A bacterial cell membrane / dna tetrahedron complex, and a preparation method and use thereof

Abstract

The application provides a bacterial cell membrane / DNA tetrahedron complex and a preparation method and use thereof, and belongs to the technical field of biological medicines. The complex is obtained by taking bacterial cell membranes as drug carriers and loading DNA tetrahedrons. The application is based on DNA tetrahedrons, takes bacterial cell membranes as drug carriers, utilizes the natural chemotaxis of bacterial cell membranes to bacterial tumor biofilms, carries DNA tetrahedron drugs to bacterial biofilms that specifically gather around tumors, simultaneously releases the drugs, and enables the drugs to be accurately applied to tumor cells. The bacterial cell membranes are preferably Streptococcus cell membranes. The application not only has good tumor biofilm chemotaxis, greatly enhances the infiltration of drugs to solid tumors, and the bacterial carriers can affect immune cells in the tumor microenvironment, play an immune enhancement role, and ultimately enhance the inhibition of tumor growth by the drugs.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to a bacterial cell membrane / DNA tetrahedral complex, its preparation method, and its uses. Background Technology

[0002] Numerous studies have shown that self-assembled DNA tetrahedra, as a novel type of nucleic acid nanomedicine, can promote the regeneration of various stem cells (adipose stem cells, bone marrow stem cells, neural stem cells, etc.), and their inherent anti-inflammatory and antioxidant functions also make them applicable to the treatment of various diseases. Furthermore, this three-dimensional tetrahedral structure possesses excellent biocompatibility and flexible editability, and can be taken up by most cells via endocytosis. Therefore, DNA tetrahedra are frequently used as drug carriers, with various nucleic acid sequences (aptamers, siRNA, microRNA, etc.) and chemotherapeutic drugs (paclitaxel, doxorubicin, etc.) loaded onto them through incubation, chemical linkage, or other methods, and then carried into cells in large quantities to exert their respective effects.

[0003] Patent application CN109663134B discloses a method using DNA tetrahedrons to carry 5-fluorouracil and AS1411, which exhibits significantly stronger anticancer effects than 5-fluorouracil and better safety. 5-Fluorouracil, the most common antimetabolite of uracil, is converted into fluorouracil deoxynucleotide (F-dUMP) after entering the body. It covalently binds to the active site of thymidine synthase, inhibiting its enzyme activity and thus leading to a deoxynucleotide deficiency, affecting DNA synthesis. Furthermore, fluorouracil nucleoside (FUMP) can be incorporated into DNA and RNA as a pseudometabolite, affecting cellular function and producing cytotoxicity. AS1411 is a 26-base guanine-rich oligonucleotide sequence that can form G-tetramers. It can specifically bind to nucleolin, which is highly expressed on the nuclear membrane of tumor cells, acting as a nucleic acid aptamer. It can also form a complex with nucleolin, causing nucleolin redistribution, inhibiting ribosome biosynthesis, and inducing apoptosis in tumor cells. AS1411 and 5-fluorouracil-modified DNA tetrahedra possess both tumor cell targeting and killing properties. First, AS1411 targets tumor cells, and the DNA tetrahedra carry 5-fluorouracil into the tumor cells and release it, affecting the synthesis of tumor cell DNA and RNA, thereby exerting a tumor cell killing effect.

[0004] New research indicates that microbes exist within tumor cells and immune cells, suggesting that these microbes can influence the state of the tumor microenvironment. The microbes in tumor tissues differ significantly from those in normal tissues, and certain microbes specifically aggregate in tumor tissues, making them potent therapeutic targets.

[0005] Tumors are closely related to their tumor microenvironment. Tumors can influence their microenvironment by releasing cellular signaling molecules, promoting angiogenesis and inducing immune tolerance. Conversely, immune cells within the tumor microenvironment can affect the growth and development of cancer cells. The tumor microenvironment refers to the microenvironment surrounding tumor cells, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, various signaling molecules, and the extracellular matrix. The tumor microenvironment typically presents an overall hypoxic and acidic environment, leading to extensive apoptosis of tumor and surrounding tissue cells, resulting in inflammatory infiltration and the secretion of inflammatory factors. Furthermore, the development and progression of the tumor itself trigger corresponding immune responses. T cells, as the main group of cellular immunity, play a crucial role in the killing of tumor cells by immune cells. Within the tumor microenvironment, CD8-positive T subtypes can kill tumor cells.

[0006] The DNA tetrahedron co-modified with AS1411 and 5-fluorouracil is a novel DNA nanomedicine that simultaneously targets and kills tumor cells. First, the DNA aptamer AS1411 targets tumor cells. The DNA tetrahedron drug delivery carrier then carries and releases 5-fluorouracil into the tumor cells, affecting the synthesis of DNA and RNA in these cells and thus exerting a killing effect. In vitro, it exhibits a relatively ideal killing effect on tumor cells. However, in vivo, its infiltration into tumors is poor, resulting in low drug concentrations in tumor tissues and thus a less effective inhibitory effect on solid tumor growth. Furthermore, the AS1411 and 5-fluorouracil co-modified DNA tetrahedron does not affect the immune cells in the tumor microenvironment, thus failing to enhance immunity. Summary of the Invention

[0007] Although 5-fluorouracil and AS1411-modified DNA tetrahedra exhibit promising in vitro tumor cytotoxicity, their efficacy in treating solid tumors in vivo is poor due to limited tumor invasiveness. To address this issue, this invention provides a bacterial cell membrane / DNA tetrahedron complex, its preparation method, and its uses. This invention addresses the problem of poor tumor invasiveness and unsatisfactory in vivo efficacy of DNA tetrahedron drugs by using a bacterial cell membrane as a carrier. Leveraging the natural chemotaxis of the bacterial cell membrane to bacterial biofilms aggregated outside the tumor, tetrahedron drugs are delivered to tumor tissues and precisely target tumor cells, thereby improving the in vivo therapeutic effect of tetrahedron drugs.

[0008] This invention provides a bacterial cell membrane / DNA tetrahedral complex, which is a complex obtained by loading DNA tetrahedra onto a bacterial cell membrane as a drug carrier.

[0009] Furthermore, the complex is a complex obtained by mixing and incubating bacteria and DNA tetrahedra.

[0010] Furthermore, the bacterial concentration during incubation is 10. 8 ~10 10 CFU / ml; and / or, the concentration of the DNA tetrahedron is 500–4000 nM.

[0011] Furthermore, the bacterial concentration during incubation is 10. 9 CFU / ml; and / or, the concentration of the DNA tetrahedron is 2000 nM.

[0012] Furthermore, the incubation temperature is 37°C; and / or the incubation time is 10–30 min;

[0013] Preferably, the incubation time is 15 minutes.

[0014] Furthermore, the incubation process also includes purification, with the following steps: centrifugation, collection of precipitate, fixation with glutaraldehyde solution, washing, and centrifugation again;

[0015] Preferably, the centrifugation conditions are 8000-10000 rpm for 5-10 min;

[0016] And / or, the concentration of the glutaraldehyde solution is 2-5%;

[0017] And / or, the fixed time is 10 to 30 minutes.

[0018] Furthermore,

[0019] The bacteria in question are Streptococcus mutans.

[0020] And / or, the DNA tetrahedron is synthesized by self-assembly of four DNA single strands; the nucleotide sequences of the four DNA single strands are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.5, respectively; the 5' end of the nucleotide sequence shown in SEQ ID NO.3 is linked to 5-fluorouracil.

[0021] Furthermore, the method for synthesizing the DNA tetrahedron includes the following steps: adding four single strands of DNA to TM buffer, maintaining at 95°C for 10 min, cooling to 4°C and maintaining for more than 30 min, to obtain the product;

[0022] Preferably, the four DNA single strands are four DNA single strands in an equimolar ratio.

[0023] The present invention also provides a method for preparing the aforementioned bacterial cell membrane / DNA tetrahedral complex, comprising the following steps:

[0024] (1) Adjust the bacterial concentration to 10 8 ~1010 CFU / ml;

[0025] (2) Adjust the concentration of DNA tetrahedrons to 500–4000 nM;

[0026] (3) Mix the two together and incubate to obtain the product;

[0027] Preferably, in step (1), the bacterial concentration is adjusted to 10. 9 CFU / ml;

[0028] And / or, in step (2), the concentration of DNA tetrahedrons is adjusted to 2000 nM;

[0029] And / or, in step (3), the incubation temperature is 37°C; and / or, the incubation time is 10 to 30 minutes;

[0030] And / or, in step (3), the incubation process further includes purification, the steps of which are as follows: centrifugation, collection of precipitate, fixation with glutaraldehyde solution, washing, and centrifugation again;

[0031] More preferably, in step (3), the incubation time is 15 minutes;

[0032] And / or, in step (3), the centrifugation conditions are 8000-10000 rpm for 5-10 min;

[0033] And / or, the concentration of the glutaraldehyde solution is 2-5%;

[0034] And / or, the fixed time is 10 to 30 minutes.

[0035] The present invention also provides the use of the aforementioned bacterial cell membrane / DNA tetrahedral complex in the preparation of antitumor drugs;

[0036] Preferably, the tumor is oral squamous cell carcinoma.

[0037] The present invention also provides an antitumor drug, which is prepared by using the aforementioned bacterial cell membrane / DNA tetrahedral complex as the active ingredient, plus pharmaceutically acceptable excipients.

[0038] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0039] This invention is based on DNA tetrahedral nanomedicine, using bacterial cell membranes as drug carriers. Utilizing the natural chemotaxis of bacterial cell membranes to bacterial tumor biofilms, the DNA tetrahedral nanomedicine is delivered to bacterial biofilms specifically aggregated around tumors, simultaneously releasing the drug and enabling precise application to tumor cells. The bacterial cell membrane used in this invention is preferably that of *Streptococcus mutans*. This invention not only possesses excellent tumor biofilm chemotaxis, significantly enhancing the drug's invasiveness to solid tumors, but also allows the bacterial carrier to influence immune cells in the tumor microenvironment, exerting an immune-enhancing effect and ultimately strengthening the drug's inhibitory effect on tumor growth.

[0040] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0041] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0042] Figure 1 The identification results of DNA nanomedicine carried by Streptococcus mutans cell membrane are as follows: a) Results of selecting the optimal reaction time by quantitative fluorescence method; b) Results of co-localization of Streptococcus mutans and DNA nanomedicine by immunofluorescence staining; c) Results of selecting the working concentration of AT5 by flow cytometry; d) Zeta potential results of AT5, simple bacteria (Sm), and Sm-AT5; e) Characterization results of material morphology by transmission electron microscopy and immunoelectron microscopy; f) Characterization results of material size by dynamic light scattering.

[0043] Figure 2 Results of efficacy and safety of DNA nanomedicine carried by *Streptococcus mutans* cell membrane: a) Results of metabolic time of different materials in mice as detected by in vivo imaging in small animals; b) Distribution of materials in vivo as observed by colony culture; c) Statistical graph of distribution of materials in vivo as observed by colony culture; d) Results of hemolysis test to detect hemolysis of materials; e) Statistical graph of hemolysis results.

[0044] Figure 3 To demonstrate the uptake of the drug of the present invention in tumor cells in in vitro experiments: a) Immunofluorescence detection of Sm-AT5 chemotaxis on tumor biomembranes and AT5 release results; b) Transwell model used for in vitro studies; c) Immunofluorescence observation of Sm-AT5 uptake in human oral squamous cell carcinoma cells (SCC25).

[0045] Figure 4 The results of cytotoxicity assays for DNA nanomedicine carried on the cell membrane of Streptococcus mutans are as follows: a) Transwell model used for in vitro studies; b) CCK-8 assay results; c) Statistical results of cell cycle assay by flow cytometry; d) Statistical results of cell apoptosis assay by flow cytometry; e) Protein expression results of cells detected by Western blotting; f and g) Statistical bar charts of protein expression results of cells detected by Western blotting; h and i) Protein expression levels of cells detected by immunofluorescence staining.

[0046] Figure 5 The results are as follows: a) Flow cytometry results of cell cycle detection; b) Flow cytometry results of apoptosis detection.

[0047] Figure 6 The following are the animal experimental results of the drug of the present invention: a is a schematic diagram of the tumor-bearing animal treatment model; b is the result of immunofluorescence observation of the tumor infiltration of the drug; c is the result of solid tumor imaging measurement; d is the result of tumor volume calculation; e is the result of mouse survival rate; f is the result of H&E staining; g is the result of TUNEL staining.

[0048] Figure 7 The following are the results of tumor microenvironment studies for the drug of this invention: a) Results of flow cytometry sorting of mature dendritic cells; b) Statistical bar chart of flow cytometry sorted mature dendritic cells; c) Immunofluorescence staining results of CD11c expression; d) Flow cytometry sorting of mature dendritic cells with antigen-presenting capacity; e) Statistical graph of flow cytometry sorted mature dendritic cells with antigen-presenting capacity; f) Flow cytometry sorting of tumor-infiltrating CD3 and CD8 cells; g) Flow cytometry sorting of tumor-infiltrating CD3 and CD8 cells. Cellular statistics; h shows the results of CD4 and CD8 expression in tumor infiltration observed by immunofluorescence staining; i shows the results of CD4 and CD8 expression in tumor-infiltrating lymph nodes observed by immunofluorescence staining; j shows the proportion of effector memory T cells (Tem) and central memory T cells (Tcm) in mouse peripheral blood sorted by flow cytometry; k shows the statistical graph of the proportion of effector memory T cells (Tem) and central memory T cells (Tcm) in mouse peripheral blood sorted by flow cytometry; l shows the infiltration of neutrophils in tumors observed by immunofluorescence staining. Detailed Implementation

[0049] The raw materials and equipment used in the specific embodiments of the present invention are all known products, obtained by purchasing commercially available products.

[0050] Example 1: Preparation of the bacterial cell membrane / DNA tetrahedral complex of the present invention

[0051] 1. Preparation of DNA tetrahedra modified with 5-fluorouracil and AS1411

[0052] Prepared according to the method described in patent announcement number CN109663134B, the method is as follows:

[0053] Four single-stranded DNA tetrahedra were prepared as S1, S2, S3, and S4-AS1411; among them, the 5' end of the S3 strand was ligated with 5-fluorouracil (5-FU); S4-AS1411 was obtained by ligating AS1411 to the 5' end of the S4 strand. The sequences of the single-stranded DNA tetrahedra S1, S2, S3, S4, S4-AS1411, and AS1411 are shown in Table 1, where the lowercase part of the sequence is the AS1411 sequence.

[0054] Table 1. Sequence List

[0055]

[0056] One μL each of four 1M single-stranded DNA tetrahedra (S1, S2, S3, and S4-AS1411) were added to 96 μL of a buffer solution containing MgCl2 and Tris at pH 8.0 (0.605 g of Tris-base and 5.075 g of MgCl2·6H2O dissolved in 50 mL of ddH2O). The entire 100 μL system was heated to 95 °C for 10 minutes, then cooled to 4 °C for 30 minutes to synthesize a 100 μL volume of 1000 nM 5-fluorouracil and AS1411-modified DNA tetrahedron (DNA nanomedicine, AT5).

[0057] By linking Cy5 to the 5' end of the S1 strand and labeling it as S1-Cy5, and replacing S1 with S1-Cy5 according to the above method, a Cy5-labeled DNA tetrahedral drug can be prepared.

[0058] 2. Synthesis of the Streptococcus mutans membrane / DNA tetrahedral complex

[0059] Collect Streptococcus mutans in the logarithmic growth phase and adjust the concentration to 10. 9 The DNA nanomedicine (AT5) prepared in step 1 was concentrated and resuspended in DPBS to 2000 nM. After incubating 1 mL of Streptococcus mutans and 1 mL of AT5 in a 37°C incubator for 15 min, the mixture was centrifuged at 10000 rpm for 5 min, the supernatant was discarded, the precipitate was collected, fixed with 2% glutaraldehyde solution for 30 min, washed three times with DPBS, and centrifuged again to obtain DNA nanomedicine (Sm-AT5) carried by the cell membrane of Streptococcus mutans at a concentration of 2000 nM.

[0060] 3. Identification

[0061] (1) AT5 was incubated with Streptococcus mutans using the method described above, with varying incubation times. The optimal incubation time for the DNA nanodrug and Streptococcus mutans was selected using quantitative real-time fluorescence. By calculating the DNA concentration in the supernatant at different time points and comparing it with the initial DNA concentration of 2000 nM, the binding rate of the DNA drug at different reaction times was calculated.

[0062] The results are as follows Figure 1 As shown in Figure a, at 15 min, up to approximately 57.5% of AT5 can be bound to the cell membrane of Streptococcus mutans, therefore 15 min is considered the optimal binding time.

[0063] (2) Immunofluorescence staining was used to observe the co-localization of Streptococcus mutans and DNA nanomedicine: AT5 was labeled with Cy5 (Cy5 was used to label the S1 chain, and then Cy5-labeled AT5 was prepared according to the above method), and Streptococcus mutans was stained with SYTO 9. AT5 and Streptococcus mutans were incubated according to the above method, and the binding of bacteria and drugs was observed at 15 min, 1 h and 4 h of incubation.

[0064] The results are as follows Figure 1 As shown in b: at 15 min, the most observable colocalization of AT5 with Streptococcus mutans was observed. Figure 1 The result is consistent.

[0065] (3) Flow cytometry to select the working concentration of AT5: Cy5-labeled AT5 was prepared in concentration gradients of 500, 1000, 2000 and 4000 nM. The complex was prepared according to the above method, and the binding ratio of the complex to the cell membrane of Streptococcus mutans was detected.

[0066] The results are as follows Figure 1 As shown in c: When AT5 is concentrated to 2000 nM, its binding rate with bacteria is as high as 82.8%, therefore 2000 nM is chosen as the working concentration.

[0067] (4) Detect the zeta potential of AT5, Streptococcus mutans simplex (Sm) and Sm-AT5.

[0068] The results are as follows Figure 1 As shown in d: AT5, as a DNA material, exhibits a negative potential, Streptococcus mutans, as a bacterium, also exhibits a negative charge, while Sm-AT5 is weakly negatively charged, representing the combination of the two.

[0069] (5) Transmission electron microscopy and immunoelectron microscopy were used to characterize the morphology of the material.

[0070] The results are as follows Figure 1As shown in e, AT5 has an approximately triangular shape under transmission electron microscopy, with a particle size in the range of 20 nm. The morphology of bacteria (Sm) and Sm-AT5 is similar, with a particle size of about 1000 nm.

[0071] (6) Dynamic light scattering is used to characterize the size of materials.

[0072] The results are as follows Figure 1 As shown in f: Consistent with the results of transmission electron microscopy, the particle size of AT5 is 19.15 nm, while the particle sizes of bacteria (Sm) and Sm-AT5 are 1125.09 nm and 1132.18 nm, respectively.

[0073] 4. Effectiveness and safety

[0074] (1) Detection of the metabolic time of different materials (Cy5-labeled AT5 and Cy5-labeled Sm-AT5) in mice using small animal in vivo imaging: After local injection of Cy5-labeled drugs (balb / c male tumor-bearing mice, local injection at a concentration of 500 nM into the buccal mucosa at the corner of the mouth), the mice were placed in a small animal in vivo imaging instrument.

[0075] The results are as follows Figure 2 As shown in Figure a: AT5 is rapidly metabolized to the liver within 4 hours of local injection and excreted from the body after 12 hours; while Sm-AT5 is partially metabolized to the liver after 12 hours of local injection, but some remains in the tumor.

[0076] (2) Colony culture to observe the distribution of materials in vivo: Using balb / c male tumor-bearing mice, a concentration of 500 nM was injected locally into the buccal mucosa at the corner of the mouth of the mice. After 5 injections (the CTRL group was injected with physiological saline), on the 17th day of the treatment, the heart, liver, spleen, lungs, kidneys and tumors of the mice were collected and prepared into homogenates, mixed with bacterial solid culture medium, and cultured on bacterial agar plates for 24 hours. The number of bacterial colonies was counted and statistically analyzed.

[0077] The results are as follows Figure 2 As shown in b and 2c: local injection resulted in most of the material accumulating within the tumor tissue, followed by more in the kidneys, with less in other organs, indicating the drug's biocompatibility with other organs.

[0078] (3) Hemolysis test to detect the hemolytic activity of the materials: A quantitative colorimetric method was used to determine the total blood hemoglobin (TBH) and plasma free hemoglobin levels after blood contact with nanoparticles (Cy5-labeled AT5 and Cy5-labeled Sm-AT5). The content of hemoglobin (PFH) was measured. Hemoglobin and its inducing agents, except for thiohemoglobin, are oxidized to methemoglobin by ferricyanide in strong alkali. Methemoglobin then reacts further with cyanide (Drabkin's solution) to form cyanogenic methemoglobin. Cyanogenic methemoglobin can be detected at 540 nm using a spectrophotometer. A standard curve was established using hemoglobin standards, with concentrations ranging from 0.025 to 0.80 mg / mL. Quality control samples at low (0.0625 mg / mL), medium (0.125 mg / mL), and high (0.625 mg / mL) concentrations were prepared to monitor the experiment. The experimental results are expressed as the percentage of hemolysis and can be used to evaluate the in vitro acute hemolytic properties of the nanoparticles.

[0079] The results are as follows Figure 2 As shown in d and 2e: Compared with the positive control (PC), AT5 and Sm-AT5 showed virtually no hemolytic activity.

[0080] The following specific experimental examples demonstrate the beneficial effects of the present invention.

[0081] Experimental Example 1: In vitro experiments demonstrate the uptake of the complex of the present invention in tumor cells.

[0082] The preparation methods of AT5 and Sm-AT5 used in this experimental example are the same as in Example 1.

[0083] (1) Immunofluorescence detection of Sm-AT5 chemotaxis and AT5 release in tumor biofilms: Extracellular polysaccharides of biofilms were labeled with Alexa Fluor 647, AT5 was labeled with AMCA, and bacteria were labeled with SYTO 9. Sm-AT5 labeled with AMCA and SYTO 9 was added to the biofilm, and the chemotaxis of Sm-AT5 to the biofilm and the gradual release of AT5 were observed under a confocal microscope from 2h, 6h to 12h and 24h.

[0084] The results are as follows Figure 3 As shown in Figure a: the green fluorescence representing Sm-AT5 can be seen to accumulate on the surface of the tumor biofilm, and the blue fluorescence representing AT5 gradually increases, indicating the gradual release of AT5, proving that the release amount gradually increases from 2h to 24h.

[0085] (2) Figure 3 b represents the Transwell model used for in vitro studies.

[0086] (3) Immunofluorescence observation of Sm-AT5 uptake by human oral squamous cell carcinoma cells (SCC25): Tumor biofilms were cultured in the upper chamber of Transwell, and cells were seeded in the lower well plate of Transwell. After 24 h, Cy5-labeled Sm-AT5 was added to the upper biofilm, and the uptake of the drug by cells in the lower well plate was observed under a confocal microscope to obtain the uptake of Sm-AT5 by SCC25 cells after 6 h, 18 h and 30 h.

[0087] The results are as follows Figure 3 As shown in Figure c, the increase in the number of red fluorescent cells from 6h to 30h indicates that the uptake of Sm-AT5 by cells gradually increases, reaching its peak at 30h.

[0088] The above experimental results show that the complex of the present invention has the characteristic of being able to chemotact with the bacterial biofilm around the tumor and gradually release the drug, thereby increasing the uptake of the drug by the tumor cells.

[0089] Experimental Example 2: In vitro cell assay of the complex of the present invention

[0090] The preparation methods of AT5 and Sm-AT5 used in this experimental example are the same as in Example 1.

[0091] (1) CCK-8 assay to detect the toxic effects of drugs on tumor cells at different time points: Using the Transwell model, the upper chamber contained the tumor biofilm and the drug added later, while the lower chamber contained tumor cells (SCC25). The cells were treated with the drug for 24h, 48h and 72h, respectively. Then, CCK-8 assay solution was added to the lower chamber cells and incubated for 1-4h. The absorbance of each well was measured at a wavelength of 450nm using an ELISA reader.

[0092] The results are as follows Figure 4 As shown in b: According to statistics, the drug treatment with Sm-AT5 showed the most significant effect on SCC25 cells at 72h.

[0093] (2) Cell cycle detection by flow cytometry: After the cells were treated with the drug for 72 h, the cells in the lower chamber were collected, fixed with pre-cooled ethanol at -20 ℃ for 1 h, then resuspended with 100 μl RNase A, incubated at 37 ℃ for 20 min, centrifuged and washed, and incubated with 400 μl PI at 4 ℃ in the dark for 20 min. The cell cycle was detected by flow cytometry at a wavelength of 488 nm.

[0094] The results are as follows Figure 4 As shown in c and 5a: After the machine test, the results are as follows Figure 5 a, according to statistics Figure 4 c. It can be seen that after treatment with Sm-AT5, the number of cells in the S phase, which represents the cell proliferation state, was significantly reduced, indicating the cytotoxicity of Sm-AT5.

[0095] (3) Flow cytometry detection of apoptosis: After the cells were treated with the drug for 72 hours, the cells were collected, 5 μl Annexin V and PI were mixed with binding buffer, stained in the dark for 15 minutes, and then detected by flow cytometry.

[0096] The results are as follows Figure 4 As shown in d and 5b: Based on the results obtained after the machine test. Figure 5 b, obtained from statistics Figure 4 As shown in d, the two quadrants representing early and late apoptosis (LR+UR) had the highest proportion in the Sm-AT5 group, further demonstrating the promoting effect of Sm-AT5 on cell apoptosis.

[0097] (4) Immunoblotting to detect protein expression in cells: After 48 hours of drug treatment, cells were collected, total protein was extracted, and proteins were separated by 15% SDS-PAGE electrophoresis. After membrane blocking, the cells were incubated overnight with β-actin, annti-caspase-3 (ab32351) and anti-cleaved caspase-3 (#9661S), respectively. The next day, the cells were incubated with secondary antibody for one hour, washed with TBST, and exposed to the light.

[0098] The results are as follows Figure 4 As shown in e, 4f, and 4g: The band analysis results show that the Sm-AT5 group had the lowest expression of caspase-3, which represents the apoptosis pathway, and the highest expression of cleaved caspase-3, which proves the apoptosis-promoting effect of Sm-AT5.

[0099] (5) Immunofluorescence staining to detect the protein expression level of cells: After the cells were treated with the drug for 48 hours, the culture medium was discarded, the cells were fixed with paraformaldehyde for 25 minutes, 0.5% Triton X-100 was used to punch holes, sheep serum was used to block the cells, and the cells were incubated with the primary antibody at 4°C overnight. The cells were warmed for one hour the next day and then incubated with the secondary antibody. The cell nuclei were stained with DAPI, and the cells were mounted with glycerol and then observed and imaged using a laser confocal microscope.

[0100] The results are as follows Figure 4 As shown in h and 4i: it can be seen that the fluorescence of caspase-3, which represents the apoptosis pathway, is weakest in the Sm-AT5 group, while the fluorescence of the corresponding cleaved caspase-3 is strongest, which proves the apoptosis-promoting effect of Sm-AT5.

[0101] The above experimental results indicate that the complex of the present invention has a strong cytotoxic effect on tumor cells.

[0102] Test Example 3: Animal Tests of the Compound of the Present Invention

[0103] The preparation methods of AT5 and Sm-AT5 used in this experimental example are the same as in Example 1.

[0104] (1) Schematic diagram of tumor-bearing animal treatment model: Mouse oral squamous cell carcinoma cells (SCC7) were implanted under the buccal mucosa at the corner of the mouth of mice. The tumor volume increased to 1 cm. 3 Local injection of the drug (at a concentration of 500 nM, 100 μl / time, injected around the tumor) was initiated, once every 4 days for a total of 5 times. Samples were collected after 20 days.

[0105] (2) Immunofluorescence observation of tumor invasiveness of drugs: Sm-AT5 stained with syto 9 and Cy5-labeled, and Sm-AT5 co-labeled with syto 9 and Cy5 were injected locally around the tumor for 24 hours. The tumors were collected, sections were stained with DAPI, and the whole process was kept out of the light.

[0106] The results are as follows Figure 6 As shown in b: Red fluorescence is concentrated at the tumor edge, indicating that AT5 infiltrates the tumor superficially and accumulates little inside the tumor. Green fluorescence is deposited in large quantities on the tumor surface, indicating that the bacteria have less internal infiltration. However, in the Sm-AT5 group, both red and green fluorescence appear inside the tumor, indicating that AT5 has better internal infiltration.

[0107] (3) Solid tumor imaging measurement

[0108] The results are as follows Figure 6 As shown in c: After comparison, it can be found that the tumor size in the Sm-AT5 drug treatment group was significantly smaller than that in other drug treatment groups, followed by AT5.

[0109] (4) Tumor volume calculation: Based on the length and width of the tumor measured in the previous step, the tumor volume is calculated as: length x width 2 xπ / 6.

[0110] The results are as follows Figure 6 As shown in Figure d: With increasing treatment time, the tumor volume in the Sm-AT5 treatment group showed a slight increase. The tumor growth in the AT5 treatment group was more significant, while the tumor volume in the other treatment groups all showed a significant increase.

[0111] (5) Mouse survival rate: The time of death of each group of mice was recorded until only one mouse survived.

[0112] The results are as follows Figure 6 As shown in e, by day 75, half of the mice in the Sm-AT5 group were still alive, while the survival rate of the AT5 group reached 28.6% on day 75. In the other treatment groups, only half of the mice survived between days 18 and 30.

[0113] (6) H&E staining: After fixing, embedding and sectioning the tumor, hematoxylin and eosin staining was performed, and the images were observed under a light microscope.

[0114] The results are as follows Figure 6 As shown in f, obvious squamous cell carcinoma keratin beads could be observed in the saline, simple tetrahedral, and 5-fluorouracil treatment groups. Obvious nuclear atypia could be observed in the simple bacterial group, and atypia was reduced in the AT5 group. In contrast, the Sm-AT5 group showed obvious tissue necrosis and lymphocyte infiltration.

[0115] (7) TUNEL staining: After dewaxing, dehydrating and treating paraffin sections with proteinase K, TUNEL detection solution was added and observed under a fluorescence microscope.

[0116] The results are as follows Figure 6 As shown in g: FITC, representing DNA breaks, is visible in the AT5 and Sm-AT5 treatment groups, with more green fluorescence in Sm-AT5.

[0117] The above experimental results show that the compound of the present invention, when applied to mice, can infiltrate tumor tissue extensively and deeply, thereby inhibiting tumor growth. At the same time, the compound of the present invention has good safety when used.

[0118] Experimental Example 4: Tumor Microenvironment Study of the Complex of the Present Invention

[0119] The AT5 and Sm-AT5 used in this experiment were prepared using the same methods as in Example 1. Mice were treated according to the method described in Example 3, and the following studies were conducted.

[0120] (1) Flow cytometry sorting of mature dendritic cells: lymph nodes infiltrated by tumors in mice in each treatment group were collected, ground and sieved, and then the isolated lymphocytes were stained with CD45, CD11c, CD86, and MHC II flow cytometry antibodies.

[0121] The results are as follows Figure 7 As shown in a and 7b: The results can be obtained by computer. Figure 7 a. According to statistics, the CD45+ / CD11c+ ratio, which represents mature dendritic cells, was the highest in the Sm-AT5 treatment group, indicating that Sm-AT5 activates tumor-infiltrating dendritic cells.

[0122] (2) Immunofluorescence staining to observe CD11c expression: lymph nodes infiltrated by tumors in mice in each treatment group were taken, fixed, embedded, sectioned and stained.

[0123] The results are as follows Figure 7 As shown in c: it can be seen that CD11c has the highest proportion in the Sm-AT5 treatment group, and the conclusion is the same as above.

[0124] (3) Flow cytometry sorting of mature dendritic cells with antigen-presenting ability

[0125] The results are as follows Figure 7 As shown in d and 7e: Results obtained after machine testing Figure 7 d. Statistical analysis showed that the proportion of CD86+ / MHC II, which represents the antigen presentation capacity of mature dendritic cells, was the highest in the Sm-AT5 treatment group, further illustrating the activation of tumor-infiltrating dendritic cells by Sm-AT5.

[0126] (4) Flow cytometry sorting of tumor-infiltrating CD3 and CD8 cells: Collect tumors from mice in each treatment group, digest and grind them, centrifuge at differential speed, extract the infiltrating lymphocytes, and stain the isolated lymphocytes with CD3 and CD8 flow cytometry antibodies.

[0127] The results are as follows Figure 7 As shown in f and 7g: CD3+ T cells represent all immune cells infiltrating the tumor, and their proportion was significantly increased in the simple bacterial and Sm-AT5 groups, indicating that the presence of bacteria plays an immune activation role in the immune cells in the tumor tissue. CD8+ T cells represent cytotoxic T cells, that is, T cells that kill tumor cells, and their proportion was the highest in the Sm-AT5 group, indicating the immune killing effect of Sm-AT5.

[0128] (5) Immunofluorescence staining to observe the expression of CD4 and CD8 in tumor infiltration: tumors from mice in each treatment group were collected, and the sections were stained with CD4 and CD8 antibodies.

[0129] The results are as follows Figure 7 As shown in h: CD4 and CD8, representing memory T and cytotoxic T cells, were expressed at the highest levels in the Sm-AT5 treatment group, demonstrating the immune activation effect of tumor itself after Sm-AT5 treatment.

[0130] (6) Immunofluorescence staining to observe the expression of CD4 and CD8 in tumor-infiltrating lymph nodes: Tumor-infiltrating lymph nodes were collected from mice in each treatment group, and the sections were stained with CD4 and CD8 antibodies.

[0131] The results are as follows Figure 7 As shown in i, CD4 and CD8 were expressed at the highest levels in the Sm-AT5 treatment group, demonstrating the immune activation effect of the tumor itself after Sm-AT5 treatment.

[0132] (7) Flow cytometry sorting of the ratio of effector memory T cells (Tem) and central memory T cells (Tcm) in mouse peripheral blood: peripheral blood was collected from mice by ocular blood sampling. After red blood cell lysis, the isolated lymphocytes were stained with CD3, CD8, CD44, and CD62L flow cytometry antibodies.

[0133] The results are as follows Figure 7 As shown in j and 7k: Tem (CD44+, CD62L-) located in peripheral blood rapidly secretes virulence factors to exert toxic effects upon stimulation, while Tcm (CD44+, CD62L+) is converted into Tem to exert toxic effects. Instrumental analysis revealed that Sm-AT5 treatment significantly increased Tem while correspondingly decreasing Tcm, indicating the activating effect of Sm-AT5 on immune cells in peripheral blood.

[0134] (8) Immunofluorescence staining to observe the infiltration of neutrophils in tumors: Tumors from mice in each treatment group were collected, and the sections were stained with CD11b and Ly6G antibodies.

[0135] The results are as follows Figure 7 As shown in Figure 1, when bacteria invade, neutrophils act as the first line of defense and rapidly exert phagocytic activity. In the bacterial-only group and the Sm-AT5-treated group, the expression of proteins representing neutrophils (CD11b and Ly6G) was significantly increased, indicating the activating effect of Sm-AT5 on neutrophils.

[0136] The above experimental results indicate that the complex of the present invention activates the immune response in tumor invasion and the tumor microenvironment.

[0137] In summary, this invention, based on DNA tetrahedral nanomedicine, uses bacterial cell membranes as drug carriers. Leveraging the natural chemotaxis of bacterial cell membranes to bacterial tumor biofilms, the DNA tetrahedral nanomedicine is delivered to bacterial biofilms specifically aggregated around tumors, simultaneously releasing the drug and enabling precise application to tumor cells. The bacterial cell membrane used in this invention is preferably that of *Streptococcus mutans*. This invention not only possesses excellent tumor biofilm chemotaxis, significantly enhancing the drug's invasiveness to solid tumors, but the bacterial carrier can also influence immune cells in the tumor microenvironment, exerting an immune-enhancing effect and ultimately strengthening the drug's inhibitory effect on tumor growth.

Claims

1. A bacterial cell membrane / DNA tetrahedral complex for antitumor purposes, characterized in that: It is a complex obtained by using a bacterial cell membrane as a drug carrier to load DNA tetrahedra; the DNA tetrahedra are synthesized by the self-assembly of four DNA single strands; the nucleotide sequences of the four DNA single strands are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.5, respectively; the 5' end of the nucleotide sequence shown in SEQ ID NO.3 is linked to 5-fluorouracil; the bacteria is Streptococcus mutans; The complex is obtained by incubating bacteria and DNA tetrahedra; the bacterial concentration during incubation is 10. 9 CFU / ml; the concentration of the DNA tetrahedron is 2000 nM; the incubation temperature is 37°C; and the incubation time is 15 min.

2. The bacterial cell membrane / DNA tetrahedral complex according to claim 1, characterized in that: The incubation process also includes purification, with the following steps: centrifugation, collection of precipitate, fixation with glutaraldehyde solution, washing, and centrifugation again.

3. The bacterial cell membrane / DNA tetrahedral complex according to claim 2, characterized in that: The centrifugation conditions are 8000~10000 rpm for 5~10 min; And / or, the concentration of the glutaraldehyde solution is 2-5%; And / or, the fixed time is 10~30 minutes.

4. The bacterial cell membrane / DNA tetrahedral complex according to claim 1, characterized in that: The method for synthesizing the DNA tetrahedron includes the following steps: adding four single strands of DNA to TM buffer, maintaining at 95°C for 10 min, cooling to 4°C and maintaining for more than 30 min, to obtain the tetrahedron.

5. The bacterial cell membrane / DNA tetrahedral complex according to claim 4, characterized in that: The four DNA single strands are in an equimolar ratio.

6. A method for preparing the bacterial cell membrane / DNA tetrahedral complex according to any one of claims 1 to 5, characterized in that: It includes the following steps: (1) Adjust the bacterial concentration to 10 9 CFU / ml; (2) Adjust the DNA tetrahedral concentration to 2000 nM; (3) Mix the two and incubate to obtain the product; in step (3), the incubation temperature is 37°C; and / or the incubation time is 15 min.

7. The method according to claim 6, characterized in that: In step (3), the incubation process also includes purification, which involves the following steps: centrifugation, collection of precipitate, fixation with glutaraldehyde solution, washing, and centrifugation again.

8. The method according to claim 7, characterized in that: In step (3), the centrifugation conditions are 8000~10000 rpm for 5~10 min; And / or, the concentration of the glutaraldehyde solution is 2-5%; And / or, the fixed time is 10~30 minutes.

9. Use of the bacterial cell membrane / DNA tetrahedral complex according to any one of claims 1 to 5 in the preparation of an antitumor drug; wherein the tumor is human oral squamous cell carcinoma.

10. An antitumor drug, characterized in that: It is prepared using the bacterial cell membrane / DNA tetrahedral complex as described in any one of claims 1 to 5 as the active ingredient, plus pharmaceutically acceptable excipients.

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