A tumor local acute inflammation inducer, and a preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses a tumor local acute inflammation inducer, a preparation method and application thereof. Specifically, extracted bacterial outer membrane vesicles are loaded into prepared temperature-sensitive hydrogel for in-situ injection treatment of common clinical solid tumors, such as breast cancer, melanoma, colorectal cancer and the like. The temperature-sensitive hydrogel loaded with bacterial outer membrane vesicles constructed in the application induces acute inflammation at the tumor site, effectively activates anti-tumor immunity, and improves the immunosuppressive microenvironment; at the same time, the temperature-sensitive hydrogel causes rupture of tumor blood vessels, and can be combined with photothermal therapy to further eradicate tumors; and the temperature-sensitive hydrogel limits acute inflammation in the tumor, improves biological safety, and provides a new strategy for development of in-situ tumor treatment preparations.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically a novel local acute inflammation inducer for tumors, namely a method for preparing a temperature-sensitive hydrogel carrying bacterial outer membrane vesicles and its application in the preparation of clinical treatment agents for in situ solid tumors. Background Technology

[0002] Cancer is a major disease that seriously affects human health. In recent years, tumor immunotherapy has gradually emerged, but its clinical efficacy is limited by the immunosuppressive microenvironment of solid tumors. Among these factors, the presence of a chronic inflammatory environment in tumors is a key factor, which can promote tumor development, angiogenesis, metastasis, and drug resistance. Inducing acute inflammation in tumors can rapidly recruit immune cells, trigger anti-tumor immunity, and thus overcome the immunosuppression caused by chronic inflammation. Omniform vesicles (OMVs) are spherical vesicles with a lipid bilayer secreted by Gram-negative bacteria, with a particle size of 20–250 nm. They contain various endogenous immunostimulatory components and pathogen-associated molecular patterns from parental cells and can serve as effective activators of acute inflammation. However, they have significant systemic toxicity, and intravenous injection can easily cause a cytokine storm.

[0003] Based on this, this invention utilizes a localized temperature-sensitive hydrogel to encapsulate bacterial outer membrane vesicles secreted by *Escherichia coli* DH5α, constructing a novel in situ tumor vaccine, OMVs-gel. This activates an effective anti-tumor immune response, induces local acute inflammation to eliminate the tumor, and confines the inflammation to the tumor site, avoiding systemic toxicity. This approach demonstrates high feasibility and application potential. Currently, there are no reported studies on using induced local acute inflammation to treat clinical solid tumors. Summary of the Invention

[0004] This invention provides a novel acute inflammation inducer for tumors that can release bacterial outer membrane vesicles locally on the tumor, recruiting a large number of neutrophils locally. These neutrophils selectively kill tumor cells by secreting elastase and release a large number of cytokines to amplify the acute inflammatory response, activating subsequent non-specific and specific anti-tumor immunity. The bacterial outer membrane vesicles induce tumor blood vessel rupture and red blood cell extravasation and coagulation, causing the tumor to turn black. Combined with photothermal therapy, this further eradicates solid tumors and prevents tumor metastasis.

[0005] Before describing the technical solution of this invention, the abbreviations used herein are defined as follows:

[0006] The abbreviation "OMVs" stands for bacterial outer membrane vesicles.

[0007] The abbreviation "Blank gel" stands for Blank temperature-sensitive hydrogel.

[0008] The abbreviation "OMVs-gel" stands for: Novel local acute inflammation inducer for tumors, namely temperature-sensitive hydrogel carrying bacterial outer membrane vesicles.

[0009] To address the aforementioned technical problems, one objective of this invention is to provide a method for preparing a tumor local acute inflammation inducer, comprising bacterial outer membrane vesicles and temperature-sensitive hydrogels; wherein the bacterial outer membrane vesicles are secreted by Escherichia coli DH5α.

[0010] The preparation method described above contains 10 bacterial outer membrane vesicles per 1000 μl of temperature-sensitive hydrogel. 9 -10 12 One. Further preferred: 10 10 particles / ml.

[0011] The preparation method described above, specifically the preparation process of the bacterial outer membrane vesicles, is as follows: After culturing Escherichia coli DH5α for 16-24 hours, the bacterial culture is centrifuged at 10000g for 10-20 minutes, the precipitate is discarded, the supernatant is filtered through a 0.22μm filter membrane, the filtrate is then centrifuged at 150000g-200000g for 4-6 hours, the supernatant is discarded, the precipitate is washed with PBS, and then centrifuged again at 150000g-200000g for 4-6 hours, the supernatant is discarded, and the precipitate is the purified Escherichia coli DH5α bacterial outer membrane vesicle.

[0012] The preparation method described above states that the temperature-sensitive hydrogel is composed of pluronic F-127 and sodium alginate.

[0013] The temperature-sensitive hydrogel described in the preparation method is obtained by dissolving 15-20 wt% pluronic F-127, 1-2 wt% sodium alginate, and the remainder pure water at 4-10°C by stirring and mixing.

[0014] The mass fraction ratio of pluronic F-127 and sodium alginate constituting the temperature-sensitive hydrogel includes: 25:2, 20:2, 18:2, 15:2, 25:1, 20:1, 18:1, 15:1, and more preferably: 20:1.

[0015] The preparation method involves adding bacterial outer membrane vesicles to a temperature-sensitive hydrogel, mixing the two thoroughly, incubating, and allowing them to stand to remove air bubbles; thus, the product is obtained.

[0016] The bacterial outer membrane vesicles and temperature-sensitive hydrogels are mixed, incubated, and left to stand for at least 12 hours, all within a low temperature environment of 4-10℃.

[0017] The preparation method described in this invention preferably includes the following steps:

[0018] (A) Escherichia coli DH5α was cultured, and OMVs in the bacterial culture medium were extracted and purified by differential ultra-high speed centrifugation. The particle size and concentration were measured by dynamic light scattering instrument, the microstructure was observed by transmission electron microscopy, and the protein composition was analyzed by gel electrophoresis. The culture was stored at -80℃ for long-term use.

[0019] The results showed that the average particle size of the extracted and purified OMVs was 101.2 ± 2.2 nm, and the particle concentration was 1.49 × 10⁻⁶. 11 The protein content was [number] particles / mL, and transmission electron microscopy revealed a spherical vesicle structure with a distinct lipid bilayer. The total protein exhibited clear band distributions at 17 kDa, 35 kDa, and 60 kDa.

[0020] (B) The temperature-sensitive hydrogel was formed by stirring pluronic F-127 (20% wt%) and sodium alginate (1% wt%) in pure water at 4°C for 12 hours. Its microstructure was observed using scanning electron microscopy, and its gelation temperature and time were determined using the inverted test tube method. The drug release curve was plotted using the Transwell plate method. The results showed that the gelation temperature of this temperature-sensitive hydrogel was 18±1°C, and the gelation time was 39s±2s. Scanning electron microscopy revealed a porous network structure, and the hydrogel gradually released the drug in vitro over 72 hours.

[0021] (C) Mix the purified gel from (A) with the gel from (B) at 4°C, incubate, and allow to stand to remove air bubbles. Adjust the OMVs concentration to 10. 10 The novel local acute inflammation inducer for tumors was obtained by measuring 1 particle / ml and stored at 4°C for later use.

[0022] A second objective of this invention is to provide a prepared local acute inflammation inducer for tumors.

[0023] The OMVs-gel of this invention comprises: OMVs secreted by Escherichia coli DH5α, and a temperature-sensitive hydrogel composed of pluronic F-127, sodium alginate, and the balance water. The average particle size of the OMVs is 101.2±2.2 nm, the gelation temperature of the temperature-sensitive hydrogel is 18±1℃, and the gelation time is 39±2 s.

[0024] A third objective of this invention is to provide an application of the aforementioned tumor local acute inflammation inducer, used to prepare a preparation that induces acute inflammation at the tumor site, further recruits neutrophils to secrete elastase, selectively kills tumor cells, and secretes a large number of pro-inflammatory factors to amplify acute inflammation, induce specific anti-tumor immunity, treat the primary tumor and prevent its metastasis, especially for preparing preparations for in situ injection therapy of common clinical solid tumors or preparations for inhibiting lung metastasis of primary tumors.

[0025] A fourth objective of this invention is to provide another application of the aforementioned tumor local acute inflammation inducer for the preparation of a formulation that induces tumor vascular rupture and red blood cell extravasation, resulting in a darkening of the tumor's appearance, and further for the preparation of a formulation for use in combination with photothermal therapy to treat clinical solid tumors.

[0026] Furthermore, the solid tumors mentioned include: breast cancer, melanoma, and colorectal cancer.

[0027] This invention selects a temperature-sensitive hydrogel that transforms into a gel state at body temperature, thereby trapping bacterial outer membrane vesicles within the tumor to induce acute inflammation without triggering a systemic cytokine storm. Furthermore, the intratumoral distribution and degradability of OMVs-gel were investigated, with the specific steps as follows:

[0028] This invention verifies the acute inflammation-inducing effect and potential mechanism of this novel local acute inflammation inducer on solid tumors, using triple-negative breast cancer as an example. The specific steps are as follows:

[0029] A BalB / c mouse triple-negative breast cancer orthotopic model was established, and the tumor volume grew to 200 mm. 3 When DiR-labeled OMVs-gel was injected into the tumor, the fluorescence intensity was recorded using a small animal in vivo imaging system at 24, 48, 72, and 96 hours. It was found that OMVs-gel remained in the tumor for a long time, and the intratumoral retention and sustained release effect of OMVs-gel was good.

[0030] Simultaneously, an orthotopic model of triple-negative breast cancer in BALB / C mice was established, and the tumor volume grew to 200 mm. 3 At that time, a temperature-sensitive hydrogel carrying OMVs was injected into the tumor. After 24 hours, some mice were sacrificed, and the tumor tissue was embedded, sectioned, stained, and analyzed for inflammatory factors in the tumor. A large number of neutrophil infiltrations, significant elastase secretion, and upregulated inflammatory factor levels were found. After 24 hours, the tumor on the surface of the mice was observed to soften and turn black. After 48 hours, a scab formed and fell off in 7-14 days, and the tumor disappeared.

[0031] The advantages of this invention are as follows: The DH5a-OMVs used in this invention possess multiple anti-tumor mechanisms. They can act as exogenous immune activators to activate acute tumor inflammation, triggering anti-tumor immunity, recruiting neutrophils to the tumor site, secreting elastase to selectively kill tumor cells, and releasing a large number of cytokines to amplify acute inflammation and activate specific anti-tumor immunity. Simultaneously, they cause tumor blood vessel rupture, resulting in a darkening of the tumor's appearance. Combined with photothermal therapy, this eradicates the tumor and prevents metastasis. The temperature-sensitive hydrogel used in this invention has excellent intratumoral retention and degradation capabilities, confining acute inflammation within the tumor, greatly improving biosafety, and expanding the application scenarios of bacterial outer membrane vesicles. This invention has significant translational value and application prospects in anti-tumor immunotherapy. Attached Figure Description

[0032] Figure 1 Characterization results of OMVs, Blankgel, and OMVs-gel in Examples 1 and 2 of this invention;

[0033] Figure 2 : Intratumoral retention performance diagram of OMVs-gel in Example 3 of this invention;

[0034] Figure 3 Figure 4 shows the evaluation results of the in vivo safety and tumor angiogenesis rupture induced by OMVs-gel in Examples 4 and 5 of this invention.

[0035] Figure 4 Figure 6 shows the feasibility verification results of OMVs-gel combined with photothermal therapy in Example 6 of this invention.

[0036] Figure 5 : Neutrophil infiltration following acute tumor inflammation induced by OMVs-gel;

[0037] Figure 6 : Intratumoral elastase and pro-inflammatory factor levels after OMVs-gel induces acute inflammation in tumors;

[0038] Figure 7 Evaluation results of the activation of specific anti-tumor immunity after OMVs-gel induces acute inflammation in tumors;

[0039] Figure 8 The statistical results of the antitumor efficacy of OMVs-gel combined with photothermal therapy against 4T1 breast tumors are the experimental results of the embodiments of the present invention.

[0040] Figure 9 Photographic record of the antitumor efficacy of OMVs-gel combined with photothermal therapy on 4T1 breast tumors;

[0041] Figure 10Statistical results of the antitumor efficacy of OMVs-gel combined with photothermal therapy against B16F10 melanoma and CT26 colorectal tumors;

[0042] Figure 11 Photographic record of the antitumor efficacy of OMVs-gel combined with photothermal therapy against B16F10 melanoma;

[0043] Figure 12 Photographic record of the antitumor efficacy of OMVs-gel combined with photothermal therapy on CT26 colorectal tumors;

[0044] Figure 13 Evaluation results of OMVs-gel combined with photothermal therapy inhibiting lung metastasis of 4T1 breast tumors. Detailed Implementation

[0045] The following examples are intended to further illustrate the present invention, but not to limit it.

[0046] To more accurately describe the purpose, features, and functions of the present invention, the present invention is further illustrated below with reference to preferred embodiments. However, these embodiments are only used for more detailed and specific illustration, and the scope of protection of the present invention is not limited to the following specific embodiments.

[0047] The structures, proportions, sizes, etc., shown in the accompanying drawings of this invention are merely for the understanding and reading of those skilled in the art, and are not intended to limit the conditions of this invention. Any changes in proportions or adjustments or modifications to sizes, without affecting the main functions and objectives of this invention, should still fall within the scope of the technical content disclosed in this invention. Furthermore, the relational terms used in this invention are merely for ease of description and are not intended to limit the scope of this invention. Adjustments to their order and relative relationships, without substantial changes at the technical level, also fall within the scope of this invention.

[0048] The term "embodiment" as used in this section refers to the features of the method and the specific results obtained in at least one implementation of the present invention. "Embodiments" appearing in different places in the present invention are not the same embodiment, nor are they mutually exclusive embodiments.

[0049] This section provides a general description of the materials and experimental methods used in this invention. While many of the materials and methods used in this invention are well known in the art, they are described in as much detail as possible. Those skilled in the art will understand that, unless otherwise specified, the materials used in this context are conventional commercial products, and the methods are well known in the art.

[0050] The reagents and instruments used in the examples are as follows:

[0051] Escherichia coli DH5α was purchased from Beina Biotechnology, LB broth medium was purchased from Hangzhou Microbiology, BCA protein kit and SDS-PAGE gel preparation kit were purchased from Boster Biological, Coomassie Brilliant Blue was purchased from Solarbio, methanol, glacial acetic acid and phosphotungstic acid were purchased from Sinopharm Group, pluronic F-127 was purchased from Sigma-Aldrich, sodium alginate was purchased from Aladdin, and DiR dye was purchased from Shanghai Yusheng Biotechnology.

[0052] The biosafety cabinet was purchased from Likang Company, the biochemical incubator from Shanghai Yuejin Company, the low-temperature ultracentrifuge from Beckman Coulter, the NS300 nanoparticle size tracking analyzer from Malvern Company, the small animal in vivo imaging system from Thermo Fisher Scientific, the scanning electron microscope from FEI (Czech Republic), the transmission electron microscope from Hitachi Company, and the microplate reader from Tecan (Switzerland).

[0053] The specific steps are as follows:

[0054] Example 1

[0055] This example is used for the extraction, purification and characterization of Escherichia coli DH5α-OMVs.

[0056] (1) Extraction and purification of OMVs: *Escherichia coli* DH5α glycerol bacteria, revived from storage at -80℃, were inoculated onto Columbia blood agar plates and cultured at 37℃ for 24 hours. After two subcultures, a single colony was picked using a disposable inoculation loop and inoculated into 20 mL of LB broth. The culture was incubated overnight at 37℃ for 12 hours. The culture was then diluted 1:100 with LB broth and transferred to a 500 mL sterile Erlenmeyer flask, and cultured at 37℃ for another 24 hours. When the OD500 of the bacterial culture was detected using a microplate reader... 600 When the value is 1, it can be used to extract OMVs. OD 600 800 ml of DH5α bacterial culture with a value of 1 was collected and aliquoted into 50 mL high-speed round-bottom centrifuge tubes. The tubes were centrifuged at 10,000 g at 4°C for 10 minutes to remove most bacterial debris. The supernatant was filtered through a 0.22 μm filter to remove residual bacteria. The collected filtrate was then concentrated by ultrafiltration through a 100 kDa filter to remove most non-OMV protein components and concentrate the volume. Finally, the concentrated filtrate was ultracentrifuged at 150,000 g at 4°C for 4 hours. The supernatant was discarded, and the OMVs were resuspended in PBS. The OMVs were then ultracentrifuged again at 150,000 g at 4°C for 4 hours. The precipitate was dissolved in PBS to obtain purified OMVs, which were stored at -80°C for later use.

[0057] (2) The particle size and concentration of OMVs were determined by a particle tracking analyzer (NTA), the potential was determined by a dynamic light scattering analyzer (DLS), the morphology was characterized by a transmission electron microscope, and the protein expression was characterized by gel electrophoresis.

[0058] In the particle size distribution diagram ( Figure 1 A) shows that the OMVs particle size is 101.2 ± 2.2 nm and the concentration is 1.49 × 10⁻⁶. 11 The number of particles / ml (the results in the figure are measured after a 100-fold dilution) was -30.25 ± 0.85 mV. Figure 1 A), Transmission electron microscopy results showed that OMVs were spherical structures with a lipid bilayer. Figure 1 B), gel electrophoresis results showed that OMV protein expression had distinct band distributions around 17kDa, 35kDa, and 60kDa. Figure 1 C).

[0059] Example 2

[0060] This embodiment illustrates the preparation of the blank temperature-sensitive hydrogel and the tumor local acute inflammation inducer in this invention.

[0061] (1) Preparation of blank temperature-sensitive hydrogel: Accurately weigh 2g of pluronic F-127 powder and 200mg of sodium alginate powder, place them in a clean beaker, add 16ml of water, and thoroughly wet the powder with a 1000μl disposable tip. Stir thoroughly at 4℃ for 4 hours until the powder is completely dissolved and there are no visible lumps. Place at 4℃ for at least 12 hours to allow air bubbles to completely disappear. Filter the above liquid through a 0.22μm filter membrane under ice bath conditions in a clean bench to remove bacteria, place in a 15ml centrifuge tube, and seal for later use.

[0062] (2) Preparation of OMVs-gel, an acute inflammatory inducer for tumors: OMVs were extracted and purified according to the method described in Example 1 to obtain OMVs stock solution; the concentration of the OMVs stock solution was adjusted to 1×10⁻⁶ with PBS. 11 The working solution for OMVs was prepared using particles / ml. In a clean bench, 100 μl of the OMVs working solution and 900 μl of blank temperature-sensitive hydrogel were accurately measured. Under ice bath conditions, the 100 μl OMVs working solution was first placed in a 10 ml sterile EP tube, followed by the addition of 900 μl of temperature-sensitive hydrogel. The mixture was thoroughly mixed and incubated for 2 hours, then allowed to stand for at least 12 hours to remove air bubbles, yielding OMVs-sol (4℃). This solution converted to OMVs-gel at 37℃. The microstructure of Blankgel and OMVs-gel was observed using a scanning electron microscope. The gelation temperature (LCST) and gelation time of both were determined using the inverted tube method. The cumulative release of OMVs from the OMVs-gel was determined using the transwell plate method.

[0063] The prepared tumor local acute inflammation inducer is a clear solution (OMVs-sol) at 4°C and a clear gel (OMVs-gel) at 37°C. Figure 1 D). The gelation temperature of Blank gel was 18±1℃ and the gelation time was 39±2s, as determined by the inverted test tube method; the gelation temperature of OMVs-gel was 18±1℃ and the gelation time was 38±1s. Figure 1 E). Scanning electron microscopy results showed that the microstructure of the prepared Blankgel and OMVs-gel was a porous network structure. Figure 1 F). The drug release curve shows that OMVs-gel basically released all OMVs within 72 hours. Figure 1 G).

[0064] Example 3

[0065] This embodiment is used to illustrate the intratumoral retention performance of the OMVs-gel of the present invention.

[0066] (1) Mix 10 μl of membrane dye DiR (1 mg / ml) with the OMVs working solution (10 μl) described in Example 3. 10 1000 μl of the sample (particles / ml) was incubated in the dark for 30 minutes, then centrifuged at 2500g using a 100 kDa ultrafiltration tube for 10 minutes to remove unbound free DiR, yielding DiR-labeled free OMVs (DiR-OMVs), which were stored at 4°C for later use. This DiR-OMVs were used to prepare the aforementioned OMVs-gel, resulting in a temperature-sensitive hydrogel loaded with DiR-labeled OMVs.

[0067] (2) Orthotopic xenograft model was constructed using the 4T1 breast cancer cell line: BALB / C mice (♀, 6 weeks old) were injected with 2×10⁻⁶ cells under the fat pad of the left mammary gland. 5 4T1 cells; until the tumor volume reaches 150 mm. 3 Subsequently, participants were randomly divided into two groups: a control group (DiR-OMVs group) and a temperature-sensitive hydrogel group loaded with DiR-labeled OMVs (DiR-OMVs-gel group). 100 μl of each of the different formulations (OMVs concentration of 10) was injected intratumorally. 10 After (particles / ml), the fluorescence distribution was observed at 0, 24, 48, 72, and 96 hours using a small animal in vivo imaging system.

[0068] Imaging results showed that DiR-OMVs-gel had the strongest retention effect within the tumor and the slowest degradation, taking approximately 4 days; DiR-OMVs, on the other hand, degraded completely within one day, and no fluorescence was observed at the tumor site after 4 days. Figure 2 AE). However, fluorescent signals were observed in the tumor-draining lymph nodes ( ). Figure 2The presence of fluorescence (FG) indicates that OMVs can enter the tumor draining lymph nodes and stimulate subsequent immune responses. Furthermore, the DiR-OMVs-gel group showed no fluorescent distribution in the liver, demonstrating its safety.

[0069] Example 4

[0070] This example illustrates how OMVs-gel enhances the in vivo safety of OMVs.

[0071] (1) Construct the 4T1 breast tumor orthotopic mouse model as described in Example 3, and wait until the tumor grows to 150 mm. 3 Subsequently, the mice were randomly divided into four groups, with six mice in each group: control group (PBS group), free OMVs solution injected via tail vein (FreeOMVs in s.v. group), OMVs solution injected intratumorally (FreeOMVs in s.t. group), and OMVs-gel injected intratumorally (OMVs-gel in s.t. group). Different formulations (OMVs concentration of 10) were injected intratumorally. 10 (particles / ml), and within 24 hours after administration, the body temperature changes and mortality rates of all mice were recorded. After 24 hours, 3 mice from each group were sacrificed, and serum was collected for analysis of pro-inflammatory factor levels (IL-6, TNF-α). After 72 hours, the remaining mice from each group were sacrificed, and the major organs (heart, liver, spleen, lung, kidney) were removed, sectioned, embedded in paraffin, and stained with H&E.

[0072] Mortality statistics showed that OMVs solution injected via the tail vein had the highest toxicity, causing drastic changes in body temperature in mice, and all mice died within 12 hours; the intratumoral injection of free OMVs was the second most toxic; OMVs-gel had the lowest toxicity, with no mouse deaths and no significant changes in body temperature. Figure 3 AB). Section staining results showed no lesions in the major organs. Figure 3 C); Serum inflammatory factor level analysis showed that the levels of inflammatory factors were highest in mice injected via the tail vein and lowest in the OMVs-gel group. Figure 3 D), combined with mortality statistics, it can be proven that OMVs-gel greatly improves biosafety.

[0073] Example 5

[0074] This embodiment is used to illustrate the ability of the OMVs-gel of the present invention to induce tumor blood vessel rupture.

[0075] The 4T1 breast cancer orthotopic tumor-bearing mouse model of Example 3 was constructed, and the tumor volume was allowed to grow to 150 mm. 3 Subsequently, they were randomly divided into two groups: a control group (PBS group) and an experimental group (OMVs-gel group). 100 μl of each of the different formulations (OMVs concentration of 10) was injected intratumorally. 10After obtaining the samples (particles / ml), the tumor's appearance and color changes were observed at 0, 12, and 24 hours. Mice were sacrificed after 24 hours, the tumors were removed, fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and immunofluorescence stained with CD31 antibody to observe the integrity of blood vessels.

[0076] The results showed that the experimental group had a significantly darker tumor appearance compared to the control group. Figure 3 E); the results of section staining showed that the integrity of tumor blood vessels in the experimental group was significantly disrupted. Figure 3 F) demonstrates that OMVs can cause tumor blood vessel rupture. The resulting darkening of the tumor's appearance can be combined with photothermal therapy for further tumor treatment.

[0077] Example 6

[0078] This embodiment is used to illustrate the feasibility of the OMVs-gel combined photothermal therapy of the present invention.

[0079] (1) Construct the 4T1 breast tumor orthotopic mouse model as described in Example 3, and wait until the tumor grows to 150 mm. 3 Then, the mice were randomly divided into two groups, with six mice in each group: control group (PBS+NIR) 808 Group ), Experimental group (OMVs-gel+NIR) 808 Group). Intratumoral injection of 100 μl of each of the different formulations (OMVs concentration of 10). 10 (particles / ml), after 24 hours the tumor appearance turned black, at which point 808nm near-infrared light was applied for 5 minutes (power 0.8W / cm). 2 The tumor surface temperature was monitored every minute using an infrared imager, and statistical analysis was performed.

[0080] The results showed that, compared with the control group, OMVs-gel+NIR 808 The tumor surface temperature in the mice in this group gradually increased to 55℃ over time, reaching the effective tumor-killing temperature. Figure 4 A, 4B); and the heating area is concentrated in the area where the tumor has turned black, without damaging the surrounding normal tissue. Figure 4 A).

[0081] Example 7

[0082] This embodiment illustrates the acute inflammation-inducing effect and anti-tumor mechanism of the OMVs-gel of the present invention on tumors. It should be specifically noted that this embodiment uses 4T1 breast cancer, B16F10 melanoma, and CT26 colorectal tumor as models, but the present invention exhibits significant tumor-suppressing effects on other solid tumors.

[0083] (1) Construct the 4T1 breast tumor orthotopic mouse model as described in Example 3, and wait until the tumor grows to 150 mm.3 Afterwards, the mice were randomly divided into two groups, with six mice in each group: a control group (PBS group) and an experimental group (OMVs-gel group). The experimental group received an intratumoral injection of 100 μl of OMVs-gel prepared in Example 2 (OMVs concentration of 10). 10 The control group received an equal volume of PBS (100 μl) and was administered the drug once.

[0084] (2) After 24 hours, three mice from each group were sacrificed. Tumors were collected and embedded in 4% paraformaldehyde, sectioned, and stained with H&E. Immunohistochemical staining was performed for neutrophil elastase ELANE and histone H1.0. The levels of intratumoral inflammatory factors (IL-1β, IL-6, TNF-α, IFN-γ, IL-12p70, IL-17A) and chemokines (CXCL1, CCL2) were also measured. After 72 hours, the remaining mice from each group were sacrificed. The spleens and tumor drainage lymph nodes were removed and prepared into single-cell suspensions. Flow cytometry was then used to analyze DC cells (CD80). + CD86 + ) and T cells (CD3) + CD8 + Phenotypic analysis.

[0085] H&E staining results showed that 24 hours after intratumoral injection of OMVs-gel, a large number of neutrophil infiltrations and significant tumor cell death were observed. Figure 5 Immunohistochemical staining results showed that a large number of elastase ELANE and histone H1.0 were present in the tumor microenvironment. Figure 6 A); Analysis of intratumoral inflammatory factor levels demonstrated that a significant acute inflammatory response occurred within the tumor after OMVs-gel injection, with significantly elevated levels of pro-inflammatory and chemokines. Figure 6 B); The proportions of mature DC cells and T cells in the spleen and tumor-draining lymph nodes were highest in the OMVs-gel group, confirming that subsequent specific anti-tumor immunity had been activated. It was observed that after OMVS-gel treatment, the proportions of DC cells in the spleen and tumor-draining lymph nodes increased by 24% and 20%, respectively, compared to the control group. Figure 7 A, 7B), the proportion of T cells increased by 35% ( Figure 7 C).

[0086] Example 8

[0087] This embodiment illustrates the efficacy of the OMVs-gel combined with photothermal therapy for tumors according to the present invention. It should be specifically noted that this embodiment uses 4T1 breast cancer tumors, B16F10 melanoma, and CT26 colorectal tumors as models, but the present invention exhibits significant tumor-suppressing effects on other solid tumors.

[0088] (1) The 4T1 breast tumor orthotopic tumor-bearing mouse model from Example 3 was constructed, and another batch of bioluminescent mouse orthotopic tumor models was constructed using 4T1-Luc cells. Subcutaneous tumor models were constructed using B16F10 cells (for C57BL6j mice) and CT26 cells (for BALB / C mice). The tumors were allowed to grow to 150 mm². 3 All tumor models were randomly divided into 5 groups: control group (PBS group), blank temperature-sensitive hydrogel group (Blankgel group), single photothermal therapy group (Blankgel+NIR group). 808 Group), OMVs-gel group, photothermal therapy combined group (OMVs-gel+NIR) 808 Group). 100 μl of the corresponding preparation was injected intratumorally. Twenty-four hours later, both the single-photothermal therapy group and the combined photothermal therapy group were irradiated with 808 nm near-infrared light for 5 minutes (power 0.8 W / cm²). 2 ).

[0089] (2) After administration, the growth of 4T1-Luc tumors was monitored using a small animal in vivo imaging system (bioluminescence), and the changes in the volume of other tumors were measured. The changes in the volume of tumors in each group were statistically analyzed. After 14 days, the mice were sacrificed, and the tumors were removed for 4% paraformaldehyde, paraffin embedding, sectioning, H&E staining, TUNEL staining, and Ki67 staining. The lung tissue was removed for 4% paraformaldehyde, paraffin embedding, sectioning, and H&E staining to observe the inhibitory effect of OMVs-gel on lung metastasis of 4T1 tumors.

[0090] The efficacy results showed that the OMVs-gel group and the OMVs-gel+NIR group of different models 808 Tumor growth in the group of mice was significantly inhibited. In the 4T1 orthotopic tumor model, according to... Figure 8 Dosing regimen A, regardless of whether the tumor cells carried the fluorescent gene or the normal tumor cell model, resulted in a rapid reduction in tumor volume within 14 days after administration. Figure 8 B, Figure 9 The volume of the control group continued to increase (), while the volume of the control group continued to increase (). Figure 8 (C, 8D) Tumor volume removed from euthanized mice was significantly reduced, and tumor growth was significantly inhibited or even completely eliminated. Figure 8 E, 8F); there was no significant change in the body weight of the mice after treatment ( Figure 8 G), significantly prolonged survival ( Figure 8 H). Tumor volume changes and post-treatment tissue section staining in each group of mice demonstrated the potent efficacy of OMVs-gel. Figure 8 I, 8J). The combination of OMVs-gel with photothermal therapy after causing tumor surface darkening further enhances the tumor-eliminating ability of OMVs-gel. In melanoma and colorectal tumor models, according to... Figure 10After administration of regimen A, the tumor volume in mice was found to be ( Figure 10 B, 10C), weight ( Figure 10 The trends of D and 10E were consistent with the 4T1 model, showing a significant decrease; mouse survival was significantly prolonged. Figure 10 F, 10G); and the tumor surface also turned black (F, 10G); Figure 11 ,12). Furthermore, the immune response induced by OMVs-gel can inhibit lung metastases of the primary tumor ( Figure 13 The above results strongly demonstrate that OMVs-gel can effectively eradicate solid tumors in situ by inducing acute inflammation in the tumor site.

[0091] Although embodiments have been described to some extent in this invention, the various conditions and parameters in the embodiments may be appropriately varied without departing from the spirit and scope of the invention. It is understood that the invention is not limited to the embodiments and test conditions described, but falls within the scope of the claims, including substitutions for each factor or parameter.

Claims

1. Use of a tumor local acute inflammation inducer, characterized in that, The preparation method of the agent for inducing acute inflammation at the tumor site includes the following steps: It is prepared from bacterial outer membrane vesicles and temperature-sensitive hydrogels; the bacterial outer membrane vesicles are secreted by Escherichia coli DH5α. The temperature-sensitive hydrogel is obtained by stirring and dissolving 15-20 wt% pluronic F-127, 1-2 wt% sodium alginate, and the balance pure water at 4-10°C. 10 bacterial outer membrane vesicles per 1000 μl of temperature-sensitive hydrogel 9 -10 12 individuals; The preparation process of the bacterial outer membrane vesicles is as follows: After culturing Escherichia coli DH5α for 16-24 hours, the bacterial culture was centrifuged at 10000g for 10-20 minutes, the precipitate was discarded, and the supernatant was filtered through a 0.22μm filter membrane. The filtrate was then centrifuged at 150000g-200000g for 4-6 hours, the supernatant was discarded, the precipitate was washed with PBS, and centrifuged again at 150000g-200000g for 4-6 hours, the supernatant was discarded, and the precipitate was the purified Escherichia coli DH5α outer membrane vesicles. Add bacterial outer membrane vesicles to a temperature-sensitive hydrogel, mix the two thoroughly, incubate, and allow to stand to remove air bubbles to obtain the desired product. Bacterial outer membrane vesicles and temperature-sensitive hydrogels were mixed, incubated, and left to stand for at least 12 hours, with the entire process kept at a low temperature of 4-10℃. The tumor is breast cancer; the inducing agent is an intratumoral injection preparation.

2. Use according to claim 1, characterized in that, This preparation is used to develop agents that recruit neutrophils to secrete elastase, selectively kill tumor cells, and secrete large amounts of pro-inflammatory factors to amplify acute inflammation, induce specific anti-tumor immunity, treat primary tumors, and prevent their metastasis.

3. Use according to claim 2, characterized in that, Prepare formulations for in situ injection therapy of common solid tumors in clinical practice or for inhibiting lung metastasis of primary tumors.

4. Use according to claim 1, characterized in that, It is used to prepare a formulation that induces rupture of tumor blood vessels and extravasation of red blood cells, resulting in a darkening of the tumor's appearance.

5. Use according to claim 4, characterized in that, This formulation is used to prepare a combination of photothermal therapy for the treatment of clinical solid tumors.

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