Oncolytic adenovirus for targeted therapy of brain tumors as well as preparation method and application of oncolytic adenovirus

Abstract

The invention discloses an oncolytic adenovirus for targeted therapy of brain tumors as well as a preparation method and application of the oncolytic adenovirus. The oncolytic adenovirus is modified by cholesterol, and the cholesterol is modified on the surface of the oncolytic adenovirus in a non-covalent binding mode. According to the invention, the oncolytic adenovirus is non-covalently modified by cholesterol, and an apolipoprotein E-mediated pathway is utilized, so that the blood-brain barrier and brain tumor targeting are efficiently spanned, the virus activity and the biological safety are maintained, and the advantages of immune remodeling and easiness in preparation are achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, in particular to an oncolytic adenovirus for targeted treatment of brain tumors and a preparation method and application thereof. BACKGROUND

[0002] Glioblastoma (GBM) is the most common and most invasive primary brain tumor, which is difficult to treat clinically, and the median survival of patients is generally less than two years. The current standard treatment methods mainly include surgical resection, radiotherapy and chemotherapy, but due to the high heterogeneity and diffuse infiltrative growth characteristics of the tumor, it is prone to recurrence and has very limited efficacy. In addition, the blood-brain barrier (BBB) forms an important obstacle to drug delivery, further exacerbating the difficulty of treatment.

[0003] Oncolytic viruses (OVs), such as oncolytic adenovirus, are a class of viruses that can selectively replicate in tumor cells and lyse tumor cells, and can also induce the body to produce an anti-tumor immune response, so they are considered a promising candidate for treating brain tumors. Due to the plasticity of genetic modification and better safety, they have attracted widespread attention. However, systemically administered oncolytic adenovirus is rapidly cleared by the immune system in the body, and has low efficiency in crossing the BBB, which severely limits its practical application in the treatment of brain tumors.

[0004] In order to improve the targeting of oncolytic adenovirus in the brain, existing technologies attempt to introduce specific targeting ligands through genetic engineering or chemical modification. For example, some studies insert a peptide segment that recognizes the transferrin receptor (TfR) into the virus capsid in order to improve its ability to cross the BBB. However, this strategy still has several shortcomings: first, chemical coupling or genetic modification may change the structure of the virus, thereby reducing the infection ability of the virus; second, after the virus enters the body, the targeting ligand on the surface of the virus is easily covered by the "protein crown" formed by plasma proteins, which shields the targeting ligand on the surface of the virus, resulting in reduced or lost targeting ability and thus reduced treatment effect; third, the modification process often involves complex process steps, increasing the difficulty and cost of preparation. In addition, existing technologies also attempt to modify oncolytic adenovirus with cell membranes to enhance its ability to break through the blood-brain barrier and target tumor cells. However, this technology also has some drawbacks: it relies on specific cell membrane sources, and different tumor types may require different cell membrane modification strategies, which increases the complexity of research and development; using cell membrane modification may introduce additional immunogenicity problems, thereby triggering an immune response; the cell membrane modification process may involve complex biochemical operations, including cell membrane separation, purification and binding with viruses, which has high industrial replication costs.

[0005] Therefore, the existing oncolytic adenovirus still faces problems such as low delivery efficiency and limited preparation process in brain tumor treatment, and new solutions are urgently needed. SUMMARY

[0006] In view of the defects in the prior art, the present application provides an oncolytic adenovirus for targeted treatment of brain tumors, a preparation method and application thereof. The present application can actively guide the adsorption of the oncolytic adenovirus to specific endogenous apolipoprotein E (ApoE) in the body through simple cholesterol physical modification, thereby converting the disadvantage of the "protein crown" into a targeting advantage. This "surface modification-protein enrichment-receptor mediated" biomimetic strategy ingeniously utilizes the body's inherent lipoprotein transport system to achieve efficient brain tumor targeting of the virus, and provides a new technology for targeted treatment of central nervous system diseases.

[0007] The present application provides an oncolytic adenovirus for targeted treatment of brain tumors, which is modified by cholesterol, wherein the cholesterol is modified on the surface of the oncolytic adenovirus in a non-covalent binding manner.

[0008] Cholesterol is a natural hydrophobic molecule that can be adsorbed on the surface of viral capsid proteins through non-covalent interaction, avoiding damage to the viral capsid and maintaining its infectivity and replication characteristics. At the same time, cholesterol molecules can enhance the interaction between the virus and the cell membrane, and improve its ability to cross the blood-brain barrier.

[0009] In some embodiments, the oncolytic adenovirus is an adenovirus type 5, which lacks the E1B-55kD gene and the 78.3-85.8 mu fragment of the E3 region.

[0010] The present application preferably uses adenovirus type 5 (Ad5) as an oncolytic vector, wherein the deletion of the E1B-55kD gene makes the virus unable to efficiently replicate in normal cells, but can selectively proliferate in tumor cells with defective p53 pathways, thereby enhancing its specificity.

[0011] In some embodiments, the oncolytic adenovirus has a hydration particle size of 150-200 nm; this particle size is not only conducive to maintaining appropriate circulation stability in the body, but also suitable for crossing the blood-brain barrier; particles that are too small are easily cleared by the kidneys, and particles that are too large are difficult to enter brain tissue, so the particle size in this range helps to improve brain targeting efficiency.

[0012] In some embodiments, the oncolytic adenovirus surface is combined with apolipoprotein E; when the virus surface is combined with ApoE, it can enter the brain through the receptor-mediated transendothelial transport pathway, thereby significantly improving the brain targeting delivery efficiency of the virus.

[0013] In the present application, the binding of apolipoprotein E can be naturally formed in vivo (cholesterol modification promotes ApoE binding) or achieved by pre-incubating ApoE in vitro.

[0014] The present application also provides a preparation method of the oncolytic adenovirus, comprising the following steps: The oncolytic adenovirus and the cholesterol solution are mixed, incubated at 0-6℃ for 8-16 h, and filtered to obtain the oncolytic adenovirus.

[0015] The present application does not require complex chemical modification or genetic engineering, and by incubation under suitable conditions, cholesterol molecules are non-covalently bound to the surface of the virus capsid, and then unbound cholesterol molecules are removed by a filtration step to obtain a uniform and stable modified virus preparation.

[0016] In some embodiments, the concentration of the oncolytic adenovirus is 10 10 ~10 12 VP / mL; The concentration of the cholesterol solution is 1-5 mmol / L; In the present application, the use amount of cholesterol solution corresponding to 1×10 12 VP oncolytic adenovirus is 2-4 μmol.

[0017] In the present application, the use amount of oncolytic adenovirus and cholesterol solution is limited to facilitate the sufficiency and controllability of cholesterol modification.

[0018] The present application also provides a virus complex comprising the oncolytic adenovirus.

[0019] The present application also provides a pharmaceutical composition comprising the oncolytic adenovirus and a pharmaceutically acceptable carrier.

[0020] In some embodiments of the present application, the pharmaceutically acceptable carrier can be normal injection carriers such as physiological saline, PBS buffer, etc. The pharmaceutical composition can be prepared into an injection dosage form, administered by intravenous injection, so as to achieve systemic delivery.

[0021] The present application also provides the use of any one of the oncolytic adenovirus, the virus complex and the pharmaceutical composition in the preparation of a drug for treating brain tumors.

[0022] In some embodiments, the brain tumor comprises glioblastoma.

[0023] In summary, compared with the prior art, the present application achieves the following technical effects: 1. Strong targeting: The present application utilizes the endogenous ApoE-LDLR / LRP1 pathway to achieve double active targeting of the blood-brain barrier and tumor cells, and the penetration efficiency is significantly higher than that of passive penetration or traditional exogenous ligand modification strategies.

[0024] 2. Biomimetic intelligence: The present application fully utilizes the natural "protein crown" effect in the body to transform it from an unfavorable factor into a beneficial targeting tool, and proposes an intelligent targeting strategy for in vivo self-assembly, which is unique and innovative.

[0025] 3. High safety: The present application uses human endogenous cholesterol as a modification molecule and combines through non-covalent physical adsorption, avoiding the problems of virus inactivation or increased immunogenicity caused by chemical coupling; the modification strategy has good biocompatibility.

[0026] 4. Simple preparation: The preparation method of the present application only needs to mix the oncolytic adenovirus and cholesterol solution under suitable conditions, incubate and filter, which is simple and controllable, easy to mass produce, and has good clinical transformation prospects. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments, and it should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0028] Figure 1 Figure 1 is the characterization results of the oncolytic adenovirus-cholesterol complex of the present application embodiment 1; a is the dynamic light scattering (DLS) result; b is the transmission electron microscope (TEM) image.

[0029] Figure 2 Figure 2 is the characterization results and experimental results of the oncolytic adenovirus-cholesterol complex of the present application embodiment 1; a is the Zeta potential analysis result; b is the change result of the particle size and polydispersity index (PDI) of the oncolytic adenovirus-cholesterol after incubation in PBS for different times; c is the Dot Blot detection result; d is the MST test result.

[0030] Figure 3 Figure 3 is the experimental results of the oncolytic adenovirus-cholesterol complex of the present application embodiment 1; a is the statistical result of the total protein content stained by Coomassie brilliant blue; b is the content statistical result of apolipoprotein E; c is the Western blot result of apolipoprotein E in the oncolytic adenovirus and the oncolytic adenovirus-cholesterol; d is the quantitative analysis of the relative protein level of apolipoprotein E; e is the MST test result.

[0031] Figure 4 The in vitro anti-tumor activity detection results of the oncolytic adenovirus @ cholesterol of the present application example 3; a is the survival rate detection results of GL261 cells after being treated by different concentrations of oncolytic adenovirus; b is the survival rate detection results of GL261 cells after being treated by different concentrations of cholesterol; c is the survival rate of GL261, C6 and T98G cells under different treatment conditions for 24 h and 48 h.

[0032] Figure 5 The fluorescence microscope images of living cells (green) and dead cells (red) of GL261 cells in different treatment groups after Calcein-AM / PI staining of the present application example 3.

[0033] Figure 6 The flow cytometry detection results of the present application example 3.

[0034] Figure 7 The scratch test results of the present application example 3.

[0035] Figure 8 The blood-brain barrier penetration ability experiment results of the oncolytic adenovirus @ cholesterol-apoE complex of the present application example 4; a is the fluorescence microscope images of GL261 cells after being treated by oncolytic adenovirus and oncolytic adenovirus @ cholesterol-apoE at different time points (2H, 4H, 8H, 12H, 24H); b is the construction process of the in vitro blood-brain barrier model; c is the fluorescence intensity of different treatment groups (Control, oncolytic adenovirus, oncolytic adenovirus @ cholesterol, oncolytic adenovirus-apoE and oncolytic adenovirus @ cholesterol-apoE) analyzed by flow cytometry.

[0036] Figure 9 The blood-brain barrier penetration ability experiment results of the oncolytic adenovirus @ cholesterol-apoE complex of the present application example 4; a is the penetration of bEnd.3 to GL261 cells at different time points (1h, 6h) analyzed by flow cytometry; b is the number of penetrated cells of bEnd.3 to GL261 cells after 1 hour; c is the number of penetrated cells of bEnd.3 to GL261 cells after 6 hours; d is the number of penetrated cells of GL261 to GL261 cells after 1 hour; e is the number of penetrated cells of GL261 to GL261 cells after 6 hours.

[0037] Figure 10 The blood-brain barrier penetration ability experiment results of the oncolytic adenovirus @ cholesterol-apoE complex of the present application example 4; a is the confocal laser scanning microscope image; b is the three-dimensional multicellular tumor spheroid model; c is the fluorescence distribution of GL261 cells after being treated by oncolytic adenovirus and oncolytic adenovirus @ cholesterol-apoE at different depths (-10μm to 0μm).

[0038] Figure 11 Results of distribution and penetration ability of oncolytic adenovirus @ cholesterol complex in vivo and in vitro in Example 5 of the present application; a is the result of in vivo fluorescence imaging; b is the result of quantitative analysis of fluorescence intensity at different time points in a.

[0039] Figure 12 Results of distribution and penetration ability of oncolytic adenovirus @ cholesterol complex in vivo and in vitro in Example 5 of the present application; a is the comparison of in vivo fluorescence imaging, distribution of oncolytic adenovirus and oncolytic adenovirus @ cholesterol in different organ sections (heart, liver, spleen, lung, kidney); b is the comparison of fluorescence imaging, distribution of indocyanine green, oncolytic adenovirus and oncolytic adenovirus @ cholesterol in different organ sections (heart, liver, spleen, lung, kidney); c is the result of quantitative analysis of fluorescence intensity of different organ sections in b.

[0040] Figure 13 Results of distribution and penetration ability of oncolytic adenovirus @ cholesterol complex in vivo and in vitro in Example 5 of the present application; a is in vivo photoacoustic imaging; b is the result of quantitative analysis of photoacoustic intensity in a.

[0041] Figure 14 Results of different treatment methods on Luc + -GL261 tumor cell implanted mice in Example 6 of the present application; a is the result of tumor bioluminescence imaging of different treatment groups on the 7th, 10th, 13th, 16th and 19th day after implantation; b is the result of body weight change; c is the survival curve drawn by the Kaplan-Meier method; d is the total fluorescence intensity change of each group during treatment; e is the relative tumor growth rate; f is the statistical result of tumor inhibition rate; g is the tumor tissue section H&E staining chart; h is the statistical result of tumor / brain size ratio. DETAILED DESCRIPTION

[0042] In order to enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application are described clearly and completely. Obviously, the described embodiments are only a part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should be within the scope of protection of the present application.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials, reagents, etc. used can be obtained from commercial channels.

[0044] The preparation steps of the oncolytic adenovirus @ cholesterol complex of the present embodiment are as follows: The oncolytic adenovirus (10 μL, 1×10 12Virus particles (VPs) and cholesterol (50 μL, 3 mmol / L) were incubated in 40 μL PBS at 4 °C for 12 h. Then the mixture was sterile filtered (0.22 μm membrane) and stored at 4 °C. The morphology of oncolytic viruses was observed using transmission electron microscopy (ht7800, Hitachi, Japan). Dynamic light scattering instrument (Zetasizer nano zs90, Malvern, UK) was used to measure the hydrated particle size, Zeta potential and polydispersity index (PDI).

[0045] Dynamic light scattering (DLS) results showed that the hydrated particle size of oncolytic adenovirus increased from 78.8 nm to 164.2 nm after cholesterol modification, indicating the formation of "cholesterol coupled oncolytic adenovirus complex" (oncolytic adenovirus@cholesterol). In the simulated physiological fluid environment, the protein corona formed on the surface of naked oncolytic adenovirus increased its particle size to 141.8 nm (oncolytic adenovirus-PC), while oncolytic adenovirus@cholesterol further increased to 190.1 nm (oncolytic adenovirus@cholesterol-PC), indicating that cholesterol modification can enhance the adsorption capacity of viruses to proteins Figure 1 Transmission electron microscopy (TEM) characterization showed that both oncolytic adenovirus and oncolytic adenovirus@cholesterol exhibited good monodispersity Figure 1

[0046] Zeta potential analysis results showed that cholesterol modification significantly increased the surface charge of oncolytic adenovirus, again confirming the successful combination of cholesterol on the virus surface. Oncolytic adenovirus-protein corona and oncolytic adenovirus@cholesterol-protein corona had a displacement after the formation of the protein corona Figure 2

[0047] In order to further evaluate the colloidal stability of oncolytic adenovirus@cholesterol, it was incubated in phosphate buffered saline (PBS) at 37 °C, and its hydrated particle size and PDI were monitored by DLS within 8 days. The results showed that oncolytic adenovirus@cholesterol remained highly stable in all tested media, without obvious aggregation or PDI increase Figure 2

[0048] In this example, dot blot detection and microscale thermophoresis (MST) methods were used to characterize the protein binding properties of oncolytic adenovirus@cholesterol.

[0049] First, oncolytic adenovirus or oncolytic adenovirus@cholesterol (1 × 10 10 ​​​VPs were spotted onto a nitrocellulose membrane (NC membrane, 2 μL / spot, Solarbio). The membrane was incubated overnight at 4°C with recombinant anti-adenovirus 5 hexagonal antibody (ab316852, 1:1000 dilution). After washing three times with TBST (10 min each time), HRP-conjugated secondary antibody (Huabiotech, 1:5000, PBS) was added at room temperature under the same washing conditions, and the membrane was incubated for 2 h. The membrane was then treated with HRP-conjugated anti-rabbit IgG (CST, 7077 / 7076). ECL luminescence imaging was then used, and hexon protein was detected using a chemical imaging system (Bio-Rad). The results showed that Dot Blot analysis confirmed the presence of cholesterol-modified viral particles. Figure 2 (c).

[0050] The interaction between oncolytic adenovirus or oncolytic adenovirus@cholesterol and apolipoprotein E was analyzed using the MST method. The specific steps were as follows: Protein, apolipoprotein E, and fluorescent dye were mixed in a 1:1 ratio and incubated at room temperature for 30 min. The mixture was then centrifuged at 15000×g for 10 min at 4°C, and the supernatant was collected. Subsequently, a mixture containing an equal amount of virus particles (1×10⁻⁶) was prepared. 8 Oncolytic adenovirus (ADF) or ADF@cholesterol was mixed with an equal volume of apolipoprotein E solution (200 nM). The resulting mixture was aspirated via capillary tube and subjected to MST detection at 40% power. The direct binding of apolipoprotein E and ADF was reflected in the thermophoretic changes of the fluorescent binding proteins during complex formation. Data were analyzed using Mo affinity analysis software (version 2.3). Experimental results showed that the MST data indicated that ADF@cholesterol remained stably bound to cholesterol at both 4℃ and 37℃, and that a strong thermodynamic interaction existed (…). Figure 2 d).

[0051] In further in vivo assays, oncolytic adenovirus@cholesterol was incubated with plasma from glioma model mice at 4°C for 1 h. After separation by ultracentrifugation, the total protein adsorbed on the particle surface was detected and analyzed using Coomassie brilliant blue staining. Figure 3The results showed that the level of apolipoprotein E in the serum of glioma mice was increased, and the total protein content adsorbed on the surface of the oncolytic adenovirus@cholesterol was significantly higher than that of the naked oncolytic adenovirus group. Further detection of the apolipoprotein E content in the surface adsorbed protein showed that the oncolytic adenovirus@cholesterol group was significantly higher than the oncolytic adenovirus group. In order to quantitatively characterize the binding properties of apolipoprotein E and nanoparticles, MST was used again for affinity analysis. The results showed that compared with the naked oncolytic adenovirus, the oncolytic adenovirus@cholesterol had higher binding affinity with apolipoprotein E. The calculation results of the binding free energy further confirmed that the cholesteroid modification significantly enhanced the thermodynamic interaction between apolipoprotein E and nanoparticles.

[0052] The results of this example showed that the cholesteroid modification not only achieved effective modification of the virus particles, but also significantly enhanced the selective binding ability of the nanoparticles to apolipoprotein E, thereby improving the protein crown formation characteristics of the nanoparticles in the pathological state, and provided favorable conditions for subsequent targeted delivery applications.

[0053] In this example, the anti-tumor activity of oncolytic adenovirus@cholesterol was verified by various in vitro experiments.

[0054] First, GL261 cells were seeded in a 96-well plate at a density of 8000 cells per well, and pre-incubated at 37°C for 12 h. Then, the drugs of each treatment group were prepared in a culture medium containing 2% FBS, and added to the cells according to different multiplicity of infection (MOI) levels. After 48 h of incubation at 37°C, 10 μL of CCK-8 reagent was added to each well, and after 1 h of incubation at 37°C, the absorbance was measured at 450 nm wavelength to obtain the cell viability data.

[0055] Figure 4 The results of a~b showed the cytotoxicity of oncolytic adenovirus to GL261 glioma cells and bend.3 normal cells at different time points (24 hours) under different infection multiples (MOI). The results showed that oncolytic adenovirus exhibited significant killing effect on GL261 cells at 10 8 PFU / mL (plaque forming unit per milliliter) oncolytic adenovirus showed significant killing effect on GL261 cells, and there was no significant difference in cell viability between the cholesteroid treatment group and the control group in the concentration range of 0~10 mM, indicating that the used cholesteroid concentration had no significant effect on cell growth.

[0056] GL261, C6 and T98G and cells were seeded in a 96-well plate at a density of 8000 cells per well, and pre-incubated at 37°C for 12 h. The drugs (containing oncolytic adenovirus 10 8PFU / mL) were prepared in 2% FBS-containing medium and added to cells. After 24 h and 48 h incubation at 37 °C, 10 μL CCK-8 reagent was added to each well, and after 1 h incubation at 37 °C, the absorbance was measured at 450 nm wavelength to obtain cell viability data.

[0057] Figure 4 The results showed that GL261, C6 and T98G tumor cells all exhibited significant differences under different treatments at 24 h and 48 h. The inhibitory effect of oncolytic adenovirus-cholesterol, oncolytic adenovirus-apoE and oncolytic adenovirus-cholesterol-apoE on cell survival was significantly stronger than that of oncolytic adenovirus, and the inhibition of oncolytic adenovirus-cholesterol-apoE was the most significant.

[0058] After 48 h incubation at 37 °C and washing with serum-free DMEM, a staining solution was prepared by diluting calcein AM and propidium iodide (PI) (Beyotime Biotechnology, China) at a ratio of 1:1000, and the cells were stained at 37 °C for 30 min. After washing with PBS for three times, the live cells showed green fluorescence and the dead cells showed red fluorescence, and the cells were observed under a fluorescence microscope (Thermo Fisher Scientific, USA). The staining results further verified the above conclusion, and more red PI positive cells appeared in the oncolytic adenovirus-cholesterol and oncolytic adenovirus-cholesterol-apoE groups, indicating that they had stronger ability to induce cell death. Figure 5 ).

[0059] GL261 cells (2 x 10 5 / well) were seeded in 6-well plates, and after 24 h, each group was given drug treatment, and after 48 h incubation at 37 °C, the cells were collected and washed with pre-cooled PBS. Then, Annexin V-FITC and PI staining (Beyotime Biotechnology, China) was performed, and after 5 min incubation at room temperature in the dark, the cells were detected by flow cytometry (Agilent, Beijing, China). The flow cytometry results showed that the proportion of Annexin V-FITC and PI double positive cells in the oncolytic adenovirus-cholesterol and oncolytic adenovirus-cholesterol-apoE treatment groups increased significantly, especially in the oncolytic adenovirus-cholesterol-apoE group, the proportion of apoptotic cells was as high as more than 35%, which was much higher than that in the oncolytic adenovirus group (<10%), indicating that cholesterol modification and apoE binding can significantly enhance the ability of virus to induce cell apoptosis. Figure 6 ).

[0060] In the cell migration experiment, CT26 cells (5 x 10 4Cells were seeded in the upper chamber of a Transwell chamber (1640 medium + 10% FBS) and 500 μL of FBS-free 1640 medium was added to the lower chamber. After 24 h of culture, the lower chamber medium was replaced with 1640 medium containing 20% ​​FBS, and the upper chamber was treated with different drugs. After another 24 h of culture, the medium was removed, and the cells were fixed with 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet for 20 min. After removing non-migrated cells, images of migrating cells were taken under an optical microscope (ThermoFisher Scientific, USA).

[0061] The results showed that the number of migrating cells in the oncolytic adenovirus@cholesterol and oncolytic adenovirus@cholesterol-apolipoprotein E groups was significantly reduced, with the cell migration ability of the oncolytic adenovirus@cholesterol-apolipoprotein E group being almost completely inhibited. Figure 7 ).

[0062] The experimental results above indicate that oncolytic adenovirus modified with cholesterol (oncolytic adenovirus@cholesterol) exhibits enhanced anti-proliferative and pro-apoptotic effects in various tumor cell models. The combination of oncolytic adenovirus@cholesterol with apolipoprotein E significantly enhances its inhibitory effects on cell activity, promotes apoptosis, and inhibits cell migration compared to oncolytic adenovirus or the oncolytic adenovirus-apolipoprotein E group. Cholesterol modification not only improves the stability of viral particles but also endows them with stronger anti-tumor efficacy. Further binding with apolipoprotein E exerts a synergistic enhancing effect, demonstrating that cholesterol modification can enhance the anti-tumor activity and targeting properties of nanoparticles.

[0063] To evaluate the blood-brain barrier penetration ability of oncolytic adenovirus@cholesterol-apolipoprotein E, an in vitro blood-brain barrier model was constructed, such as... Figure 3 As shown in b, the specific method is as follows: bEnd.3 cells (1×10) 5 Cells per well were seeded onto Transwell inserts with a 0.4 μm polyester membrane and allowed to develop until the transepithelial electrical resistance (TEER) reached 200 Ω·cm. 2 After the above, GL261 cells (4 × 10⁶) were added to the inferior vena cava. 5 (cells / well). After incubation with the drug for 8 hours, GL261 cells were collected from the lateral basement cavity, and the fluorescence signal penetrating the cavity was detected by flow cytometry and confocal microscopy (CLSM) to assess the penetration efficiency.

[0064] The results showed that the fluorescence intensity of the oncolytic adenovirus@cholesterol-apolipoprotein E treatment group gradually increased, indicating that its intracellular accumulation was greater than that of the oncolytic adenovirus treatment group. This suggests that oncolytic adenovirus@cholesterol-apolipoprotein E can effectively cross the blood-brain barrier model and be taken up by inferior vena cava tumor cells. Figure 8a).

[0065] The blood-brain barrier is a highly selective semi-permeable membrane boundary formed by endothelial cells, which severely limits the delivery of drugs from the circulation to the brain, leading to poor treatment efficacy of glioblastoma. To solve this problem, the present embodiment uses bEnd.3 cells to establish an in vitro BBB model. When the transendothelial TEER value is stable at 200 Ω-cm 2 The above indicates that the BBB model has been successfully constructed Figure 8 b). By comparing the BBB penetration efficiency of the four preparations, oncolytic adenovirus @ cholesterol, oncolytic adenovirus-ApoE and oncolytic adenovirus @ cholesterol-apoE, it is found that the transcytosis efficiency of the lower cavity is significantly higher in the oncolytic adenovirus @ cholesterol-apoE-treated group than in the other groups Figure 8 c). Further evaluation of the transcytosis of oncolytic adenovirus @ cholesterol-apoE from bEnd.3 to GL261 cells or between GL261 cells. The samples are pretreated with bEnd.3 or GL261 cells for 4 hours, and then GL261 cells stained with Hoechst-33342 are added for different time periods. The oncolytic adenovirus group shows negligible transcytosis, as the fluorescence changes little over time. In particular, oncolytic adenovirus @ cholesterol-apoE shows enhanced transcytosis during the extended incubation period Figure 9 a~c). After 6 hours of incubation, oncolytic adenovirus @ cholesterol-apoE reaches 78.59% and 73.15% respectively (bEnd.3→GL261) and (GL261→GL261) compared with oncolytic adenovirus Figure 9 d~e). Fluorescence imaging results show that the oncolytic adenovirus @ cholesterol-apoE-treated group exhibits stronger fluorescence signals in both the insert and bottom chambers, further confirming its excellent BBB penetration performance Figure 10 a).

[0066] To further evaluate the penetration ability of nanoparticles in solid tumors, a three-dimensional multicellular tumor spheroid (MCTS) model derived from GL261 cells is established Figure 10 b). CLSM Z-stack imaging results show that after 8 hours, significant Cy5 fluorescence signals of oncolytic adenovirus @ cholesterol-apoE are observed at all scanning depths (especially 0 µm), while the non-targeted oncolytic adenovirus-treated group only shows weak fluorescence in the superficial area Figure 10 c). These results fully confirm that oncolytic adenovirus @ cholesterol-apoE can effectively penetrate the BBB and penetrate deep into tumor tissues, which is mainly attributed to its specific interaction with ApoE and the LDLR family receptors, which are highly expressed on the surface of BBB endothelial cells and tumor cells.

[0067] (1) Construction of orthotopic glioma mouse model To verify the antitumor activity of oncolytic adenovirus cholesterol in vivo, an orthotopic glioma mouse model was established. The specific steps are as follows: Female C57BL / 6 mice (6-8 weeks old, purchased from Hangzhou Qizhen Experimental Animal Technology Co., Ltd.) were selected. In order to construct the brain tumor model of C57BL / 6 mice, the mice were placed under gaseous anesthesia and fixed on the brain stereotaxic instrument. After craniotomy, 2 μL of 5.0 × 10 4 GL261 cells expressing luciferase (Luc + ) were slowly injected into the right striatum of the mice, and the injection coordinates were 0.5 mm posterior to the Bregma point, 2.0 mm to the right, and 2.5 mm ventral. The tumor growth in the brain was monitored by bioluminescence signal using an in vivo fluorescence imaging system (IVIS Spectrum, PerkinElmer).

[0068] (2) In vivo distribution and targeting research After successfully verifying the orthotopic glioma mouse model, free ICG-NHS, oncolytic adenovirus or oncolytic adenovirus cholesterol (1 × 10 10 VPs) were injected into mice, and their distribution was recorded by IVIS fluorescence imaging at different time points (2, 4, 8, 12 and 24 hours). Euthanasia was performed 8 hours after injection, and then the main organs and tumor tissues were collected. The in vivo organ distribution was evaluated by IVIS imaging. The tumor tissue was sectioned, DAPI stained, and ICG fluorescence was detected using CLSM. Quantitative analysis of fluorescence intensity in tissues provided data for in vivo distribution and tumor targeting efficiency. The distribution of oncolytic adenovirus and oncolytic adenovirus cholesterol (100 μL, 1 × 10 10 VPs) in nude mice was evaluated using in vivo photoacoustic imaging (LOIS-3D system, Tomowave, Suzhou, China). Under isoflurane anesthesia, the drugs were injected, and 8 hours later, photoacoustic imaging (PAI) was performed at 880 nm. Then the mice were sacrificed and the brain tissue was collected for further examination.

[0069] Based on their excellent BBB penetration ability and tumor targeting properties, oncolytic adenovirus@cholesterol was further evaluated for their biodistribution and behavior in vivo in vitro experiments. After the Luc+-GL261 orthotopic glioma mouse model was constructed and the successful establishment of the model was verified using the in vivo imaging system (IVIS), a systematic in vivo distribution study was conducted. Oncolytic adenovirus and oncolytic adenovirus@cholesterol will be combined with ICG-NHS drugs intravenously injected into tumor-bearing mice. In vivo imaging shows that the oncolytic adenovirus@cholesterol group is compared with the oncolytic adenovirus at all time points, indicating that the targeting of oncolytic adenovirus is enhanced by cholesteryl modification. It is worth noting that oncolytic adenovirus@cholesterol is still very strong after 24 hours, while those in the oncolytic adenovirus group have largely disappeared, indicating that the oncolytic adenovirus group has a longer in vivo circulation time oncolytic adenovirus@cholesterol (a). Analysis shows that oncolytic adenovirus@cholesterol is compared with free ICG and oncolytic adenovirus at 8 hours after injection (b), which is mainly due to its immune escape and ApoE-mediated targeting. To elucidate whether the tumor targeting of oncolytic adenovirus@cholesterol is based on ApoE adsorption, the groups were compared after 8 hours of free ICG, oncolytic adenovirus and oncolytic adenovirus@cholesterol treatment. The results show that only the oncolytic adenovirus@cholesterol-treated group presents a clear fluorescent signal in the brain tumor area, while the free ICG group has no obvious tumor targeting, ruling out the possibility of ICG self-targeting (a). Organ distribution studies show that oncolytic adenovirus@cholesterol presents strong fluorescent signals in the liver and kidneys, indicating that it is mainly metabolized and cleared through the liver-kidney pathway, which is consistent with the mechanism of reticuloendothelial system capture and clearance of nanoparticles (b~c). In addition, the photoacoustic imaging (PAI) performance of oncolytic adenovirus@cholesterol was evaluated. In vivo PAI results show that the photoacoustic signal at the tumor site is significantly enhanced after 8 hours. Isolated brain tissue PAI results are consistent with in vitro imaging, further confirming the specificity of PAI to accumulate oncolytic adenovirus@cholesterol at the tumor site (a~b). Figure 11 Figure 11 Figure 12 Figure 12 Figure 13

[0070] ​​​​​All animal experiments in this example were performed and approved by the Animal Ethics Committee. One week after inoculation of Luc+-GL261 tumor cells, all mice were randomly divided into four groups (n=6), (1) control group (PBS treatment); (2) cholesterol; (3) oncolytic adenovirus; (4) oncolytic adenovirus@cholesterol. Each group was given 100 μL of drug (containing 10 μL of oncolytic adenovirus) on days 8, 10, 12, 14 and 16. In order to monitor the inhibition of glioma by different drugs, intraperitoneal injection of luciferin potassium (10 μL / g) was performed on days 7, 10, 13, 16 and 19 after implantation. The bioluminescence intensity of the tumor was dynamically observed by IVIS-lumina S5 system. After the relative photon flux of the tumor was unified, the fluorescence intensity curve was drawn.

[0071] The calculation formula of tumor inhibition rate of each group is: tumor inhibition rate (TIR) = (I PBS -I therapy ) / I PBS ×100%, where I PBS is the bioluminescence intensity of the PBS group on the same day, and I therapy is the bioluminescence intensity of the nanodrug treatment group. At the same time, the tumor bioluminescence growth rate before and after treatment = I Day19 / I Day7 . During the treatment, the body weight of the mice was recorded every two days, and the Kaplan-Meier survival curve was drawn.

[0072] Evaluation of the in vivo anti-tumor efficacy of the drug oncolytic adenovirus@cholesterol, this example established a Luc⁺-GL261 orthotopic glioma model. Seven days after stereotactic injection into the right striatum of female C57BL / 6 mice, tumor implantation was confirmed by IVIS imaging. Then the mice were randomly divided into four groups and received intravenous injection of PBS, Cho, oncolytic adenovirus or oncolytic adenovirus@cholesterol once every two days. After intraperitoneal injection of D-luciferin on days 7, 10, 13, 16 and 19, the tumor growth was dynamically monitored using in vivo bioluminescence imaging (a) of FIG. 10. The results showed that the PBS and Cho-treated control mice showed a tendency of rapid growth, while the oncolytic adenovirus and oncolytic adenovirus@cholesterol-treated groups showed different degrees of tumor growth inhibition (e) of FIG. 10. Notably, the oncolytic adenovirus@cholesterol group showed the weakest bioluminescence signal on day 19, and quantitative analysis showed that the tumor bioluminescence intensity of the PBS, Cho and oncolytic adenovirus groups increased by 70.38%, 64.94% and 58.86%, respectively. While the oncolytic adenovirus@cholesterol group only increased by 16.18% (d) of FIG. 10. The tumor inhibition rate (TIR) of oncolytic adenovirus@cholesterol reached 86%, which was significantly higher than that of other treatment groups (f) of FIG. 10. Figure 14 Figure 14 Figure 14 ​​​Figure 14 Fig. 6. Kaplan-Meier survival analysis of Luc-GL261 orthotopic glioma model treated with different groups. The significant anti-tumor effect of Ad-Chol was observed in the Kaplan-Meier survival analysis (Fig. 6f). This significant anti-tumor effect might be due to the specific targeting of glioma by the enhanced oncolytic adenovirus modified by cholesterol. Kaplan-Meier survival analysis further confirmed that oncolytic adenovirus@cholesterol treatment significantly prolonged the median survival of the bearing mice (Fig. 6f). Figure 14 Fig. 7. Body weight changes of mice treated with different groups. The treatment-related toxicity was evaluated by monitoring the changes in body weight of mice during the treatment. The results showed that the body weight of mice in each group showed a slow downward trend, but there was no significant difference between groups (Fig. 7c). This indicated that oncolytic adenovirus@cholesterol had good biosafety. Figure 14 Fig. 8. Pathological analysis of brain tissues of mice treated with different groups. The results of gross observation showed that the tumor volume of the oncolytic adenovirus@cholesterol group was the smallest, which was consistent with the biopsy results (Fig. 8b). H&E staining showed that the cell structure of the oncolytic adenovirus@cholesterol group was obviously loose, and the tissue damage was extensive, while the tumor cells in the control group were intact (Fig. 8d). This indicated that oncolytic adenovirus@cholesterol had good biosafety (Fig. 8b). The slow decline in body weight might be related to the dysfunction of the central nervous system caused by the rapid progression of glioma. After the mice were euthanized on day 19, brain tissues were taken for pathological analysis. Gross observation showed that the tumor volume of the oncolytic adenovirus@cholesterol group was the smallest, which was consistent with the biopsy results (Fig. 8b). H&E staining showed that the cell structure of the oncolytic adenovirus@cholesterol group was obviously loose, and the tissue damage was extensive, while the tumor cells in the control group were intact (Fig. 8d). This indicated that oncolytic adenovirus@cholesterol had good biosafety (Fig. 8b). Figure 14 Fig. 9. The mechanism of oncolytic adenovirus@cholesterol in Luc-GL261 orthotopic glioma model. In summary, oncolytic adenovirus@cholesterol showed significant anti-tumor effect in Luc-GL261 orthotopic glioma model, and the mechanism might involve multiple aspects such as enhancing tumor targeting, inducing tumor cell apoptosis, and inhibiting tumor cell proliferation. + Fig. 9. The mechanism of oncolytic adenovirus@cholesterol in Luc-GL261 orthotopic glioma model. In summary, oncolytic adenovirus@cholesterol showed significant anti-tumor effect in Luc-GL261 orthotopic glioma model, and the mechanism might involve multiple aspects such as enhancing tumor targeting, inducing tumor cell apoptosis, and inhibiting tumor cell proliferation.

[0073] The above merely describes preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An oncolytic adenovirus for targeted therapy of brain tumors, characterized in that, The oncolytic adenovirus is modified with cholesterol, wherein the cholesterol is modified on the surface of the oncolytic adenovirus in a non-covalent manner.

2. The oncolytic adenovirus according to claim 1, characterized in that, The oncolytic adenovirus is adenovirus type 5, which lacks the E1B-55kD gene and the 78.3-85.8 mu segment of the E3 region.

3. The oncolytic adenovirus according to claim 1, characterized in that, The hydrated particle size of the oncolytic adenovirus is 150~200 nm.

4. The oncolytic adenovirus according to claim 1, characterized in that, The oncolytic adenovirus has apolipoprotein E bound to its surface.

5. The method for preparing oncolytic adenovirus according to any one of claims 1 to 4, characterized in that, Includes the following steps: The oncolytic adenovirus was mixed with a cholesterol solution and incubated at 0–6°C for 8–16 h. The mixture was then filtered to obtain the oncolytic adenovirus.

6. The preparation method according to claim 5, characterized in that, The concentration of the oncolytic adenovirus was 10. 10 ~10 12 VP / mL; The concentration of the cholesterol solution is 1~5 mmol / L; Among them, each 1×10 12 The amount of cholesterol solution used for VP oncolytic adenovirus is 2-4 μmol.

7. A viral complex, characterized in that, The viral complex comprises the oncolytic adenovirus according to any one of claims 1 to 4.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the oncolytic adenovirus as described in any one of claims 1 to 4 and a pharmaceutically acceptable vector.

9. The use of any one of the oncolytic adenoviruses of claims 1 to 4, the viral complex of claim 7, and the pharmaceutical composition of claim 8 in the preparation of a medicament for treating brain tumors.

10. The application according to claim 9, characterized in that, The brain tumors include glioblastoma.

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