Preparation method and application of preparation based on platelet targeting oncolytic virus delivery

Abstract

The invention provides a preparation method and application of a platelet-based targeting oncolytic virus delivery preparation, effective loading of oncolytic viruses is realized by infecting platelets with the oncolytic viruses, and meanwhile, the cellular structure of the platelets can be used as a physical barrier, so that the platelet-based targeting oncolytic virus delivery preparation is prepared. The tumor targeting drug delivery system shields the neutralizing and removing effects of in-vivo neutralizing antibodies / complements / removing cells on the oncolytic viruses, so that the oncolytic viruses can enter the body in a systematic drug delivery mode, tumor targeting drug delivery of the oncolytic viruses is achieved through the tumor targeting capacity of platelets, and the success of intravenous drug delivery and tumor targeting of the oncolytic viruses is achieved. The invention provides a technical support and a new idea for wider and more effective treatment of cancers, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of drug delivery, and in particular, relates to a preparation method and application of a platelet-targeted oncolytic virus delivery preparation. Background Art

[0002] Oncolytic virus is a new type of immunotherapy that can specifically kill tumor cells and spare normal cells through direct oncolytic effects and inducing anti-tumor immune responses. It is regarded as a potential new star in tumor immunotherapy. Currently, many oncolytic virus products have been approved for marketing around the world, but these approved products are all intratumorally injected, which limits the expansion and promotion of product indications. Oncolytic viruses administered intravenously can be applied to a wider range of tumor lesions and do not require complex clinical interventional procedures. However, currently approved products are difficult to achieve the goal of intravenous injection due to the antibodies and complement-mediated neutralization produced in the body after repeated administration, as well as the difficulty in achieving effective targeting of tumor lesions.

[0003] An invention submitted by the applicant is entitled: Engineered cells for targeted delivery of oncolytic viruses, their preparation methods and applications. In the invention patent with application publication number CN118460469A, it is specifically disclosed that the engineered cells for targeted delivery of oncolytic viruses do not affect the activity of oncolytic viruses, have strong resistance to antibody neutralization, have good long circulation ability, target tumor lesions and inhibit tumors compared with simple oncolytic virus preparations. However, the engineered cells are tumor cells. After being infected with oncolytic viruses, the tumor cells need to be immersed in liquid nitrogen for freezing treatment to make them lose their proliferation ability and pathogenicity. On the one hand, it increases the complexity of the operation and the cost of the experiment. On the other hand, the treatment is accidental and cannot completely guarantee that the tumor cells have completely lost their pathogenicity. Even if it is a very small possibility, there is a certain safety risk. Therefore, in order to further improve the safety of the experiment, reduce the complexity of the operation and the cost of the experiment, further exploration is still needed.

[0004] Therefore, it is urgent to find a method for preparing an oncolytic virus delivery vector, which can protect oncolytic cells in the blood circulation, prevent them from being neutralized by antibodies, and realize the intravenous injection of oncolytic viruses; at the same time, the vector can also effectively target tumor lesions, and realize targeted oncolytic virus delivery to circulating tumor cells and tumor metastases. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a preparation method and application of a platelet-targeted oncolytic virus delivery preparation. By infecting platelets with oncolytic viruses, effective loading of oncolytic viruses is achieved. At the same time, the cell structure of platelets can serve as a physical barrier to shield the neutralization and clearance of oncolytic viruses by neutralizing antibodies / complement / scavenging cells in the body, so that oncolytic viruses can enter the body through systemic administration, and tumor-targeted drug delivery of oncolytic viruses can be achieved through the tumor targeting ability of platelets themselves.

[0006] In order to achieve the above object, the present invention adopts the following scheme:

[0007] In one aspect, the present invention provides a cell complex for targeted delivery of oncolytic viruses, comprising platelets and oncolytic viruses; the oncolytic viruses are loaded on platelets by infecting platelets.

[0008] In some embodiments, the present invention successfully prepares a platelet-targeted oncolytic virus delivery preparation, so that the platelets are successfully loaded with oncolytic viruses, and the oncolytic viruses are basically all loaded in the platelets, and still have good activity. At the same time, the surface morphology of the platelets is intact and has not been activated, and still has the function of being activated, aggregating, and adhering to tumor cells. At the same time, the prepared P-OV also has tumor targeting, successfully realizing tumor-targeted drug delivery of oncolytic viruses.

[0009] Furthermore, the oncolytic cells infect platelets by co-culturing with platelets.

[0010] In some embodiments, the methods of oncolytic virus infection of platelets were screened, and the experimental results showed that the method of co-incubation of oncolytic virus and platelets to infect platelets is preferred. At this time, while the infection efficiency is the highest, the anti-tumor ability of the prepared P-OV is also the highest.

[0011] Furthermore, the oncolytic virus includes any one or more combinations of oncolytic adenovirus, vaccinia virus, herpes simplex virus, Newcastle virus, parvovirus, vesicular stomatitis virus, reovirus or poliovirus.

[0012] On the other hand, the present invention provides a platelet-targeted oncolytic virus delivery preparation, which comprises the cell complex described in any one of the above technical solutions.

[0013] In yet another aspect, the present invention provides a kit comprising the cell complex described in any one of the above technical solutions.

[0014] On the other hand, the present invention provides a method for preparing a cell complex as described in any one of the above technical solutions, comprising the following steps: (1) co-incubating oncolytic virus and platelets; (2) after secondary centrifugation, removing free virus particles to obtain the cell complex.

[0015] Furthermore, in the step (1), the ratio of the oncolytic virus titer to the platelet count is 1:1-1:1000, and the co-incubation time is 1-12 h.

[0016] In another aspect, the present invention provides a use of the cell complex described in any one of the above technical solutions for preparing a preparation for improving the ability to target tumor lesions or anti-tumor.

[0017] In some embodiments, the anti-tumor ability of platelet-targeted oncolytic virus delivery preparations was compared and tested, and the experimental results showed that: 6 hours after the tail vein inoculation of tumor cells, different preparations were used for treatment. Among the groups treated differently, the P-OV group showed the best tumor inhibition effect, and the tumor growth was the slowest; while the platelet and oncolytic virus mixed group and the naked virus group also showed partial anti-tumor efficacy, but the inhibition efficiency was much lower than that of the P-OV group, and there were significant differences. At the same time, nearly 65% ​​of the mice treated with P-OV survived for more than 30 days. In contrast, all the other three groups of mice died within 30 days. It can be seen that the platelet-targeted oncolytic virus delivery preparation prepared by the present invention can significantly inhibit tumor growth and has extremely strong anti-tumor ability.

[0018] In another aspect, the present invention provides a use of the cell complex as described in any one of the above technical solutions for preparing a preparation for improving the long circulation ability of a drug in vivo or extending the half-life of a drug in vivo.

[0019] In some embodiments, the pharmacokinetic properties of platelet-targeted oncolytic virus delivery formulations in mice were explored, and the experimental results showed that compared with naked oncolytic viruses, P-OV significantly prolonged the half-life of oncolytic viruses in mice from the initial 7 minutes to 69 minutes, indicating that P-OV has a stronger long-circulation ability in vivo.

[0020] On the other hand, the present invention provides a use of platelets for preparing a preparation for improving the adhesion efficiency of cell complexes to tumors or the neutralization ability of antibodies in vivo, wherein the cell complex comprises platelets and oncolytic viruses; the oncolytic viruses are loaded on platelets by infecting platelets; and the oncolytic cells infect platelets by co-incubation and culture with platelets.

[0021] The beneficial effects of the present invention are:

[0022] 1. The present invention provides a method for preparing a platelet-targeted oncolytic virus delivery preparation, which achieves effective loading of oncolytic viruses by infecting platelets with oncolytic viruses. On the one hand, the cell structure of platelets acts as a physical barrier to shield the neutralizing and scavenging effects of neutralizing antibodies / complement / scavenging cells in the body on oncolytic viruses, allowing oncolytic viruses to enter the body through systemic administration; on the other hand, tumor-targeted drug delivery of oncolytic viruses is achieved through the tumor targeting ability of platelets themselves.

[0023] 2. Compared with simple oncolytic virus preparations, the platelet-targeted oncolytic virus delivery preparation prepared by the present invention has the following characteristics: 1. It does not affect the activity of oncolytic viruses, nor does it affect the cellular functions and surface molecular activities of platelets; 2. The platelet structure acts as a physical barrier to shield the neutralization and clearance effects of antibodies in the body on oncolytic viruses, thereby achieving intravenous administration of oncolytic viruses; 3. The tumor targeting ability of platelets themselves enables oncolytic viruses to have a stronger ability to target tumor lesions; 4. It has better long circulation ability and significantly improves the half-life of oncolytic viruses in mice; 5. It significantly improves the anti-tumor ability.

[0024] 3. The platelet-targeted oncolytic virus delivery preparation provided by the present invention successfully achieves intravenous administration and tumor targeting, provides technical support and new ideas for a wider and more effective treatment of cancer, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a scanning electron micrograph of a platelet (P-OV) loaded with an oncolytic virus according to an embodiment of the present invention, with a scale bar of 250 nanometers.

[0026] Figure 2 This is a super-resolution confocal microscopy image of platelets loaded with Cy5-labeled oncolytic virus in an embodiment of the present invention, with a scale of 1 micron. The dotted line indicates a single platelet.

[0027] Figure 3 TEM images of P-OV and natural platelets in an embodiment of the present invention. The scale bar of the left image is 1 micron, and the scale bar of the right image is 200 nanometers.

[0028] Figure 4 This is the ratio of platelets loaded with Cy5-labeled oncolytic virus detected by flow cytometry in an embodiment of the present invention.

[0029] Figure 5 This is a transmission electron microscopy image of the supernatant released by platelets loaded with oncolytic virus in an embodiment of the present invention.

[0030] Figure 6In the examples of the present invention, it was verified that infection of platelets with oncolytic adenovirus Ad5 in the presence of PGE1 for 4 hours did not significantly cause platelet activation.

[0031] Figure 7 To verify that platelets can adhere to tumor cells, flow cytometry gate images, fluorescence microscopy and scanning electron microscopy images were taken.

[0032] Figure 8 The protective effect of platelets on oncolytic viruses in the presence of neutralizing antibodies in the embodiment of the present invention

[0033] Fig. 9 : In vivo pharmacokinetic curves of P-OV and naked OV in the examples of the present invention.

[0034] Fig.10 A is the small animal fluorescence imaging results and quantitative statistical graph of the difference in enrichment of lung tumor metastases between P-OV treated with liquid nitrogen frozen cells and naked OV after tail vein administration in an embodiment of the present invention.

[0035] Fig.10 B is the flow cytometry detection result of the difference in enrichment of P-OV and naked OV in lung tumor metastases after tail vein administration in the embodiment of the present invention.

[0036] Fig.10 C is the targeting result of P-OV on circulating tumor cells in the blood.

[0037] Fig.11A Bioluminescent images of tumors in each group of mice treated with different preparations.

[0038] Fig. 11B The bioluminescence statistical analysis chart of the tumor sites of each group of mice after treatment.

[0039] Fig. 11C The survival of mice after receiving different preparations treatment. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below in conjunction with the examples. It should be pointed out that the examples described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on the present invention.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0042] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0043] Unless otherwise specified, the oncolytic viruses used in the embodiments of the present invention are all oncolytic adenovirus Ad5, and the tumor cells used in the embodiments are all B16 or 4T1 cells.

[0044] The instruments used in the examples of the present invention are shown in Table 1, and the experimental reagents used in the examples are shown in Table 2.

[0045] Table 1 Instruments used in the examples

[0046]

[0047] Table 2 Experimental reagents used in the examples

[0048]

[0049]

[0050] Example 1 Preparation method of engineered cells for targeted delivery of oncolytic viruses

[0051] 1. Optimal preparation of platelets loaded with oncolytic viruses (P-OV)

[0052] In a 1 μM PGE1 DPBS solution, the oncolytic virus titer and platelet number were incubated at a ratio of 1:1-1000:1 for 1-12 hours. In this embodiment, the optimal ratio between the oncolytic virus titer and platelet was 10:1, and the optimal incubation time was 4 hours. After the oncolytic virus infection entered the platelets, the platelets were centrifuged at 1000 g for 10 minutes, and the platelets loaded with oncolytic virus were in the sedimentation layer, and the supernatant was removed. After two centrifugation operations to remove the free virus particles, a platelet-delivered oncolytic virus system was obtained.

[0053] The types of oncolytic viruses include, but are not limited to, oncolytic adenovirus, vaccinia virus, herpes simplex virus, Newcastle virus, parvovirus, vesicular stomatitis virus, reovirus, poliovirus and other viruses that can infect platelets.

[0054] 2. Verification of successful oncolytic virus loading of platelets and functional testing of platelets and P-OV

[0055] 1. Results under scanning electron microscope

[0056] The prepared platelets loaded with oncolytic virus (P-OV) were observed under a scanning electron microscope. The specific results are as follows Figure 1 As shown, Figure 1 Figure 1 is a scanning electron microscope image of a platelet (P-OV) loaded with an oncolytic virus in an embodiment of the present invention, with a scale of 250 nanometers. P-OV is the surface morphology of a platelet loaded with an oncolytic virus after the platelet is infected with an oncolytic virus; platelet is the surface morphology of a normal platelet. Figure 1 It can be seen that after platelets are infected with oncolytic viruses, the surface morphology of platelets loaded with oncolytic viruses is similar to that of natural platelets, which indicates that the oncolytic virus is loaded inside the cells and the amount of virus outside the cells is not significant.

[0057] 2. Cy5 labeling results

[0058] Before preparation, platelets were stained with FITC and oncolytic viruses were labeled with Cy5. After P-OV was prepared, co-localization of platelets and Cy5-labeled oncolytic viruses showed that oncolytic viruses were successfully loaded into platelets. Figure 2 As shown, Figure 2 This is a super-resolution confocal microscopy image of platelets loaded with Cy5-labeled oncolytic virus in an embodiment of the present invention, with a scale of 1 micron. The dotted line marks a single platelet.

[0059] 3. Results of P-OV slices under transmission electron microscope

[0060] The platelets loaded with oncolytic viruses were fixed and sliced, and transmission electron microscopy results showed that there were a large number of virus particles in the platelets. Figure 3 As shown, Figure 3 TEM images of P-OV and natural platelets in an embodiment of the present invention. The scale bar of the left image is 1 micron, and the scale bar of the right image is 200 nanometers.

[0061] 4. Flow cytometry test results

[0062] When platelets were infected with Cy5-labeled oncolytic virus, flow cytometry was used to detect the proportion of cells with Cy5 signals. The specific results are as follows Figure 4 As shown, Figure 4 is the ratio of platelets loaded with Cy5-labeled oncolytic virus detected by flow cytometry in the embodiment of the present invention, wherein control is natural platelets. Figure 4 It can be seen that almost all platelets are infected by oncolytic viruses.

[0063] 5. Transmission electron microscopy results of oncolytic virus released by P-OV

[0064] In this example, platelets loaded with oncolytic virus were collected and cultured in vitro with shaking for 12 h. The supernatant was collected by centrifugation at a centrifugal speed of 1000 g, and transmission electron microscopy images were taken. The specific results are as follows: Figure 5 As shown, Figure 5 Figure 1 is a transmission electron micrograph of the supernatant released from platelets loaded with oncolytic virus in an embodiment of the present invention. Figure 5It can be seen that P-OV can release oncolytic viruses in two forms: ① Release in the form of pure oncolytic virus; ② Release in the form of oncolytic virus encapsulated in platelet granules. This may be because oncolytic viruses may exist in a free form in the cytoplasm of platelets or in the granular structure inside platelets. The advantage of having two forms of release is that even if the oncolytic virus is released from the platelets, the platelet granules can still protect the oncolytic virus from being cleared by neutralizing antibodies.

[0065] 6. Verification that platelets are not activated

[0066] Platelets were infected with oncolytic virus at a PEG1 (prostaglandin E1; inhibitor of platelet activation) concentration of 1 nM, and then the expression of platelet activation indicator CD62p was detected by flow cytometry. The specific results are as follows Figure 6 As shown, Figure 6 In the present invention, it was verified that the infection of platelets with oncolytic adenovirus Ad5 in the presence of PGE1 for 4 hours did not significantly cause platelet activation. Figure 6 It can be seen that there is no significant difference in the expression of CD62p between P-OV and natural platelets, which indicates that oncolytic virus infection of platelets does not cause significant activation of platelets, making the use of the preparation of the present invention similar to normal platelet transfusion. At the same time, infusion of unactivated platelets into the body can avoid the risk of thrombosis.

[0067] 7. Verification of tumor targeting of both platelets and P-OV

[0068] After CFSE-labeled platelets and cell tracker deep red-labeled B16 tumor cells were co-cultured in a non-adherent culture plate for 4 hours, the cells were centrifuged at 200 g for 5 minutes, the supernatant was removed, and the cells were resuspended in PBS and centrifuged again to harvest the cell pellets. The cell pellets were characterized by flow cytometry, fluorescence microscopy, and scanning electron microscopy. The specific results are shown in Figure 2. Figure 7 As shown, Figure 7 To verify that platelets can adhere to tumor cells, flow cytometry gate images, fluorescence microscopy and scanning electron microscopy images all prove that platelets can adhere to tumor cells in vitro. Therefore, platelets have tumor targeting properties.

[0069] At the same time, the tumor targeting of P-OV was also verified in this example. Similarly, CFSE-labeled P-OV and cell tracker deep red-labeled B16 tumor cells were co-cultured in a non-adhesive culture plate for 4 hours, centrifuged at 200g, the supernatant was removed for 5 minutes, and the cell pellet was harvested after resuspending with PBS. The cell pellet was characterized by flow cytometry, fluorescence microscopy and scanning electron microscopy, and the experimental results showed that P-OV can adhere to tumor cells in vitro, so P-OV also has tumor targeting.

[0070] 8. Testing of oncolytic virus activity in P-OV

[0071] In order to verify that the process of oncolytic virus loading on platelets does not affect the activity of oncolytic virus, P-OV and naked oncolytic virus of the same titer were used to infect HEK 293 cells (Pusano Life Science Co., Ltd., CL-0001), respectively. The oncolytic cells can express EGFP protein, and the expression of protein in HEK 293 cells is detected to indicate active virus. The results show that P-OV has similar activity as naked oncolytic virus.

[0072] It can be seen that in the P-OV prepared by the optimal preparation method in this embodiment, the platelets are successfully loaded with oncolytic viruses, and the oncolytic viruses are basically all loaded in the platelets, and still have good activity. At the same time, the surface morphology of the platelets is intact and has not been activated, and still has the function of normal platelets. At the same time, the prepared P-OV also has tumor targeting, and successfully realizes tumor-targeted drug delivery of oncolytic viruses.

[0073] Example 2 Pharmacokinetic Experimental Test of Platelet-Targeted Oncolytic Virus Delivery Preparation

[0074] 1. Testing of platelets as a protective barrier

[0075] One of the biggest problems with intravenous administration of oncolytic viruses is that they are easily neutralized by antibodies, complement or scavenger cells in the circulatory system. In the present invention, the OV group and the P-OV group containing the same virus content were used to infect tumor cells in the presence of neutralizing antibodies. The P-OV group showed higher EGFP expression, indicating that the platelets in the P-OV group can protect the oncolytic virus in the presence of neutralizing antibodies.

[0076] Specific results such as Figure 8 As shown, Figure 8 The protective effect of platelets on oncolytic viruses in the presence of neutralizing antibodies in the embodiments of the present invention. The antibodies used in the present invention are extracted from the serum of mice that have been immunized with viruses in advance.

[0077] It can be seen that the cell structure of platelets can serve as a physical barrier to shield the neutralizing and clearing effects of neutralizing antibodies / complement / scavenging cells on oncolytic viruses in the body, allowing oncolytic viruses to enter the body through systemic administration;

[0078] 2. Comparative testing of P-OV pharmacokinetics

[0079] In this example, the pharmacokinetic properties of P-OV and naked oncolytic viruses in mice were further explored, and it was found that the P-OV group exhibited better long-circulation ability and significantly improved the half-life of oncolytic viruses in mice. The specific steps of the pharmacokinetic experiment are as follows: C57BL / 6 mice were divided into OV group and P-OV group. Each group of mice was injected with the corresponding preparation at 0h, and 50 microliters of blood was collected from the eye sockets at 5min, 30min, 1h, 2h, and 4h, respectively. The genome of these 50 microliters of blood was extracted and the virus content in the blood was analyzed using virus-specific primers. The specific results are as follows: Fig. 9 As shown, Fig. 9 In vivo pharmacokinetic curves of P-OV and naked OV in the embodiment of the present invention (OV: oncolytic virus), Fig. 9 It can be seen that the pharmacokinetic properties of the virus in the P-OV group in the blood are significantly better than those in the OV group.

[0080] It can be seen that compared with naked oncolytic virus, P-OV significantly prolonged the half-life of oncolytic virus in mice, from the initial 7 minutes to 69 minutes, indicating that P-OV has a stronger long-circulation ability in vivo.

[0081] Example 3 Testing of the ability of platelet-targeted oncolytic virus delivery preparations to accumulate at tumor sites

[0082] Most of the oncolytic virus products at this stage are limited by the effective enrichment of the tumor site, making it difficult to exert their efficacy in intravenous administration. Therefore, in this embodiment, the ability of the P-OV preparation to improve the enrichment of oncolytic viruses in the tumor site was tested, and the experimental steps were as follows: OV and P-OV containing equal amounts of virus were injected into the tail vein, respectively, where OV was labeled with cy5.5, and the lungs of mice in the OV group and P-OV group were removed after 12 hours, and the fluorescence of OV in the lungs was analyzed by the small animal imaging system. The lungs were then digested into a single cell suspension for flow cytometry analysis, and the content of OV cells in the lungs was analyzed by the fluorescence of cy5.5. In order to verify the targeting effect of P-OV on circulating tumor cells in the blood, 2 million CFSE-labeled B16 tumor cells were injected into mice, and then P-OV preparations containing 200 million platelets were injected into mice, where platelets were labeled with cell tracker deepred, and the blood of mice was collected, and the platelets attached to the tumor cells were analyzed by flow cytometry after red lysis.

[0083] Specific results such as Fig.10 A- Fig.10 C shows the difference in the enrichment of P-OV and naked OV in lung tumor metastases after tail vein administration in the embodiment of the present invention, as well as the adhesion of P-OV to circulating tumor cells in the blood. Fig.10 A is the small animal fluorescence imaging results and its quantitative statistical chart, Fig.10 B is the result of flow cytometry. Fig.10 C is the targeting result of P-OV on circulating tumor cells in the blood, as shown by flow cytometry results.

[0084] from Fig.10 As shown in A, after tail vein injection of mice in different groups, the fluorescence signal of Cy5.5 in the control group was weaker than that in the P-OV group, indicating that the P-OV group significantly increased the enrichment of the virus in the tumor site.

[0085] from Fig.10 As can be seen from B, the proportion of cy5.5-labeled virus particles in the lung cells of the P-OV group was significantly higher than that of the OV group, indicating that the P-OV group increased the enrichment of the virus in the lungs containing tumors.

[0086] from Fig.10 As can be seen in C, the CFSE-positive cell population is circled from the blood cells, representing the tumor cells in the blood. Subsequently, it was found that there were double-positive areas for CFSE and Cell tracker deep red, which means that the P-OV preparation injected in the experiment adhered to the previously injected tumor cells, indicating that the P-OV preparation of the present invention can also target circulating tumor cells in the blood.

[0087] It can be seen that compared with naked oncolytic viruses, platelet-targeted oncolytic virus delivery preparations significantly enhance the enrichment ability of oncolytic viruses at tumor sites and can capture circulating tumor cells in the blood, thereby greatly improving the efficacy of intravenous administration.

[0088] Example 4 Testing of the Anti-tumor Ability of Platelet-Targeted Oncolytic Virus Delivery Preparation

[0089] In this example, the anti-tumor ability of the P-OV preparation to improve the oncolytic virus was tested, and the specific experimental steps are as follows:

[0090] Babl / c mice were taken and 500,000 4T1 breast cancer cells were injected into them through the tail vein. Then they were randomly divided into four groups, namely blank group (PBS), OV group, and Mix group (here Mix group refers to the mixture of platelets and oncolytic virus but the oncolytic virus is not loaded on the platelets). The corresponding drugs of the groups were injected at 4 hours, and then the corresponding drugs were injected every two days until four drug injections were completed and the drug treatment course was ended. At the same time, the small animal imaging system was used to detect tumor signals on the fourth day, eighth day, twelfth day, sixteenth day, and twentieth day, respectively.

[0091] Specific results such as Figures 11A to 11C As shown, the in vitro efficacy data of the embodiments of the present invention are shown. Among them, Fig.11A Bioluminescent imaging of tumors in each group of mice treated with different preparations; Fig. 11B The bioluminescence statistical analysis diagram of the tumor sites of each group of mice after treatment; Fig. 11C The survival of mice after receiving different preparations treatment.

[0092] from Figures 11A to 11C It can be seen that 6 hours after the tumor cells were inoculated into the tail vein, different preparations were used for treatment. Among the groups with different treatments, the P-OV group showed the best tumor inhibition effect, and the tumor grew the slowest; the platelet and oncolytic virus mixed group and the naked virus group also showed partial anti-tumor efficacy, but the inhibition efficiency was much lower than that of the P-OV group, and there were significant differences. At the same time, nearly 65% ​​of the mice treated with P-OV survived for more than 30 days. In contrast, all the mice in the other three groups died within 30 days.

[0093] It can be seen from this that the platelet-targeted oncolytic virus delivery preparation prepared by the present invention can significantly inhibit tumor growth and has extremely strong anti-tumor ability.

[0094] Example 5 Screening of oncolytic virus delivery vectors

[0095] In order to verify that platelets are the best delivery carriers of oncolytic viruses in the present invention, different carriers were compared and screened in this example, and platelets, engineered cells (TC-1 tumor cells), and engineered cells (HCT116 cells) were selected as carriers, respectively. According to the best preparation method described in Example 1, different oncolytic virus delivery preparations were prepared for comparison (the engineered cells are specifically the engineered cells mentioned in the aforementioned comparative documents, the name of the invention is: Engineered cells for targeted delivery of oncolytic viruses, preparation methods and applications thereof, and the application publication number is CN118460469A).

[0096] In terms of tumor adhesion efficiency, platelets can efficiently adhere to tumor cells when used as carriers. In contrast, when engineered cells (TC-1 tumor cells, HCT116 cells) are used as carriers, their tumor adhesion efficiency is significantly lower than that of platelets. This may be because platelets themselves have special physiological characteristics and surface molecular structures, which enable them to more effectively identify and bind to tumor cells, thereby improving their enrichment ability at the tumor site, providing a basis for oncolytic viruses to better function.

[0097] Considering the long-term circulation ability in the body, platelets have good long-term circulation ability in the body, which can keep the oncolytic virus active for a long time in the body and extend its half-life from the initial 7 minutes to 69 minutes. When engineered cells are used as carriers, the circulation and survival time in the body may be affected by many factors, such as the body's immune recognition and clearance mechanism, resulting in their long-term circulation ability not as good as platelets.

[0098] In summary, from the perspectives of tumor adhesion efficiency, long-term circulation ability in vivo and other aspects, in the present invention, platelets as delivery vectors of oncolytic viruses have obvious advantages over engineered cells and are the best delivery vectors for oncolytic viruses.

[0099] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A cell complex for targeted delivery of oncolytic viruses, characterized in that: It includes platelets and oncolytic viruses; the oncolytic viruses are loaded on platelets by infecting platelets.

2. The cell complex according to claim 1, characterized in that The oncolytic cells infect platelets by co-incubating with platelets.

3. The cell complex according to claim 2, characterized in that The oncolytic virus includes any one or more combinations of oncolytic adenovirus, vaccinia virus, herpes simplex virus, Newcastle virus, parvovirus, vesicular stomatitis virus, reovirus or poliovirus.

4. A platelet-targeted oncolytic virus delivery preparation, characterized in that: It comprises the cell complex according to any one of claims 1 to 3.

5. A kit, characterized in that: It comprises the cell complex according to any one of claims 1 to 3.

6. A method for preparing a cell complex according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) co-culturing oncolytic virus and platelets; (2) after secondary centrifugation, removing free virus particles to obtain the cell complex.

7. The cell complex according to claim 6, characterized in that In the step (1), the ratio of the oncolytic virus titer to the platelet count is 1:1-1:1000, and the co-incubation time is 1-12 hours.

8. Use of the cell complex according to any one of claims 1 to 3 for preparing a preparation for improving tumor lesion targeting or anti-tumor ability.

9. Use of the cell complex according to any one of claims 1 to 3 for preparing a preparation for improving the long circulation ability of a drug in vivo or extending the half-life of a drug in vivo.

10. Use of platelets for preparing a preparation for improving the adhesion efficiency of cell complexes to tumors or the neutralization ability of antibodies in vivo, characterized in that: The cell complex includes platelets and oncolytic viruses; the oncolytic viruses are loaded on platelets by infecting platelets; and the oncolytic cells infect platelets by co-incubating and culturing with platelets.

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