Method for synthesizing metal nanoparticle MgO-coated HSA and application of metal nanoparticle MgO-coated HSA

Abstract

The invention relates to a method for synthesizing metal nanoparticles MgO-coated HSA and application of the metal nanoparticles MgO-coated HSA. The metal nanoparticles are synthesized by taking magnesium glycinate and human serum albumin (HSA) as raw materials, and a toxic material and a complex synthesis route are avoided; it is found for the first time that in the presence of serum albumin, Mg < 2 + > serves as a metal core, albumin wraps the surface of the metal core to form a protein crown, MgO and HSA NPs are assembled to form MgO and HSA NPs, and the lattice spacing of the particle surface is 0.32 nm. The synthesized nano-particles are low in toxic and side effects, have excellent tumor site targeting capacity, have remarkable killing and inhibiting effects on liver cancer cells after being loaded with chemotherapeutic drug docetaxel (DTX), and have wide 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, which is characterized by high malignancy and poor prognosis for patients. Traditional treatments for liver cancer include surgical resection, chemotherapy, and transarterial chemoembolization. Surgical resection is currently the main treatment, but the chronic inflammation caused by surgery will increase the recurrence rate of liver cancer. Sorafenib, lenvatinib, and regorafenib are all commonly used drugs for the treatment of liver cancer in clinical practice, but these drugs are non-specifically distributed in human tissues. While fighting tumors, they will kill normal cells. 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, it is necessary to develop a delivery vector with excellent targeting and safety for humans to fight liver cancer.

[0003] Zhang et al. prepared NaYF as the outer shell 4 、The core is SiO 2 Coated magnetic Fe 3 O 4 The nanospheres have a hollow core-shell structure, and DOX is loaded in the hollow structure between the core and shell of the nanospheres (DOX-MUC-F-NR). In vivo experiments have found that DOX-MUC-F-NR can significantly inhibit tumor growth, but 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 UVA-1 to treat human head and neck squamous cell carcinoma (HNSCC) and primary oral mucosal cells pOMCs. Compared with HNSCC cells that were not treated with ZnO nanoparticles or only treated with UVA-1, the photocatalytic effect of ZnO nanoparticles on the HNSCC cells was significantly reduced after 15 min of UVA-1 irradiation. 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 but have no effect on normal cells. However, these studies are limited to in vitro studies, and 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] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for synthesizing metal nanoparticles MgO@HSA (MgO@HSA NPs for short) and its application. The present invention avoids the traditional cumbersome chemical synthesis route, the raw materials used are environmentally friendly and safe, the synthesized metal nanoparticles have stable physical and chemical properties, and have little side effects on normal tissues. In particular, MgO@HSA NPs can specifically target liver cancer tissues, and loading docetaxel (DTX) can significantly promote the apoptosis of liver cancer cells, providing valuable inspiration 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: In a first aspect, the present invention provides a method for synthesizing metal nanoparticles MgO@HSA, comprising the following steps: Step 1, preparing an albumin stock solution with a concentration of 20-60 g / L; 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; 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; Step 4, centrifuge, discard the supernatant, and 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.

[0007] Preferably, in step 1, the albumin mother solution concentration 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.

[0008] 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.

[0009] More preferably, in step 2, the concentration of the magnesium glycinate solution is 0.2 mg / mL.

[0010] Preferably, in step 3, the volume ratio of the magnesium glycinate solution to the albumin mother solution is 1:5-15, preferably 1:10.

[0011] Preferably, in step 3, the shaking incubation conditions are 37±1°C, 220±80 rpm, and the time is 24 h.

[0012] More preferably, in step 3, the shaking incubation conditions are 37°C, 220 rpm, and the time is 24 h.

[0013] Preferably, in step (4), the centrifugation is ultra-high speed centrifugation, and the ultra-high speed centrifugation conditions are: 4°C, 35000±5000rpm, and time is 2±0.5 h.

[0014] In a second aspect, the present invention also protects the metal nanoparticles MgO@HSA obtained by the method described above.

[0015] 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, so that the metal nanoparticles MgO@HSA are specifically enriched in the tumor site and the controllable release of chemotherapy drugs in the tumor site is achieved.

[0016] In a third aspect, the present invention also protects the use of the metal nanoparticles MgO@HSA described above in the preparation of drugs targeting liver cancer.

[0017] Preferably, the drug is delivered by metal nanoparticles MgO@HSA.

[0018] Preferably, the drug is docetaxel.

[0019] 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.

[0020] Beneficial effects of the present invention: The structure of the nanoparticles is characterized in the present invention. The size distribution of MgO@HSA NPs observed under transmission electron microscopy is uniform and there is no agglomeration. The red blood cell fragmentation rate of MgO@HSA NPs at high concentrations is only about 2%, proving that it is stable during in vivo transportation. CCK8 proliferation toxicity experiment shows that the cell survival rate of MgO@HSA NPs at a concentration of 100 μg / mL reaches 90%, proving that MgO@HSA NPs is a safe drug delivery carrier. Through acridine orange (AO) / ethidium bromide (EB) staining, laser confocal observation of DTX-loaded MgO@HSA NPs promoting apoptosis of liver cancer cells, the control group cells are large and stretched, the morphology is clear, the cell nucleus is large, the chromatin and nucleolus are clearly visible, the DTX MgO@HSA NPs group cells are reduced in volume without complete cell morphology, and the cell nucleus is fragmented and condensed; DNA ladder shows that DTX-loaded DTX MgO@HSA NPs show the most obvious apoptotic effect on liver cancer cells. Finally, small animal visible light imaging demonstrated that MgO@HSA NPs had excellent tumor targeting properties.

[0021] 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 little side effects on normal tissues. In particular, MgO@HSA NPs can specifically target liver cancer tissues, and loading 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

[0022] Figure 1 Figure A is 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.

[0023] Figure 2 Agarose gel electrophoresis experiment of MgO@HSA NPs.

[0024] Figure 3 MgO@HSA NPs hemolysis experiment.

[0025] 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, 80μg / mL, 100μg / mL) for 24 h (mean±SD, n=3, *P<0.05, **P<0.01, ***P<0.001).

[0026] 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 effect of MgO@HSA NPs loaded with DTX (MgO@HSA NPs with DTX) in promoting apoptosis of MHCC 97-H liver cancer cells.

[0027] Figure 6 In vivo tumor targeting detection of MgO@HSA NPs in subcutaneous liver cancer tumors. DETAILED DESCRIPTION

[0028] The technical solution provided by the present invention is described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention. The reagents or instruments used without indicating the manufacturer are regarded as conventional products that can be purchased on the market.

[0029] Example 1

[0030] In order to synthesize green and environmentally friendly nanoparticles that can efficiently target liver cancer sites, we used magnesium glycinate and human serum albumin (HSA) as raw materials to prepare nanomaterials. The preparation process is as follows: 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 fully dissolved, slowly add the prepared magnesium glycinate solution to the albumin mother solution, incubate on a shaker at 220 rpm and 37 °C for 24 h to stop the reaction, divide it into 5 mL ultracentrifuge tubes, heat seal it and centrifuge it 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 found to be 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).

[0031] Example 2

[0032] 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 agarose gel electrophoresis (1.5% agarose, 80 V for 2 h) was performed with a loading volume of 50 μL. Then, an IVIS Spectrum GFP was used to take a picture. The results are shown in Figure 2. Figure 2 As shown in the figure, as time goes by, MgO@HSA NPs can adsorb more HSA, showing a shorter running distance in agarose gel at the same time.

[0033] Example 3

[0034] In order to evaluate the lysis of red blood cells by MgO@HSA NPs obtained in Example 1, PBS was used as Yangshen, H 2 O was used as the negative reference. 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 and incubated at 37 °C for 4 h. The samples were centrifuged at 3000 rpm for 15 min. The samples were placed on the same horizontal line and photographed. The supernatant of the samples was aspirated and the absorbance of the samples at 542 nm was detected by an ELISA reader. The hemolysis rate (%) was calculated as (OD 样本 -OD PBS ) / (OD H2O -OD PBS ) × 100%. The result is Figure 3 As shown, the red blood cell fragmentation rate was only about 2% at high concentrations, proving that it was stable during in vivo transport.

[0035] Example 4

[0036] In order to evaluate the cytotoxicity of 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. Nanoparticles of different concentrations (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), and then 10 uL CCK8 reagent was added to each well. After culturing for 1 h, the OD value at 450 nm was detected using an enzyme reader. The results are 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.

[0037] Example 5

[0038] In order to evaluate the killing effect of docetaxel (DTX) loaded on MgO@HSA NPs obtained in Example 1 on liver cancer cells, in this example, 1×10 4 After 24 h, 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 their nuclei were fragmented and condensed.

[0039] Cells in the logarithmic growth phase were seeded in 6-well plates at 37°C and 5% CO 2 The cells were cultured in an incubator environment. On the second 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: The culture medium was collected into a 15 mL centrifuge tube, the cells were washed with 1 mL PBS, and 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, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded.

[0040] Extract the cell pellet with 50 uL DNA ladder extraction buffer for 10 s, centrifuge at 4500 rpm for 5 min, transfer the supernatant to a new tube, add 5 uL proteinase K to the supernatant, vortex gently, incubate at 37 °C for 10 min, add 5 uL RNase, incubate at 50 °C for 30 min or longer (can be overnight), add 5 uL ammonium acetate solution to each sample and mix well, add 100 uL isopropanol, mix well, and keep at -20 °C for 30 min. Centrifuge at maximum speed (13000 rpm) for 10 min to precipitate DNA.

[0041] Discard the supernatant, wash the DNA precipitate with 0.5 mL 70% ethanol, vortex and centrifuge, centrifuge again at maximum speed (13000 rpm), remove trace ethanol, and air-dry at room temperature for 10 min.

[0042] The DNA precipitate was dissolved in 30 uL DNA suspension buffer, 30 uL DNA was loaded onto 1.5% agarose gel, and electrophoresis was performed at 100 V for 1 h before taking photos and saving the images.

[0043] Example 6

[0044] In order to study the tumor targeting effect of MgO@HSA NPs obtained in Example 1, a MHCC97-H liver cancer subcutaneous tumor model was constructed in this example, and the near-infrared fluorescent dye indocyanine green (ICG) was induced to connect to the surface of MgO@HSANPs through a cross-linking reaction system. The connection method is as follows: 0.03 g MgO@HSA NPs were dissolved in 1 mL sterile deionized water, 0.006 g 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) was dissolved in 500 uL DMSO, the solution was aspirated and dissolved in 0.003 g N-hydroxysuccinimide (NHS), and then the solution was added to 0.01 g indocyanine green (ICG) and mixed thoroughly and then transferred to the nanoparticle solution, and finally the volume was adjusted to 10 mL with sterile deionized water and placed in a 360° rotating mixer at room temperature and in the dark for full reaction for 12 h, and the supernatant was aspirated at 1000 rcf for 10 min. 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 in 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 the MgO@HSA NPs we synthesized have excellent tumor targeting ability and long-term retention ability in the tumor site.

[0045] 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 attached 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 drop the magnesium glycinate solution prepared in step 2 into the albumin mother solution prepared in step 1, and incubate on a shaker for 24±2 h before stopping the reaction; Step 4: Centrifuge and discard the supernatant. The precipitate is the synthesized nanoparticles MgO@HSA.

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 the step 3, the volume ratio of the magnesium glycinate solution to the albumin mother solution is 1:5-15.

5. The method for synthesizing metal nanoparticles MgO@HSA according to claim 1, characterized in that: In step 3, the shaking incubation conditions are 37±1°C, 220±80 rpm, and the incubation time is 24 h.

6. The method for synthesizing metal nanoparticles MgO@HSA according to claim 1, characterized in that: In step 4, the centrifugation conditions are 4°C, 35000±5000 rpm, and the time is 2±0.5 h.

7. The metal nanoparticles MgO@HSA obtained by the method for synthesizing metal nanoparticles MgO@HSA according to any one of claims 1 to 6.

8. Use of the metal nanoparticle MgO@HSA according to claim 7 in the preparation of a drug targeting liver cancer.

9. The use according to claim 8, characterized in that: The drug is delivered by metal nanoparticles MgO@HSA.

10. Use of the metal nanoparticle MgO@HSA according to claim 9 in preparing a drug delivery carrier targeting liver cancer.

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