A method for synthesizing metal nanoparticles MgO@HSA and its application

Abstract

The present invention relates to a method for synthesizing metal nanoparticles MgO@HSA and its application. The metal nanoparticles of the present invention are synthesized using magnesium glycinate and human serum albumin (HSA) as raw materials, bypassing toxic materials and complex synthesis routes; the present invention is the first to discover that in the presence of serum albumin, MgO@HSA can be synthesized using magnesium glycinate and human serum albumin (HSA). 2+ The nanoparticles, composed of a metal core and an albumin-coated corona, form MgO@HSA NPs. The lattice spacing on the particle surface is 0.32 nm. The nanoparticles synthesized in this invention not only have low toxicity and side effects but also possess excellent tumor targeting capabilities. When loaded with the chemotherapy drug docetaxel (DTX), they exhibit significant anti-tumor effects on liver cancer cells, suggesting broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a method for synthesizing metal nanoparticles MgO@HSA and an application thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver, characterized by high malignancy and poor prognosis. Traditional treatments for HCC include surgical resection, chemotherapy, and transarterial chemoembolization. Surgical resection is currently the main treatment, but the chronic inflammation caused by surgery can increase the recurrence rate of HCC. Sorafenib, lenvatinib, and regorafenib are all commonly used drugs for the treatment of HCC in clinical practice. However, these drugs are non-specifically distributed in human tissues, killing normal cells while fighting tumors. In addition, side effects and drug resistance seriously affect the quality of life of patients. The distribution of drugs in the body and their effective effects are closely related to the delivery vector. Therefore, the development of delivery vectors with excellent targeting and safety is necessary for the human fight against HCC.

[0003] Zhang et al. prepared nanospheres with a hollow core-shell structure and a magnetic Fe3O4 shell with a SiO2-coated core. DOX was loaded in the hollow structure between the core and shell of the nanosphere (DOX-MUC-F-NR). In vivo experiments found that DOX-MUC-F-NR can significantly inhibit tumor growth. However, the high-temperature heat treatment and reverse microemulsion method involved in the preparation scheme have added difficulties to the large-scale preparation of DOX-MUC-F-NR in practical applications, limiting clinical transformation. Rudolf et al. used the photocatalytic effect of ZnO nanoparticles and combined them with UVA-1 to treat human head and neck squamous cell carcinoma (HNSCC) and primary oral mucosal cells pOMCs. Compared with HNSCC cells not treated with ZnO nanoparticles or only treated with UVA-1, DOX showed a significant inhibitory effect on the growth of tumors after irradiation with UVA-1 for 15 min. After 10 min, the viability of HNSCC cancer cells treated with ZnO nanoparticles was significantly reduced, while the activity of pOMCs cells treated with ZnO nanoparticles was not affected under UVA-1 irradiation. These studies show that ZnO nanoparticles can inhibit the cell viability of cancer cells without affecting normal cells. However, these studies are limited to in vitro studies. The whereabouts, biocompatibility, potential toxic side effects and half-life of ZnO nanoparticles in the body are still unknown.

[0004] The search has not yet found a method for synthesizing metal nanoparticles MgO@HSA using albumin (HSA) and magnesium glycinate. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method for synthesizing metal nanoparticles MgO@HSA (MgO@HSA NPs) and their applications. This method avoids the traditional, cumbersome chemical synthesis route, utilizes environmentally safe raw materials, and produces stable physicochemical properties with minimal side effects on normal tissues. Notably, MgO@HSA NPs can specifically target liver cancer tissue, and loading them with docetaxel (DTX) significantly promotes apoptosis in liver cancer cells, providing valuable insights for the preparation of drug carriers targeting liver cancer.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a method for synthesizing metal nanoparticles MgO@HSA, comprising the following steps:

[0008] Step 1, preparing an albumin stock solution with a concentration of 20-60 g / L;

[0009] Step 2: Accurately weigh 0.01-0.5 mg of magnesium glycinate and dissolve it in 1 mL of sterile deionized water until fully dissolved to prepare a 0.1-0.4 mg / mL magnesium glycinate solution;

[0010] Step 3: slowly dropwise add the magnesium glycinate solution prepared in step 2 to the albumin mother solution prepared in step 1, and incubate on a shaker for 24±2 h before stopping the reaction;

[0011] Step 4: Centrifuge and discard the supernatant. The resulting precipitate is the synthesized metal nanoparticles MgO@HSA. In a specific embodiment, the nanoparticles in step 4 are dissolved in sterile deionized water and then freeze-dried to eliminate the interference of the solvent.

[0012] Preferably, in step 1, the concentration of the albumin mother solution is 35-51 mg / mL, which is also the normal concentration of human serum albumin. Too much albumin will produce excess products and affect observation under an electron microscope.

[0013] Preferably, in step 2, the concentration of the magnesium glycinate solution is 0.1-0.4 mg / mL. 2+ It will cause protein denaturation and precipitation, which is not conducive to the reaction.

[0014] More preferably, in step 2, the concentration of the magnesium glycinate solution is 0.2 mg / mL.

[0015] Preferably, in step 3, the volume ratio of the magnesium glycinate solution to the albumin mother solution is 1:5-15, preferably 1:10.

[0016] Preferably, in step 3, the shaking incubation conditions are 37±1°C, 220±80 rpm, and the time is 24 h.

[0017] More preferably, in step 3, the shaking incubation conditions are 37° C., 220 rpm, and the time is 24 h.

[0018] Preferably, in step (4), the centrifugation is ultracentrifugation, and the ultracentrifugation conditions are: 4°C, 35000±5000 rpm, and time is 2±0.5 h.

[0019] In a second aspect, the present invention also protects the metal nanoparticles MgO@HSA obtained by the method described above.

[0020] The metal nanoparticles MgO@HSA synthesized in the present invention have excellent tumor targeting ability and can be used as drug delivery carriers for tumor targeting. Specifically, albumin on the surface of the metal nanoparticles MgO@HSA interacts with the albumin receptor GP60 highly expressed on the surface of liver cancer cells, allowing the metal nanoparticles MgO@HSA to be specifically enriched at the tumor site and achieve controllable release of chemotherapy drugs at the tumor site.

[0021] In a third aspect, the present invention also protects the use of the aforementioned metal nanoparticles MgO@HSA in the preparation of drugs targeting liver cancer.

[0022] Preferably, the drug is delivered by metal nanoparticles MgO@HSA as a drug delivery carrier.

[0023] Preferably, the drug is docetaxel.

[0024] In a fourth aspect, the present invention also protects the use of the metal nanoparticles MgO@HSA described above in the preparation of a drug delivery carrier targeting liver cancer.

[0025] Beneficial effects of the present invention:

[0026] The nanoparticles were structurally characterized. Transmission electron microscopy revealed uniform size distribution and absence of agglomeration of MgO@HSA NPs. The red blood cell fragmentation rate of MgO@HSA NPs at high concentrations was only approximately 2%, demonstrating their stability during in vivo delivery. CCK8 proliferation toxicity assays showed that at a concentration of 100 μg / mL, MgO@HSA NPs achieved a 90% cell viability, confirming their safety as a drug delivery vehicle. Laser confocal microscopy and acridine orange (AO) / ethidium bromide (EB) staining revealed that DTX-loaded MgO@HSA NPs promoted apoptosis in liver cancer cells. In the control group, cells were larger and more expanded, with clear morphology, enlarged nuclei, and clearly visible chromatin and nucleoli. In the DTX-MgO@HSA NP group, cells were reduced in size, lacking intact morphology, and exhibited fragmented and condensed nuclei. A DNA ladder analysis revealed that DTX-loaded MgO@HSA NPs exhibited the most pronounced apoptotic effect on liver cancer cells. Finally, small animal visible light imaging demonstrated that MgO@HSA NPs had excellent tumor targeting properties.

[0027] The present invention utilizes magnesium glycinate and albumin to prepare MgO@HSA NPs, avoiding the traditional cumbersome chemical synthesis route. The raw materials used are environmentally friendly and safe. The synthesized MgO@HSA NPs have stable physical and chemical properties and minimal side effects on normal tissues. In particular, MgO@HSA NPs can specifically target liver cancer tissue, and loading with DTX can significantly promote the apoptosis of liver cancer cells. The present invention provides valuable inspiration for the preparation of drug carriers targeting liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Figure A shows the morphology of MgO@HSA NPs under transmission electron microscopy, scale bar = 200 nm; Figure 1 Figure B shows the lattice fringes of MgO@HSA NPs under transmission electron microscopy, scale bar = 2 nm; Figure 1 Figure C shows the particle size distribution of MgO@HSA NPs; Figure 1 Figure D in the figure shows the Zeta potential distribution of MgO@HSA NPs.

[0029] Figure 2 Agarose gel electrophoresis experiment of MgO@HSA NPs.

[0030] Figure 3 This is the hemolysis experiment of MgO@HSA NPs.

[0031] Figure 4Cell viability of MHCC 97-H cells after treatment with different concentrations of MgO@HSA NPs (5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, 100 μg / mL) for 24 h (mean±SD, n=3, *P<0.05, **P<0.01, ***P<0.001).

[0032] Figure 5 Figure A is a confocal image of MHCC 97-H liver cancer cells stained with AO / EB; Figure 5 Figure B shows the DNALadder verification of the apoptosis-promoting effect of MgO@HSA NPs loaded with DTX (MgO@HSA NPs with DTX) on MHCC 97-H liver cancer cells.

[0033] Figure 6 In vivo tumor targeting detection of MgO@HSA NPs in subcutaneous liver cancer tumors. DETAILED DESCRIPTION

[0034] The technical solutions provided by the present invention are described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention. Reagents or instruments used without manufacturer's indication are considered to be conventional products that can be purchased on the market.

[0035] Example 1

[0036] To synthesize green, environmentally friendly nanoparticles that can efficiently target liver cancer, we used magnesium glycinate and human serum albumin (HSA) as raw materials to prepare nanomaterials. The preparation process is as follows:

[0037] Weigh 0.4 g HSA and dissolve it in 10 mL sterile deionized water to prepare albumin mother solution. Then accurately weigh 0.2 mg magnesium glycinate and dissolve it in 1 mL sterile deionized water. After it is fully dissolved, slowly add the prepared magnesium glycinate solution dropwise to the albumin mother solution. Incubate on a shaker at 220 rpm and 37 °C for 24 h to stop the reaction. Dispense into 5 mL ultracentrifuge tubes, heat seal and centrifuge at 35000 rpm and 4 °C for 2 h. The precipitate is the synthesized nanoparticles MgO@HSA NPs. In order to eliminate the interference of the solvent, the precipitate can be dissolved in sterile deionized water and then freeze-dried for storage. After completion, observe the shape and size under a transmission electron microscope. The results are as follows: Figure 1 As shown in Figure A, they are round particles with uniform size distribution; the lattice spacing is equal to 0.32 nm, the hydrated particle size is about 40 nm, and the zeta potential peak is equal to -15.9 mV ( Figure 1 Figure B in - Figure 1 Figure D in the figure).

[0038] Example 2

[0039] In this example, the magnesium glycinate solution was incubated with albumin for 0.5 h, 3 h, 6 h, 12 h, and 24 h, and then subjected to agarose gel electrophoresis (1.5% agarose, 80 V for 2 h). The sample volume was 50 μL. The IVIS Spectrum GFP was then used to photograph the results. Figure 2 As shown in the figure, as time goes by, MgO@HSA NPs can adsorb more HSA, showing that the running distance in agarose gel becomes shorter at the same time.

[0040] Example 3

[0041] In order to evaluate the lysis of red blood cells by MgO@HSA NPs obtained in Example 1, PBS was used as the yang ginseng and H2O was used as the yin ginseng. 1 mL of MgO@HSA NPs (25 μg / mL, 50 μg / mL, 75 μg / mL, 100 μg / mL) was mixed with 20 μL of mouse blood cells. After incubation at 37°C for 4 h, the mixture was centrifuged at 3000 rpm for 15 min. The samples were placed on the same horizontal line and photographed. The supernatant of the sample was aspirated and the absorbance of the sample at 542 nm was detected by a microplate reader. The hemolysis rate (%) was calculated as (OD 样本 -OD PBS ) / (OD H2O -OD PBS ) × 100%. The result is as follows Figure 3 As shown, the red blood cell fragmentation rate is only about 2% at high concentrations, proving that it is stable during in vivo transportation.

[0042] Example 4

[0043] In order to evaluate the cytotoxicity of the MgO@HSA NPs obtained in Example 1, the CCK8 method was used to detect the cytotoxicity of MgO@HSA NPs on human highly migratory hepatocellular carcinoma cells MHCC 97-H. Different concentrations of nanoparticles (5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, 100 μg / mL) were incubated with 10,000 cells for 24 h (the above concentrations and cell numbers are the final concentrations in the culture medium, and the culture medium volume is 100 uL / well). Then, 10 uL of CCK8 reagent was added to each well. After incubation for 1 h, the OD value at 450 nm was measured using a microplate reader. The results are shown as follows: Figure 4 As shown in the figure, at a concentration of 100 μg / ml, the cell survival rate can still reach 90%, proving that MgO@HSA NPs is a safe drug delivery carrier.

[0044] Example 5

[0045] In order to evaluate the killing effect of liver cancer cells by MgO@HSA NPs loaded with docetaxel (DTX) obtained in Example 1, 1×10 4 After 24 h, the cells were stained with AO / EB dye (eg Figure 5 ), it was found that the cells in the control group were larger and stretched, with clear morphology, large nuclei, and clearly visible chromatin and nucleoli, while the cells in the MgO@HSA NPs with DTX group were smaller in size and had no complete cell morphology, and the nuclei were fragmented and condensed.

[0046] Cells in the logarithmic growth phase were seeded in 6-well plates in advance and cultured in a 37°C, 5% CO2 incubator. The next day, the cells were treated with MgO@HSA NPs, DTX, and MgO@HSA NPs with DTX for 24 h, and then apoptotic DNA was extracted using an apoptotic DNA ladder extraction kit. The specific steps were as follows:

[0047] The culture medium was collected into a 15 mL centrifuge tube, and the cells were washed with 1 mL PBS. Then, the cells were digested with 0.5 mL trypsin at 37°C. After the digestion was terminated, the cells were collected into a centrifuge tube and centrifuged at 1000 rpm for 5 min to precipitate the cells. The supernatant was discarded.

[0048] Extract the cell pellet with 50 μL of DNA ladder extraction buffer for 10 seconds, centrifuge at 4500 rpm for 5 minutes, transfer the supernatant to a new tube, add 5 μL of proteinase K to the supernatant, gently vortex to mix, and incubate at 37°C for 10 minutes. Then add 5 μL of RNase A and incubate at 50°C for 30 minutes or longer (can be overnight). Add 5 μL of ammonium acetate solution to each sample and mix thoroughly. Add 100 μL of isopropanol, mix thoroughly, and incubate at -20°C for 30 minutes. Centrifuge at maximum speed (13,000 rpm) for 10 minutes to precipitate DNA.

[0049] Discard the supernatant and wash the DNA pellet with 0.5 mL of 70% ethanol. Vortex and centrifuge, centrifuge again at maximum speed (13,000 rpm), remove traces of ethanol, and air-dry at room temperature for 10 min.

[0050] The DNA precipitate was dissolved in 30 μL DNA suspension buffer, and 30 μL DNA was loaded onto a 1.5% agarose gel. After electrophoresis at 100 V for 1 h, the images were photographed and saved.

[0051] Example 6

[0052] To investigate the tumor-targeting efficacy of the MgO@HSA NPs obtained in Example 1, a subcutaneous MHCC97-H liver cancer model was constructed. The near-infrared fluorescent dye indocyanine green (ICG) was induced to attach to the surface of MgO@HSANPs via a cross-linking reaction system. The attachment method was as follows: 0.03 g of MgO@HSA NPs was dissolved in 1 mL of sterile deionized water, and 0.006 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) was dissolved in 500 μL of DMSO. The solution was aspirated and dissolved in 0.003 g of N-hydroxysuccinimide (NHS). This solution was then added to 0.01 g of indocyanine green (ICG), mixed thoroughly, and transferred to the nanoparticle solution. Finally, the solution was diluted to 10 mL with sterile deionized water and placed in a 360° rotary mixer at room temperature in the dark for 12 hours. The mixture was centrifuged at 1000 rcf for 10 minutes, and the supernatant was aspirated. Then, ICG-MgO@HSA NPs were injected into the tail vein of tumor-bearing nude mice at a dose of 1 mg / kg, and the near-infrared fluorescence distribution of the tumor site of the tumor-bearing nude mice was analyzed using a small animal visible light imaging system 48 hours later. Figure 6 As shown: 48 h after tail vein injection, the fluorescence of ICG-MgO@HSA NPs was concentrated in the tumor site, indicating that our synthesized MgO@HSA NPs have excellent tumor targeting ability and long-term retention ability in the tumor site.

[0053] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the appended claims.

Claims

1. A method for synthesizing metal nanoparticles MgO@HSA, characterized in that: The steps include: Step 1: Prepare albumin stock solution with a concentration of 20-60 g / L; Step 2: Weigh 0.01-0.5 mg of magnesium glycinate and dissolve it in 1 mL of sterile deionized water until fully dissolved to prepare a 0.01-0.5 mg / ml magnesium glycinate solution; Step 3: Slowly add the magnesium glycinate solution prepared in step 2 to the albumin mother solution prepared in step 1, and incubate on a shaker for 24±2 hours before stopping the reaction; Step 4: Centrifuge and discard the supernatant. The precipitate is the synthesized nanoparticles MgO@HSA. Wherein, in step 3, the incubation conditions on a shaking table are 37±1°C, 220±80 rpm, and the incubation time is 24 h; in step 4, the centrifugation conditions are 4°C, 35000±5000 rpm, and the time is 2±0.5 h.

2. The method for synthesizing metal nanoparticles MgO@HSA according to claim 1, characterized in that: In step 1, the concentration of the albumin stock solution is 35-51 mg / mL.

3. The method for synthesizing metal nanoparticles MgO@HSA according to claim 1, characterized in that: In step 2, the concentration of the magnesium glycinate solution is 0.1-0.4 mg / mL.

4. The method for synthesizing metal nanoparticles MgO@HSA according to claim 1, characterized in that: In step 3, the volume ratio of the magnesium glycinate solution to the albumin mother solution is 1:5-15.

5. Metal nanoparticles MgO@HSA obtained by the method for synthesizing metal nanoparticles MgO@HSA according to any one of claims 1 to 4.

6. Use of the metal nanoparticles MgO@HSA according to claim 5 in the preparation of a drug targeting liver cancer, wherein the drug uses the metal nanoparticles MgO@HSA as a drug delivery carrier.

7. Use of the metal nanoparticles MgO@HSA according to claim 5 in preparing a drug delivery carrier targeting liver cancer.

Images