Preparation method and application of mesoporous cerium dioxide nano-drug based on gold nanostar loaded indocyanine green

Abstract

The preparation method comprises the following steps: synthesizing gold nanostars (GNS) by using a seed growth method, coating mesoporous cerium dioxide (mCeO2) by using a template method, and loading a photosensitizer indocyanine green (ICG), so as to form a nano enzyme diagnosis and treatment system with chemical activity, photoactivity and photothermal effects. In the system, the gold nanostars have a good photo-thermal effect due to the multi-branched structure of the gold nanostars; on one hand, the mesoporous cerium dioxide coated on the outer layer has a chemical effect due to the nano-enzyme effect, and on the other hand, the mesoporous structure of the mesoporous cerium dioxide can better load the photosensitizer indocyanine green. All the components have good stability and biological safety, also have peroxidase simulation activity and a drug loading function, can improve the level of reactive oxygen species (ROS) in a tumor microenvironment and load photosensitizer drugs, and have an excellent anti-tumor effect and small side effects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and health technology, and particularly relates to a preparation method and application of a mesoporous cerium dioxide nano-drug based on gold nanorods loaded with indocyanine green. Background Art

[0002] Nanozymes are nanomaterials with enzyme-like properties, which have attracted extensive attention due to their obvious advantages over natural enzymes, such as high stability in harsh environments, easy synthesis, and tunable catalytic activity. Cerium dioxide nanoparticles are an inorganic nanomaterial with cerium redox states on their surfaces. Based on different valence ratios, they exhibit peroxidase-like activity and can convert excessive H2O2 in the tumor microenvironment into ·OH. Gold nanorods (GNS) have the advantages of strong near-infrared absorption, high photothermal conversion efficiency, good photothermal stability, and good biocompatibility, and are photothermal materials with broad application prospects in the biomedical field in recent years.

[0003] Although cerium dioxide is a highly potential nanozyme carrier material, there are still many challenges to realizing its application in the preparation of tumor therapeutic drugs: poor water dispersibility, insufficient obvious effect of generating reactive oxygen species simply by Ce 3+ insufficient obvious effect of generating reactive oxygen species, poor cerium dioxide loading capacity, and inability to achieve good therapeutic effects with single treatment, etc. Therefore, to solve such problems, this nano-drug system is designed to simultaneously perform photothermal therapy (PTT), photodynamic therapy (PDT), and chemodynamic therapy (CDT), which has great advantages in tumor treatment.

[0004] Based on this, this application is developed. Summary of the Invention

[0005] The main purpose of the present invention is to overcome the defects of the prior art and provide a preparation method of a mesoporous cerium dioxide nano-drug carrier loaded with gold nanorods and indocyanine green. The nano-drug carrier modified with hyaluronic acid prepared by this method has good water solubility, high safety, and small size.

[0006] The present invention also provides a preparation method and application of the above-mentioned mesoporous cerium dioxide nano-drug based on gold nanorods loaded with indocyanine green. The mesoporous cerium dioxide material and indocyanine green can cooperate to achieve chemodynamic and photodynamic therapies.

[0007] To achieve the above-mentioned invention purpose, the present invention adopts the following technical solutions: A preparation method of a mesoporous cerium dioxide-coated gold nanorod nano-carrier, which comprises the following steps: Step 1: Add chloroauric acid tetrahydrate or its aqueous solution into water and mix well, heat it in an oil bath to boiling, add sodium citrate or its aqueous solution and stir for 8 - 20 min, and immediately put it into ice water to obtain solution A; Step 2: Mix chloroauric acid tetrahydrate or its aqueous solution with dilute hydrochloric acid, then add the above Solution A. While stirring, add silver nitrate and ascorbic acid aqueous solution, stir, centrifuge, and dry to obtain Product B (GNS). Step 3: At room temperature, mix the above Product B with cetyltrimethylammonium bromide aqueous solution, and adjust the pH to 9 - 11 to obtain Product C. Step 4: At room temperature, dropwise add the methanol solution of tetraethyl orthosilicate to the above Product C, then continue stirring for 10 - 15 h, centrifuge, wash, and dry to obtain Product D. Step 5: Disperse the above Product D into the ethanol solution of ammonium nitrate, reflux at 50 - 70 °C for 4 - 6 h, centrifuge, wash, and dry to obtain Product E (GNS@SiO2, abbreviated as GS). Step 6: Disperse the above Product E in water, add cetyltrimethylammonium bromide, hexamethylenetetramine, and cerium nitrate hexahydrate aqueous solution, heat to 80 - 95 °C and react for 4 - 8 h, centrifuge, wash, and dry to obtain the nano - carrier F (GNS@mCeO2, abbreviated as GC).

[0008] Specifically, in Step 1, the mass ratio of chloroauric acid tetrahydrate to sodium citrate can be 4 - 5:15 - 16; in Step 2, taking dilute hydrochloric acid as HCl, the molar ratio of chloroauric acid tetrahydrate, dilute hydrochloric acid, sodium citrate, silver nitrate, and ascorbic acid can be 30:90 - 110:25 - 30:1 - 5:50. Further preferably, in Step 2, the concentration of chloroauric acid tetrahydrate aqueous solution is 0.01 M, and the concentration of dilute hydrochloric acid is 1 M; in Step 4, the concentration of the methanol solution of tetraethyl orthosilicate is 0.9 M; in Step 5, the concentration of the ethanol solution of ammonium nitrate is 6 mg / mL.

[0009] Specifically, in Step 3, the mass ratio of Product B to cetyltrimethylammonium bromide can be 1:3 - 5; in Step 4, the mass ratio of Product C to tetraethyl orthosilicate can be 1:1.5 - 2.

[0010] Specifically, in Step 5, the mass ratio of Product D to ammonium nitrate can be 1 - 5:300; in Step 6, the mass ratio of Product E, cetyltrimethylammonium bromide, hexamethylenetetramine, and cerium nitrate hexahydrate can be 13:1 - 3:6 - 9:2 - 3.

[0011] The present invention provides a mesoporous cerium dioxide - coated gold nanostar nano - carrier prepared by the above preparation method.

[0012] The present invention also provides the application of the above mesoporous cerium dioxide - coated gold nanostar nano - carrier in drug loading, such as the nano - carrier is used for preparing drugs, especially for preparing anti - tumor drugs, etc.

[0013] The present invention provides a method for preparing a hyaluronic acid-modified mesoporous cerium dioxide nano-drug based on gold nanostar-loaded indocyanine green using the above-mentioned nanocarrier, which comprises the following steps: Step a: Under room temperature conditions, first disperse the nanocarrier in water, then add indocyanine green, stir in the dark for 24 - 48 h, centrifuge, wash, and freeze-dry to obtain product G (GNS@CeO2-ICG, hereinafter referred to as GCI); Step b: Under room temperature conditions, dissolve product G in water, then add an aqueous solution of hyaluronic acid and stir evenly in the dark, centrifuge, wash, and freeze-dry to obtain the final product H (GNS@CeO2-ICG-HA, hereinafter referred to as GCIH).

[0014] Furthermore, in step a, the mass ratio of product F to indocyanine green can be 1:1 - 2; in step b, the mass ratio of product G to hyaluronic acid can be 2 - 3:1.

[0015] The present invention also provides a hyaluronic acid-modified mesoporous cerium dioxide nano-drug based on gold nanostar-loaded indocyanine green prepared by the above method. This nano-drug can be applied in aspects such as hyperthermia, photodynamic therapy, and combined chemo-photodynamic therapy functions. That is, the present invention provides a drug with combined hyperthermia-photodynamic-chemo-dynamic therapy functions, which can be applied to the combined treatment of photodynamic therapy, photothermal therapy, and chemodynamic therapy for cancer.

[0016] The present invention also provides the application of the above-mentioned hyaluronic acid-modified mesoporous cerium dioxide nano-drug based on gold nanostar-loaded indocyanine green as an anti-tumor drug.

[0017] The present invention synthesizes gold nanostars (GNS) by the seed growth method, then coats mesoporous cerium dioxide (mCeO2) by the template method and loads the photosensitizer indocyanine green (ICG) to form a nanozyme diagnosis and treatment system with chemo-dynamic, photo-dynamic, and photothermal effects. In this system, the gold nanostars play a good photothermal effect due to their multi-branched structure; on the one hand, the outer-coated mesoporous cerium dioxide has a chemo-dynamic effect due to its nanozyme action, and on the other hand, its mesoporous structure can better load the photosensitizer indocyanine green. Each component of the present invention not only has good stability and biological safety, but also has peroxidase mimicking activity and drug-loading function, can increase the level of reactive oxygen species (ROS) in the tumor microenvironment, load photosensitizer drugs, and has excellent anti-tumor effects and less side effects.

[0018] The present invention can achieve a synergistic effect by loading indocyanine green into mesoporous cerium dioxide, greatly promoting photodynamic therapy. Compared with the prior art, the mesoporous cerium dioxide-coated gold nanostar nano-drug loaded with indocyanine green of the present invention has the following advantages and beneficial effects: 1) The present invention uses a photosensitizer indocyanine green, mesoporous cerium dioxide, and gold nanorods as raw materials to obtain a nanomedicine of mesoporous cerium dioxide-coated gold nanorods loaded with indocyanine green. The raw materials used in the present invention have low toxicity and high safety.

[0019] 2) Coating mesoporous cerium dioxide on the surface of gold nanorods in the present invention can not only be used for drug loading but also participate in the treatment. Among them, cerium dioxide can act as a nanozyme with peroxidase-like activity to provide reactive oxygen species for the tumor microenvironment.

[0020] 3) Finally, the present invention modifies hyaluronic acid on the outermost layer to further improve the hydrophilicity of the prepared mesoporous cerium dioxide-coated gold nanorods loaded with indocyanine green in aqueous solution and the affinity with tumor cells.

[0021] 4) The surface potential of the mesoporous cerium dioxide-coated gold nanorods loaded with indocyanine green in the present invention is negative. The negative potential is beneficial to the dispersion of the nanomedicine carrier and is beneficial to circulation in the organism.

[0022] 5) The present invention can effectively coordinate the chemodynamic and photothermal therapy effects of the mesoporous cerium dioxide-coated gold nanorods loaded with indocyanine green with the photodynamic effect of the photosensitizer indocyanine green to enhance the toxicity to tumor cells; the mesoporous cerium dioxide-coated gold nanorods loaded with indocyanine green after hyaluronic acid modification have less toxicity to normal cells and greater toxicity to tumor cells, and can achieve photothermal / photodynamic / chemodynamic synergistic therapy for tumors.

[0023] 6) The present invention has the characteristics of simple operation, high safety, mild reaction conditions, controllable reaction process, and low cost. The prepared mesoporous cerium dioxide-coated gold nanorods loaded with indocyanine green have good hydrophilicity, small size, specific targeting, which is beneficial to circulation in the body, and the reagents used in the preparation process have low toxicity. Description of the Drawings

[0024] Figure 1 Transmission electron microscope (TEM) images of product GNS (a) and product H (GCIH) in Example 1, elemental mapping images of C, N, O, Au, and Ce in product H (GCIH), and SEM image (c); Figure 2 Ultraviolet absorption spectra of product B (GNS), product E (GS), product F (GC), product G (GCI), and product H (GCIH) in Example 1; Figure 3 Zeta potential diagrams of product B (GNS), product E (GS), product F (GC), product G (GCI), and product H (GCIH); Figure 4Hydration particle size diagrams of product B (GNS), product E (GS), product F (GC), product G (GCI), and product H (GCIH); Figure 5 Hemolysis photos (left) and hemolysis percentages (right) of different concentrations of product H (GCIH) in Example 1; Figure 6 Infrared thermal imaging diagrams of different concentrations of product H (GCIH) in Example 1 irradiated with 808 nm laser (1.5 W / cm 2 ) for 6 min; Figure 7 Temperature change diagrams of different concentrations of product H (GCIH) in Example 1 irradiated with 808 nm laser (1.5 W / cm 2 ) for 6 min; Figure 8 Temperature distribution diagrams within 4 laser on / off cycles when 100 μg / mL of product H (GCIH) in Example 1 was irradiated with 808 nm laser (1.5 W / cm 2 ) for 6 min; Figure 9 Intracellular reactive oxygen species generation diagrams of product H (GCIH) in Example 1; Figure 10 Survival rate diagrams of 4T1 cells before and after laser irradiation with products E (GS), F (GC), G (GCI), and H (GCIH) in Example 1. Detailed implementation manners

[0025] To further understand the present invention, the present invention will be further described below in conjunction with the accompanying drawings and detailed implementation manners. However, it should be understood that these descriptions are only for further explaining the features and advantages of the present invention rather than limiting the protection scope of the present invention.

[0026] Names and models of experimental instruments: Perkin-Elmer Lambda-850 ultraviolet spectrophotometer, USA; JEOL JEM-200CX transmission electron microscope, Japan; BioTek multifunctional microplate reader, USA; LEICA TCS SP8+STED laser confocal microscope, Germany; BD Facsverse flow cytometer, USA; LABCONCO-FreeZone 6L freeze-drying system, USA; Cytiva AI800flour ultrasensitive multifunctional imager, USA.

[0027] In the examples, all raw materials used are common commercially available products that can be directly purchased, or can be prepared by conventional techniques in the art.

[0028] Unless otherwise specified, room temperature refers to 25 ± 5 °C; freeze-drying refers to freeze-drying at -60 °C for 12 h.

[0029] Example 1 A preparation method of a mesoporous cerium dioxide-coated gold nanosphere nanocarrier. The preparation steps described in this example are as follows: Step 1: Add 1 mL of 0.01 M chloroauric acid tetrahydrate aqueous solution to water and mix well. Heat it to boiling in an oil bath at 100 °C, add 1.5 mL of 0.04 M sodium citrate aqueous solution, stir for 10 min, and immediately place it in ice water to obtain a wine-red solution A.

[0030] Step 2: Mix 6 mL of 0.01 M chloroauric acid tetrahydrate aqueous solution with 200 μL of 1 M dilute hydrochloric acid, then add the above solution A. Under vigorous stirring, sequentially add 400 μL of 0.01 M silver nitrate and 1 mL of 0.1 M ascorbic acid aqueous solution, stir vigorously, centrifuge at 5000 rpm, and dry to obtain a blue-green product B (GNS).

[0031] Step 3: At room temperature, take 20 mg of the above product B and mix it evenly with 5 mL of 15 mg / mL cetyltrimethylammonium bromide aqueous solution by stirring for 10 min. Add sodium hydroxide aqueous solution to adjust the pH to about 10 to obtain product C.

[0032] Step 4: At 30 °C, add a total of 960 μL of 0.9 M tetraethyl orthosilicate methanol solution dropwise to the above 95 mg of product C in three portions, with a half-hour interval between each addition. Then continue to heat and stir for 12 h, centrifuge at 8000 rpm, wash, and dry to obtain product D.

[0033] Step 5: Disperse 3 mg of the above product D into 50 mL of 6 mg / mL ammonium nitrate ethanol solution, reflux at 60 °C for 5 h, centrifuge at 7000 rpm, wash, and dry to obtain product E (GNS@SiO2, abbreviated as GS).

[0034] Step 6: Disperse 13 mg of the above product E in water, add 25 μL of 0.2 M cetyltrimethylammonium bromide, 0.5 mL of 0.1 M hexamethylenetetramine, and 0.5 mL of 0.01 M cerium nitrate hexahydrate aqueous solution, stir gently, place it in a polytetrafluoroethylene-lined autoclave, heat to 85 °C and react for 5 h, centrifuge, wash, and dry to obtain the nanocarrier product F (GNS@mCeO2, abbreviated as GC).

[0035] The present invention also provides a method for preparing a mesoporous cerium dioxide-coated gold nanosphere nanomedicine modified with hyaluronic acid and loaded with a photosensitizer indocyanine green by using the above-mentioned nanocarrier, which comprises the following steps: Step a: At room temperature, first disperse 4.5 mg of the product F of the nanocarrier in water, then add 9 mg of indocyanine green (ICG), stir in the dark for 24 h, centrifuge, wash, and freeze-dry to obtain product G (GNS@CeO2-ICG, abbreviated as GCI).

[0036] Step b: At room temperature, dissolve 12 mg of the product G in water, then add 2 mL of a 3 mg / mL aqueous solution of hyaluronic acid (HA), stir evenly in the dark, centrifuge, wash, and freeze-dry to obtain the final product H (GNS@CeO2-ICG-HA, abbreviated as GCIH).

[0037] The size and dispersion degree of the above synthetic products were characterized by transmission electron microscopy (TEM), and the results are shown in Figure 1 . From Figure 1 it can be observed that: The initially prepared gold nanospheres (GNS) have a multi-branched structure, laying a structural foundation for good photothermal effects (Figure a); The prepared nanomedicine product H (GCIH) has a mesoporous structure, and the structure is clear and the size is small, with a size of 100-130 nm and a uniform morphology (see Figure 1 b and c therein). The EDS map shows the elemental mappings of C, N, O, Au, and Ce from left to right (Figure c), and it can be seen that the signal uniformly passes through the selected area of the sample, further confirming the synthesis of GCIH.

[0038] The nanomedicine delivery system was scanned at full wavelength using a UV-near infrared absorption spectroscopy instrument, and the results are shown in Figure 2 ; Figure 2 is the UV absorption peak spectra of product B (GNS), product E (GS), product F (GC), product G (GCI), and product H (GCIH) in Example 1 of the present invention. From Figure 2 it can be seen that: GNS has a maximum absorption at 808 nm. After coating with SiO2 and CeO2, there is a small red shift in the maximum absorption peak, indicating that the coating has a certain effect on the nanoparticles. And after loading ICG and coating with HA, there is no significant impact on the absorption peak, which also provides a basic condition for subsequent photothermal and photodynamic therapy.

[0039] The Zeta potential of each component of the nanoparticles was studied using a Zeta potentiometer, and the results are shown in Figure 3 . Figure 3 is the Zeta potential map of product B (GNS), product E (GS), product F (GC), product G (GCI), and product H (GCIH) of the present invention.Figure 3 It can be seen that: the potential of product B (GNS) is -6.7 mV; the potential of product E (GS) becomes -3.6 mV; the potential value of product F (GC) after cerium dioxide coating becomes 19.5 mV; the potential value of product G (GCI) after loading indocyanine green becomes -1.2 mV; the potential value of product H (GCIH) after hyaluronic acid coating becomes -1.7 mV.

[0040] The Zeta potentiometer was used to study the hydrodynamic diameters of the components of the nanoparticles, and the results are shown in Figure 4 . Figure 4 It is the hydrodynamic diameter diagram of product B (GNS), product E (GS), product F (GC), product G (GCI) and product H (GCIH) of the present invention. Figure 4 It can be seen that: the hydrodynamic diameter of product B (GNS) is 164 nm; the hydrodynamic diameter of product F (GC) after cerium dioxide coating becomes 220 nm; the hydrodynamic diameter of product G (GCI) after loading indocyanine green becomes 250 nm; the hydrodynamic diameter of product H (GCIH) after hyaluronic acid coating becomes 295 nm.

[0041] Figure 5 This is the hemolysis rate safety experiment of the nano-drug product H of hyaluronic acid-modified mesoporous cerium dioxide-coated and indocyanine green-loaded gold nanospheres. The specific process is as follows: ① Take 0.5 mL of fresh mouse eyeball blood in a heparin sodium blood collection tube to prevent blood coagulation (take and use immediately). ② Centrifuge the mouse whole blood at 2000 rpm for 5 minutes, and then wash it 2-3 times with normal saline until the supernatant is colorless, and discard the supernatant. Prepare a 4% red blood cell normal saline suspension from the lower layer of red blood cells for later use. ③ Take 7 1.5 mL EP tubes to prepare nano-drug groups with different concentrations (100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL), a negative control group (red blood cell suspension prepared with normal saline), and a positive control group (red blood cell suspension of ultrapure water). ④ Incubate the red blood cell suspensions treated with the nano-drugs of each concentration group numbered above at 37°C in the dark for 1 h. After the incubation, centrifuge at 2000 rpm for 5 minutes, take 100 μL of the supernatant and add it to a 96-well culture plate. Each concentration is repeated in three replicates. Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the OD value of hemoglobin in the supernatant at 540 nm and calculate the hemolysis rate. The results are shown in Figure 5 .

[0042] From Figure 5 It can be seen that: the hemolysis rate of product H (GCIH) at 500 μg / mL is still less than 5%, indicating that this nano-drug does not have a hemolytic effect, has high safety, and can be applied to biological evaluation.

[0043] The obtained hyaluronic acid-modified mesoporous cerium dioxide-coated gold nanostars loaded with indocyanine green nanopharmaceutical product H (GCIH) was formulated into solutions with different concentrations (25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL) using water, and then irradiated with near-infrared light at 808 nm and 1.5 W / cm 2 . The FLIR thermal imager was used to take pictures and statistically record the change in the solution temperature. The thermal image is shown in Figure 6 , and the change in the solution temperature is shown in Figure 7 . It can be seen from Figure 6 and Figure 7 that after different concentrations of GCIH were irradiated with laser, the higher the concentration, the faster the temperature increased, and the temperature also increased with the increase in concentration, finally rising to 48.6 °C, 52.8 °C, 59.2 °C, 66.2 °C, 69.2 °C respectively, while the blank control group only rose to 40.9 °C, directly indicating that GCIH has good photothermal conversion ability, and at the same time indicating that the photothermal performance of gold nanostars is not affected by the outer coating material and drug loading.

[0044] Figure 8 To evaluate the photothermal stability of the hyaluronic acid-modified mesoporous cerium dioxide-coated gold nanostars loaded with indocyanine green nanopharmaceutical product H (GCIH) through multiple laser on / off treatments. In each cycle, an aqueous solution of GCIH (100 μg / mL) was irradiated with an 808 nm laser (1.5 W / cm 2 ) for 6 min, and then naturally cooled to room temperature after turning off the laser. The on / off cycle was repeated 4 times, and the change curve of temperature with irradiation time was plotted to record the temperature change curve during these cycles. The results showed that within 4 on / off cycles, although the highest temperature decreased slightly with the increase in the number of cycles, there was still a good heating effect, indicating that the product GCIH had a relatively stable structure and good photothermal stability after several laser irradiations.

[0045] To verify the ability of the product GCIH with different concentrations (0, 25, 50, 150, 300 μg / mL) to generate ROS in 4T1 cells, the concentration of reactive oxygen species in 4T1 cells was detected using a ROS kit, and the operation was carried out according to the instructions. The results are shown in Figure 9 . Figure 9 The results showed that with the increase in concentration, the number of generated ROS increased, and the ROS generated by the laser-irradiated group (photodynamic + chemodynamic) was more than that of the non-laser-irradiated group (chemodynamic), proving that the nanopharmaceutical has the ability to generate reactive oxygen species and can inhibit tumor cells through the generation of reactive oxygen species.

[0046] The MTT assay was used to detect the toxicity of the hyaluronic acid-modified mesoporous cerium dioxide-coated and indocyanine green-loaded gold nanostar nanopharmaceutical product H (GCIH) to tumor cells. The in vitro activities of GS, GC, GCI, GCIH, and ICG were detected using the MTT method. Well-cultured 4T1 cells were digested with trypsin, centrifuged in a centrifuge, the supernatant was discarded, 1 mL of DMEM medium (containing 1% penicillin-streptomycin and 10% fetal bovine serum, the same below) was added to resuspend the cells evenly, an appropriate amount of the cell suspension was taken for dilution so that the number of cells per well was approximately 4,500, and they were inoculated into 96-well plates and cultured at 37 °C for 24 h. GS, GC, GCI, GCIH, and ICG were diluted with the medium to different concentrations, each concentration had 4 replicate wells, and a blank control group without inoculating cells and a negative control group without adding drugs were set up, and the culture was continued for 24 hours. The laser control group experiment was divided into 2 groups (GCIH+L and ICG+L groups). In the laser group, 808 nm laser (1.5 W / cm 2 ) was used to irradiate for 6 min 4 h after drug administration, and the culture was continued until 24 h; in the non-laser group, the culture was continued until 24 h after drug administration. 50 μL of 1 mg / mL MTT solution was added to each well and the culture was continued for 4 h. The liquid in the wells was aspirated, 100 μL of DMSO was added to each well, and it was placed on a shaker for 10 min. The absorbance value A at 570 nm was measured using a multifunctional microplate reader; through the formula: cell inhibition rate × 100% = (average OD value of the negative group - average OD value of the experimental group) / (average OD value of the negative group - average OD value of the blank group), the inhibition rate of each concentration of the sample on the cells was calculated. The results are shown in Figure 10 .

[0047] As Figure 10 shown, the results of the 4T1 cell proliferation rate showed that when the concentrations of the GS and GC groups were as high as 300 μg / mL, the cell survival rate was still relatively high, about 60% or so, indicating that the carrier had almost no toxicity to 4T1 cells, and it could be proved that GC was a nanomaterial with high biosafety. Then, the toxicity of the GCIH and ICG groups under laser irradiation to 4T1 cells was tested. The results showed that the cell proliferation rate of the GCIH group decreased under laser irradiation compared with that of the non-irradiated laser group, indicating that the nanopharmaceutical also had significant photothermal and photodynamic effects. In addition, the cell proliferation rate of the GCIH+L group decreased significantly to about 7%, which was significantly lower than that of the ICG+L group, showing higher cytotoxicity, indicating that the nanopharmaceutical had a better tumor inhibitory effect than the pure photosensitizer. This was mainly because the GCIH+L group carried out phototherapy (photothermal therapy and photodynamic therapy) through near-infrared laser irradiation and synergistically achieved a significant tumor inhibitory effect with chemodynamic therapy.

Claims

1. A preparation method of a mesoporous cerium dioxide-coated gold nanosphere nanocarrier, characterized in that, It includes the following steps: Step 1: Add chloroauric acid tetrahydrate or its aqueous solution into water, mix well, heat to boiling, add sodium citrate hydrate or its solution, stir for 8 - 20 min, and place it in ice water to obtain solution A; Step 2: Mix chloroauric acid tetrahydrate or its aqueous solution with dilute hydrochloric acid, then add the above solution A, add silver nitrate and ascorbic acid aqueous solution under stirring, stir, centrifuge and dry to obtain product B; Step 3: Under room temperature conditions, mix the above product B with cetyltrimethylammonium bromide aqueous solution, and adjust the pH to 9 - 11 to obtain product C; Step 4: Dropwise add the methanol solution of tetraethyl orthosilicate into the above product C at room temperature, then continue to stir for 10 - 15 h, centrifuge, wash and dry to obtain product D; Step 5: Disperse the above product D into the ethanol solution of ammonium nitrate, reflux at 50 - 70 °C for 4 - 6 h, centrifuge, wash and dry to obtain product E; Step 6: Disperse the above product E in water, add cetyltrimethylammonium bromide, hexamethylenetetramine and cerium nitrate hexahydrate aqueous solution, heat to 80 - 95 °C and react for 4 - 8 h, centrifuge, wash and dry to obtain the product.

2. The preparation method of the mesoporous cerium dioxide-coated gold nanosphere nanocarrier according to claim 1, wherein, In Step 1, the mass ratio of chloroauric acid tetrahydrate to sodium citrate is 4 - 5:15 - 16; in Step 2, based on HCl for dilute hydrochloric acid, the molar ratio of chloroauric acid tetrahydrate, dilute hydrochloric acid, sodium citrate, silver nitrate, ascorbic acid is 30:90 - 110:25 - 30:1 - 5:

50.

3. The preparation method of the mesoporous cerium dioxide-coated gold nanosphere nanocarrier according to claim 1, wherein, In Step 3, the mass ratio of product B to cetyltrimethylammonium bromide is 1:3 - 5; in Step 4, the mass ratio of product C to tetraethyl orthosilicate is 1:1.5 - 2.

4. The preparation method of the mesoporous cerium dioxide-coated gold nanosphere nanocarrier according to claim 1, characterized in that In Step 5, the mass ratio of product D to ammonium nitrate is 1 - 5:300; in Step 6, the mass ratio of product E, cetyltrimethylammonium bromide, hexamethylenetetramine and cerium nitrate hexahydrate is 13:1 - 3:6 - 9:2 - 3.

5. A mesoporous cerium dioxide - coated gold nanosphere nanocarrier prepared by the preparation method according to any one of claims 1 to 4.

6. Application of the mesoporous cerium dioxide - coated gold nanosphere nanocarrier according to claim 5 in drug loading.

7. A method for preparing a mesoporous cerium dioxide nanodrug modified with hyaluronic acid and based on indocyanine green-loaded gold nanospheres using the nanocarrier described in claim 5, characterized in that, It includes the following steps: Step a: Under room temperature conditions, first disperse the nanocarrier according to claim 5 in water, then add indocyanine green, stir in the dark for 24 - 48 h, centrifuge, wash, and freeze - dry to obtain product G; Step b: Under room temperature conditions, dissolve the product G in water, then add hyaluronic acid aqueous solution and stir evenly in the dark, centrifuge, wash, and freeze - dry to obtain the final product H.

8. The method for preparing the hyaluronic acid-modified mesoporous cerium dioxide nanomedicine based on gold nanostar-loaded indocyanine green according to claim 7, wherein, In Step a, the mass ratio of product F to indocyanine green is 1:1 - 2; in Step b, the mass ratio of product G to hyaluronic acid is 2 - 3:

1.

9. A hyaluronic acid - modified mesoporous cerium dioxide nanodrug based on gold nanosphere - loaded indocyanine green prepared by the method according to any one of claims 7 to 8.

10. Application of the hyaluronic acid - modified mesoporous cerium dioxide nanodrug based on gold nanosphere - loaded indocyanine green according to claim 9 as an antitumor drug.

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