Anti-angiogenic composite gene vector and preparation method and application thereof

Abstract

The application discloses an anti-vascular composite gene vector and a preparation method and application thereof. The anti-vascular composite gene vector is prepared by coating the cationized tussah silk fibroin which is modified by an angiogenesis inhibitor, endostatin, on the surface of an adenovirus by electrostatic adsorption. The anti-vascular composite gene vector is in the form of spherical nano-particles, can effectively infect umbilical vein endothelial cells and liver cancer cells, express anticancer genes, inhibit the proliferation of vascular endothelial cells and tumor cells, and block the growth and metastasis of tumors. Different anticancer genes are coded in the vector, so that the anti-vascular composite gene vector can play the therapeutic effect of resisting tumors and preventing metastasis for different malignant tumors. Therefore, the anti-vascular composite gene vector can be used for preparing a medicine for treating tumors.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical organic polymer materials, and particularly relates to an anti-vascular composite gene vector and a preparation method and application thereof. BACKGROUND

[0002] Gene therapy is a treatment for malignant tumors from the root of genes, and has great application potential and clinical value. The introduction of exogenous therapeutic genes into tumor cells must overcome complex cell and tissue barriers to deliver genetic information without destroying cell regulation mechanisms. Therefore, the design and construction of suitable delivery vectors to safely and effectively transport therapeutic genes into the lumen of tumor cells and stably and efficiently express them are the key to the wide application of gene therapy in clinical practice.

[0003] Artificially modified adenovirus is the most commonly used viral vector in tumor gene therapy, and can infect both dividing and non-dividing cells, with a transgene efficiency much higher than that of non-viral vectors. In addition, it has the advantages of strong therapeutic gene insertion ability and no integration into the host genome, but the strong immune response of the host and the natural tendency of the coxsackie receptor are the main obstacles to the clinical application of adenovirus vectors. The main strategy adopted in clinical practice is to modify the adenovirus capsid, which can not only avoid direct contact between adenovirus and host and reduce immunogenicity, but also enhance the recognition and internalization of the vector by tumor cells.

[0004] The cationic polymers currently used more mainly include non-reducible cationic polymers (such as polyethyleneimine, polylysine and chitosan) and bioreducible polymers (bioreducible branched polyethyleneimine, poly N-(2-hydroxypropyl) methacrylamide, etc.). However, the nanocomposites formed by coating adenovirus with cationic polymers have the problems of poor serum stability and cytotoxicity.

[0005] Gene therapy has a short-term effect, but long-term problems of tumor metastasis still exist, because tumor vascular networks are formed during tumor development, which provide oxygen and nutrients required for tumor growth, and remove waste from tumor tissues, providing a pathway for tumor metastasis. SUMMARY

[0006] The present application aims to overcome the deficiencies of the prior art, and provides an anti-vascular composite gene vector and a preparation method and application thereof. The adenovirus vector modified by the cationized tussah silk fibroin used in the present application has excellent biocompatibility and biodegradability, can significantly reduce the immunogenicity risk of the adenovirus-associated gene vector, improve the coxsackie receptor tropism, and further improve the transgene efficiency. At the same time, the cationized tussah silk fibroin is side-linked to an endogenous angiogenesis inhibitor, endostatin, which can induce tumor cell apoptosis, effectively inhibit the formation of new blood vessels in tumor tissues, and block the development and metastasis of tumors.

[0007] Technical solutions: The purpose of the application is achieved by the following technical solutions:

[0008] The application provides an anti-angiogenic composite gene vector, wherein cationized tussah silk fibroin is modified by an angiogenesis inhibitor endostatin, and is coated on the surface of an adenovirus by electrostatic adsorption to prepare the anti-angiogenic composite gene vector.

[0009] In a preferred embodiment of the application, the adenovirus carries one or two of tumor suppressor gene p53, retinoblastoma gene RB gene, multiple tumor suppressor gene p16, phosphatase and tensin homolog gene PTEN, growth inhibitor 4 (ING4) or interleukin-24 (IL-24).

[0010] The application further provides a preparation method of the anti-angiogenic composite gene vector, comprising the following steps:

[0011] (1) endostatin is dissolved in a sodium carbonate buffer solution, sodium periodate-activated heparin is added, and the reaction is carried out at 0-4 DEG C in the dark for 24-48 h; after the reaction is completed, glycine is added to terminate the reaction, and the solution is purified to obtain purified endostatin modified heparin, which is freeze-dried to obtain endostatin modified heparin freeze-dried powder;

[0012] (2) the endostatin modified heparin freeze-dried powder prepared in step (1) is dissolved in a MES buffer solution to prepare an endostatin-MES buffer solution with a concentration of 0.05-1 mg / mL, N-hydroxysuccinimide (NHS) and carbodiimide (EDC) are sequentially added to activate the carboxyl groups in the heparin, after 30-40 min, cationized tussah silk fibroin solution is added, and the reaction is carried out at 0-4 DEG C in the dark for 20-24 h; after ultrafiltration, endostatin modified cationized tussah silk fibroin is obtained;

[0013] (3) the endostatin modified cationized tussah silk fibroin prepared in step (2) is prepared into a solution with a concentration of 10-200 μg / mL, and is mixed with an adenovirus solution coding a gene, vortexed, and then allowed to stand to obtain the anti-angiogenic composite gene vector.

[0014] In the application, the endostatin is modified by heparin activated by high-iodine acid oxidation, which improves the in-vivo stability of the endostatin and improves the availability, and the endostatin modified by heparin is further reacted with cationized tussah silk fibroin, the carboxyl groups in the heparin are activated by adding NHS and EDC, the carboxyl groups in the heparin can be reacted with the amino groups in the cationized tussah silk fibroin, and the endostatin can be grafted to the side chain of the tussah silk fibroin.

[0015] Preferably, in step (1), the sodium periodate-activated heparin is prepared by the following method:

[0016] To the heparin aqueous solution with concentration of 1-100 mg / mL, drop 12 wt% sodium periodate solution with volume ratio of 1:9, adjust the pH of the solution to 4.8-5.0, activate the solution at 0-4℃ for 20-24 hours in dark, drop 5 wt% sodium bisulfite solution to terminate the activation, and adjust the pH of the solution to 8.8-9.0, which is the activated heparin solution.

[0017] Preferably, in step (1), the mass ratio of the endostatin to the heparin is (1-10):1.

[0018] The present application further preferably uses 4-5 kDa low molecular weight heparin, and the endostatin modified by the heparin can effectively prolong the stability and improve the bioavailability.

[0019] Preferably, in step (1), the concentration of the sodium carbonate buffer is 0.3 M, and the pH is 9.5; after adding glycine to terminate the reaction, the solution is transferred to a dialysis bag, dialyzed in phosphate buffer solution at 0-4℃ for 48-72 hours in dark, the dialysis solution is replaced every 2-4 hours, and then the purified heparin-modified endostatin is obtained by ultrafiltration centrifugation for 30-45 minutes, and then the purified heparin-modified endostatin is frozen at -20 to -80℃ for at least 24 hours, and then the freeze-dried powder is collected after freeze-drying.

[0020] Further, the molecular weight cut-off of the dialysis is 10 kDa; the concentration of the phosphate buffer solution is 10 mM, and the pH is 8.0; and the molecular weight cut-off of the ultrafiltration is 10 kDa.

[0021] Preferably, in step (2), the mass ratio of the endostatin to the cationized tussah silk fibroin is 1:(10-200); and the concentrations of the NHS and EDC in the endostatin-MES buffer solution are 10 mM and 20 mM, respectively.

[0022] Further, in step (2), the cationized tussah silk fibroin is prepared by cationization modification of the regenerated tussah silk fibroin solution using polyethyleneimine (PEI), and the modification method is as follows: polyethyleneimine powder is dissolved in water, and then added dropwise to the regenerated tussah silk fibroin solution, the pH of the mixed solution is adjusted using 0.1 M 4-morpholine ethanesulfonic acid (MES), 20% of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) based on the mass of the regenerated tussah silk fibroin is added, and the reaction is carried out in an ice bath, and then the cationized tussah silk fibroin is obtained by dialysis and centrifugal ultrafiltration.

[0023] The EDC reacts with the tussah silk fibroin to form an unstable intermediate product, which is conducive to the reaction of the tussah silk fibroin with PEI to form a new amide bond, and promotes the progress of the cationization reaction.

[0024] Further preferably, the polyethyleneimine has a molecular weight of 1.8 kDa, contains a large number of amino groups and imino groups in the low-molecular-weight polyethyleneimine, and has a cytotoxicity much lower than that of high-molecular-weight polyethyleneimine; the use of the low-molecular-weight polyethyleneimine to modify the tussah silk fibroin can enable the tussah silk fibroin to carry amino groups and imino groups on the side chain, and the surface negative charge to be reversed to a positive charge; and meanwhile, the toxic side effects on cells and tissues can be avoided.

[0025] The regenerated tussah silk fibroin solution is prepared by a conventional method.

[0026] The preparation method of the regenerated tussah silk fibroin solution is specifically preferred, and comprises the following steps.

[0027] The washed tussah raw silk is boiled and degummed in a Na2CO3 aqueous solution, and is dried in an oven at 60℃; the calcium nitrate tetrahydrate is heated and melted, filtered, and kept at a constant temperature of 105℃; the tussah silk is added to the calcium nitrate tetrahydrate, and is dissolved until no flocculation is observed; after cooling, the purified tussah silk fibroin solution is obtained by dialysis and filtration, and is the regenerated tussah silk fibroin solution.

[0028] Further, in order to completely remove the sericin, the alkaline reagent degumming method is used, the tussah raw silk is boiled and degummed in a 2.5‰ Na2CO3 aqueous solution for three times, each time for not less than 30 min, and each time after boiling, the tussah raw silk is washed with deionized water at room temperature for three times; when the tussah silk is dissolved, the tussah silk is added to the melted calcium nitrate tetrahydrate according to a bath ratio of 1:10, i.e. 1 g of tussah silk is dissolved in 10 mL of the calcium nitrate tetrahydrate; when the tussah silk fibroin solution is purified, the dialysis bag has a molecular weight cut-off of 10 kDa, and is dialyzed in deionized water at 4℃ for 2-4 days, and the deionized water is changed every 2-6 h.

[0029] In a further preferred embodiment, the concentration of the polyethyleneimine aqueous solution is 1-8 mg / mL, the concentration of the regenerated tussah silk fibroin solution is 10-20 mg / mL, the mass ratio of the polyethyleneimine to the regenerated tussah silk fibroin is 0.01-0.08:1, the pH value of the mixed solution is 7.8-8, the reaction time in the ice bath is 12-16 h, the molecular weight cut-off of the dialysis is 3 kD, and the ultrafiltration conditions are 4℃, 8000 rpm, and 3 times of ultrafiltration, each time for 30 min.

[0030] In order to improve the transgenic efficiency and reduce the usage titer and immunogenicity of the adenovirus vector, preferably, in step (3), the titer of the adenovirus in the blended solution is (1-50)×10 7 pfu / mL; the vortexing time is 30-120 s; and the standing temperature is 0-4℃, and the standing time is 20-30 min.

[0031] The application adopts polyethylene imine to perform cationization modification on tussah silk fibroin, so that the silk fibroin carries positive charges, and then grafts heparin-modified endostatin, and covers the adenovirus surface by electrostatic action, to construct an anti-angiogenic composite gene vector.

[0032] The application further provides application of the anti-angiogenic composite gene vector in preparation of a tumor treatment drug.

[0033] Beneficial effects:

[0034] 1. The application grafts endostatin with an anti-angiogenic effect on the tussah silk fibroin, and through transgenic expression, induces tumor cell apoptosis while inhibiting tumor angiogenesis, so as to effectively block the growth and metastasis of tumors.

[0035] 2. The application uses the organic polymer material tussah silk fibroin carrying positive charges to electrostatically coat the adenovirus gene vector with negative charges, so that the adenovirus capsid protein can be effectively shielded, direct contact with the host is avoided, the immunogenicity risk of the adenovirus vector is significantly reduced, and the natural cosackie receptor tropism problem is improved.

[0036] 3. The application uses the cationized tussah silk fibroin to shield the negative charges on the surface of the adenovirus vector, so that the endocytosis of the vector and cells is enhanced, the transgenic efficiency is improved, and the use titer of the adenovirus can be effectively reduced. DETAILED DESCRIPTION

[0037] Figure 1 It is a schematic diagram for preparation of the anti-angiogenic composite gene vector in the application.

[0038] Figure 2 It is a reaction mechanism for grafting heparin-modified endostatin on the cationized tussah silk, and after the carboxylic acid in heparin is activated, the amino group in the side chain of the cationized silk reacts to generate an amide bond, and the endostatin is grafted to the side chain of the silk fibroin.

[0039] Figure 3 It is a scanning electron microscope photo of the anti-angiogenic composite gene vector in the application.

[0040] Figure 4 It is a laser confocal photo of expression of green fluorescent protein of umbilical vein vascular endothelial cells after infection of the ING4-IL-24 double-gene co-expression adenovirus and the anti-angiogenic composite gene vector.

[0041] Figure 5 It is a cell survival rate of umbilical vein vascular endothelial cells after infection of the ING4-IL-24 double-gene co-expression adenovirus and the anti-angiogenic composite gene vector for 1, 3, 5 and 7 days.

[0042] Figure 6Cell survival rate of HepG2 hepatoma cells infected with ING4-IL-24 double gene co-expression adenovirus and anti-vascular composite gene vector and cultured for 1, 3, 5 and 7 days. DETAILED DESCRIPTION

[0043] The technical solutions of the present application are described in detail below through specific examples, but the protection scope of the present application is not limited to the examples.

[0044] The specific techniques or conditions not specified in the examples are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The reagents or instruments not specified by the manufacturer are conventional products that can be purchased through regular channels.

[0045] The preparation schematic diagram of the anti-vascular composite gene vector in the present application is shown in Figure 1 .

[0046] Example 1 Preparation of an anti-vascular composite gene vector

[0047] (1) 100 g of tussah raw silk was boiled in 5 L of a 2.5 ‰ Na2CO3 aqueous solution three times, each time for 45 min, and washed with deionized water three times. The washed tussah silk was dried in an oven at 60 ℃ and loosened. Calcium nitrate tetrahydrate was heated and melted and filtered, and 150 mL was heated to 105 ℃. 15 g of the loosened degummed tussah silk was added in portions and dissolved until no flocculation occurred, and the dissolution time was about 5 h. After the solution was cooled, it was transferred to a dialysis bag (with a molecular weight cut-off of 10 kDa) and dialyzed in deionized water at 4 ℃ for 48 h, and the water was changed every 2 h. After filtration, a purified tussah silk fibroin solution, i.e., a regenerated tussah silk fibroin solution, was obtained. The solution concentration was measured by weighing method to be 35 mg / mL.

[0048] (2) The regenerated tussah silk fibroin solution was diluted with triple distilled water to a concentration of 10 mg / mL, and divided into 6 groups, each group of 20 mL, and equilibrated in an ice bath for 20 min. Polyethyleneimine (molecular weight 1.8 kDa) was dissolved in triple distilled water to prepare a solution with concentrations of 1, 2, 3, 4, 6 and 8 mg / mL, respectively, and added dropwise to each group of regenerated tussah silk fibroin solution, and the volume of polyethyleneimine added was 2 mL. Slow stirring was performed, and the solution pH was adjusted to 8 with 0.1 M MES. EDC was added at a concentration of 20% of the mass of the regenerated tussah silk, and the reaction time was 12 h in an ice bath (0 ℃). The solution was transferred to a dialysis bag (with a molecular weight cut-off of 3 kDa) and dialyzed in triple distilled water at 4 ℃ for 48 h, and the triple distilled water was changed every 2 h. The solution was centrifuged and ultrafiltered at 4 ℃ and 8000 rpm for 3 times, each time for 30 min, to obtain cationized tussah silk fibroin.

[0049] (3) 50 mg of heparin (low molecular weight heparin of 4 kDa) was dissolved in 45 mL of deionized water, 5 mL of 12% sodium periodate solution was added dropwise, the pH of the solution was adjusted to about 5.0, and the solution was activated at 4°C for 24 h in the dark. The activation was terminated by adding 5% sodium bisulfite solution, and the pH of the solution was adjusted to about 9.0. 100 mg of endostatin was dissolved in 50 mL of 0.3 M, pH 9.5 sodium carbonate buffer, and 50 mL of sodium periodate-activated heparin was added. The solution was reacted at 4°C for 24 h in the dark, and glycine was added to terminate the reaction. The solution was transferred to a dialysis bag (10 kDa molecular weight cut-off) and dialyzed at 4°C in a 10 mmol / L phosphate buffer at pH 8.0 in the dark for 24 h. The dialysate was changed every 2 h, and the dialysis was completed. The solution was filtered through a 0.45 μm microporous filter, centrifuged at 8000 rpm at 4°C for 30 min (10 kDa molecular weight cut-off), and then freeze-dried to collect heparin-modified endostatin freeze-dried powder.

[0050] (4) The heparin-modified endostatin was dissolved in a 50 mM MES buffer solution to prepare a 1 mg / mL endostatin-MES buffer solution. NHS and EDC were added in sequence (the concentrations of NHS and EDC in the endostatin-MES buffer solution were 10 mM and 20 mM, respectively), and the carboxyl groups in the heparin were activated. After 30 min, an equal volume of 10 mg / mL cationized tussah silk fibroin solution was added, and the solution was reacted at 4°C for 24 h in the dark. The solution was ultrafiltered at 4°C at 8000 rpm for 30 min to obtain endostatin-modified cationized tussah silk fibroin protein.

[0051] (5) The endostatin-modified cationized silk fibroin solution was diluted with sterilized triple distilled water to a concentration of 100 μg / mL, and the titer of ING4-IL-24 dual gene coexpression adenovirus (preparation method: Cationic Antheraea pernyi Silk Fibroin-Modified Adenovirus-Mediated ING4 and IL-24 Dual Gene Coexpression Vector Suppresses the Growth of Hepatoma Carcinoma Cells, International Journal of Nanomedicine, 2019, 14: 9745-9761) was 1 × 10 7 pfu / mL. The two were mixed in equal volumes, vortexed for 30 s, and left to stand at 0°C for 20 min to obtain an anti-angiogenic composite gene vector.

[0052] Figure 2The reaction mechanism of grafting heparin modified endostatin to cationized tussah silk fibroin is that the carboxylic acid in heparin is activated and reacts with the amino group in the side chain of cationized silk fibroin to form an amide bond, and endostatin is grafted to the side chain of silk fibroin.

[0053] The surface charge of the cationized tussah silk fibroin after reacting the regenerated tussah silk fibroin in Example 1 with polyethyleneimine of different concentrations is tested by a nanoparticle size potential instrument, and the results are shown in Table 1.

[0054] Table 1 Surface Zeta potential of the cationized protein prepared in Example 1

[0055] Polyethyleneimine concentration (mg / mL) Surface Zeta potential of cationized silk (mV) 1 +1.97 2 +4.11 3 +9.45 4 +12.39 6 +12.08 8 +12.16

[0056] As can be seen from Table 1, with the increase of the concentration of polyethyleneimine, the positive charge distribution density on the surface of the modified tussah silk fibroin increases, and when the concentration of polyethyleneimine reaches 4 mg / mL, i.e., the mass ratio of regenerated tussah silk fibroin to polyethyleneimine is 100:4, the surface charge distribution of the cationized tussah silk fibroin reaches a peak, and the surface Zeta potential thereof is about +12.39 mV.

[0057] Figure 3 It is a scanning electron microscope photo of the anti-vascular composite gene carrier in the application, wherein the concentration of polyethyleneimine used in the cationized silk fibroin is 4 mg / mL. As can be seen, the anti-vascular composite gene carrier is in the form of approximately spherical nanoparticles, and the size is between 300-470 nm.

[0058] Example 2 Application of an anti-vascular composite gene carrier

[0059] (1) 100 g of tussah raw silk is boiled in 5 L of a Na2CO3 aqueous solution with a mass concentration of 2.5 ‰ for three times, each time for 45 min, and washed with deionized water for three times. The washed tussah silk is dried in an oven at 60 ℃ and loosened. Calcium nitrate tetrahydrate is heated and melted and filtered, and 150 mL thereof is heated to 105 ℃, and 15 g of the loosened degummed tussah silk is added in portions and dissolved until no flocculation occurs, and the dissolution time is about 5 h. After the solution is cooled, it is transferred to a dialysis bag (with a molecular weight cut-off of 10 kDa) and dialyzed in deionized water at 4 ℃ for 48 h, and the water is changed every 2 h. After filtration, a purified tussah silk fibroin solution, i.e., a regenerated tussah silk fibroin solution, is obtained. The concentration of the solution is measured by weighing method to be 35 mg / mL.

[0060] (2) Dilute the regenerated silk fibroin solution to 20 mg / mL with triple distilled water, take 20 mL, and equilibrate in an ice bath for 30 min; prepare 2 mL of a 8 mg / mL polyethyleneimine (1.8 kDa molecular weight) aqueous solution, and add dropwise to the regenerated silk fibroin solution, slowly stirring, and adjust the solution pH to 7.8 with 0.1 M MES, add EDC at 20% of the mass of the regenerated silk fibroin, and react in an ice bath (0°C) for 16 h, transfer to a dialysis bag (3 kDa molecular weight cut-off), and dialyze in triple distilled water in an ice bath (0°C) for 48 h, changing the triple distilled water every 2 h, and centrifugal ultrafiltration at 4°C and 8000 rpm for 3 times, 30 min each time, to obtain cationized silk fibroin.

[0061] (3) Dissolve 50 mg of heparin (4 kDa low molecular weight heparin) in 45 mL of deionized water, add dropwise 5 mL of a 12% sodium periodate solution, adjust the solution pH to about 5.0, activate at 4°C for 24 h in the dark, add 5% sodium bisulfite solution dropwise to terminate the activation, and adjust the solution pH to about 9.0; weigh 50 mg of endostatin and dissolve in 50 mL of 0.3 M, pH 9.5 sodium carbonate buffer, add 50 mL of sodium periodate-activated heparin, react at 4°C in the dark for 24 h, and add glycine to terminate the reaction. Transfer the solution to a dialysis bag (10 kDa molecular weight cut-off), dialyze in 4°C, pH 8.0, 10 mmol / L phosphate buffer in the dark for 24 h, change the dialysis solution every 2 h, filter with a 0.45 μm microporous filter at the end of dialysis, centrifugal ultrafiltration at 4°C and 8000 rpm for 30 min (10 kDa molecular weight cut-off), and freeze-dry to collect heparin-modified endostatin freeze-dried powder.

[0062] (4) Dissolve the heparin-modified endostatin in a 50 mM MES buffer solution, prepare a 1 mg / mL endostatin-MES buffer solution, sequentially add NHS and EDC (NHS and EDC concentrations in the endostatin-MES buffer solution are 10 mM and 20 mM, respectively), and activate the carboxyl groups in the heparin; after 30 min, add an equal volume of 20 mg / mL cationized silk fibroin solution, react at 4°C in the dark for 24 h, and ultrafiltrate at 4°C and 8000 rpm for 30 min to obtain endostatin-modified cationized silk fibroin protein.

[0063] (5) Adjust the concentration of the endostatin-modified cationized silk fibroin solution to 50, 100, and 150 μg / mL with sterilized triple distilled water, and the titer of the ING4-IL-24 double-gene co-expressed adenovirus is 2 x 10 7pfu / mL, and the two were blended in equal volume, vortexed for 30 s, and left at 0 ℃ for 20 min to obtain three anti-vascular composite gene vectors, denoted as CASF-ES 50, CASF-ES 100, and CASF-ES 150.

[0064] (6) Human umbilical vein endothelial cells HUVEC (ATCC) were seeded at a density of 1×10 5 The cells were seeded in a six-well plate at a density of 1×10

[0065] The expression of green fluorescent protein by the cells was observed by laser confocal microscopy, and the results are shown in FIG. 3. Figure 4 As can be seen, the naked adenovirus and the three anti-vascular composite vectors can effectively infect umbilical vein vascular endothelial cells and express green fluorescence. The number of cells expressing green fluorescence is higher, and the fluorescence intensity is higher, when the endothelial cell growth inhibitor-modified cationized silk fibroin is used to modify the adenovirus to infect the umbilical vein vascular endothelial cells, indicating that the transgene level of the anti-vascular composite gene vector is higher than that of the naked adenovirus.

[0066] Example 3 Application of an Anti-vascular Composite Gene Vector

[0067] (1) 100 g tussah silk was boiled in 5 L of 2.5‰ Na2CO3 aqueous solution for three times, each time for 45 min, and washed with deionized water for three times. The washed tussah silk was dried in an oven at 60℃ and loosened. Calcium nitrate tetrahydrate was heated and melted, and 150 mL of the melted solution was heated to 105℃. 15 g of the loosened degummed tussah silk was added to the solution in portions and dissolved for about 5 h until no flocculation was observed. The solution was cooled and transferred to a dialysis bag (10 kDa molecular weight cut-off) and dialyzed in deionized water at 4℃ for 48 h, and the water was changed every 2 h. The purified tussah silk fibroin solution, i.e., regenerated tussah silk fibroin solution, was obtained by filtration. The concentration of the solution was measured by weighing method to be 35 mg / mL.

[0068] (2) The concentration of the regenerated tussah silk fibroin solution was diluted to 10 mg / mL with triple distilled water, and 20 mL of the solution was taken and equilibrated in an ice bath for 30 min. A 2 mL solution of polyethyleneimine (1.8 kDa molecular weight) with a concentration of 4 mg / mL was prepared and added dropwise to the regenerated tussah silk fibroin solution, which was slowly stirred, and the pH of the solution was adjusted to 7.8 with 0.1 M MES. EDC was added in an amount of 20% of the mass of the regenerated tussah silk, and the solution was reacted in an ice bath for 12 h. The solution was transferred to a dialysis bag (3 kDa molecular weight cut-off) and dialyzed in triple distilled water at 4℃ for 48 h, and the triple distilled water was changed every 2 h. The cationized tussah silk fibroin was obtained by centrifugal ultrafiltration at 4℃ and 8000 rpm for 30 min for three times.

[0069] (3) 10 mg of heparin (low molecular weight heparin of 4 kDa) was dissolved in 18 mL of deionized water, and 2 mL of 12% sodium periodate solution was added dropwise. The pH of the solution was adjusted to about 5.0, and the solution was activated at 4℃ in the dark for 24 h. The activation was terminated by adding 5% sodium bisulfite solution, and the pH of the solution was adjusted to about 9.0. 100 mg of endostatin was dissolved in 20 mL of 0.3 M sodium carbonate buffer with a pH of 9.5, and 50 mL of the activated heparin sodium periodate was added. The solution was reacted at 4℃ in the dark for 24 h, and glycine was added to terminate the reaction. The solution was transferred to a dialysis bag and dialyzed in a phosphate buffer with a pH of 8.0 and a concentration of 10 mmol / L at 4℃ in the dark for 24 h, and the dialysis solution was changed every 2 h. After dialysis, the solution was filtered with a 0.45 μm microporous filter, centrifugal ultrafiltration was performed at 4℃ and 8000 rpm for 30 min, and the heparin-modified endostatin was collected by freeze-drying.

[0070] (4) The heparin-modified endostatin was dissolved in 50 mM MES buffer solution to prepare endostatin-MES buffer solutions with concentrations of 1, 0.1, and 0.05 mg / mL. Then, NHS and EDC were added in sequence (the concentrations of NHS and EDC in the endostatin-MES buffer solution were 10 mM and 20 mM, respectively) to activate the carboxyl groups in heparin. After 30 min, an equal volume of 10 mg / mL cationized tussah silk fibroin solution was added, and the mixture was reacted at 4°C in the dark for 24 h. Then, the mixture was ultrafiltered at 4°C and 8000 rpm for 30 min to obtain three groups of endostatin-cationized tussah silk fibroin proteins with different modification ratios, which were denoted as C1, C2, and C3.

[0071] (5) The three groups of endostatin-cationized silk fibroin solutions were diluted with sterile distilled water to a concentration of 100 μg / mL, and the titer of the ING4-IL-24 double-gene co-expressed adenovirus was 2×10 7 pfu / mL. Then, the two solutions were mixed in equal volumes and vortexed for 30 s, and the mixture was allowed to stand at 4°C for 30 min to obtain three anti-angiogenic composite gene vectors, which were denoted as C1 / Ad, C2 / Ad, and C3 / Ad.

[0072] (6) Human umbilical vein endothelial cells HUVEC and human hepatoma cells HepG2 (ATCC) were inoculated in 96-well plates at a density of 4×10 3 cells / well, and five replicate wells were set up. The cells were cultured in a 37°C, 5% CO2 environment for 24 h in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The culture medium was removed, and naked adenovirus (Ad) and the three anti-angiogenic composite gene vectors C1 / Ad, C2 / Ad, and C3 / Ad were added. After 8 h of infection, the silk fibroin / adenovirus composite and naked adenovirus were removed, and 200 μL of serum-containing medium was added to each well. The cells were cultured for 1, 3, 5, and 7 days. At the four time points, the survival rates of the two types of cells were determined by the CCK-8 method, and the results are shown in FIGS. 2 and 3. Figure 5 、 Figure 6

[0073] Figure 5 As shown in FIGS. 2 and 3, the three anti-angiogenic composite gene vectors can significantly inhibit the proliferation of vascular endothelial cells, and the C1 / Ad vector with a higher grafting concentration of endostatin has a more obvious inhibitory effect on vascular endothelial cells. The survival rates of the vascular endothelial cells infected with the three composite vectors are significantly lower than those of the vascular endothelial cells infected with naked adenovirus, indicating that grafting endostatin and modifying adenovirus can effectively inhibit the proliferation of vascular endothelial cells.

[0074] Figure 6 ​The cell survival rate of HepG2 infected by the composite vector can be seen, the naked adenovirus and the three composite vectors all have inhibitory effect on the proliferation of liver cancer cells, the survival rate of liver cancer cells infected by the composite vector is significantly lower than that of liver cancer cells infected by the naked adenovirus, therefore, the composite vector has higher transgenic efficiency and more obvious anticancer effect.

[0075] Figure 5 and Figure 6 The results show that the endothelin-cationized silk fibroin modified double gene co-expression adenovirus can effectively inhibit the proliferation of vascular endothelial cells and tumor cells, and has obvious anti-vascular and anti-tumor activity.

[0076] As described above, although the present application has been indicated and expressed with reference to specific preferred embodiments, it must not be interpreted as a limitation of the present application itself. Various changes can be made in form and details without departing from the spirit and scope of the present application defined by the appended claims.

Claims

1. A method for preparing an anti-angiogenic composite gene vector, characterized by, The method comprises the following steps: (1) dissolving endothelin into sodium carbonate buffer solution, adding sodium periodate activated heparin, and reacting at 0-4℃ in the dark for 24-48 hours; after the reaction is completed, adding glycine to terminate the reaction; purifying the solution to obtain purified heparin modified endothelin; freeze-drying to obtain heparin modified endothelin freeze-dried powder; The mass ratio of endothelin to heparin is (1-10):1; (2) dissolving the heparin modified endothelin freeze-dried powder prepared in step (1) into MES buffer solution to prepare endothelin-MES buffer solution with a concentration of 0.05-1 mg / mL; sequentially adding N-hydroxysuccinimide (NHS) and carbodiimide (EDC); after 30-40 minutes, adding cationized tussah silk fibroin solution; reacting at 0-4℃ in the dark for 20-24 hours; after ultrafiltration, endothelin modified cationized tussah silk fibroin is obtained; The mass ratio of endothelin to cationized tussah silk fibroin is 1:(10-200); the concentrations of NHS and EDC in the endothelin-MES buffer solution are 10 mM and 20 mM, respectively; The endothelin modified cationized tussah silk fibroin prepared in step (2) is prepared into a solution with a concentration of 10-200 μg / mL; the solution is mixed with an adenovirus solution encoding a gene; after vortexing and standing, the anti-vascular composite gene vector is obtained. The adenovirus is an ING4-IL-24 double gene co-expression adenovirus.

2. The production method according to claim 1, characterized by, In step (1), the sodium periodate activated heparin is prepared by the following method: A 12wt% sodium periodate solution is added dropwise into a heparin aqueous solution with a concentration of 1-100 mg / mL; the volume ratio of the sodium periodate solution to the heparin aqueous solution is 1:9; the pH of the solution is adjusted to 4.8-5.0; the solution is activated at 0-4℃ in the dark for 20-24 hours; 5wt% sodium bisulfite solution is added dropwise to terminate the activation; the pH of the solution is adjusted to 8.8-9.0, and the activated heparin solution is obtained.

3. The preparation method according to claim 1, characterized in that, In step (1), the concentration of the sodium carbonate buffer solution is 0.3 M, and the pH is 9.5; after adding glycine to terminate the reaction, the solution is transferred into a dialysis bag and dialyzed in phosphate buffer solution at 0-4℃ in the dark for 48-72 hours; the dialysis solution is replaced every 2-4 hours; the purified heparin modified endothelin is obtained after ultrafiltration centrifugation for 30-45 minutes; the freeze-dried powder is collected after freeze-drying at -20 to -80℃ for at least 24 hours.

4. The method of claim 1, wherein, In step (2), the cationized tussah silk fibroin is prepared by cationizing and modifying a regenerated tussah silk fibroin solution with polyethyleneimine (PEI); the modification method is as follows: polyethyleneimine powder is dissolved in water and added to the regenerated tussah silk fibroin solution; the pH of the mixed solution is adjusted to 0.1 M 4-morpholine ethanesulfonic acid (MES); 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) accounting for 20% of the mass of the regenerated tussah silk fibroin is added; the reaction is carried out in an ice bath; after dialysis and centrifugal ultrafiltration, the cationized tussah silk fibroin is obtained.

5. The preparation method according to claim 1, characterized in that, In step (3), the titer of adenovirus in the blended solution is (1-50) x 10 7 pfu / mL; the vortexing time is 30-120 s; and the temperature for standing is 0-4°C, and the standing time is 20-30 min.

6. The anti-angiogenic composite gene vector prepared by the method according to any one of claims 1-5.

7. Use of the anti-angiogenic composite gene vector of claim 6 for the preparation of a medicament for treating tumors, characterized in that, The tumor is liver cancer.

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