Preparation method and application of lectin-modified nano-selenium composite material

Abstract

The present invention first discloses a method for preparing a lectin-modified nano-selenium composite material, including the steps of mixing AANL with sodium selenite for the first incubation, mixing with ascorbic acid for the second incubation, coating, centrifuging and discarding the supernatant, resuspending, washing, etc. The present invention also discloses the application of the composite material in sugar binding, especially cell agglutination and / or in anti-oxidation and / or in the preparation of drugs for inhibiting lung cancer cell proliferation. The method of the present invention has low cost, environmental protection, high efficiency, simple preparation method, simple required equipment, and is easy to promote. The composite material provided has good dispersibility, is more stable, has good biocompatibility and solubility, and has significant application effect. It can be better used in the field of glycobiology as a sugar tool; it has better biological activity, strong antioxidant activity and ability to inhibit lung cancer cell proliferation, and has good application prospects in the field of biomedicine.

Description

[Technical field]

[0001] The invention relates to the technical field of biomedicine, and in particular to a preparation method and application of a lectin-modified nano-selenium composite material. [Background technology]

[0002] Lectins are a class of proteins that can specifically recognize carbohydrates on the surface of cell membranes or proteins and lipids in the extracellular matrix. They have multiple biological functions such as cell recognition, anti-bacterial and anti-viral infection, immunomodulatory activity and anti-tumor. Different lectins recognize and bind to proteins or lipids with different glycosylation modifications through their specific sugar recognition domains. The glycosylation modification on the surface of tumor cells is significantly abnormal compared with normal cells, and lectins mainly inhibit the proliferation of tumor cells by recognizing sugar complexes on the surface of tumor cells or through their immunomodulatory activity. Therefore, lectins have gradually become a hot topic in the screening of anti-tumor drugs. For example, the lectin PFL from Praecitrullus fistulosus induces apoptosis of tumor cells by changing the expression of apoptotic proteins through the caspase-3 dependent pathway, prolonging the survival time of tumor-bearing mice; the Panax notoginseng tuber lectin KRL can effectively inhibit the proliferation of colon cancer cells; the lectin extracted from okra induces apoptosis of human U87 glioma cells by mediating the caspase pathway and inhibits the proliferation of glioma cells.

[0003] Agrocybe aegerita lectin 2 (AANL) is a lectin with a molecular weight of 43.175 kDa identified from Agrocybe aegerita. It can induce apoptosis of liver cancer cells in vitro and inhibit the growth of liver cancer cells in tumor-bearing mice in vivo. AANL has six carbohydrate binding regions, which can highly selectively bind to sugar chain structures with N-acetylglucosamine (GlcNAc) at the end, and AANL has a higher specificity for GlcNAc- than other GlcNAc-binding lectins. The sugar-binding property of lectin AANL can be used as a GlcNAc-enrichment tool for glycobiology and identification of tumor diagnostic markers. The inventors previously used AANL to enrich serum samples from patients with early non-small cell lung cancer and healthy controls, and the enriched glycoproteins / glycopeptides were used as tumor markers for early diagnosis of lung cancer; serum samples from myeloma patients were also enriched, and the enriched glycopeptides were used for the screening of myeloma diagnostic markers.

[0004] Selenium is an essential trace element for the human body. It is a component of the amino acid selenocysteine ​​and is necessary for the biosynthesis of selenoproteins. It participates in the metabolism of peroxides and iodine in the body, regulates the levels of iodine and free radicals in the body, and can enhance the body's ability to prevent and resist diseases. It plays an important role in many physiological processes. The effective and toxic doses of selenium are very close, and it is difficult to control the effective dose. When elemental selenium exists in the nano state, its toxicity is lower than that of inorganic selenium and organic selenium, but its properties are unstable and its biological activity is poor. When it reaches a certain nanoscale, it forms a colloid in the water phase, which has biological activity and nanoscale effects.

[0005] Nano-selenium (SeNPs) is formed by the aggregation of elemental selenium (Se0). It has the advantages of high loading rate, low toxicity, good activity, easy synthesis and easy storage, and has broad application prospects in the field of biomedicine. Well-dispersed nano-selenium can effectively remove free radicals produced by the body's metabolism and has antioxidant properties. Nano-selenium particles also have anti-tumor activity, especially against melanoma, liver cancer and breast cancer, and reduce the toxic side effects such as kidney and bone marrow damage caused by chemotherapy drugs. Nano-selenium has significant anti-tumor and antioxidant effects on tumor-bearing mice, and has great potential in improving cyclophosphamide-induced toxicity and improving the effect of chemotherapy, which may have a beneficial effect on patients and chemotherapy patients.

[0006] However, nano-selenium is easy to aggregate in aqueous solution and has poor dispersibility, which greatly reduces its bioactivity and bioavailability. Studies have shown that coating nano-selenium with a capping agent is an effective strategy to improve the bioactivity and stability of nano-selenium. Using polysaccharides as capping agents can effectively improve the biocompatibility and stability of nano-selenium in aqueous solution. The inventors used polysaccharides as capping agents to embed SeNPs into β-(1,3)-D-glucan extracted from black fungus, namely BFP. The physical adsorption of BFP and SeNPs in water formed well-dispersed and stable hollow BFP-se nanotubes. The nanocomposite (BFP-Se) can kill acute myeloid leukemia (AML) cells and prolong the survival of AML mice. Lectins have good bioactivity when coupled with nanomaterials as capping agents, and have shown good biomedical application prospects in carbohydrate enrichment, tumor diagnosis and treatment, and anti-inflammatory and antibacterial. For example, nanogold is coupled with a variety of lectins to enrich a variety of sugar chains. Lectins are coupled to nanomaterials to identify sugar chains on the surface of tumor cell exosomes for the diagnosis of pancreatic cancer. Lectin-functionalized fluorescently labeled silica nanoparticles can be used for early detection of colon cancer. Magnetic nanoparticles combined with wheat germ agglutinin (WGA) significantly promote apoptosis of three prostate cancer cells (LNCaP, PC-3, DU-145) and prevent tumor growth in tumor-bearing mice. Drug-encapsulated nanomaterials are coupled to concanavalin A (ConA) to maintain drug release and effectively resist Helicobacter pylori infection.

[0007] In general, as mentioned above, the biological activity of nano-selenium is not high and the bioavailability is low, while the lectin-modified nanomaterials have good biological activity, but there are few research reports on the coupling of lectin and nano-selenium. The present invention uses the redox method to couple the high-affinity GlcNAc lectin AANL with nano-selenium, and encapsulates them into lectin AANL-modified nano-selenium complexes (AANL-SeNPs) to improve the biocompatibility and stability of nano-selenium, and detects the nano-characterization of AANL-SeNPs in vitro, detects its sugar binding activity, and provides a new tool for sugar enrichment; detects its antioxidant activity and anti-tumor activity, and provides a basis for anti-tumor drug screening. [Summary of the invention]

[0008] The purpose of the present invention is to overcome the above-mentioned defects of the prior art and provide a method for preparing a lectin-modified nano-selenium composite material with simple operation and significant application effect.

[0009] To achieve the above object, the present invention provides a method for preparing a lectin-modified nano-selenium composite material, which is characterized by comprising the following steps:

[0010] 1) Weigh the freeze-dried lectin AANL and dissolve it in the sodium selenite solution, rotate to mix, and perform the first incubation;

[0011] 2) Add ascorbic acid solution, rotate and mix, incubate for the second time, and coat;

[0012] 3) After the second incubation, centrifuge and discard the supernatant to obtain a precipitate, then resuspend the precipitate in 0.01 M PBS and wash twice to obtain the lectin-modified nano-selenium complex material AANL-SeNPs.

[0013] Preferably, in step 1), the ratio of lectin AANL to sodium selenite is AANL: sodium selenite = 10 mg: 0.25 mmol, the conditions for the rotary mixing operation are 25° C., 5 rpm, and the first incubation is 30 min;

[0014] The volume of the ascorbic acid solution added in step 2) is 5 times the volume of the sodium selenite solution in step 1), and the molar ratio of ascorbic acid to sodium selenite is 4:1; the second incubation conditions are 25° C., 5 rpm, and the coating time is 12 h;

[0015] The centrifugation in step 3) is 12000 rpm for 4 min.

[0016] Another object of the present invention is to provide the use of the above nano-selenium complex material in cell aggregation and / or anti-oxidation and / or in the preparation of drugs for inhibiting lung cancer cell proliferation.

[0017] The beneficial effects of the present invention are: low cost, environmental protection, high efficiency, simple preparation method, simple required equipment, easy promotion, the provided complex has good dispersibility, more stability, good biocompatibility and solubility, significant application effect, and can be better used in the field of glycobiology as a sugar tool; it has better biological activity, strong antioxidant activity and ability to inhibit lung cancer cell proliferation, and has good application prospects in the biomedical field.

Brief Description of the Drawings

[0018] Figure 1 This is the colorimetric appearance of AANL-SeNPs;

[0019] Figure 2 This is the detection diagram of the time gradient result of AANL-SeNPs coating;

[0020] Figure 3 is the nanoparticle size distribution diagram of AANL-SeNPs;

[0021] Figure 4 This is the Zeta potential detection diagram of AANL-SeNPs;

[0022] Figure 5 This is the UV wavelength detection diagram of AANL-SeNPs;

[0023] Figure 6 This is the intrinsic fluorescence detection diagram of AANL-SeNPs;

[0024] Figure 7a This is the detection diagram of SeNPs agglutination red blood cells activity;

[0025] Figure 7b This is the detection diagram of AANL-SeNPs agglutination red blood cells activity;

[0026] Figure 8 This is the result of DPPH experiment to detect the free radical scavenging ability of AANL-SeNPs;

[0027] Fig. 9 This is the result of T-AOC experiment to detect the total antioxidant capacity of AANL-SeNPs;

[0028] Fig.10 This is the result of CCK8 experiment to detect the cell viability of H157 cells after 24 hours of treatment with AANL-SeNPs. [Specific implementation method]

[0029] In order to better understand the present invention, the following embodiments are described in detail in conjunction with the accompanying drawings. It should be understood that the embodiments are only used to illustrate the present invention and cannot limit the scope of the present invention.

[0030] Unless otherwise specified, the technical means adopted in this embodiment are conventional technical means in the art.

[0031] Example 1 Preparation method and nano-characterization of lectin AANL-modified nano-selenium complex

[0032] 1. Reagents and Materials

[0033] (1) Experimental Materials

[0034] Lectin AANL [extracted and prepared according to the method disclosed in the patent "Agrocybe aegerita lectin AAL-2 and its encoding gene, preparation method and application thereof" (ZL 201110218000.5)], ascorbic acid (analytical grade), sodium selenite (analytical grade).

[0035] (2) Reagent preparation:

[0036] ① Ascorbic acid solution: weigh 3.52 g of ascorbic acid and dissolve it in 50 ml of distilled water to prepare a 400 mM ascorbic acid solution;

[0037] ② Sodium selenite solution: Weigh 4.3 g of sodium selenite and dissolve it in 50 ml of distilled water to prepare a 500 mM sodium selenite solution.

[0038] 2. Experimental Methods

[0039] ①Material preparation of AANL-SeNPs

[0040] Weigh 10 mg of freeze-dried lectin AANL and dissolve it in 500 μL 500 mM sodium selenite solution. Rotate at 5 rpm with a DH-II mixer at 25°C, mix well, incubate for 30 minutes, add 5 times the volume of sodium selenite solution, i.e. 2.5 ml 400 mM ascorbic acid, the molar ratio of ascorbic acid to sodium selenite is 4:1, and then add 7 ml of distilled water to a total system of 10 ml. The solution turns red and is incubated at 25°C with a rotating mixer at 5 rpm for 12 hours overnight. After incubation, centrifuge at 12000 rpm for 4 minutes, discard the supernatant to obtain a precipitate, and then resuspend the precipitate with 0.01 M PBS and wash twice to obtain the AANL-SeNPs composite material.

[0041] ②Nano ZS Malvern particle size analyzer to detect the particle size and Zeta potential distribution of AANL-SeNPs

[0042] Select a clean sample pool, slowly inject 1 ml of AANL-SeNPs to avoid bubbles, seal the sample pool with a lid, place the sample into the injection slot, and start the Zetasizer software for detection.

[0043] ③UV wavelength detection of AANL-SeNPs

[0044] Select a clean cuvette, slowly inject 1 ml of AANL-SeNPs sample, place the sample in the injection slot, and detect it with a UV detector. Select spectral scanning, set the wavelength and blank control, and measure the sample.

[0045] ④Intrinsic fluorescence detection of AANL-SeNPs

[0046] The endogenous fluorescence of the sample was detected by a fluorescence spectrophotometer. The wavelength of the excitation light was set to 295 nm, the emission start wavelength was set to 300 nm, the emission end wavelength was set to 500 nm, the scanning speed was set to 1200 nm / min, and the voltage was set to 700 V. Scanning was performed to obtain the fluorescence spectrum data, and then the spectrum was plotted using Grapher software.

[0047] 3. Experimental results

[0048] The AANL-SeNPs nanomaterial complex was prepared by redox method. The prepared solution has uniform color and good solubility ( Figure 1 ), the loading effect was better after coating for 12h than after coating for 3h and 6h, and the protein loss in the supernatant was the lowest ( Figure 2 ), the nanoparticle size test revealed that the composite particle size was uniform, 1464nm, and concentrated in a narrow area ( Figure 3 The Zeta potential test results showed that the Zeta potential of AANL-SeNPs was -34.5 mV, and the Zeta potential of SeNPs was -10.4 mV, which was higher than that of SeNPs ( Figure 4 ), indicating that AANL-SeNPs are more stable and have better dispersion than nano-selenium systems. The UV spectrum results show that the UV absorption spectrum of the composite is red-shifted, with a small peak at 500-600nm ( Figure 5 ), indicating that the secondary structure of the complex has changed, the molecular conjugation ability has been enhanced, and it is more stable. The intrinsic fluorescence spectrum also shows that the spectrum of AANL-SeNPs has red-shifted, and the Trp microenvironment has changed to hydrophilic ( Figure 6 ), the hydrophilicity is enhanced.

[0049] Example 1 shows that we have successfully prepared a new material of lectin AANL-coated nano-selenium complex, which is stable, has good dispersibility, good solubility, and has a good nano-scale, and improves the solubility and stability of nano-elemental selenium.

[0050] Example 2 Application of AANL-SeNPs—Reduction of Hemocyte Agglutination Activity

[0051] 1. Reagents and Materials

[0052] (1) Experimental Materials

[0053] Citric acid, sodium citrate, glucose, sodium chloride (all analytical grade), BCA protein quantification kit.

[0054] (2) Reagent preparation:

[0055] ① Anticoagulant (ACD): weigh 0.48 g citric acid, 1.32 g sodium citrate, and 1.47 g glucose and dissolve them in 100 ml distilled water.

[0056] ②Normal saline: weigh 0.9g NaCl and dissolve it in 100ml distilled water.

[0057] 2. Experimental Methods

[0058] ① Preparation of red blood cell suspension

[0059] Use a disposable distal blood collection needle to collect finger blood and drip it into an EP tube containing a small amount of anticoagulant. Add 1 ml of normal saline and centrifuge at 1500 rpm and 4°C for 3 minutes. Discard the upper layer and resuspend the red blood cells with normal saline. After washing twice, resuspend the red blood cells with normal saline and dilute.

[0060] ② Red blood cell agglutination test

[0061] Take a clean coagulation plate and add 50 μL of saline to each well from front to back except the first well in each row. The concentrations of AANL and AANL-SeNPs are both 600 μg / ml. The protein concentration is detected by BCA protein quantification kit and read at 562 nm using an enzyme reader. Add 100 μL of sample to the first well of the coagulation plate and dilute it by 2 times in a doubling ratio, that is, take 50 μL from the previous well to the next well in each well, and the last well is a saline control. Add 50 μL of diluted red blood cell suspension to each well of the coagulation plate and mix well. Let it stand at room temperature until the red blood cells in the control group sink to the bottom of the well, and scan the coagulation plate.

[0062] 3. Experimental results

[0063] The activity of AANL-SeNPs in agglutinating human ABO red blood cells showed that SeNPs had no coagulation activity ( Figure 7a ), all red blood cells naturally settled. Compared with AANL, the activity of AANL-SeNPs in agglutinating red blood cells of four blood types was weakened ( Figure 7b ).

[0064] Example 3 Application of AANL-SeNPs—Enhanced Antioxidant Activity

[0065] 1. Reagents

[0066] DPPH kit; Total antioxidant capacity (T-AOC) kit.

[0067] 2. Experimental Methods

[0068] ① Free radical scavenging ability test

[0069] The DPPH kit was used to detect the free radical scavenging ability, and the total antioxidant capacity of positive control ascorbic acid, SeNPs, AANL and AANL-SeNPs was determined. The sample concentrations were 0.2 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.8 mg / ml and 1.0 mg / ml, respectively. The samples were added according to the kit instructions as shown in Table 1, mixed, and kept at room temperature 25°C in the dark for 30 minutes, centrifuged at 4000 rpm for 5 minutes, and the absorbance of each tube was measured under a microplate reader at a wavelength of 517 nm. The total antioxidant capacity was calculated by substituting the formula, and the graph was drawn using GraphPadPrsim 8.0 software.

[0070] Formula: DPPH free radical scavenging rate (%) = (1-(A determination-A control) ÷ A blank) × 100%

[0071] Table 1 DPPH experiment sample addition

[0072]

[0073] ②Total antioxidant capacity test

[0074] The total antioxidant capacity (T-AOC) assay kit was used to determine the total antioxidant capacity of positive control ascorbic acid, SeNPs, AANL and AANL-SeNPs, with a sample concentration of 2 mg / ml. Samples were added according to the kit instructions, as shown in Table 2. After adding reagent 3, mix thoroughly, incubate in a 37°C water bath for 30 min, and then add reagent 4 and the sample to be tested.

[0075] Table 2 T-AOC experiment sample addition

[0076]

[0077] Mix well, place at room temperature for 10 min, and measure the absorbance of each tube with a microplate reader at a wavelength of 520 nm. Substitute the formula into the total antioxidant capacity and plot it using GraphPad Prsim 8.0 software.

[0078] Formula: Total antioxidant capacity (U / mgprot) = (absorbance of the test group - absorbance of the control group) ÷ 0.01 ÷ 30 min × V total reaction volume ÷ V sampling volume ÷ protein concentration)

[0079] 3. Experimental results

[0080] The results of DPPH kit test showed that both VC and SeNPs had strong free radical scavenging ability. AANL-SeNPs had stronger free radical scavenging ability than AANL protein itself. With the increase of concentration, the free radical scavenging ability was significantly enhanced. At 1.0 mg / ml, the free radical scavenging activity of AANL-SeNPs reached 56.5% ( Figure 8 ).

[0081] The results of T-AOC kit test showed that both VC and SeNPs had strong antioxidant capacity, while AANL protein had almost no antioxidant capacity. AANL-SeNPs had significantly stronger antioxidant capacity than AANL ( Fig. 9 ), indicating that after the lectin AANL was coated with nano-selenium, nano-selenium significantly improved the antioxidant activity of AANL.

[0082] Example 4 Application of AANL-SeNPs—Enhanced ability to inhibit lung cancer cell proliferation

[0083] 1. Reagents and Materials

[0084] Human lung squamous cell carcinoma H157 cells, CCK8 kit.

[0085] 2. Experimental Methods

[0086] ① Cell culture and plating

[0087] The lung squamous cell carcinoma H157 cells were revived and cultured in DMEM medium containing 10% FBS. The cells in the logarithmic growth phase were seeded in a 96-well plate, with 5000 cells per well.

[0088] ②CCK8 detection of the effect of drug treatment on H157 cell proliferation

[0089] After the adherent cells were plated overnight in a 96-well plate, fresh culture medium was replaced and SeNPs, AANL and AANL-SeNPs were added. 10 μl of drug was added to each well and culture was continued. After 24h or 48h of drug treatment, 10 μl of CCK8 reagent was added respectively, mixed and cultured in a 37°C incubator. The OD value at a wavelength of 450nm was measured with an enzyme marker within 1-4h, and the OD value was read every 1h. The measured values ​​were used to calculate the cell viability using an Excel table, and the data were analyzed using Graphpad Prism 8.0 software. P < 0.05 was considered statistically significant.

[0090] Cell viability (%) = (OD value of drug-added well - OD value of blank well) ÷ (OD value of control group - OD value of blank well) × 100%)

[0091] 3. Experimental results

[0092] The CCK8 kit detected that AANL-SeNPs and AANL had a significant inhibitory effect on H157 cell proliferation after 24 hours of treatment, and the effect of AANL-SeNPs on inhibiting lung cancer cell proliferation was more significant than AANL, with a cell survival rate of 64% ( Fig.10 ).

[0093] The present invention provides a lectin AANL modified nano-selenium complex material, a preparation method and an application thereof. The lectin AANL with high affinity for GlcNAc is coupled with nano-selenium by redox method to prepare a lectin AANL modified nano-selenium complex (AANL-SeNPs). The nanoparticle size, Zeta potential, ultraviolet spectrum and intrinsic fluorescence spectrum indicate that the complex provided by the present invention has better dispersibility and stability. The results of the erythrocyte agglutination experiment show that the sugar binding activity of AANL-SeNPs has changed, and the specificity for GlcNAc is stronger. The present invention provides a new feasible tool for sugar enrichment. The results of the antioxidant and CCK8 experiments show that the antioxidant activity of AANL-SeNPs is enhanced and the proliferation of lung cancer cells is significantly inhibited, indicating that the complex material provided by the present invention has good biological applications. The new material preparation method of the present invention is simple to operate and has significant application effect, and can be used as a tool to be better used in the fields of glycobiology and biomedicine.

Claims

1. A method for preparing a lectin-modified nano-selenium composite material, characterized in that The steps include: 1) Weigh the freeze-dried Agrocybe aegerita lectin AANL and dissolve it in the sodium selenite solution, rotate and mix well, and perform the first incubation; 2) Add ascorbic acid solution, rotate to mix, incubate for the second time, and coat; 3) After the second incubation, centrifuge, discard the supernatant to obtain a precipitate, resuspend the precipitate in 0.01 M PBS, and wash twice to obtain the lectin-modified nano-selenium complex material AANL-SeNPs; The ratio of lectin AANL to sodium selenite in step 1) is AANL: sodium selenite = 10 mg: 0.25mmol, the conditions for rotary mixing operation were 25℃, 5 rpm, and the first incubation was 30 min; The volume of the ascorbic acid solution added in step 2) is 5 times the volume of the sodium selenite solution in step 1), and the molar ratio of ascorbic acid to sodium selenite is 4:1; the second incubation conditions are 25°C, 5 rpm, and the coating time is 12 h; The centrifugation in step 3) is 12000 rpm, 4 min.

2. Use of the lectin-modified nano-selenium complex material obtained by the preparation method according to claim 1 in the preparation of a red blood cell agglutination reagent or in the preparation of a drug for inhibiting the proliferation of lung cancer cells.

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