Composition for intratumor delivery, anti-tumor vaccine and application thereof

Abstract

The invention provides a composition for intratumoral delivery, an anti-tumor vaccine and application thereof, and the intratumoral delivery carrier can realize in-situ delivery expression, simultaneously wrap mRNA, circRNA, siRNA, small molecule compounds and other drugs, and realize simultaneous delivery of a plurality of drugs. The multi-cytokine z therapeutic drug prepared by the method can realize in-vivo and in-vitro T cell activation, and has a remarkable anti-tumor effect in intratumor injection. The small nucleic acid medicine prepared by the method can realize in-vivo and in-vitro target gene knock-down, and the expression level of the target gene of tumor cells is obviously reduced after intratumor injection. The lipid nanoparticles of the intratumor delivery compound prepared by the method can kill multiple tumor cells in vivo and in vitro, have a remarkable anti-tumor effect after intratumor injection, have good prospects and practical value, and provide a new thought for development of intratumor treatment drugs in the field of tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the field of delivery vectors and relates to the preparation and application of lipid nanoparticles for intratumoral delivery of nucleic acids, chemical drugs, etc. Background Art

[0002] With the deepening research and understanding of tumor immunotherapy, intratumoral injection of therapeutic drugs has gradually become an important technical approach in the field of cancer treatment. It is a method of injecting drugs directly into the tumor for treatment. Compared with traditional drug therapy or chemoradiotherapy, localized tumor injection can provide higher drug concentrations, achieving better therapeutic effects while significantly reducing damage to surrounding healthy tissue. In clinical practice, interventional therapies such as radiofrequency ablation, radionuclide interventional therapy, tumor embolization, and transarterial chemoembolization have been used to place chemotherapy drugs or radionuclides in the localized area of the tumor to achieve localized therapeutic effects. These clinical treatments have the advantages of being minimally invasive, low-risk, and having a short recovery period, but they still carry certain risks of side effects and complications. Therefore, a major research area for intratumoral injection of therapeutic drugs is the improvement of drug delivery technology. Using safe and effective delivery vehicles such as nanolipid particles and polymers, the efficiency and stability of drug delivery within the tumor can be increased to enhance therapeutic efficacy and reduce side effects.

[0003] Current intratumoral therapeutic strategies in cancer immunotherapy primarily focus on modifying the tumor's immunosuppressive microenvironment to enhance its sensitivity to immunotherapy. The tumor microenvironment is a complex ecosystem composed of multiple components, including tumor cells, blood vessels, immune cells, stromal cells, and extracellular mechanisms, and plays a crucial role in tumor growth, metastasis, and drug resistance. However, an immunosuppressive tumor microenvironment often responds poorly to immunotherapy, so transforming "cold tumors" into "hot tumors" through intratumoral delivery strategies is crucial. BIONTECH's BNT131 pipeline technology utilizes intratumoral injection of mRNA expressing cytokines that modulate the tumor microenvironment, aiding the immune system in recognizing and attacking cancer cells. Both administration alone and in combination with immune checkpoint inhibitors has demonstrated significant anti-tumor responses in mouse tumor models. However, this method, which uses naked RNA for delivery, results in low delivery efficiency, and the use of delivery vectors is discouraged due to concerns about potential safety risks associated with delivery diffusion. Jiachen Xihai's JCXH-211 is a novel drug based on self-replicating RNA encoding human interleukin-12 (hIL-12). It is also the first self-replicating RNA expressing a cytokine for the treatment of advanced solid tumors. It is injected intratumorally via lipid nanoparticles encapsulating circular RNA to achieve long-term in vivo expression of IL-12. Phase I clinical trials are underway domestically and internationally, and preclinical studies have shown promising tumor-killing effects in both mouse and PDX models. CUREVAC's CV-8102 pipeline is a single-stranded, noncoding RNA-based agonist of TLR-7, TLR-8, and RIG-1. Intratumoral administration simultaneously activates both innate and adaptive immunity in the tumor microenvironment, enabling tumor immunotherapy. Phase I clinical studies evaluated CV-8102 alone or in combination with PD-1 therapy in patients with advanced melanoma, cutaneous squamous cell carcinoma, head and neck squamous cell carcinoma, or adenoid cystic carcinoma. Data demonstrated promising efficacy, and CV-8102 was safe and well-tolerated when used alone or in combination with PD-1 therapy.

[0004] In addition, intratumoral delivery of chemotherapy drugs and small nucleic acid drugs is also a research hotspot in this field. DOX (doxorubicin) is a widely used anthracycline anticancer drug that can insert into DNA, inhibit topoisomerase II, produce reactive oxygen species and interfere with lipid metabolism, leading to cancer cell death. However, DOX itself has no cancer cell specificity, so it has strong side effects, especially damaging myocardial cells, hematopoietic cells and hair follicle cells, leading to severe cardiac toxicity, bone marrow suppression and hair loss. However, the clinical use of DOX can be combined with nanocarriers to form composite nanoparticles with certain targeting and triggered release properties, thereby improving the targeting of the drug, reducing side effects and drug resistance. Currently, a variety of DOX drugs in the form of liposome preparations have been approved by the FDA for marketing, such as PEGylated liposomes. or liposomes Therefore, researchers have envisioned that intratumoral co-delivery of DOX and small nucleic acid drugs could further improve the effectiveness of tumor treatment. For example, Professor Wang Guobin of Tongji Medical College of Huazhong University of Science and Technology studied the treatment of mouse gastric cancer models by co-delivering DOX and shRNA-STAB1 through thermosensitive and magnetic cationic liposomes, which showed good anti-tumor and survival-prolonging effects. Michael J. Mitchell's laboratory at the University of Pennsylvania successfully delivered DOX to target cells and achieved target gene knockdown by linking DOX to nanolipid particles and encapsulating siRNA targeting BLC-2. Intratumoral administration effectively inhibited tumor growth in the Raji lymphoma mouse model.

[0005] In summary, nanolipid particles can deliver a variety of drug molecules to tumor cells, including small molecule drugs, siRNA, mRNA, lncRNA (long non-coding RNA), and circular RNA. The key to intratumoral delivery strategies is how to precisely deliver drugs to tumor cells while preventing them from leaking into normal tissue cells to minimize side effects. Therefore, the screening and development of nanolipid particles with these properties is crucial. Summary of the Invention

[0006] This patent provides a method for preparing and using nanolipid particles for intratumoral delivery, which can be used for the delivery of drug molecules such as small molecule compounds, small nucleic acids, mRNA and circular RNA. We have developed a delivery vector that can be efficiently delivered intratumorally without spillage through screening. This vector greatly ensures the safety of drug use while achieving functional delivery. In vitro functional studies of cytokine combination therapy expressed by this vector have shown that the combined factors can effectively activate the function and proliferation activity of T cells, showing a dose-dependent effect. Moreover, this delivery technology can realize a multi-factor RNA mixed packaging process, efficiently complete the delivery and expression of multiple mRNAs, and has a significant tumor control effect in tumor models. In addition, it can also efficiently deliver small nucleic acids and small molecule compounds, effectively achieving target gene knockdown and tumor cell death in tumor cells in vivo and in vitro.

[0007] In order to achieve the purpose of the present invention, the following technical solutions are adopted:

[0008] In one aspect, the present invention provides a composition for intratumoral delivery, comprising lipid nanoparticles encapsulating RNA molecules encoding target proteins with tumor therapeutic activity, therapeutic siRNA molecules, or small molecule compounds.

[0009] As a preferred embodiment of the present invention, the RNA molecule is selected from a linear RNA molecule, a circular RNA molecule or a siRNA molecule.

[0010] As a preferred embodiment of the present invention, the lipid nanoparticles include ionizable lipids, helper lipids, cholesterol lipids, and PEG lipids, wherein the ionizable lipids are selected from bile acid lipids and dipeptide-like lipids. Preferably, the lipid nanoparticles are composed of ionizable lipids, helper lipids, cholesterol lipids, and PEG lipids.

[0011] As a preferred embodiment of the present invention, the compound of the lipid nanoparticles includes at least one structural formula of the following formula (I-1):

[0012]

[0013] or the compound is a pharmaceutically acceptable salt, prodrug or isomer thereof;

[0014] in,

[0015] Q is independently selected from O or N;

[0016] m and n are independently selected from integers between 2 and 10;

[0017] o and n2 are independently selected from integers between 4 and 12;

[0018] n1 is independently selected from integers between 2 and 12;

[0019] q is independently selected from integers between 0 and 5;

[0020] R2 is independently selected from the following structures: -H, -SL3, -C(=O)OL3, -OC(=O)L3, -C(=O)NL3, -OL3; wherein L3 is independently selected from the following structures: wherein m1, m2 and o are independently selected from integers between 3 and 12;

[0021] R1 is independently selected from the following structures: -R3XR4M, -R3XR4Y-H, -R3M, -R3YR5, -M, -R3XR4(Z)M; R3, R4 and R5 are independently selected from the following structures: C1-C5 alkane, C2-C5 alkene;

[0022] X is independently selected from the following structures: -O-, -N(R x )-, -S-, -SS-, -OC(=O)-, -C(=O)O-, -OP(=O)O-, -OS(=O)O-, -C(=O)N-, -N(C(=O))-;

[0023] R x Independently selected from the following structures: -H, -(CH2) m -OH, -(CH2) m -CN, -(CH2)m H and alicyclic hydrocarbon, wherein m is independently selected from 0, 1, 2, 3, 4 or 5;

[0024] M is independently selected from the following structures: -(CH2) m -OH, -(CH2) m -CN, -(CH2) m -N(R**)R*, wherein m is independently selected from 0, 1, 2, 3, 4 or 5;

[0025] Y is independently selected from the following structures: -(CH2) m -G-(CH2) n -, wherein m and n are independently selected from 0, 1, 2, 3, 4 or 5;

[0026] Z is independently selected from the following structures: -(CH2) m -OH, -(CH2) m -CN, -(CH2) m -N(R**)R*, -(CH2) m -G-(CH2) n -H, wherein m and n are independently selected from 0, 1, 2, 3, 4 or 5;

[0027] G is independently selected from a benzene ring or a heterocyclic ring;

[0028] R* and R** are independently selected from the following structures: H, C1-C3 alkane, -(CH2) m Q, wherein m is independently selected from 1, 2, 3 or 4;

[0029] Q is independently selected from the following structures: -OH, -CN,

[0030] Independently selected from the following structures: Benzene ring, heterocyclic compounds;

[0031] R A 、R B 、R C Independently selected from the following structures: -H, -(CH2) m -H;

[0032] m is independently selected from 1, 2 and 3;

[0033] R d Independently selected from the following structures: -H, -(CH2) n -H-CN, -NO2, -NH2, -N(CH3)CH3;

[0034] n is independently selected from 1, 2 and 3;

[0035] The bile acid lipids include at least one structural formula as follows:

[0036]

[0037] or a pharmaceutically acceptable salt thereof, including stereoisomers, tautomers, solvates, chelates, non-covalent compounds or prodrugs.

[0038] Specifically, the "pharmaceutically acceptable salt thereof" refers to an acid addition salt or a base addition salt.

[0039] As a preferred embodiment of the present invention, the compound is preferably the following compound:

[0040]

[0041]

[0042] The bile acid lipid compound is preferably the following compound:

[0043]

[0044] As a preferred embodiment of the present invention, the small molecule compound is selected from doxorubicin, paclitaxel, and curcumin.

[0045] As a preferred embodiment of the present invention, the RNA molecule is used to express a protein or polypeptide having an immune-activating effect. Preferably, the protein or polypeptide is an immunostimulant. More preferably, the immunostimulant is an immune-activating cytokine.

[0046] As a preferred embodiment of the present invention, the RNA molecule is a mixture for expressing multiple cytokines, and the mixture can activate T cell killing activity and stimulate T cell proliferation in vitro and in vivo.

[0047] As a preferred embodiment of the present invention, the siRNA molecule is an siRNA molecule that can be used to knock down target genes in tumor cells and inhibit tumor growth.

[0048] In one aspect, the present invention provides a method for preparing a composition for intratumoral delivery, wherein the method comprises a mixed packaging method of multiple RNAs.

[0049] As a preferred embodiment of the present invention, a single RNA composition for intratumoral delivery is mixed in a certain proportion to obtain a composition containing multiple RNAs; or a microfluidic device is used to mix multiple RNA nucleic acid solutions with lipid nanoparticles to obtain a composition for intratumoral delivery of multiple RNAs.

[0050] The method for preparing the composition for intratumoral delivery provided by the present invention comprises the following steps:

[0051] (a) dissolving the ionizable lipid, DSPC (helper lipid) cholesterol, and PEG-lipid of the above technical solution in ethanol at a molar ratio of 50:10:38.5:1.5 to obtain lipid nanoparticles in an organic phase;

[0052] (b) dissolving the RNA in citrate buffer to obtain an aqueous RNA nucleic acid solution, and mixing the RNA nucleic acid solution with lipid nanoparticles using a microfluidic device at a flow rate of 6-30 mL / min, and mixing the lipid nanoparticles and RNA nucleic acid solution in a volume ratio of 1:3 to obtain a single RNA composition for intratumoral delivery;

[0053] (c) Mixing single intratumoral delivery RNA compositions in a certain ratio to obtain a composition containing multiple RNAs.

[0054] Preferably, in step (b), the flow rate is preferably 6 mL / min, 12 mL / min, 15 mL / min or 30 mL / min.

[0055] The method for preparing the composition for intratumoral delivery provided by the present invention comprises the following steps:

[0056] (a) dissolving the ionizable lipid, DSPC (helper lipid), cholesterol, and PEG-lipid of the above technical solution in ethanol at a molar ratio of 50:10:38.5:1.5 to obtain lipid nanoparticles in an organic phase;

[0057] (b) Multiple RNAs are dissolved in citrate buffer to obtain an aqueous phase, and the multiple RNA nucleic acid solutions are mixed with lipid nanoparticles using a microfluidic device at a flow rate of 6-30 mL / min. The lipid nanoparticles and the multiple RNA nucleic acid solutions are mixed in a volume ratio of 1:3 to obtain a composition for intratumoral delivery of multiple RNAs.

[0058] Preferably, in step (b), the flow rate is preferably 6 mL / min, 12 mL / min, 15 mL / min or 30 mL / min.

[0059] In one aspect, the present invention provides an anti-tumor vaccine prepared by the above-mentioned technical solution for intratumoral delivery of an RNA composition. Preferably, the anti-tumor vaccine comprises an anti-tumor mRNA vaccine.

[0060] In one aspect, the present invention provides a composition for intratumoral delivery according to the above technical solution, or use of the anti-tumor vaccine according to the above technical solution for preventing or treating tumors.

[0061] As a preferred embodiment of the present invention, the administration methods for preventing or treating tumors include oral, intraperitoneal, intravenous, intraarterial, intramuscular, intradermal, subcutaneous, transdermal, nasal, rectal, intratumoral injection, etc. Preferably, the administration method is intratumoral injection.

[0062] As a preferred embodiment of the present invention, the tumor is a solid tumor. Preferably, the tumor includes but is not limited to melanoma, colon cancer, breast cancer or liver cancer, etc.

[0063] In one aspect, the present invention provides a composition for intratumoral delivery according to the above technical solution, or an anti-tumor vaccine according to the above technical solution for combined use with a radiotherapy agent, a chemotherapeutic agent, an immunotherapy agent or an immune checkpoint inhibitor.

[0064] As a preferred embodiment of the present invention, immune checkpoint inhibitors include PD-1, PD-L1 and CTLA4 used in combination.

[0065] Compared with the prior art, the present invention has at least the following beneficial effects:

[0066] 1. This intratumoral delivery vector achieves in situ delivery without spilling into other normal tissues, greatly improving the safety of intratumoral drug delivery.

[0067] 2. This delivery vector can encapsulate drugs such as mRNA, circRNA, and siRNA, significantly improving the delivery efficiency of RNA molecules to tumor cells or tumor tissues, and improving the clinical tumor immunotherapy effect of RNA molecules.

[0068] 3. This delivery vector can achieve mixed packaging of multiple nucleic acid molecules and simultaneous delivery of multiple drugs.

[0069] 4. The multifactor vaccine drug prepared by this method can activate T cells in vivo and in vitro, and has a significant anti-tumor effect when injected into the tumor.

[0070] 5. The small nucleic acid drugs prepared by this method can achieve knockdown of target genes in vivo and in vitro, and significantly reduce the expression level of target genes in tumor cells after intratumoral injection.

[0071] 6. The compound preparation prepared by this method can kill multiple tumor cells in vitro and has a significant anti-tumor effect after intratumor injection.

[0072] 7. The intratumoral therapeutic drugs prepared by this method can be combined with immunotherapy methods such as tumor vaccines and immune checkpoint inhibitors to exert high-dimensional anti-tumor effects.

[0073] Animal testing results from the present invention demonstrate that the mRNA-LNP vaccine can provide a robust anti-tumor immune response in corresponding protein vaccine models, significantly inhibiting tumor growth in both melanoma and colon cancer models in mice. The mRNA vaccine of the present invention exhibits potent and effective tumor suppression in multiple solid tumor models, demonstrating its universal applicability, high immunogenicity, and broad application. It can be used as a neoadjuvant therapy in combination with clinical surgery and other anti-tumor drugs to enhance efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 is the size of the mRNA electrophoresis band;

[0075] Figure 2 is the size of circular RNA electrophoresis band;

[0076] Figure 3 The results of in vitro expression testing of multi-factor mRNA delivered to 293T cells by LNP formulations;

[0077] Figure 4 Expression test results of circRNA after LNP formulation delivery to 293T cells;

[0078] Figure 5 This is the knockdown result of target gene by in vitro transfection with siRNA;

[0079] Figure 6 In vivo and organ imaging of mice undergoing intratumoral drug administration;

[0080] Figure 7 The fluorescence ratio of different organs in mice with intratumoral drug administration;

[0081] Figure 8 To detect the secretion of IFNγ by T cells stimulated with different concentrations of cytokines by ELISPOT;

[0082] Figure 9 To detect IFNγ secretion by T cells stimulated with different concentrations of cytokines by ELISA;

[0083] Figure 10 The results of T cell proliferation stimulated by cytokines in vitro were detected by flow cytometry;

[0084] Figure 11 Serum test results of intratumoral administration of multi-factor vaccine in TC-1 tumor model;

[0085] Figure 12 The results of the pharmacodynamic study of the multi-factor vaccine administered intratumorally in the TC-1 tumor model are presented;

[0086] Figure 13 In vivo imaging of intratumoral delivery of siRNA-Luc using different LNP formulations;

[0087] Figure 14 This is a diagram showing the therapeutic effect of intratumoral delivery of paclitaxel by LNP formulation in the TC-1 tumor model in mice;

[0088] Figure 15 This is a diagram showing the therapeutic effect of multi-factor vaccine combined with PD-1 in the TC-1 tumor model of mice. DETAILED DESCRIPTION

[0089] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be understood as limiting the scope of implementation of the present invention. Example 1: Preparation of mRNA and circular RNA

[0090] Using T7 RNA polymerase, a linear double-stranded DNA containing the T7 promoter sequence is used as a template and NTPs are used as substrates to transcribe the DNA sequence downstream of the promoter to obtain single-stranded RNA. Finally, lithium chloride is used to purify the mRNA to remove proteins and most free nucleotides. The size of the mRNA electrophoresis band is as follows: Figure 1 shown.

[0091] The specific steps include: preparing precursor RNA by in vitro transcription through a vector, and cyclizing the precursor RNA molecule to obtain circular RNA. The size of the circular RNA band is as follows: Figure 2 shown.

[0092] Example 2: Encapsulation methods and characterization of different LNP formulations

[0093] The compound provided herein, DSPC, cholesterol, and PEG-lipid were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5, and the mRNA was diluted in 10 mM to 50 mM citrate buffer, pH 4. LNPs were prepared using a microfluidic device, mixing the lipid ethanol solution with the respective mRNA aqueous solution at a volume ratio of 1:3 at a flow rate in the range of 6 mL / min, with a total lipid to mRNA weight ratio of approximately 10:1 to 35:1. The ethanol was removed by dialysis and replaced with PBS. Finally, the lipid nanoparticles were filtered through a 0.22 μm sterile filter, and after quantification, the LNPs synthesized from the respective mRNAs were mixed in a specific ratio. As shown in Table 1, the lipid nanoparticle size was mostly around 100 nm, the polydispersity index varied around 0.1, and the encapsulation efficiency was mostly above 90%.

[0094] Table 1: Characterization of lipid nanoparticles

[0095]

[0096] Example 3: In vitro expression detection of multi-factor mRNA delivery by different LNP formulations

[0097] In Example 1, mRNA-1, mRNA-2, mRNA-3, and mRNA-4 were obtained, and SM102-mRNA-1, SM102-mRNA-2, SM102-mRNA-3, SM102-mRNA-4, LNP1-mRNA-1, LNP1-mRNA-2, LNP1-mRNA-3, and LNP1-mRNA-4 were prepared by Example 2. SM102-mRNA-1-mRNA-4mix and LNP1-mRNA-1-mRNA-4mix were prepared by mixing the mRNA. 293T cells were inoculated into a six-well plate and divided into five groups for cell transfection the next day. The supernatant was collected 24 hours after transfection for ELISA cytokine detection. The experimental results are shown in FIG. Figure 3 As shown, the expression effects of LNP transfection and LIP3000 transfection were similar, and LNP encapsulated mixed mRNA transfection and single mRNA encapsulation both had certain transfection expression effects.

[0098] Example 4: In vitro expression detection of multi-factor circRNA delivery by LNP formulations

[0099] Circular RNA Circ-1 and Circ-2 were obtained in Example 1, and LNP2-Circ-1 and LNP2-Circ-2 were prepared by Example 2. 293T cells were inoculated into six-well plates and divided into two groups for cell transfection the next day, with each group divided into two wells: Group 1: lip3000+Circ-1, lip3000+Circ-2; Group 2: LNP2-Circ-1, LNP2-Circ-1. Cell supernatants were collected from each well after 24 hours and 48 hours for ELISA cytokine GM-CSF, IL-15, IFN-α, and IL-12 detection. The experimental results are shown in the figure. Figure 4 As shown in the figure, 293T cells were transfected with circRNAs via LIP3000, and GM-CSF, IL-15, IFN-α, and IL-12 cytokines were all expressed. The cytokines expressed for 48 hours were higher than those expressed for 24 hours. However, when 293T cells were transfected with LNPs, the expression concentrations of different cytokines increased and decreased over time, and the expression concentrations of cytokines in LNP-transfected cells were all higher than those in LIP3000-transfected cells.

[0100] Example 5: In vitro transfection of siRNA target gene knockdown

[0101] Luciferase-overexpressing 293T cells were seeded into 96-well plates and divided into three groups for cell transfection the next day, with each group consisting of three wells: Group 1: SM102-siRNA; Group 2: LNP1-siRNA, LNP2-siRNA. Luciferase reporter gene luminescence assay was performed in each well 24 hours later. Figure 5 As shown in the figure, siRNA at various transfection concentrations interfered with Luciferase expression in 293T cells, and the interference effect increased with increasing transfection concentration. LNP1 and LNP2, at various concentrations, produced interference effects similar to or slightly better than SM102. This suggests that LNP1 and LNP2 have similar siRNA delivery capabilities as SM102.

[0102] Example 6: Detection of intratumoral delivery effects of different LNP formulations in mouse tumor models

[0103] A preparation containing fluorescent expression of Luc-mRNA was prepared according to Example 2. EG-7 cells were subcutaneously inoculated into C57 mice to establish a tumor model. 7 days after modeling, 5 μg of LNP-Luc mRNA was reinfused into the tumor. 6 hours after injection and reinfusion, in vivo imaging was performed, and the tumor, liver, and spleen of the mice were dissected to observe the expression and distribution of P19. The experimental results are shown in Figure 2. Figure 6-7 As shown in Figure 4, different LNPs were expressed after intratumoral administration.

[0104] Example 7: In vitro T cell function assay using multi-factor conditioned medium

[0105] Take the cell supernatant of 293T transfected with LNP2 in Example 2 and cultured for 48 hours, and dilute it with culture medium to form conditioned medium with cell supernatant content of 10%, 1%, 0.1%, and 0%. Grind the mouse spleen into a single cell suspension, add 5 ml of red blood cell lysis solution and lyse and centrifuge at room temperature, discard the supernatant, and add CD8+ magnetic beads to retrieve CD8+T cells. Use different proportions of conditioned medium to resuspend the mouse spleen CD8+T cells and add them to ELISPOT pretreated well plates and 96-well cell culture dishes. Use conditioned medium to stimulate CD8+T cells to secrete IFNγ. The effect of cytokines on CD8+T cells is determined by quantifying the number of spots by enzyme-linked reaction and detecting the secretion concentration of IFNγ in the cell supernatant in 96-well cell culture dishes by ELISA. The experimental results are as follows. Figure 8-9 As shown in Figure 2, the higher the cytokine concentration in the culture medium, the more IFNγ secretion by CD8+ T cells, which verifies the functionality of the cytokine on T cells in vitro.

[0106] Example 8: In vitro T cell proliferation activity activation assay using multi-factor conditioned medium

[0107] The 10% conditioned medium and CD8+ T cells in Example 7 were taken and pre-stained with eFluo670. The experimental system was a 96-well cell culture dish. The experiment was divided into two groups, with two replicates in each group. The NC group was cultured with 1640 complete medium for 72 hours, and the experimental group was cultured with 10% conditioned medium for 72 hours. After the culture was completed, the cells were collected and the cell division generation was detected by flow cytometry. The experimental results are shown in Figure 2. Figure 10 As shown, cytokine-conditioned medium successfully stimulated CD8+ T cell proliferation.

[0108] Example 9: Detection of serum expression after intratumoral administration of multifactor vaccine in mouse tumor model

[0109] Tumor models were established in C57 mice using TC-1 cells subcutaneously inoculated. On day 8 of tumor growth, 10 μg of multifactor mRNA was injected intratumorally. The experiment was divided into three groups: PBS, SM102-mRNA-1-mRNA-4 mix, and LNP1-mRNA-1-mRNA-4 mix. Serum was collected 6 hours after administration for ELISA cytokine detection. The experimental results are shown in the figure below. Figure 11 As shown in Figure 3, SM102 intratumorally injected has a high degree of escape, so the expression of cytokines was detected in the serum, while the degree of escape of LNP1 is low, and the expression of cytokines is not detected in the serum.

[0110] Example 10: Pharmacodynamics study of multifactor vaccines

[0111] A tumor model was established by subcutaneously inoculating C57 mice with TC-1 cells. On days 8, 12, and 16 of tumor growth, 10 μg of multifactor mRNA was injected intratumorally. The experiment was divided into three groups: PBS, SM102-mRNA-1-mRNA-4mix, and LNP1-mRNA-1-mRNA-4mix. Tumors were measured every other day after administration, and the data were finally used to plot a tumor volume growth curve. The experimental results are shown in the figure below. Figure 12 As shown in Figure 3, intratumoral delivery of multi-factor mRNA by SM102 and LNP1 can significantly inhibit tumor growth.

[0112] Example 11: Detection of the effect of intratumoral siRNA delivery in mouse tumor models using different LNP formulations

[0113] LNP1-siRNA-Luc and LNP3-siRNA-Luc preparations were prepared according to Example 2. EG7-Luc cells were subcutaneously inoculated into C57 mice to establish a tumor model. Seven days after modeling, the mice were divided into three groups: PBS, LNP1-siRNA-Luc, and LNP3-siRNA-Luc. Each group was injected intratumorally with 20 μg of the corresponding preparations. In vivo imaging was performed 6 hours later. The experimental results are shown in Figure 2. Figure 13As shown, different LNP-based intratumoral siRNA injections can significantly reduce the expression of Luc, demonstrating the effectiveness of intratumoral siRNA delivery using this patented method.

[0114] Example 12: Pharmacodynamics Study of Intratumoral Delivery of Small Molecule Compounds in Mouse Tumor Models Using Different LNP Formulations

[0115] Reference Example 2 prepared LNP1-paclitaxel formulation, encapsulating the small molecule compound paclitaxel. TC-1 cells were subcutaneously inoculated into C57 mice to establish a tumor model. LNP1-paclitaxel was injected intratumorally on days 8, 12, and 16 of tumor growth in the mice. The experiment was divided into two groups: PBS and LNP1-paclitaxel. Tumors were measured every other day after administration, and the final data was used to plot a tumor volume growth curve. The experimental results are shown in Figure 2. Figure 14 As shown, LNP1-paclitaxel has a significant inhibitory effect on tumor growth, proving the effectiveness of intratumoral delivery of small molecule compounds under this patented method.

[0116] Example 13: Pharmacodynamics study of multi-factor vaccine combined with PD-1

[0117] A tumor model was established by subcutaneously inoculating C57 mice with TC-1 cells. On days 8, 12, and 16 of tumor growth, 10 μg of a multifactor mRNA LNP formulation and PD1 were injected intratumorally. The experiment was divided into four groups: PBS, LNP1-mRNA-1-mRNA-4mix, PD1, and LNP1-mRNA-1-mRNA-4mix+PD1. Tumors were measured every other day after administration, and the tumor volume growth curve was plotted using statistical data. The experimental results are shown in Figure 2. Figure 15 As shown, intratumoral delivery of multifactor mRNA and PD-1 by LNP1 significantly inhibited tumor growth, but the combined administration group was most effective, significantly reducing tumor burden. This suggests that this vaccine can be used in combination with immune checkpoint inhibitors such as PD-1 for treatment.

[0118] Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention, and that those skilled in the art may make changes, modifications and variations to the above embodiments within the scope of the present invention.

Claims

1. A composition for intratumoral delivery, characterized in that The composition comprises lipid nanoparticles encapsulating RNA molecules encoding target proteins with tumor therapeutic activity, therapeutic siRNA molecules or small molecule compounds.

2. The composition for intratumoral delivery according to claim 1, characterized in that The RNA molecule is selected from a linear RNA molecule, a circular RNA molecule or a siRNA molecule.

3. The composition for intratumoral delivery according to claim 1, characterized in that The lipid nanoparticles include ionizable lipids, helper lipids, cholesterol lipids, and PEG lipids, wherein the ionizable lipids are selected from bile acid lipids and dipeptide-like lipids; The dipeptide-like lipid comprises at least one structural formula as shown in the following formula (I-1): or a pharmaceutically acceptable salt, prodrug or isomer thereof; in, Q is independently selected from O or N; m and n are independently selected from integers between 2 and 10; o and n2 are independently selected from integers between 4 and 12; n1 is independently selected from integers between 2 and 12; q is independently selected from integers between 0 and 5; R2 is independently selected from the following structures: -H, -SL3, -C(=O)OL3, -OC(=O)L3, -C(=O)NL3, -OL3; wherein L3 is independently selected from the following structures: wherein m1, m2 and o are independently selected from integers between 3 and 12; R1 is independently selected from the following structures: -R3XR4M, -R3XR4Y-H, -R3M, -R3YR5, -M, -R3XR4(Z)M; R3, R4 and R5 are independently selected from the following structures: C1-C5 alkane, C2-C5 alkene; X is independently selected from the following structures: -O-, -N(R x )-, -S-, -SS-, -OC(=O)-, -C(=O)O-, -OP(=O)O-, -OS(=O)O-, -C(=O)N-, -N(C(=O))-; R x Independently selected from the following structures: -H, -(CH2) m -OH, -(CH2) m -CN, -(CH2) m H and alicyclic hydrocarbon, wherein m is independently selected from 0, 1, 2, 3, 4 or 5; M is independently selected from the following structures: -(CH2) m -OH, -(CH2) m -CN, -(CH2) m -N(R**)R*, wherein m is independently selected from 0, 1, 2, 3, 4 or 5; Y is independently selected from the following structures: -(CH2) m -G-(CH2) n -, wherein m and n are independently selected from 0, 1, 2, 3, 4 or 5; Z is independently selected from the following structures: -(CH2) m -OH, -(CH2) m -CN, -(CH2) m -N(R**)R*, -(CH2) m -G-(CH2) n -H, wherein m and n are independently selected from 0, 1, 2, 3, 4 or 5; G is independently selected from a benzene ring or a heterocyclic ring; R* and R** are independently selected from the following structures: H, C1-C3 alkane, -(CH2) m Q, wherein m is independently selected from 1, 2, 3 or 4; Q is independently selected from the following structures: -OH, -CN, Independently selected from the following structures: Benzene ring, heterocyclic compounds; R A 、R B 、R C Independently selected from the following structures: -H, -(CH2) m -H; m is independently selected from 1, 2 and 3; R d Independently selected from the following structures: -H, -(CH2) n -H-CN, -NO2, -NH2, -N(CH3)CH3; n is independently selected from 1, 2 and 3; The bile acid lipids include at least one structural formula as follows: R1=hydrogen atom, alkane, alkene, substituted carbonyl, Alkanes containing nitrogen atoms, alkenes containing nitrogen atoms R2=hydrogen atom, alkane, alkene, substituted carbonyl, Alkanes containing nitrogen atoms, alkenes containing nitrogen atoms R3=hydroxyl, halogen, alkane, alkene R4 = hydrogen atom, alkane, alkene, alkane containing nitrogen atom, Olefins containing nitrogen atoms, cyclic compounds containing nitrogen atoms X=NH or O linker = alkane, SS, S, or a pharmaceutically acceptable salt thereof, including stereoisomers, tautomers, solvates, chelates, non-covalent compounds or prodrugs.

4. The composition for intratumoral delivery according to claim 3, characterized in that The dipeptide lipid-like compound includes one or more of compounds 1-6; or The bile acid lipid compound includes one or more of compounds 7-11.

5. The composition for intratumoral delivery according to claim 1, characterized in that The small molecule compound is selected from doxorubicin, paclitaxel, and curcumin.

6. The composition for intratumoral delivery according to claim 1, characterized in that RNA molecules are used to express proteins or polypeptides with immune activation effects.

7. The composition for intratumoral delivery according to claim 6, characterized in that The RNA molecule is a mixture for expressing multiple cytokines, which can activate T cell killing activity and stimulate T cell proliferation in vitro and in vivo.

8. The composition for intratumoral delivery according to claim 2, characterized in that siRNA molecules are siRNA molecules that can be used to knock down target genes in tumor cells and inhibit tumor growth.

9. A method for preparing an RNA composition for intratumoral delivery according to any one of claims 1 to 8, characterized in that: The method is a mixed packaging method of multiple RNAs.

10. The method for preparing the composition for intratumoral delivery according to claim 9, characterized in that: A single RNA composition for intratumoral delivery is mixed in a certain proportion to obtain a composition containing multiple RNAs; or a microfluidic device is used to mix multiple RNA nucleic acid solutions with lipid nanoparticles to obtain a composition for intratumoral delivery of multiple RNAs.

11. An anti-tumor vaccine prepared by the RNA composition for intratumoral delivery according to claims 1-8.

12. Use of the composition for intratumoral delivery according to any one of claims 1 to 8, or the anti-tumor vaccine according to claim 11, for preventing or treating tumors.

13. The use for preventing or treating tumors according to claim 12, wherein the administration methods for preventing or treating tumors include oral, intraperitoneal, intravenous, intraarterial, intramuscular, intradermal, subcutaneous, transdermal, nasal, rectal, and intratumoral injection.

14. The use for preventing or treating tumors according to claim 12, wherein the tumor is a solid tumor, including melanoma, colon cancer, breast cancer, and liver cancer.

15. The composition for intratumoral delivery according to any one of claims 1 to 8, or the anti-tumor vaccine according to claim 11, is used in combination with a radiotherapy agent, a chemotherapeutic agent, an immunotherapy agent or an immune checkpoint inhibitor.

16. The method according to claim 9 for combined use of a radiotherapeutic agent, a chemotherapeutic agent, an immunotherapeutic agent or an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor comprises PD-1, PD-L1 and CTLA4.

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