Oral biomimetic mineralized oncolytic adenovirus, preparation method thereof and application of oral biomimetic mineralized oncolytic adenovirus in preparation of antitumor drugs

Abstract

The invention discloses an oral biomimetic mineralized oncolytic adenovirus, a preparation method thereof and an application of the oral biomimetic mineralized oncolytic adenovirus in preparation of antitumor drugs. The preparation method comprises the following steps: 1) culturing homologous tumor cells; 2) preparing a membrane extraction buffer solution; 3) preparing a homologous tumor cell membrane; 4) preparing the homologous tumor cell membrane coated oncolytic adenovirus particles; 5) preparing a suspension; (6) centrifuging the suspension; removing supernatant to obtain precipitate; (7) adding absolute ethyl alcohol into the precipitate, resuspending, and centrifuging; supernate is removed; and 8) repeating the step 7) to obtain the oral biomimetic mineralized oncolytic adenovirus. The mineralized shell of the product can help the oncolytic adenovirus to still have complete activity when the oncolytic adenovirus reaches the intestinal tract after oral administration, the oncolytic adenovirus is wrapped by a homologous tumor cell membrane, the tumor targeting property of the oncolytic adenovirus is improved, the anti-tumor effect of the oncolytic adenovirus is enhanced, and experiments prove that the product has a good application prospect. The oral biomimetic mineralized oncolytic adenovirus disclosed by the invention has an inhibition effect on human pancreatic cancer cells and human colon cancer cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering, and in particular relates to a biomimetic mineralized oncolytic adenovirus and a preparation method and application thereof. Background Art

[0002] In recent years, oncolytic virus therapy has made rapid progress. Natural or modified oncolytic viruses can target and kill tumor cells without affecting the functions of normal cells and tissues. Oncolytic viruses that enter tumor cells can regulate certain key cellular physiological processes, such as apoptosis, inflammation, cell cycle, and angiogenesis. In addition, oncolytic viruses can enhance the host's anti-tumor immunity through a variety of mechanisms. For example, based on the specific replication ability of oncolytic viruses, tumor cells are lysed, pathogen-related molecules and damage-related molecules are released, immune cells are recruited, and ultimately the body's T cell immune response is activated through tumor-associated antigens. In recent years, a number of clinical experimental studies on oncolytic adenoviruses or in combination with chemotherapy / immunotherapy have shown that oncolytic adenoviruses are promising anticancer biotherapeutics.

[0003] However, the clinical efficacy of intravenous administration of oncolytic adenovirus is limited due to the body's natural antiviral immunity and pre-existing anti-adenovirus antibodies, as well as the occurrence of severe liver damage. In addition, neutralizing antibodies present in patients with previous viral exposure or repeated injections limit the efficacy of oncolytic adenovirus. And studies have shown that some primary tumors with low expression of coxsackievirus and adenovirus combined receptors (CAR receptors) are resistant to oncolytic adenovirus therapy. Therefore, in order to meet these challenges, there is an urgent need for an oncolytic adenovirus modification strategy that is simple to operate, stable, and biocompatible. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an oral biomimetic mineralized oncolytic adenovirus.

[0005] The second object of the present invention is to provide a method for preparing an oral biomimetic mineralized oncolytic adenovirus.

[0006] The third object of the present invention is to provide the use of oral biomimetic mineralized oncolytic adenovirus in the preparation of anti-tumor drugs.

[0007] The fourth object of the present invention is to provide a second oral biomimetic mineralized oncolytic adenovirus.

[0008] The fifth object of the present invention is to provide a second method for preparing an oral biomimetic mineralized oncolytic adenovirus.

[0009] The sixth objective of the present invention is to provide a second oral biomimetic mineralized oncolytic adenovirus for use in the preparation of anti-tumor drugs.

[0010] The technical solution of the present invention is summarized as follows:

[0011] The preparation method of oral biomimetic mineralized oncolytic adenovirus comprises the following steps:

[0012] 1) Cultivation of homologous tumor cells:

[0013] 2) mixing tris(hydroxymethyl)aminomethane, disodium ethylenediaminetetraacetic acid, mannitol, sucrose and water in a mass ratio of 30-45:1.6-2.2:400-420:80-100:9500-10000 to prepare a membrane extraction buffer;

[0014] 3) According to the proportion, 1-2 mL of the membrane extraction buffer was mixed with 1-2×10 8 The homologous tumor cells are mixed, and ultrasonicated at 30-40W using an ultrasonic disruptor for 3s, then stopped for 7s, for a total of 30s, and centrifuged at 0-4°C to obtain homologous tumor cell membranes;

[0015] 4) According to the ratio, the homologous tumor cell membrane obtained in step 3) was mixed with 100-200 μL of a titer of 1×10 10 GFU / mL of oncolytic adenovirus is added to 2-3 ml of PBS buffer with a pH of 7.4 and resuspended to obtain a resuspension 1; the resuspension 1 is squeezed and pushed by a liposome extruder to obtain a resuspension 2; the resuspension 2 contains homologous tumor cell membrane-coated oncolytic adenovirus particles with a size in the range of 100-150 nm, and the particles are referred to as MV-OA;

[0016] 5) Mix the resuspension 2, cyclohexane, 1-n-hexanol and triton X-100 in a volume ratio of 1:1.5-2.5:15-18:3.5-5.5, add calcium chloride to a final concentration of 2 mM, stir at 0-4°C, 500-1000 rpm for 20-40 min, then add sodium carbonate to a final concentration of 40-50 mM, and stir for 6-10 h;

[0017] 6) Centrifuging the suspension obtained in step 5) at 5000-8000 rpm and 0-4° C. for 20-40 min; removing the supernatant; and obtaining a precipitate;

[0018] 7) adding anhydrous ethanol to the precipitate obtained in step 6) to resuspend, centrifuging at 5000-8000 rpm, 0-4° C. for 10-20 min; removing the supernatant; the amount of anhydrous ethanol added is 1-1.5 times the volume of the suspension obtained in step 5);

[0019] 8) Repeat step 7) 2-3 times to obtain an oral biomimetic mineralized oncolytic adenovirus, which is referred to as MV@CaCO3-OA.

[0020] The oral biomimetic mineralized oncolytic adenovirus prepared by the above preparation method.

[0021] The use of the above-mentioned oral biomimetic mineralized oncolytic adenovirus in the preparation of anti-tumor drugs.

[0022] The tumor is selected from pancreatic cancer or colorectal cancer.

[0023] The second method for preparing an oral biomimetic mineralized oncolytic adenovirus comprises the following steps:

[0024] 1) Cultivation of homologous tumor cells:

[0025] 2) mixing tris(hydroxymethyl)aminomethane, disodium ethylenediaminetetraacetic acid, mannitol, sucrose and water in a mass ratio of 30-45:1.6-2.2:400-420:80-100:9500-10000 to prepare a membrane extraction buffer;

[0026] 3) According to the proportion, 1-2 mL of the membrane extraction buffer was mixed with 1-2×10 8 The homologous tumor cells are mixed, and ultrasonicated at 30-40W using an ultrasonic disruptor for 3s, then stopped for 7s, for a total of 30s, and centrifuged at 0-4°C to obtain homologous tumor cell membranes;

[0027] 4) Resuspend the homologous tumor cell membrane obtained in step 3) in 2-3 mL of PBS buffer with pH = 7.4 according to the proportion, use an ultrasonic disruptor, sonicate at 60-80 W, run for 1 s, stop for 1 s, and run for a total of 30-60 s to obtain a resuspension 3; add 100-200 μL of a 1×10 10 GFU / mL of oncolytic adenovirus, mix well to obtain a mixed solution, and use an ultrasonic water bath to perform water bath sonication on the mixed solution under ice water bath conditions, with a power of 60-80 W and ultrasound for 30-60 min to obtain a resuspension 4, wherein the resuspension 4 contains homologous tumor cell membrane-coated oncolytic adenovirus particles with a size ranging from 100-150 nm, and the particles are referred to as MV-OA;

[0028] 5) The resuspension 4, cyclohexane, 1-n-hexanol and triton X-100 were mixed at a volume ratio of 1:1.5-2.5:15-18:3.5-5.5, calcium chloride was added to make a final concentration of 2 mM, and the mixture was stirred at 0-4°C and 500-1000 rpm for 20-40 min, and sodium carbonate was added to make a final concentration of 40-50 mM, and the mixture was stirred for 6-10 h;

[0029] 6) Centrifuging the suspension obtained in step 5) at 5000-8000 rpm and 0-4° C. for 20-40 min; removing the supernatant; and obtaining a precipitate;

[0030] 7) adding anhydrous ethanol to the precipitate obtained in step 6) to resuspend, centrifuging at 5000-8000 rpm, 0-4° C. for 10-20 min; removing the supernatant; the amount of anhydrous ethanol added is 1-1.5 times the volume of the suspension obtained in step 5);

[0031] 8) Repeat step 7) 2-3 times to obtain an oral biomimetic mineralized oncolytic adenovirus, which is referred to as MV@CaCO3-OA.

[0032] The oral biomimetic mineralized oncolytic adenovirus prepared by the second preparation method mentioned above.

[0033] The present invention only describes the application of oral biomimetic mineralized oncolytic adenovirus in the preparation of anti-tumor drugs.

[0034] The tumor is selected from pancreatic cancer or colorectal cancer.

[0035] Advantages of the present invention:

[0036] The mineralized shell of the oral biomimetic mineralized oncolytic adenovirus of the present invention can help the oncolytic adenovirus to still have complete vitality when it reaches the intestine after oral administration. In addition, the present invention uses homologous tumor cell membranes to encapsulate the oncolytic adenovirus, which improves the tumor targeting of the oncolytic adenovirus, is conducive to enhancing the anti-tumor effect of the oncolytic adenovirus, and improves its biosafety. Based on the modified structure on the surface of the virus, the oral biomimetic mineralized oncolytic adenovirus of the present invention can not only effectively avoid the body's antiviral immune clearance, increase the enrichment of oncolytic adenovirus at the tumor site, but also can be used for the treatment of colorectal cancer in situ through an oral route. Experiments have shown that the oral biomimetic mineralized oncolytic adenovirus of the present invention has an inhibitory effect on Patu-8988 (human pancreatic cancer cells) and HT29 cells (human colon cancer cells). The oral biomimetic mineralized oncolytic adenovirus of the present invention is stable and has good biocompatibility. The preparation method of the present invention is simple, convenient and stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Particle size characterization of MV@CaCO3-OA prepared in Examples 1 to 6;

[0038] Figure 2 Potential characterization of MV@CaCO3-OA prepared in Examples 1 to 6;

[0039] Figure 3 Protein electrophoresis of MV@CaCO3-OA prepared in Example 2;

[0040] Figure 4 Characterization of the temporal stability of MV@CaCO3-OA prepared in Example 2;

[0041] Figure 5pH stability characterization of MV@CaCO3-OA prepared in Example 2;

[0042] Figure 6 The infection and replication ability of MV@CaCO3-OA prepared in Example 2 on Patu-8988 tumor cells;

[0043] Figure 7 The apoptosis-inducing ability of MV@CaCO3-OA prepared in Example 2 on Patu-8988 tumor cells;

[0044] Figure 8 The infection and replication ability of MV@CaCO3-OA prepared in Example 2 on HT29 tumor cells;

[0045] Fig. 9 The ability of MV@CaCO3-OA prepared in Example 2 to induce apoptosis in HT29 tumor cells;

[0046] Fig.10 The killing effect of MV@CaCO3-OA prepared in Example 2 on Patu-8988 tumor cells, wherein, ns, not significant; *p<0.05; **p<0.01; ***p<0.001; *****p<0.0001; the same below;

[0047] Fig.11 The killing effect of MV@CaCO3-OA prepared in Example 2 on HT29 tumor cells;

[0048] Fig.12 Viability detection of normal L929 cells by MV@CaCO3-OA prepared in Example 2;

[0049] Fig.13 The MV@CaCO3-OA prepared in Example 2 was intragastrically administered to mice, and the expression of the E1a gene in the circulating blood was detected at five time points, namely, 0, 1, 2, 4, and 6 h.

[0050] Fig.14 The MV@CaCO3-OA prepared in Example 2 was gavaged into mice, and intestinal endoscopy was performed to take photos. The yellow arrows marked the lesions.

[0051] Fig.15 After mice were gavage-treated with the MV@CaCO3-OA prepared in Example 2, the retention of oncolytic adenovirus in the intestine was detected by small animal imaging. DETAILED DESCRIPTION

[0052] The experimental methods in the following examples of the present invention, where no specific conditions are specified, are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0053] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0054] In some embodiments, the oral biomimetic mineralized oncolytic adenovirus of the present invention can be combined with one or more tumor cells. The present invention is further described in detail below in conjunction with specific examples and drawings, but the embodiments of the present invention are not limited thereto.

[0055] Liposome extruder (Avanti)

[0056] Oncolytic adenovirus (OA) (Fubo Bio)

[0057] Rapid Silver Staining Kit (Beytime)

[0058] Example 1

[0059] The preparation method of oral biomimetic mineralized oncolytic adenovirus comprises the following steps:

[0060] 1) Cultivate homologous tumor cells;

[0061] Homologous tumor cells (pancreatic cancer cells Patu-8988) were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% carbon dioxide. When the cell density reached 70%-80%, they were digested with EDTA-trypsin and centrifuged at 1200 rpm for 5 min. After removing the supernatant, PBS was added for re-suspending, and the supernatant was removed by centrifugation at 1200 rpm for 5 min.

[0062] 2) mixing tris(hydroxymethyl)aminomethane, disodium ethylenediaminetetraacetic acid, mannitol, sucrose and water in a mass ratio of 30:2.2:400:100:9500 to prepare a membrane extraction buffer;

[0063] 3) Mix 1 mL of the membrane extraction buffer with 2×10 8 The cells obtained in step 1) were mixed and ultrasonicated using a sonicator, with a power of 30W, for 3 seconds, 7 seconds, and a total of 30 seconds, and centrifuged at 0°C to obtain homologous tumor cell membranes;

[0064] 4) Mix the homologous tumor cell membrane obtained in step 3) with 200 μL of a titer of 1×10 10GFU / mL of oncolytic adenovirus was added into 3 ml of PBS buffer with pH=7.4 and resuspended to obtain resuspension 1; the resuspension 1 was squeezed and pushed by a liposome extruder to obtain resuspension 2, wherein the resuspension 2 contained homologous tumor membrane-coated oncolytic adenovirus particles with a size ranging from 100 to 150 nm, and the particles were referred to as MV-OA for short;

[0065] 5) The resuspension 2, cyclohexane, 1-n-hexanol and triton X-100 were mixed at a volume ratio of 1:1.5:18:3.5, and calcium chloride was added to make a final concentration of 2 mM. The mixture was stirred at 0°C and 500 rpm for 40 min, and sodium carbonate was added to make a final concentration of 40 mM, and stirred for 6 h;

[0066] 6) Centrifuging the suspension obtained in step 5) at 5000 rpm and 0° C. for 40 min; removing the supernatant; and obtaining a precipitate;

[0067] 7) adding anhydrous ethanol to the precipitate obtained in step 6) to resuspend, centrifuging at 5000 rpm and 0°C for 20 min; removing the supernatant; the amount of anhydrous ethanol added is 1 times the volume of the suspension obtained in step 5);

[0068] 8) Repeat step 7) 3 times to obtain an oral biomimetic mineralized oncolytic adenovirus, which is referred to as MV@CaCO3-OA.

[0069] Example 2

[0070] The preparation method of oral biomimetic mineralized oncolytic adenovirus comprises the following steps:

[0071] 1) Cultivate homologous tumor cells;

[0072] Homologous tumor cells (colorectal cancer cell HT29) were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% carbon dioxide. When the cell density reached 70%-80%, they were digested with EDTA-trypsin, centrifuged at 1200 rpm for 5 min, the supernatant was removed, PBS was added for re-suspending, and centrifuged at 1200 rpm for 5 min again, and the supernatant was removed.

[0073] 2) mixing tris(hydroxymethyl)aminomethane, disodium ethylenediaminetetraacetic acid, mannitol, sucrose and water in a mass ratio of 40:2.0:410:90:9800 to prepare a membrane extraction buffer;

[0074] 3) 1.5 mL of the membrane extraction buffer was mixed with 1.5 × 10 8The cells obtained in step 1) were mixed, ultrasonicated using a sonicator, 30W, running for 3s, stopping for 7s, running for a total of 30s, and centrifuged at 2°C to obtain homologous tumor cell membranes;

[0075] 4) Mix the homologous tumor cell membrane obtained in step 3) with 150 μL of a titer of 1×10 10 GFU / mL of oncolytic adenovirus was added into 2.5 ml of PBS buffer with a pH of 7.4 and resuspended to obtain a resuspension 1; the resuspension 1 was squeezed and pushed by a liposome extruder to obtain a resuspension 2, wherein the resuspension 2 contained homologous tumor cell membrane-coated oncolytic adenovirus particles with a size ranging from 100 to 150 nm, and the particles were referred to as MV-OA;

[0076] 5) The resuspension 2, cyclohexane, 1-n-hexanol and triton X-100 were mixed at a volume ratio of 1:2:16:4.5, and calcium chloride was added to make a final concentration of 2 mM. The mixture was stirred at 2°C and 800 rpm for 30 min, and sodium carbonate was added to make a final concentration of 45 mM, and stirred for 8 h.

[0077] 6) Centrifuging the suspension obtained in step 5) at 6000 rpm and 2° C. for 30 min; removing the supernatant; and obtaining a precipitate;

[0078] 7) adding anhydrous ethanol to the precipitate obtained in step 6) to resuspend, centrifuging at 6000 rpm and 2° C. for 15 min; removing the supernatant solution; the amount of anhydrous ethanol added is 1.2 times the volume of the suspension obtained in step 5);

[0079] 8) Repeat step 7) twice to obtain an oral biomimetic mineralized oncolytic adenovirus, which is referred to as MV@CaCO3-OA.

[0080] Example 3

[0081] The preparation method of oral biomimetic mineralized oncolytic adenovirus comprises the following steps:

[0082] 1) Cultivate homologous tumor cells;

[0083] At 37°C and 5% carbon dioxide, homologous tumor cells (pancreatic cancer cell Patu-8988) were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. When the cell density reached 70%-80%, they were digested with EDTA-trypsin and centrifuged at 1200 rpm for 5 min. After removing the supernatant, PBS was added for re-suspending, and centrifuged again at 1200 rpm for 5 min, and the supernatant was removed;

[0084] 2) mixing tris(hydroxymethyl)aminomethane, disodium ethylenediaminetetraacetic acid, mannitol, sucrose and water in a mass ratio of 45:1.6:420:80:10000 to prepare a membrane extraction buffer;

[0085] 3) Mix 2 mL of the membrane extraction buffer with 1×10 8 The cells obtained in step 1) were mixed and ultrasonicated using a sonicator at 40W for 3 seconds and 7 seconds, for a total of 30 seconds, and centrifuged at 4°C to obtain homologous tumor cell membranes;

[0086] 4) Mix the homologous tumor cell membrane obtained in step 3) with 100 μL of a titer of 1×10 10 GFU / mL of oncolytic adenovirus is added into 2 ml of PBS buffer with a pH of 7.4 and resuspended to obtain a resuspension 1; the resuspension 1 is squeezed and pushed by a liposome extruder to obtain a resuspension 2, wherein the resuspension 2 contains oncolytic adenovirus particles coated with homologous tumor cell membranes with a size ranging from 100 to 150 nm, and the particles are referred to as MV-OA;

[0087] 5) The resuspension 2, cyclohexane, 1-n-hexanol and triton X-100 were mixed at a volume ratio of 1:2.5:15:5.5, and calcium chloride was added to make a final concentration of 2 mM. The mixture was stirred at 4°C and 1000 rpm for 20 min, and sodium carbonate was added to make a final concentration of 50 mM, and stirred for 10 h.

[0088] 6) Centrifuging the suspension obtained in step 5) at 8000 rpm and 4° C. for 20 min; removing the supernatant; and obtaining a precipitate;

[0089] 7) adding anhydrous ethanol to the precipitate obtained in step 6) to resuspend, centrifuging at 8000 rpm and 4° C. for 10 min; removing the supernatant solution; the amount of anhydrous ethanol added is 1.5 times the volume of the suspension obtained in step 5);

[0090] 8) Repeat step 7) twice to obtain an oral biomimetic mineralized oncolytic adenovirus, which is referred to as MV@CaCO3-OA.

[0091] Example 4

[0092] The second method for preparing an oral biomimetic mineralized oncolytic adenovirus comprises the following steps:

[0093] 1)-3) are the same as steps 1)-3) of Example 1;

[0094] 4) The homologous tumor cell membrane obtained in step 3) was resuspended in 2 mL of PBS buffer with a pH of 7.4, and ultrasonicated at 60 W for 1 s and 1 s, respectively, for a total of 60 s to obtain a resuspension 3; 100 μL of a titer of 1×10 10 GFU / mL of oncolytic adenovirus was mixed to obtain a mixed solution, and the mixed solution was subjected to water bath sonication using an ultrasonic water bath instrument under ice water bath conditions, with a power of 60 W and ultrasound for 60 min to obtain a resuspension 4, wherein the resuspension 4 contained homologous tumor cell membrane-coated oncolytic adenovirus particles with a size in the range of 100-150 nm, and the particles were referred to as MV-OA.

[0095] 5) The resuspension 4, cyclohexane, 1-n-hexanol and triton X-100 were mixed at a volume ratio of 1:1.5:18:3.5, and calcium chloride was added to make a final concentration of 2 mM. The mixture was stirred at 0°C and 500 rpm for 40 min, and sodium carbonate was added to make a final concentration of 40 mM, and stirred for 6 h;

[0096] 6)-8) are the same as steps 6)-8) of Example 1.

[0097] Example 5

[0098] The second method for preparing an oral biomimetic mineralized oncolytic adenovirus comprises the following steps:

[0099] 1)-3) are the same as steps 1)-3) of Example 2;

[0100] 4) The homologous tumor cell membrane obtained in step 3) was resuspended in 2.5 mL of PBS buffer with pH = 7.4, and ultrasonicated with an ultrasonic disruptor at 70 W for 1 s, then stopped for 1 s, for a total of 45 s to obtain a resuspension 3; 150 μL of a 1×10 10 GFU / mL of oncolytic adenovirus, mix well to obtain a mixed solution, and use an ultrasonic water bath to perform water bath sonication on the mixed solution under ice water bath conditions, with a power of 70 W and sonication for 45 min to obtain a resuspension 4, wherein the resuspension 4 contains homologous tumor cell membrane-coated oncolytic adenovirus particles with a size ranging from 100 to 150 nm, and the particles are referred to as MV-OA;

[0101] 5) The resuspension 4, cyclohexane, 1-n-hexanol and triton X-100 were mixed at a volume ratio of 1:2:16:4.5, and calcium chloride was added to make a final concentration of 2 mM. The mixture was stirred at 2°C and 800 rpm for 30 min, and sodium carbonate was added to make a final concentration of 45 mM, and stirred for 8 h;

[0102] 6)-8) are the same as steps 6)-8) of Example 2.

[0103] Example 6

[0104] The second method for preparing an oral biomimetic mineralized oncolytic adenovirus comprises the following steps:

[0105] 1)-3) are the same as steps 1)-3) of Example 3;

[0106] 4) The homologous tumor cell membrane obtained in step 3) was resuspended in 3 mL of PBS buffer with a pH of 7.4, and ultrasonicated with 80 W for 1 s and 1 s, respectively, for a total of 30 s to obtain a resuspension 3; 200 μL of a titer of 1×10 10 GFU / mL of oncolytic adenovirus, mix well to obtain a mixed solution, and use an ultrasonic water bath to perform water bath sonication on the mixed solution under ice water bath conditions, with a power of 80 W and ultrasound for 30 min to obtain a resuspension 4, wherein the resuspension 4 contains homologous tumor cell membrane-coated oncolytic adenovirus particles with a size ranging from 100 to 150 nm, and the particles are referred to as MV-OA;

[0107] 5) The resuspension 4, cyclohexane, 1-n-hexanol and triton X-100 were mixed at a volume ratio of 1:2.5:15:5.5, and calcium chloride was added to make a final concentration of 2 mM. The mixture was stirred at 4°C and 1000 rpm for 20 min, and sodium carbonate was added to make a final concentration of 50 mM, and stirred for 10 h.

[0108] 6)-8) are the same as steps 6)-8) of Example 3.

[0109] Example 7

[0110] Antitumor effect of oral biomimetic mineralized oncolytic adenovirus administered by intragastric administration

[0111] HT29 cells were injected into the cecal wall of nude mice (Zhejiang Provincial Experimental Animal Center, 20-22g, 5 weeks old, male, divided into 4 groups, 6 mice in each group) to establish an orthotopic colorectal cancer model and evaluate the antitumor activity of MV@CaCO3-OA. The specific steps are as follows:

[0112] Nude mice were surgically operated and the cecum was exposed. HT29 cells (1×10 6 Two weeks later, equal volumes of PBS, OA, MV-OA (prepared in Example 2) and MV@CaCO3-OA (prepared in Example 2) were administered by gavage. The viral OA load of the latter three was 1×10 7 GFU / mouse, intragastrically every other day for 2 weeks, and the therapeutic effect was evaluated after the treatment.

[0113] Example 8

[0114] 10 μL of the oral biomimetic mineralized oncolytic adenovirus obtained in Examples 1-6 was taken respectively, and 2 mL of PBS buffer (pH = 7.4) was added respectively. After ultrasonication for 30 min, the hydration diameter was detected in a cuvette using a nanoparticle size analyzer and a Zeta potential analyzer. The corresponding particle size distribution diagram is shown in FIG. Figure 1 The corresponding electric potential is shown as Figure 2 As shown. (OA and MV-OA were used as controls)

[0115] Example 9

[0116] The oral biomimetic mineralized oncolytic adenovirus prepared in Example 2 was mixed with 500 μL of a 0.5 mM EDTA-Na2 aqueous solution, and the protein was extracted using RIPA lysis buffer. In addition, the cell membrane + oncolytic adenovirus and MV-OA prepared in Example 2 were used as controls, and the protein was extracted by the same steps. After the protein concentration was determined, the protein loading buffer was added to prepare a protein loading sample for SDS-PAGE electrophoresis. The gel after the electrophoresis was used for rapid silver staining to detect the protein composition of each group of samples. The results showed that the protein composition of the oral biomimetic mineralized oncolytic adenovirus particles was no significantly different from that of the control group, indicating that the mineralization modification process had no significant effect on the activity of the protein (see Figure 3 ).

[0117] Example 10

[0118] Take 10 μL of the oral biomimetic mineralized oncolytic adenovirus prepared in Example 2, take OA and MV-OA prepared in Example 2 as controls, add them into 2 mL of PBS containing 10% FBS; store them in a refrigerator at 4°C. Figure 4 As shown, the particles were taken out on the 1st day, 2nd day, 3rd day, 4th day, 5th day, 6th day and 7th day, and the hydration diameter of the oral biomimetic mineralized oncolytic adenovirus particles was detected by a nanoparticle size analyzer. The results showed that there was no significant change in the particle size during the observation time, indicating that the biomimetic mineralized oncolytic adenovirus of the present invention can maintain a stable hydration radius within 7 days.

[0119] Embodiment 11

[0120] In order to simulate the pH change of the digestive tract after oral administration, PBS buffers of pH = 1.5, pH = 6.8 and pH = 7.4 were prepared, 10 μL of the oral biomimetic mineralized oncolytic adenovirus obtained in Example 2 was taken, and OA and MV-OA prepared in Example 2 were used as controls, respectively, and added to the buffers of pH = 1.5, pH = 6.8 and pH = 7.4, and incubated in a 37°C incubator for 2 h, 4 h and 4 h, respectively; after the incubation, the samples were taken out and the particle sizes of the samples at 0, 1, 2, 4, 6 and 10 h were measured by a nanoparticle size analyzer, as shown in FIG. Figure 5As shown, during the observation time, the particle size of the biomimetic mineralized oncolytic adenovirus began to gradually approach the initial particle size of MV-OA after 4 hours, indicating that the oral biomimetic mineralized oncolytic adenovirus of the present invention can gradually expose the MV-OA inside it after entering the intestine, which helps it to exert its active function in the intestine.

[0121] Example 12

[0122] Under the condition of simulating the entry of drugs into the digestive tract in vitro, the oral biomimetic mineralized oncolytic adenovirus prepared in Example 2 was treated with simulated gastric fluid and then co-cultured with tumor cells Patu-8988. The flow cytometry test results were used to evaluate whether the biomimetic mineralized oncolytic adenovirus could increase the infection rate and apoptosis rate of the oncolytic virus. OA and MV-OA of the same titer were used as controls. Figure 6 and Figure 7 The results showed that the virus infection positive rate and apoptosis rate of Patu-8988 cells treated with oral biomimetic mineralized oncolytic adenovirus were increased, indicating that the oral biomimetic mineralized oncolytic adenovirus of Example 2 can promote oncolytic adenovirus infection of Patu-8988 cells and enhance its cell killing ability.

[0123] Embodiment 13

[0124] Under the condition of simulating the entry of drugs into the digestive tract in vitro, the oral biomimetic mineralized oncolytic adenovirus prepared in Example 2 was treated with simulated gastric fluid and then co-cultured with tumor cells HT29. The results of flow cytometry were used to evaluate whether the biomimetic mineralized oncolytic adenovirus could increase the infection rate and apoptosis rate of the oncolytic virus. OA and MV-OA of the same titer were used as controls. Figure 8 and Fig. 9 The results showed that the virus infection positive rate and apoptosis rate of HT29 cells treated with biomimetic mineralized oncolytic adenovirus were increased, indicating that the biomimetic mineralized oncolytic adenovirus in Example 2 can promote oncolytic adenovirus infection of HT29 cells and enhance its cell killing ability.

[0125] Embodiment 14

[0126] Under in vitro conditions simulating the entry of drugs into the digestive tract, the oral biomimetic mineralized oncolytic adenovirus prepared in Example 2 was treated with simulated gastric juice, and then co-incubated with Patu-8988 pancreatic cancer cells, HT29 colorectal cancer cells and L929 normal fibroblasts, and the tumor cell viability after 24 hours of co-incubation was detected by Cell-Counting-Kit-8 kit. Fig.10 , Fig.11 and Fig.12 The viability of the two cell lines was detected after 24 h of co-incubation, reflecting that oral administration of biomimetic mineralized oncolytic adenovirus enhanced the killing effect of oncolytic adenovirus on tumor cells.

[0127] Embodiment 15

[0128] The oral biomimetic mineralized oncolytic adenovirus prepared in Example 2 was used to treat colorectal cancer in situ model mice by gavage. DNA in the circulating blood was extracted at 0 h, 1 h, 2 h, 4 h and 6 h after administration, and the expression level of oncolytic adenovirus E1a was detected by qPCR. Fig.13 During treatment, endoscopic imaging was performed to observe differences in intestinal lesions, such as Fig.14 After the treatment, the intestinal tissues of mice in each treatment group were removed and the fluorescence intensity in the tissues was detected. Fig.15 The results showed that the circulation time and intestinal retention capacity of oral biomimetic mineralized oncolytic adenovirus were higher than those of naked virus.

[0129] Experiments have shown that the oral biomimetic mineralized oncolytic adenovirus prepared in Examples 1, 3, 4, 5, and 6 has similar effects to the oral biomimetic mineralized oncolytic adenovirus prepared in Example 2 in terms of time stability, pH stability, ability to infect and replicate Patu-8988 tumor cells, ability to induce apoptosis of Patu-8988 tumor cells, ability to infect and replicate HT29 tumor cells, ability to induce apoptosis of HT29 tumor cells, killing effect on Patu-8988 tumor cells, killing effect on HT29 tumor cells, and oral administration to mice to detect the expression of the E1a gene in the circulating blood at 5 time points of 0, 1, 2, 4, and 6 h.

[0130] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the claims.

Claims

1. A method for preparing an oral biomimetic mineralized oncolytic adenovirus, characterized in that The steps include: 1) Cultivation of homologous tumor cells: 2) mixing tris(hydroxymethyl)aminomethane, disodium ethylenediaminetetraacetic acid, mannitol, sucrose and water in a mass ratio of 30-45:1.6-2.2:400-420:80-100:9500-10000 to prepare a membrane extraction buffer; 3) According to the proportion, 1-2 mL of the membrane extraction buffer was mixed with 1-2×10 8 The homologous tumor cells are mixed, and ultrasonicated at 30-40W using an ultrasonic disruptor for 3s, then stopped for 7s, for a total of 30s, and centrifuged at 0-4°C to obtain homologous tumor cell membranes; 4) According to the ratio, the homologous tumor cell membrane obtained in step 3) was mixed with 100-200 μL of a titer of 1×10 10 GFU / mL of oncolytic adenovirus is added to 2-3 ml of PBS buffer with a pH of 7.4 and resuspended to obtain a resuspension 1; the resuspension 1 is squeezed and pushed by a liposome extruder to obtain a resuspension 2; the resuspension 2 contains homologous tumor cell membrane-coated oncolytic adenovirus particles with a size in the range of 100-150 nm, and the particles are referred to as MV-OA; 5) Mix the resuspension 2, cyclohexane, 1-n-hexanol and triton X-100 in a volume ratio of 1:1.5-2.5:15-18:3.5-5.5, add calcium chloride to a final concentration of 2 mM, stir at 0-4°C, 500-1000 rpm for 20-40 min, then add sodium carbonate to a final concentration of 40-50 mM, and stir for 6-10 h; 6) Centrifuging the suspension obtained in step 5) at 5000-8000 rpm and 0-4° C. for 20-40 min; removing the supernatant; and obtaining a precipitate; 7) adding anhydrous ethanol to the precipitate obtained in step 6) to resuspend, centrifuging at 5000-8000 rpm, 0-4° C. for 10-20 min; removing the supernatant; the amount of anhydrous ethanol added is 1-1.5 times the volume of the suspension obtained in step 5); 8) Repeat step 7) 2-3 times to obtain an oral biomimetic mineralized oncolytic adenovirus, which is referred to as MV@CaCO3-OA.

2. The oral biomimetic mineralized oncolytic adenovirus prepared by the preparation method according to claim 1.

3. Use of the oral biomimetic mineralized oncolytic adenovirus according to claim 2 in the preparation of anti-tumor drugs.

4. The use according to claim 3, characterized in that The tumor is selected from pancreatic cancer or colorectal cancer.

5. A method for preparing an oral biomimetic mineralized oncolytic adenovirus, characterized in that The steps include: 1) Cultivation of homologous tumor cells: 2) mixing tris(hydroxymethyl)aminomethane, disodium ethylenediaminetetraacetic acid, mannitol, sucrose and water in a mass ratio of 30-45:1.6-2.2:400-420:80-100:9500-10000 to prepare a membrane extraction buffer; 3) According to the proportion, 1-2 mL of the membrane extraction buffer was mixed with 1-2×10 8 The homologous tumor cells are mixed, and ultrasonicated at 30-40W using an ultrasonic disruptor for 3s, then stopped for 7s, for a total of 30s, and centrifuged at 0-4°C to obtain homologous tumor cell membranes; 4) Resuspend the homologous tumor cell membrane obtained in step 3) in 2-3 mL of PBS buffer with pH = 7.4 according to the proportion, use an ultrasonic disruptor, sonicate at 60-80 W, run for 1 s, stop for 1 s, and run for a total of 30-60 s to obtain a resuspension 3; add 100-200 μL of a 1×10 10 GFU / mL of oncolytic adenovirus, mix well to obtain a mixed solution, and use an ultrasonic water bath to perform water bath sonication on the mixed solution under ice water bath conditions, with a power of 60-80 W and ultrasound for 30-60 min to obtain a resuspension 4, wherein the resuspension 4 contains homologous tumor cell membrane-coated oncolytic adenovirus particles with a size ranging from 100-150 nm, and the particles are referred to as MV-OA; 5) The resuspension 4, cyclohexane, 1-n-hexanol and triton X-100 were mixed at a volume ratio of 1:1.5-2.5:15-18:3.5-5.5, calcium chloride was added to make a final concentration of 2 mM, and the mixture was stirred at 0-4°C and 500-1000 rpm for 20-40 min, and sodium carbonate was added to make a final concentration of 40-50 mM, and the mixture was stirred for 6-10 h; 6) Centrifuging the suspension obtained in step 5) at 5000-8000 rpm and 0-4° C. for 20-40 min; removing the supernatant; and obtaining a precipitate; 7) adding anhydrous ethanol to the precipitate obtained in step 6) to resuspend, centrifuging at 5000-8000 rpm, 0-4° C. for 10-20 min; removing the supernatant; the amount of anhydrous ethanol added is 1-1.5 times the volume of the suspension obtained in step 5); 8) Repeat step 7) 2-3 times to obtain an oral biomimetic mineralized oncolytic adenovirus, which is referred to as MV@CaCO3-OA.

6. The oral biomimetic mineralized oncolytic adenovirus prepared by the preparation method of claim 5.

7. Use of the oral biomimetic mineralized oncolytic adenovirus according to claim 6 in the preparation of anti-tumor drugs.

8. The use according to claim 7, characterized in that The tumor is selected from pancreatic cancer or colorectal cancer.

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