybean phospholipid chloroform solution, cholesterol chloroform solution and distearoylphosphatidylethanolamine chloroform solution to form a thin film, and disperse the polyethylene glycol modified with PDGF into the nanoparticles prepared in step (4) In the solution, solution B is obtained. The solution B is added into a container containing the film and ultrasonicated to obtain the solution.

In step (1), the mass ratio of mesoporous silica and precursor containing vanadium ions is 1:0.5-1.5.

Precursors containing vanadium ions include: any one of vanadium chloride, vanadyl oxalate, and vanadyl sulfate.

In step (1), the mass ratio of the precursor containing vanadium ions to ammonia water is 1:0.5-3.

The hydrothermal reaction temperature in step (2) is 120-150°C.

In step (3), the mass ratio of 3-aminopropyltriethoxysilane and vanadium-doped mesoporous silica is 1:7-10.

In step (4), the mass ratio of Pt(IV) prodrug to vanadium-doped mesoporous silica with amino groups on the surface is 1:0.5-1.5.

Vanadium-based nanomedicine prepared by the above preparation method.

Application of the above vanadium-based nanomedicine in the preparation of drugs for the treatment of lung cancer.

The invention has the following beneficial effects:

(1) The present invention uses MSN as the basic nano-skeleton, uses the doping of vanadium ions to prepare VMSN nanoparticles with a hollow structure, and selects the morphology by regulating the mass ratio of vanadium ions to MSN in the reaction system and the quality of ammonia water. VMSN nanoparticles with uniform, strong Fenton-like catalytic properties and high drug loading rate.

(2) The present invention uses VMSN with excellent performance as a carrier, loads Pt(IV) prodrug, and grafts the targeting molecule PDGF under the action of liposomes to construct a vanadium that can reverse cisplatin resistance at multiple targets. based nanomedicines. The nanomedicine is actively or passively targeted to the tumor site through PDGF, where the released Pt(IV) competitively consumes the highly expressed GSH in the CDDP-resistant tumor site, reduces cisplatin inactivation, and can be reduced to more active forms within the tumor site. It is a toxic therapeutically active compound similar to Pt(II); the released vanadium ions can not only synergistically consume GSH, but also have strong Fenton catalytic properties; in addition, the enzyme-like activity of vanadium ions may downregulate DNA damage repair proteins (ERCC1 ) ability to ultimately achieve precise treatment of lung cancer that reverses CDDP resistance.

(3) The vanadium-based nanomedicine prepared by the present invention avoids the problems of drug resistance and side effects caused by traditional single platinum drug treatment, and provides new ideas for constructing a multi-target precision treatment plan for reversing cisplatin resistance in lung cancer. .

Description of the drawings

Figure 1 is a transmission electron microscope test photo of VMSN- _NH2 nanoparticles in Experimental Example 1, where: a-Comparative Example 1, b-Example 1, c-Example 2, d-Example 3, e-Example 4, f-Example 5;

Figure 2 is a transmission electron microscope test photo of VMSN-Pt nanoparticles in Test Example 2;

Figure 3 is a transmission electron microscope test photo of pLi-VMSN-Pt nanoparticles in Test Example 3;

Figure 4 is the infrared spectrum of pLi-VMSN-Pt nanoparticles in Test Example 3;

Figure 5 shows the absorbance test results of Test Example 4;

Figure 6 shows the test results of intracellular GSH content in Test Example 5;

Figure 7 shows the intracellular reactive oxygen species (ROS) laser confocal microscopy test results in Test Example 6;

Figure 8 shows the results of laser confocal microscopy testing of cell membrane potential in Experimental Example 7;

Figure 9 shows the laser confocal microscopy test results of the tumor targeting ability of the cells in Experiment 8;

Figure 10 shows the test results of protein bands exposed by ECL chemiluminescence method in Experiment 9;

Figure 11 shows the MMT test results in Test Example 10.

Detailed ways

The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. If the specific conditions are not specified in the examples, the conditions should be carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.

Example 1:

A method for the preparation of VMSN nanoparticles and its amino functionalization, including the following steps:

(1) Disperse 0.814g cetyltrimethylammonium bromide in 200 mL H ₂ O, and heat and reflux at 60°C for 30 minutes.

(2) When the solution becomes clear and transparent, add 15g urea and 3mL tetraethyl orthosilicate (slowly add dropwise), continue to heat and reflux for 24h, and collect MSN.

(3) Evenly disperse 0.1g of MSN prepared in step (2) and 0.05g of vanadium chloride in 20 mL of water, and stir at 25°C for 20 minutes.

(4) After the reaction is completed, add 1 mL of ammonia water and continue stirring for 5 minutes, transfer to the autoclave, and react at 150°C for 12 hours to prepare VMSN.

(5) Disperse 25 mg VMSN in 57 mL ethanol, 3 mL water, 400 μL ammonia water and 750 μL 3-aminopropyltriethoxysilane, and heat and reflux at 75°C for 12 hours to obtain VMSN-NH ₂ .

Example 2:

A method for the preparation of VMSN nanoparticles and its amino functionalization, including the following steps:

(1) Disperse 0.814g cetyltrimethylammonium bromide in 200 mL H ₂ O, and heat and reflux at 60°C for 30 minutes.

(2) When the solution becomes clear and transparent, add 15g urea and 3mL tetraethyl orthosilicate (slowly add dropwise), continue to heat and reflux for 24h, and collect MSN.

(3) Evenly disperse 0.1g of MSN prepared in step (2) and 0.075g of vanadium chloride in 20 mL of water, and stir at 25°C for 20 minutes.

(4) After the reaction is completed, add 1 mL of ammonia water and continue stirring for 5 minutes, transfer to the autoclave, and react at 150°C for 12 hours to prepare VMSN.

(5) Disperse 25 mg VMSN in 57 mL ethanol, 3 mL water, 400 μL ammonia water and 750 μL 3-aminopropyltriethoxysilane, and heat and reflux at 75°C for 12 hours to obtain VMSN-NH ₂ .

Example 3:

A method for the preparation of VMSN nanoparticles and its amino functionalization, including the following steps:

(1) Disperse 0.814g cetyltrimethylammonium bromide in 200 mL H ₂ O, and heat and reflux at 60°C for 30 minutes.

(2) When the solution becomes clear and transparent, add 15g urea and 3mL tetraethyl orthosilicate (slowly add dropwise), continue to heat and reflux for 24h, and collect MSN.

(3) Evenly disperse 0.1g of MSN prepared in step (2) and 0.1g of vanadium chloride in 20 mL of water, and stir at 25°C for 20 minutes.

(4) After the reaction is completed, add 1 mL of ammonia water and continue stirring for 5 minutes, transfer to the autoclave, and react at 150°C for 12 hours to prepare VMSN.

(5) Disperse 25 mg VMSN in 57 mL ethanol, 3 mL water, 400 μL ammonia water and 750 μL 3-aminopropyltriethoxysilane, and heat and reflux at 75°C for 12 hours to obtain VMSN-NH ₂ .

Example 4:

A method for the preparation of VMSN nanoparticles and its amino functionalization, including the following steps:

(1) Disperse 0.814g cetyltrimethylammonium bromide in 200 mL H ₂ O, and heat and reflux at 60°C for 30 minutes.

(2) When the solution becomes clear and transparent, add 15g urea and 3mL tetraethyl orthosilicate (slowly add dropwise), continue to heat and reflux for 24h, and collect MSN.

(3) Evenly disperse 0.1g of MSN prepared in step (2) and 0.125g of vanadium chloride in 20 mL of water, and stir at 25°C for 20 minutes.

(4) After the reaction is completed, add 1 mL of ammonia water and continue stirring for 5 minutes, transfer to the autoclave, and react at 150°C for 12 hours to prepare VMSN.

(5) Disperse 25 mg VMSN in 57 mL ethanol, 3 mL water, 400 μL ammonia water and 750 μL 3-aminopropyltriethoxysilane, and heat and reflux at 75°C for 12 h to obtain VMSN-NH ₂ .

Example 5:

A method for the preparation of VMSN nanoparticles and its amino functionalization, including the following steps:

(1) Disperse 0.814g cetyltrimethylammonium bromide in 200 mL H ₂ O, and heat and reflux at 60°C for 30 minutes.

(2) When the solution becomes clear and transparent, add 15g urea and 3mL tetraethyl orthosilicate (slowly add dropwise), continue to heat and reflux for 24h, and collect MSN.

(3) Disperse 0.1g MSN and 0.15g vanadium chloride evenly in 20 mL of water, and stir at 25°C for 20 minutes.

(4) After the reaction is completed, add 1 mL of ammonia water and continue stirring for 5 minutes, transfer to the autoclave, and react at 150°C for 12 hours to prepare VMSN.

(5) Disperse 25 mg VMSN in 57 mL ethanol, 3 mL water, 400 μL ammonia water and 750 μL 3-aminopropyltriethoxysilane, and heat and reflux at 75°C for 12 h to obtain VMSN-NH ₂ .

Example 6:

A synthesis method of VMSN-Pt nanoparticles, including the following steps:

(1) Disperse 0.2g cisplatin in 2mL water and 5mL hydrogen peroxide, heat at 50°C in the dark for 80 minutes, cool down naturally overnight, collect the precipitate by centrifugation, wash once with cold water, once with acetone, and three times with ether, and freeze Dry and collect hydroxytetravalent platinum.

(2) Disperse 0.1g hydroxytetravalent platinum and 0.15g succinic anhydride in 1.2mL dimethyl sulfoxide, stir at 25°C for 12 hours, freeze-dry to remove the dimethyl sulfoxide, and redisperse the precipitate in acetone solution , washed with n-hexane, chloroform, and diethyl ether in sequence, and freeze-dried to collect carboxyl tetravalent platinum.

(3) Disperse 5 mg of VMSN-NH ₂ prepared in Example 3 and 10 mg of carboxytetravalent platinum in 10 mL of water, adjust the pH to 8.5, stir at 25°C for 24 hours, centrifuge at 10,000 rpm for 10 min, and vacuum dry to obtain VMSN-Pt nanomaterials.

Example 7:

A synthesis method of PDGFB polypeptide phospholipid membrane wrapping VMSN-Pt, including the following steps:

(1) Disperse 5 mg PDGF, 50 mg 1-(3dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 50 mg N-hydroxysuccinimide in 5 mL dimethyl sulfoxide, 25 Stir for 2 hours at ℃, then add 10 mg PEGNH ₂ and continue stirring for 6 hours.

(2) After the reaction is completed, dialyze in deionized water to remove unbound products, and freeze-dry to collect PDGFPEG.

(3) Quickly add 500 μL of hydrogenated soybean lecithin chloroform solution (10 mg/mL), 30 μL cholesterol chloroform solution (10 mg/mL), and 100 μL distearoylphosphatidylethanolamine chloroform solution (10 mg/mL) into the rotary evaporator and mix. , rapidly rotary evaporate for 10 minutes at 37°C and 150 rpm to form a phospholipid film, and then disperse 1 mL of the PDGF PEG solution with a concentration of 500 μg/mL into 9 mL of the VMSN-Pt solution prepared in Example 6 with a concentration of 1 mg/mL. And add it to a rotary evaporator bottle, ultrasonic hydrate it for 20 minutes, centrifuge it at 10,000 rpm for 10 minutes, and disperse it in water to obtain the vanadium-based nanomedicine pLi-VMSN-Pt.

Comparative example 1:

A method for the preparation of VMSN nanoparticles and its amino functionalization, including the following steps:

(1) Disperse 0.814g cetyltrimethylammonium bromide in 200 mL H ₂ O, and heat and reflux at 60°C for 30 minutes.

(2) When the solution becomes clear and transparent, add 15g urea and 3mL tetraethyl orthosilicate (slowly add dropwise), continue to heat and reflux for 24h to prepare MSN.

(3) Disperse 0.1g of MSN prepared in step (2) evenly in 20 mL of water, and stir at 25°C for 20 minutes.

(4) After stirring, add 1 mL of ammonia water and continue stirring for 5 minutes, transfer to the autoclave, and react at 150°C for 12 hours to prepare VMSN.

(5) Disperse 25 mg VMSN in 57 mL ethanol, 3 mL water, 400 μL ammonia water and 750 μL 3-aminopropyltriethoxysilane, and heat and reflux at 75°C for 12 hours to obtain VMSN-NH ₂ .

Test Example 1: Morphology Characterization of VMSN- _NH2 Nanoparticles by Transmission Electron Microscopy

The morphology of the VMSN- _NH2 nanoparticles prepared in Comparative Example 1 and Examples 1-5 was characterized by transmission electron microscopy.

The characterization results are shown in Figure 1. Compared with Comparative Example 1 of MSN which was not etched by vanadium ions, the VMSN prepared in the embodiment exhibits a certain degree of hollow structure. In addition, under the etching action of a small amount of vanadium ions, the hollowness of VMSN gradually increases with the increase of vanadium ions (Figure 1b, c). When the feeding ratio of MSN to vanadium chloride is 1:1 (Figure 1d), VMSN exhibits a completely hollow structure. Subsequently, when the feeding ratio of MSN to vanadium chloride exceeded 1:1, the hollow structure of VMSN began to collapse (Figure 1e, f). Therefore, the optimal ratio of the present invention is that the mass ratio of MSN and vanadium chloride is 1:1.

Test Example 2: Morphology Characterization of VMSN-Pt Nanoparticles by Transmission Electron Microscope

The morphology of the VMSN-Pt nanoparticles prepared in Example 6 was characterized by transmission electron microscopy.

The characterization results are shown in Figure 2. It can be seen from the figure that VMSN-Pt maintains a similar hollow structure to the examples in Test Example 1, indicating that the Pt(IV) prodrug has a positive effect on the VMSN-NH ₂ nanoparticles prepared by the present invention. The morphological changes are negligible.

Test Example 3: Transmission electron microscope morphology characterization and infrared spectrum structure characterization of pLi-VMSN-Pt

The pLi-VMSN-Pt prepared in Example 7 was subjected to morphology characterization through transmission electron microscopy and infrared spectrometer testing.

The experimental results are shown in Figures 3 and 4. In the microscope photo in Figure 3, it can be seen that the surface of pLi-VMSN-Pt is coated with a layer of organic matter, which may be due to the presence of the phospholipid layer; in Figure 4, the infrared spectrum can be seen that after the structure After modification with the compound, the corresponding characteristic absorption peak changes were finally reflected in the absorption spectrum of pLi-VMSN-Pt, proving the successful preparation of pLi-VMSN-Pt.

Test Example 4: GSH depletion ability test of pLi-VMSN-Pt

Evaluating the GSH depletion ability of pLi-VMSN-Pt prepared in Example 7 includes incubating the materials at different time points, specifically including the following steps:

Incubate 2mL of pLi-VMSN-Pt (where the concentration of Pt is 20 μg/mL) in acidic PBS buffer solution (pH 6.5) at 37°C for 1 h, and add 20 μL of 10 mmol/L 5,5'-disulfide(2- Nitrobenzoic acid) and 30 μL of 10 mmol/L GSH solution. After mixing, the absorbance at 412 nm was measured by a UV-visible spectrophotometer at different time points.

The experimental results are shown in Figure 5. As the incubation time of pLi-VMSN-Pt with GSH is prolonged, the absorbance of 5,5'-disulfobis(2-nitrobenzoic acid) at 412nm decreases significantly, which indicates that pLi- VMSN-Pt has GSH depletion ability and is time-dependent.

Test Example 5: Effect of pLi-VMSN-Pt on intracellular GSH content

A549 (human lung adenocarcinoma cell line) cells were seeded in a 6-well plate at a density of 2×10 ⁵ cells/well and cultured in 1640 containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin double antibody. Culture the cells for 12 hours. After 12 hours, incubate the cells with PBS, Pt(IV), VMSN prepared in Example 3, VMSN-Pt prepared in Example 6, and pLi-VMSN-Pt prepared in Example 7 respectively for another 12 hours, and then Collect cells and use GSH content detection kit to detect intracellular GSH levels.

The experimental results are shown in Figure 6. Among the VMSN, VMSN-Pt and pLi-VMSN-Pt treatment groups, the pLi-VMSN-Pt group had the lowest intracellular GSH content.

Test Example 6: Effect of pLi-VMSN-Pt on intracellular ROS

A549 (human lung adenocarcinoma cell line) cells were seeded in confocal dishes at a density of 1×10 ⁵ cells/dish, and cultured in 1640 containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin double antibody. The cells were cultured for 12 hours. After 12 hours, the cells were incubated with PBS, Pt(IV), CDDP, VMSN prepared in Example 3, VMSN-Pt prepared in Example 6 and pLi-VMSN-Pt prepared in Example 7 respectively for 6 hours. , and then stained the cells with DCFH-DA staining reagent. After staining, confocal laser photography was performed to detect intracellular reactive oxygen species (ROS) levels.

The experimental results are shown in Figure 7. There was no obvious fluorescence in the cancer cells treated with Pt(IV) prodrug and CDDP, but there was obvious fluorescence in the cells in the VMSN, VMSN-Pt and pLi-VMSN-Pt treatment groups. And the pLi-VMSN-Pt group showed the strongest fluorescence, indicating that the pLi-VMSN-Pt nanomedicine has the strongest Fenton-like catalytic ability, can achieve efficient chemokinetic treatment of lung cancer, and is conducive to reversing CDDP resistance.

Test Example 7: Effect of pLi-VMSN-Pt on cell membrane potential

A549 (human lung adenocarcinoma cell line) cells were seeded in confocal dishes at a density of 1×10 ⁵ cells/dish, and cultured in 1640 containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin double antibody. Culture the cells for 12 hours. After 12 hours, incubate the cells with PBS, Pt(IV), CDDP, VMSN-Pt prepared in Example 6 and pLi-VMSN-Pt prepared in Example 7 respectively for another 12 hours, and then use JC-1 staining reagent. The cells were stained, and after staining, laser confocal photography was performed to detect changes in cell membrane potential.

The experimental results are shown in Figure 8. Compared with other treatment groups, the pLi-VMSN-Pt group showed the greatest degree of membrane potential transition, indicating that pLi-VMSN-Pt nanomedicine can cause significant damage to the mitochondria after entering cancer cells. , which is beneficial to promote the apoptosis of cancer cells.

Test Example 8: Test of tumor targeting ability of pLi-VMSN-Pt

The VMSN-Pt prepared in Example 6 and the pLi-VMSN-Pt prepared in Example 7 were tested to evaluate their tumor targeting ability, which specifically included the following steps:

(1) Culture of tumor cells

A549 (human lung adenocarcinoma cell line) cells were seeded in confocal dishes at a density of 1×10 ⁵ cells/dish, and cultured in 1640 containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin double antibody. base and cultured for 12h.

(2) Monitoring the uptake of nanoparticles by tumor cells

The prepared FITC-pLi-VMSN-Pt and FITC-VMSN-Pt nanoparticles loaded with fluorescein isothiocyanate (FITC) (the concentration of Pt is 10 μg/mL) were incubated in the cells, and after 6 h, the cells were incubated with 4% The cells were fixed with paraformaldehyde for 10 minutes, and then stained with DAPI dead cell nuclear staining reagent. After staining, confocal laser photography was performed to monitor the uptake of PDGF protein nanoparticles into the cells.

The experimental results are shown in Figure 9. Compared with cancer cells incubated with FITC-VMSN-Pt, tumor cells incubated with FITC-pLi-VMSN-Pt nanoparticles had higher FITC fluorescence signals, indicating that pLi combined with PDGF -VMSN-Pt nanoparticles have tumor cell targeting properties, and the number of nanoparticles taken up by cells increases.

Test Example 9: Expression of ERCC1 and GCLC proteins in cells

A549 (human lung adenocarcinoma cell line) cells were seeded in a 6-well plate at a density of 2×10 ⁵ cells/well and cultured in 1640 containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin double antibody. Culture the cells for 12 hours. After 12 hours, incubate the cells with PBS, Pt(IV), CDDP, VMSN-Pt prepared in Example 6, and pLi-VMSN-Pt prepared in Example 7 respectively for another 12 hours. Then, collect the cells and lyse them with RIPA solution. Lyse the cells, collect proteins by centrifugation, take the supernatant and measure the protein concentration by BCA method, then dilute each protein sample with water and 5× Loading buffer to the appropriate concentration, incubate at 100°C for 10 minutes, and cool to room temperature. Next, each protein sample was added to the SDS-PAGE gel well for separation, and then transferred to a PVDF membrane. The protein-loaded PVDF membrane was then blocked in a 1×TBST solution containing 5% BSA for 1 h. Then use ERCC1 and GCLC primary antibody dilutions to incubate the PVDF membrane with the target band overnight at 4°C, wash it three times with 1×TBST buffer (5 minutes each time), and continue to incubate with the corresponding secondary antibodies at room temperature. After 1 h, washing three times with 1×TBST buffer (5 min each time), the protein bands were exposed using ECL chemiluminescence method.

The experimental results are shown in Figure 10. After entering the cancer cells, the pLi-VMSN-Pt nanomedicine can inhibit the expression of GCLC, inhibit the synthesis of GSH in the cancer cells, and reduce the content of GSH, which is beneficial to reversing CDDP resistance. In addition, pLi-VMSN-Pt nanomedicine can also inhibit the expression of ERCC1, further blocking the activation of DNA repair pathways, and providing an early basis for reversing CDDP resistance.

Test Example 10: Anti-tumor effect of pLi-VMSN-Pt on cisplatin (CDDP)-resistant tumors

The pLi-VMSN-Pt prepared in Example 7 was used to evaluate its anti-tumor effect on cisplatin (CDDP)-resistant tumors, which specifically included the following steps:

(1) Culture of tumor cells

A549/CDDP (human lung adenocarcinoma-resistant cisplatin-resistant strain) cells were seeded in 96-well plates at a density of 10 ⁴ cells/well, and cultured in 1640 medium for 12 hours. After 12 hours, the cells were incubated with different concentrations of CDDP and pLi-VMSN-Pt for 24 hours.

(2) MTT detects the inhibitory effect of pLi-VMSN-Pt on the proliferation of CDDP-resistant tumors

Remove the culture medium from the 96-well plate, add a solution of culture medium and MTT in a ratio of 9:1 to each well, continue to incubate in the incubator for 4 hours, remove the liquid, and add 150 μL dimethyl sulfoxide solution to each well. Shake gently and measure the absorbance at 490nm.

The experimental results are shown in Figure 11. The experimental results show that pLi-VMSN-Pt has a concentration-dependent growth inhibitory effect on A549/CDDP cells.

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

Claims (9)

1. The preparation method of the vanadium-based nano-drug capable of reversing cisplatin resistance by multiple targets is characterized by comprising the following steps of:

(1) Dispersing mesoporous silica and a precursor containing vanadium ions in water, uniformly stirring, adding ammonia water, and continuously stirring to obtain a solution A;

(2) Carrying out hydrothermal reaction on the solution A obtained in the step (1) to obtain vanadium-doped mesoporous silica;

(3) Dispersing the vanadium doped mesoporous silica prepared in the step (2) in 3-aminopropyl triethoxysilane and ammonia water solution to prepare vanadium doped mesoporous silica with amino groups on the surface;

(4) Mixing the Pt (IV) prodrug with the vanadium-doped mesoporous silica with the amino groups on the surface, which is prepared in the step (3), and stirring in a dark place to obtain nano-particles carrying the Pt (IV) prodrug;

(5) Mixing and steaming hydrogenated soybean phospholipid chloroform solution, cholesterol chloroform solution and distearoyl phosphatidylethanolamine chloroform solution to form a film, dispersing polyethylene glycol modified with PDGF into the nanoparticle solution prepared in the step (4) to obtain a solution B, and adding the solution B into a container containing the film, and performing ultrasonic treatment to obtain the nano-particle solution.

2. The method for preparing a vanadium-based nano-drug capable of reversing cisplatin resistance with multiple targets according to claim 1, wherein the mass ratio of the mesoporous silica to the precursor containing vanadium ions in the step (2) is 1:0.5 to 1.5.

3. The method for preparing a vanadium-based nano-drug capable of reversing cisplatin resistance with multiple targets according to claim 2, wherein the precursor containing vanadium ions comprises: any one of vanadium chloride, vanadyl oxalate and vanadyl sulfate.

4. The method for preparing the vanadium-based nano-drug capable of reversing cisplatin resistance with multiple targets according to claim 1, wherein the mass ratio of the precursor containing vanadium ions to ammonia water in the step (2) is 1:0.5 to 3.

5. The method for preparing the vanadium-based nano-drug capable of reversing cisplatin resistance by multiple targets according to claim 1, wherein the hydrothermal reaction temperature in the step (3) is 120-150 ℃ and the reaction time is 10-15h.

6. The method for preparing a vanadium-based nano-drug capable of reversing cisplatin resistance with multiple targets according to claim 1, wherein the mass ratio of the 3-aminopropyl triethoxysilane to the vanadium-doped mesoporous silica in the step (4) is 1: 7-10.

7. The method for preparing a vanadium-based nano-drug capable of reversing cisplatin resistance with multiple targets according to claim 1, wherein the mass ratio of the Pt (IV) prodrug to the vanadium-doped mesoporous silica with amino groups on the surface in the step (5) is 1:0.5 to 1.5.

8. A vanadium-based nano-drug prepared by the preparation method of any one of claims 1 to 7.

9. The application of the vanadium-based nano-medicament prepared by the method in preparing medicaments for treating lung cancer.