A drug delivery system for oncolytic virus and its preparation method

Abstract

This application relates to the field of pharmaceutical materials, and particularly to a drug delivery system for oncolytic viruses and its preparation method; the drug delivery system has a core-shell structure, including a polymer complex outer shell and an oncolytic virus inner core, and the structural unit of the polymer complex is shown in Formula 1; the method includes: mixing the oncolytic virus and initiator molecules, performing an NHS chemical reaction to obtain a virus supramolecular initiator; mixing the protein supramolecular initiator, monomer 1, monomer 2 and linker molecules, performing an ATRP polymerization reaction to obtain an oncolytic virus drug delivery system; inserting a photo-responsive element NPPOC group into the structural unit shown in Formula 1, and the photo-responsive element decomposes under light irradiation, enabling the polymer complex to release the oncolytic virus, realizing the site-specific and time-controlled residue-free release of the oncolytic virus; at the same time, ethylene glycol molecules are designed in the monomer, which can increase the hydrophilicity of the polymer complex and also improve the cell uptake efficiency.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical materials, and particularly to a drug delivery system for oncolytic virus and its preparation method. Background Art

[0002] Cancer is a malignant disease threatening human health. Currently, cancer treatment methods based on viruses have been proposed. The principle is to selectively destroy cancer cells and induce lethal damage by utilizing the ability of selective replication in tumor cells, without damaging normal cells, and it has been proven effective for certain tumors, which is an alternative therapy for cancer. Among them, as a new tumor therapy, oncolytic virus therapy has the advantages of safety, high efficiency, and few side effects, and has become a potential solution.

[0003] However, the virus types targeted by existing oncolytic virus therapies are relatively common, and multiple administrations are required. Intravenous injection usually causes oncolytic virus to be agglutinated and precipitated by neutralizing antibodies in the blood system and then captured and phagocytosed by macrophages, greatly reducing the therapeutic effect. Therefore, in order to improve the drug utilization efficiency, the administration methods of current oncolytic virus products are usually intratumoral injection or intracavitary perfusion. This administration method greatly limits the popularization and application of oncolytic virus, and it is only applied in cancer types such as melanoma, cutaneous squamous cell carcinoma, and head and neck tumors that are located superficially in the skin and are easy for intratumoral injection in most cases.

[0004] Therefore, developing an efficient oncolytic virus delivery system, reducing the immunogenicity of oncolytic virus in vivo, improving its stability and cell uptake efficiency, etc., and at the same time providing a more controllable protection and release strategy for oncolytic virus, are of great significance for the development and application of oncolytic virus. Although the existing method of combining oncolytic virus with nano-delivery materials to form an oncolytic virus delivery system can alleviate the immunogenicity brought by the virus itself to a certain extent, since nano-materials generally carry the virus in an embedding manner, the biological activity of nano-delivery materials, as well as the targeting or infectivity of the virus, will be damaged to varying degrees. And because it is difficult to release the encapsulated oncolytic virus at a fixed point and time by the embedding method, which affects the delivery efficiency of oncolytic virus. Therefore, how to provide a delivery system that releases oncolytic virus at a fixed point and time to improve the delivery efficiency of oncolytic virus is a technical problem that urgently needs to be solved at present. Summary of the Invention

[0005] This application provides a drug delivery system for oncolytic virus and its preparation method to solve the technical problem that the oncolytic virus delivery system in the prior art is difficult to release at a fixed point and time.

[0006] In a first aspect, the present application provides a drug delivery system for oncolytic viruses. The drug delivery system has a core-shell structure and includes a polymer complex outer shell and an oncolytic virus core. The structural unit of the polymer complex is shown in Formula 1:

[0007]

[0008] Formula 1;

[0009] wherein, R1 is Br or Cl;

[0010] m1 ≥ 1;

[0011] m2 ≥ 1;

[0012] k ≥ 4;

[0013] n ≥ 1;

[0014] * represents the connection point between the polymer complex and the oncolytic virus.

[0015] Optionally, the raw materials of the polymer complex include: a protein initiator and a polymer complex molecule.

[0016] Optionally, the structural formula of the protein initiator is shown in Formula 2:

[0017]

[0018] Formula 2;

[0019] wherein, R1 is Br or Cl;

[0020] m3 ≥ 1.

[0021] Optionally, the structural formula of the polymer complex molecule is shown in Formula 3:

[0022]

[0023] Formula 3;

[0024] wherein, m1 ≥ 1;

[0025] m2 ≥ 1.

[0026] Optionally, the repeating structural unit of the polymer outer shell is shown in Formula 4:

[0027]

[0028] Formula 4;

[0029] wherein, R1 is Br or Cl;

[0030] m1 ≥ 1;

[0031] m2 ≥ 1;

[0032] m4 ≥ 1;

[0033] m5 ≥ 1;

[0034] i ≥ 1;

[0035] j ≥ 1;

[0036] k ≥ 4;

[0037] n ≥ 1;

[0038] * represents the connection point between the structural unit and the oncolytic virus.

[0039] Optionally, the raw materials of the polymer shell further include a first monomer and a second monomer, and the structural formula of the first monomer is as shown in Formula 5:

[0040]

[0041] Formula 5;

[0042] wherein, m4 ≥ 1;

[0043] The structural formula of the second monomer is as shown in Formula 6:

[0044]

[0045] Formula 6;

[0046] wherein, m5 ≥ 1.

[0047] In a second aspect, the present application provides a method for preparing the drug delivery system described in the first aspect, and the method includes:

[0048] Mix the oncolytic virus and the protein initiator, and carry out NHS chemical reaction to obtain a protein supramolecular initiator;

[0049] Mix the protein supramolecular initiator, the first monomer, the second monomer and the polymer complex molecule, and carry out ATRP polymerization reaction to form a polymer complex with surface charge and light-responsive effect on the surface of the oncolytic virus, so as to obtain an oncolytic virus drug delivery system.

[0050] Optionally, the molar ratio of the protein of the oncolytic virus to the protein initiator is 1:25 to 100.

[0051] Optionally, the molar ratio of the protein supramolecular initiator, the first monomer, the second monomer and the polymer complex molecule is 0.9 to 1.1:600:300:30.

[0052] Optionally, the reaction temperature of the NHS chemical reaction is 3°C to 5°C, and the pH value of the NHS chemical reaction is 7.5 to 8.5.

[0053] The above technical solutions provided by the embodiments of the present application have the following advantages compared with the prior art:

[0054] A drug delivery system for oncolytic virus provided by an embodiment of the present application, through the structural unit of the polymer complex shown in Formula 1, inserts a photo-responsive element NPPOC group ( ) in the structural unit. Utilizing the property that the NPPOC group can be removed under light illumination conditions, the polymer complex can release the oncolytic virus, realizing the fixed-point and timed residue-free release of the oncolytic virus; at the same time, ethylene glycol molecules are designed in the monomer, which can increase the hydrophilicity of the polymer complex and also improve the cell uptake efficiency. Description of the Drawings

[0055] The drawings here are incorporated into the specification and constitute a part of this specification, showing embodiments in line with the present invention, and are used together with the specification to explain the principles of the present invention.

[0056] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0057] Figure 1 It is a schematic structural diagram of the drug delivery system provided by the embodiment of the present application;

[0058] Figure 2 It is a schematic flowchart of the preparation method provided by the embodiment of the present application;

[0059] Figure 3 It is a schematic diagram of the effect of the delivery system for fixed-point and timed release in cells under light illumination conditions provided by the embodiment of the present application;

[0060] Figure 4 It is a diagram of the reaction sequence of OH2, OH2@polymer, and OH2@polymer+UV provided by the embodiment of the present application;

[0061] Figure 5 It is a comparison result diagram of OH2, OH2@polymer, and OH2@polymer+UV with a Zeta potential of 7.02 mV provided by the embodiment of the present application;

[0062] Figure 6Comparison result graphs of OH2, OH2@polymer, and OH2@polymer+UV with a Zeta potential of 13.37 mV provided by the embodiments of the present application;

[0063] Figure 7 Comparison result graphs of OH2, OH2@polymer, and OH2@polymer+UV with a Zeta potential of 13.27 mV provided by the embodiments of the present application;

[0064] Figure 8 Comparison result graphs of OH2, OH2@polymer, and OH2@polymer+UV with a Zeta potential of 8.93 mV provided by the embodiments of the present application;

[0065] Figure 9 Uptake result graphs of OH2@polymer with different Zeta potentials in cells provided by the embodiments of the present application;

[0066] Figure 10 Distribution result graphs of GFP fluorescent protein in the blank group, OH2, and OH2@polymer after reacting for 4 h with a Zeta potential of 13.37 mV for T24 cells provided by the embodiments of the present application;

[0067] Figure 11 Distribution result graphs of GFP fluorescent protein in the blank group, OH2, OH2@polymer, and OH2@polymer+UV after reacting for 8 h with a Zeta potential of 13.37 mV for the T24 cell line provided by the embodiments of the present application;

[0068] Figure 12 Distribution result graphs of GFP fluorescent protein in the blank group, OH2, OH2@polymer, and OH2@polymer+UV after reacting for 24 h with a Zeta potential of 13.37 mV for the T24 cell line provided by the embodiments of the present application;

[0069] Figure 13 Distribution result graphs of GFP fluorescent protein in the blank group, OH2, and OH2@polymer after reacting for 4 h with a Zeta potential of 13.37 mV for BIU87 cells provided by the embodiments of the present application;

[0070] Figure 14 Distribution result graphs of GFP fluorescent protein in the blank group, OH2, OH2@polymer, and OH2@polymer+UV after reacting for 8 h with a Zeta potential of 13.37 mV for the BIU87 cell line provided by the embodiments of the present application;

[0071] Figure 15The results of the distribution of GFP fluorescent protein in the blank group, OH2, OH2@polymer, and OH2@polymer+UV after 24 hours of reaction at a Zeta potential of 13.37 mV provided by the BIU87 cell line in the embodiments of the present application;

[0072] Figure 16 The results of the distribution of GFP fluorescent protein in the blank group, OH2, and OH2@polymer after 4 hours of reaction at a Zeta potential of 13.37 mV provided by the 5637 cells in the embodiments of the present application;

[0073] Figure 17 The results of the distribution of GFP fluorescent protein in the blank group, OH2, OH2@polymer, and OH2@polymer+UV after 8 hours of reaction at a Zeta potential of 13.37 mV provided by the 5637 cell line in the embodiments of the present application;

[0074] Figure 18 The results of the distribution of GFP fluorescent protein in the blank group, OH2, OH2@polymer, and OH2@polymer+UV after 24 hours of reaction at a Zeta potential of 13.37 mV provided by the 5637 cell line in the embodiments of the present application;

[0075] Figure 19 The flow cytometry results of the GFP fluorescent protein in the blank group, OH2, OH2@polymer, and OH2@polymer+UV provided by the embodiments of the present application;

[0076] Figure 20 The schematic diagram of the cell uptake mechanism provided by the embodiments of the present application. Detailed implementation manners

[0077] The present invention will be specifically described below in combination with the detailed implementation manners and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these detailed implementation manners and examples are used to illustrate the present invention, rather than to limit the present invention.

[0078] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In case of contradiction, this specification shall prevail.

[0079] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchases or can be prepared by existing methods.

[0080] The creative idea of this application is as follows: Currently, by shielding virus particles with chemically modified outer proteins, the isolation of viruses by the mononuclear phagocyte system can be effectively avoided. Chemical modification generally uses nanoparticles to modify outer proteins. One of the most studied uses of such nanoparticles in virus therapy is to modify virus vectors with various polymers / micelles, dendrimers, liposomes, and cells. The main way for nanoparticles to bind to viruses is to enhance the therapeutic effect by downregulating specific compartments of the immune system.

[0081] The last obstacle for oncolytic viruses to reach their tumor targets is the mechanism of cell entry (as Figure 20 shown). Although some viruses select natural tropism for tumor cells through tumor phenotypes (immortality, DNA instability, etc.), cell entry depends on specific signal transduction pathways. In other words, the internalization of adenovirus requires CAR expression, and glycoprotein D is an essential component for HSV-1 to enter cells. However, the mechanism by which the nanoparticle-mediated oncolytic virus delivery system enters cells is independent of virus receptors. In this regard, a responsive nanoparticle delivery mechanism can be combined. For example, a virus composite coated with external-responsive nanomaterials such as light, magnetic, pH, temperature-responsive nanomaterials can be constructed to release oncolytic viruses at the tumor site under external environmental response signals, thereby achieving the purpose of tumor targeting. Light has high spatio-temporal controllability, operability, and non-invasiveness, and has received extensive attention from scientific researchers in recent years. It has been widely used to construct light-responsive intelligent delivery systems and achieved good application effects.

[0082] Therefore, constructing a light-responsive polymer nanomaterial to effectively cover the immunogenicity of oncolytic viruses and offset the resistance of neutralizing antibodies, while releasing oncolytic viruses without damage under appropriate spatio-temporal conditions or in the tumor tissue environment, and then exerting the anti-tumor effect of oncolytic viruses themselves has important research significance and clinical application value.

[0083] The technical solution provided in the embodiments of the present invention to solve the above technical problems has the following general idea:

[0084] In one embodiment of this application, as Figure 1 shown, a drug delivery system for oncolytic viruses is provided. The drug delivery system has a core-shell structure, including an outer shell of a polymer complex and an inner core of oncolytic viruses. The structural unit of the polymer complex is shown in Formula 1:

[0085]

[0086] Formula 1;

[0087] Among them, R1 is Br or Cl;

[0088] m1≥1;

[0089] m2≥1;

[0090] k ≥ 4;

[0091] n ≥ 1;

[0092] * represents the junction of the polymer complex and the oncolytic virus.

[0093] In the embodiments of the present application, since the binding site of the polymer complex and the oncolytic virus is generally on the protein coat that encapsulates the oncolytic virus, by controlling the binding site of the linear polymer complex and the oncolytic virus, the polymer complex can encapsulate the surface of the oncolytic virus to form a delivery system.

[0094] In some alternative embodiments, the raw materials of the polymer complex include: a protein initiator and polymer complex molecules.

[0095] In the embodiments of the present application, by controlling the raw materials of the polymer complex, the polymer complex can be completely formed in the ATRP polymerization reaction involving the protein initiator and polymer complex molecules.

[0096] In some alternative embodiments, the structural formula of the protein initiator is as shown in Formula 2:

[0097]

[0098] Formula 2;

[0099] Wherein, R1 is Br or Cl;

[0100] m3 ≥ 1.

[0101] In the embodiments of the present application, an NPPOC group is introduced into the protein initiator, and an NHS group is provided at one end of the introduced NPPOC group. After the protein initiator is activated by NHS, it can specifically bind to the amino group of the protein. Since lysine contains a primary amine part with high nucleophilic activity, it is the most commonly used protein chemical modification site, and lysine has a high natural abundance on the protein surface. At the same time, this specific chemical modification targeting lysine is a non-specific modification, enabling the protein initiator to react with the protein coat of the oncolytic virus to form an amide bond and remove the NHS group, obtaining a protein supramolecular agent that binds to the protein coat of the oncolytic virus. And an NPPOC group is introduced into this protein supramolecular agent. Since an NHS group is provided at one end of the NPPOC group, the NPPOC group can be tightly connected to the amide bond, as Figure 3 shown. Under the condition of light, due to the removal of the NPPOC group, the amide bond is broken, causing the polymer complex to dissociate from the oncolytic virus.

[0102] Introducing multiple ethylene glycol molecules into the protein initiator can regulate according to the water solubility of cells in different parts or regions, making the water solubility of the protein initiator controllable and facilitating the entry of the final polymer complex product into cells.

[0103] In the embodiments of the present application, the structural formula of the polymer complex molecule is as shown in Formula 3:

[0104]

[0105] Formula 3;

[0106] wherein, m1≥1;

[0107] m2≥1.

[0108] In the embodiments of the present application, by controlling the structural formula of the polymer complex molecule forming the polymer complex, introducing NPPOC groups and ethylene glycol molecules into the polymer complex molecule, and utilizing the removal reaction of the NPPOC group under light conditions, the outer shell of the polymer complex of the delivery system can be decomposed. Together with the introduced multiple ethylene glycol molecules, it can not only increase the water solubility of the polymer complex, but also further enable the decomposition rate of the polymer complex while the NPPOC group reacts, quickly releasing the encapsulated oncolytic virus.

[0109] In some alternative embodiments, the repeating structural unit of the polymer outer shell is as shown in Formula 4:

[0110]

[0111] Formula 4;

[0112] wherein, R1 is Br or Cl;

[0113] m1≥1;

[0114] m2≥1;

[0115] m4≥1;

[0116] m5≥1;

[0117] i≥1;

[0118] j≥1;

[0119] k≥4;

[0120] n≥1;

[0121] * represents the connection point of the structural unit and the oncolytic virus.

[0122] In the embodiments of the present application, by introducing a first monomer containing an amino group and a second monomer containing a methyl group into the structural unit of the polymer shell, since the first monomer with an amino group is positively charged and the second monomer with a methyl group is uncharged, the charging situation of the polymer complex can be controlled by controlling the polymerization degree of the first monomer and the second monomer. Since the nanomaterial on the cationic surface can contribute to membrane fusion, adjusting the charging situation of the polymer complex according to different cell entry systems can enable the delivery system to directly enter the cells.

[0123] In some alternative embodiments, the raw materials of the polymer shell further include a first monomer and a second monomer, and the structural formula of the first monomer is as shown in Formula 5:

[0124]

[0125] Formula 5;

[0126] wherein, m4≥1;

[0127] The structural formula of the second monomer is as shown in Formula 6:

[0128]

[0129] Formula 6;

[0130] wherein, m5≥1.

[0131] In the embodiments of the present application, in order to further increase the water solubility of the polymer complex, ethylene glycol molecules are added to the first monomer and the second monomer, so that the polymer complex not only carries a positive charge but also increases the water solubility of the polymer complex.

[0132] Next, as Figure 2 shown, a preparation method of an oncolytic virus drug delivery system provided by the embodiments of the present application is described, and the method includes:

[0133] S1. Mix the oncolytic virus and the protein initiator, and carry out an NHS chemical reaction to obtain a protein supramolecular initiator;

[0134] S2. Mix the protein supramolecular initiator, the first monomer, the second monomer and the polymer complex molecule, and carry out an ATRP polymerization reaction to form a polymer complex with surface charge and photo-responsive effect on the surface of the oncolytic virus, and obtain an oncolytic virus drug delivery system.

[0135] In the embodiments of the present application, the specific reaction form is controlled, and the NHS group in the protein initiator undergoes an NHS reaction with the protein coat of the oncolytic virus to form an amide bond. Since one end of the NPPOC group is connected to the NHS group, the amide bond after the reaction will be tightly bound to the NPPOC group. In the light-induced removal stage, the polymer complex can be detached from the protein coat of the oncolytic virus.

[0136] Then, the ATRP polymerization reaction is in-situ initiated by the active species of the protein supramolecular initiator, enabling the polymerization of the positively charged first monomer, the uncharged second monomer, and the polymer complex molecule containing the NPPOC group, thereby forming a polymer complex with surface charge and light-responsive effects, facilitating the direct entry of the oncolytic virus into cells and releasing the oncolytic virus under light conditions.

[0137] Currently, there are electron transfer-generated activator atom transfer radical polymerization (agent-ATRP) and single electron transfer living radical polymerization (SET-LRP) in ATRP polymerization reactions. Among them, since the agent-ATRP polymerization reaction system can significantly reduce the dosage of transition metal complexes, and due to the presence of a reducing agent, even trace amounts of O2 in the system will not have a great impact on the reaction. Therefore, the ATRP polymerization reaction in the present application mainly prepares the virus polymer complex in the form of agent-ATRP.

[0138] Since the preparation method of the drug delivery system introduced in the embodiments of the present application includes the drug delivery system provided in the foregoing embodiments of the present application, the structural units and structural composition features of the drug delivery system will not be elaborated herein again. Any method including the drug delivery system of the embodiments of the present application belongs to the scope protected by the present application.

[0139] In some alternative embodiments, the molar ratio of the protein of the oncolytic virus to the protein initiator is 1:25 to 100, where the molar ratio can be 1:25, 1:50, or 1:100.

[0140] In the embodiments of the present application, the positive effect of controlling the molar ratio of the protein of the oncolytic virus to the protein initiator is that within the range of this molar ratio, the protein coat of the oncolytic virus can react completely with the protein initiator, thereby obtaining a suitable protein supramolecular initiator.

[0141] In some alternative embodiments, the molar ratio of the protein supramolecular initiator, the first monomer, the second monomer, and the polymer complex molecule is 0.9 to 1.1:600:300:30, where the molar ratio can be 0.9:600:300:30 or 1.1:600:300:30 or 1:600:300:30.

[0142] In the embodiments of the present application, the molar ratios of the protein supramolecular initiator, the first monomer, the second monomer, and the polymer complex molecule are controlled such that a delivery system in which an oncolytic virus is encapsulated by a positively charged polymer complex is finally formed.

[0143] In some alternative embodiments, the reaction temperature of the NHS chemical reaction is 3°C to 5°C, and the pH value of the NHS chemical reaction is 7.5 to 8.5. Among them, the reaction temperature can be 3°C, 4°C, or 5°C, and the pH value of the reaction is 7.5, 8.0, or 8.5.

[0144] In the embodiments of the present application, the positive effect of controlling the reaction temperature of the NHS chemical reaction to be 3°C to 5°C is that within this temperature range, the NHS chemical reaction can proceed smoothly, so that an amide bond is formed between the protein initiator and the protein coat of the oncolytic virus, and the NHS group is removed.

[0145] The positive effect of controlling the pH value of the NHS chemical reaction to be 7.5 to 8.5 is that within this pH value range, the NHS chemical reaction can proceed sufficiently, so that an amide bond is formed between the protein initiator and the protein coat of the oncolytic virus, and the NHS group is removed.

[0146] Example 1

[0147] The polymer complex molecule, the protein initiator, the first monomer, and the second monomer are synthesized separately, and the specific structural formulas are as follows:

[0148] Polymer complex molecule:

[0149]

[0150] Formula 3;

[0151] Protein initiator:

[0152]

[0153] Formula 2;

[0154] First monomer:

[0155]

[0156] Formula 5;

[0157] Second monomer:

[0158]

[0159] Formula 6.

[0160] Next, prepare the delivery system, and the specific process is as follows:

[0161] S1. Mix the oncolytic virus and the protein initiator, and carry out NHS chemical reaction to obtain a protein supramolecular initiator;

[0162] S2. Mix the protein supramolecular initiator, the first monomer, the second monomer and the polymer complex molecule, and carry out ATRP polymerization reaction to form a polymer complex with surface charge and light-responsive effect on the surface of the oncolytic virus, and obtain an oncolytic virus drug delivery system as Figure 1 shown.

[0163] The molar ratio of the protein of the oncolytic virus to the protein initiator is 1:50.

[0164] The molar ratio of the protein supramolecular initiator, the first monomer, the second monomer and the polymer complex molecule is 1:600:300:30.

[0165] The reaction temperature of the NHS chemical reaction is 4 °C, and the pH value of the NHS chemical reaction is 8.0.

[0166] Example 2

[0167] Compare Example 2 with Example 1. The difference between Example 2 and Example 1 is that:

[0168] Fusion expression is carried out using an oncolytic virus and green fluorescent protein. Among them, the green fluorescent protein is GFP, purchased from Wuhan BinHui Biotechnology Co., Ltd. The specific process of the fusion expression refers to PLoS ONE. 2014, 9(3):e93103;

[0169] Then carry out the method of this application to obtain a delivery system, and carry out cell localization tracing through a laser scanning confocal microscope (CLSM). The specific process is as follows:

[0170] Select three bladder cancer cell lines, T24, BIU87 and 5637. The oncolytic virus used is oHSV2 (Wuhan BinHui Biotechnology Co., Ltd., titer 10 8 ).

[0171] The process of using the method of this application is as Figure 4 shown, and the specific process is:

[0172] 1. OH2 pretreatment: Place oHSV2 (OH2) in a 1×PBS solution with pH = 7.8 and dialyze for 4 h under ice bath conditions.

[0173] 2. OH2@init synthesis: Take 1 mL of virus, add protein initiator, then add 50 μL of dimethyl sulfoxide (DMSO) as an auxiliary agent, and stir the reaction in an ice bath for 3 hours.

[0174] 3. OH2@polymer synthesis: The first monomer (monomer-1), the second monomer (monomer-2), the third monomer (monomer-3), the polymer complex molecule (crosslinker), CuBr2, tris(2-dimethylaminoethyl)amine (Me6TREN), and vitamin C were added to the OH2@init system according to the proportions in the table, and the reaction was carried out under a nitrogen atmosphere for 40 minutes, and then continued to react in an ice bath for 4 hours; among them, the third monomer was butyl methacrylate, CAS: 97-88-1, purchased from Jiuding Chemical, and the amount of each substance added is shown in Table 1.

[0175] Table 1 The ratio of monomer-1, monomer-2, crosslinker, CuBr2, Me6TREN and vitamin C

[0176]

[0177] 4. Potential measurement of OH2@polymer:

[0178] According to the ratio in Table 1 and the measured Zeta potential, Table 2 is obtained.

[0179] Table 2 Zeta potential of each system

[0180]

[0181] 5. Intracellular uptake experiment of OH2@polymer conjugates:

[0182] (1) Count T24 cells in an 8-well dish, with 10,000 cells per well.

[0183] (2) Add samples after standing for 10 hours: aspirate a certain amount of OH2@polymer, dilute it with 1640 culture medium, then aspirate the original culture medium in the 8-well dish, add culture medium containing OH2@polymer, 200 μL per well, and place incubator for incubation.

[0184] (3) Uncoating: After incubation for 4 h, irradiate the target wells with a 10 W UV lamp at 365 nm for 2 min.

[0185] (4) Measurement: Measure 24 hours after adding the sample, subtract the pure cell background, and use this parameter to measure the target well.

[0186] The premise of this experiment is that when constructing OH2, the GFP gene was inserted into T24 cells. After the oncolytic virus infects the cells, the infected cells can express GFP by transfection. Since GFP is a green fluorescent protein, the expression level of GFP can be observed through a laser scanning confocal microscope, which can intuitively show the efficiency of cell uptake of the virus. The stronger and more the green fluorescence, the more virus the cells take up. The results are as Figures 5 - 8 shown.

[0187] Figure 5 Figure showing the comparison of OH2, OH2@polymer, and OH2@polymer+UV with a Zeta potential of 7.02 mV provided in the embodiment of the present application;

[0188] Figure 6 Figure showing the comparison of OH2, OH2@polymer, and OH2@polymer+UV with a Zeta potential of 13.37 mV provided in the embodiment of the present application;

[0189] Figure 7 Figure showing the comparison of OH2, OH2@polymer, and OH2@polymer+UV with a Zeta potential of 13.27 mV provided in the embodiment of the present application;

[0190] Figure 8 Figure showing the comparison of OH2, OH2@polymer, and OH2@polymer+UV with a Zeta potential of 8.93 mV provided in the embodiment of the present application;

[0191] From Figures 5 to 8 it can be obtained Figure 9 the uptake situation diagram of OH2@polymer with different Zeta potentials as shown. From Figure 9 it can be seen that with the increase of charge, the uptake of OH2@polymer by cells will increase, but not continuously. After adding hydrophobic molecules, the uptake of OH2@polymer by cells will decrease. Therefore, both charge and hydrophilicity / hydrophobicity will affect the intracellular uptake efficiency of OH2@polymer, and OH2@polymer with a Zeta potential of 13.37 mV is the best.

[0192] 6. Study on the cell uptake efficiency and time of different bladder cancer cells:

[0193] T24 cell system: According to the above steps and operations, the results are as Figures 10 - 12 shown. From Figures 10 to 12 it can be seen that compared with naked OH2, the cell uptake efficiency of 2@polymer and OH2@polymer+UV after light-induced uncoating increases significantly with the extension of time.

[0194] BIU87 cell system: According to the above steps and operations, the results are as Figures 13 - 15 shown. It can be seen from Figures 13 to 15 that for 2@polymer and OH2@polymer+UV after light-induced uncoating compared with bare OH2, the cell uptake efficiency increases significantly with the prolongation of time.

[0195] 5637 cell system: According to the above steps and operations, the results are as Figures 16 - 18 shown. It can be seen from Figures 16 to 18 that for 2@polymer and OH2@polymer+UV after light-induced uncoating compared with bare OH2, the cell uptake efficiency increases significantly with the prolongation of time.

[0196] 7. GFP fluorescence intensity experiment:

[0197] The principle is the same as that of Experiment 5. The specific steps are as follows:

[0198] (1) Count T24 cells into a 96-well plate, with 10,000 cells in each well.

[0199] (2) After standing for 10 h, add samples: Pipette a certain amount of OH2@polymer, dilute it with 1640 culture medium, then aspirate the original culture medium in the 96-well plate, and add the culture medium containing OH2@polymer, 200 μL per well, and place it in an incubator for incubation.

[0200] (3) Uncoat: After incubation for 4 h, irradiate the target wells with a 10 W ultraviolet lamp at 365 nm for 2 min.

[0201] (4) Digest: Use trypsin to digest the cells to be measured 24 h after adding the samples.

[0202] (5) Measure: After deducting the pure cell background, use this reference value to measure the target wells.

[0203] The results are as Figure 19 shown. The intracellular uptake efficiency of OH2@polymer is significantly higher than that of OH2.

[0204] One or more technical solutions in the embodiments of the present application at least further have the following technical effects or advantages:

[0205] (1) The delivery system provided in the embodiments of the present application, through the structural unit of the polymer complex shown in Formula 1, inserts a photo-responsive element NPPOC group in the structural unit, and at the same time designs ethylene glycol molecules in the structural unit to increase the hydrophilicity of the polymer complex. The photo-responsive element can decompose under light irradiation, so that the polymer complex can release oncolytic virus, realizing the time-controlled and site-specific residue-free release of oncolytic virus in cells.

[0206] (2) The delivery system provided in the embodiments of the present application performs mild in situ free radical polymerization on the surface of the target oncolytic virus to prepare a polymer-protected virus complex with uniform structure and controllable quality. In addition, by introducing photosensitive groups and utilizing highly controllable and non-invasive exogenous light signals, the oncolytic virus can be released at a fixed point and at a fixed time.

[0207] (3) The delivery system provided in the embodiments of the present application can enhance the local targeting of oncolytic viruses to tumors through a light-responsive switch design, that is, a normal therapeutic effect can be achieved at a lower concentration titer, and a better therapeutic effect can be achieved at a concentration titer of conventional treatment.

[0208] (4) The method provided in the embodiments of the present application adopts an in situ polymerization strategy that has the technical advantages of strong controllability and good uniformity compared to traditional encapsulation. The designed light-controlled release mechanism is easy to operate and has strong temporal and spatial accuracy, which is of great significance for tumor treatment and gene therapy that require local precision drug delivery.

[0209] It should be noted that, in this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "include..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0210] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0211] The foregoing is merely a specific embodiment of the present invention, which enables those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A drug delivery system for oncolytic virus, characterized in that, The drug delivery system is a core-shell structure, including a polymer complex outer shell and an oncolytic virus inner core. The structural unit of the polymer complex is shown in Formula 4: Formula 4; wherein, R1 is Br or Cl; m1≥1; m2≥1; m4≥1; m5≥1; i≥1; j≥1; k≥4; n≥1; * represents the connection point between the polymer complex and the oncolytic virus.

2. The drug delivery system according to claim 1, wherein The raw materials of the polymer complex include: a protein initiator and a polymer complex molecule.

3. The drug delivery system according to claim 2, wherein The structural formula of the protein initiator is shown in Formula 2: Formula 2; wherein, R1 is Br or Cl; m3≥1。 4. The pharmaceutical delivery system according to claim 3, wherein, The structural formula of the polymer complex molecule is shown in Formula 3: Formula 3; wherein, m1≥1; m2≥1。 5. The pharmaceutical delivery system according to claim 2, characterized in that, The raw materials of the polymer outer shell further include a first monomer and a second monomer. The structural formula of the first monomer is shown in Formula 5: Formula 5; wherein, m4≥1; The structural formula of the second monomer is shown in Formula 6: Formula 6; wherein, m5≥1.

6. A method for preparing the drug delivery system as claimed in claim 5, characterized in that, The method includes: Mixing the oncolytic virus and the protein initiator, and performing an NHS chemical reaction to obtain a protein supramolecular initiator; Mixing the protein supramolecular initiator, the first monomer, the second monomer and the polymer complex molecule, and performing an ATRP polymerization reaction to form a polymer complex with surface charge and light-responsive effect on the surface of the oncolytic virus, thereby obtaining an oncolytic virus drug delivery system.

7. The method according to claim 6, characterized in that, The molar ratio of the oncolytic virus to the protein initiator is 1:25 to 100.

8. The method according to claim 6, characterized in that The molar ratio of the protein supramolecular initiator, the first monomer, the second monomer and the polymer complex molecule is 0.9 to 1.1:600:300:

30.

9. The method according to claim 6, characterized in that, The reaction temperature of the NHS chemical reaction is 3°C to 5°C, and the pH value of the NHS chemical reaction is 7.5 to 8.5.

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