CN115957336A

CN115957336A - Bacterial cell membrane/DNA tetrahedral compound and preparation method and application thereof

Info

Publication number: CN115957336A
Application number: CN202310050586.1A
Authority: CN
Inventors: 林云锋; 战雨汐; 张雨欣; 毛陈晨
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2022-10-28
Filing date: 2023-02-01
Publication date: 2023-04-14

Abstract

The invention provides a bacterial cell membrane/DNA tetrahedral complex and a preparation method and application thereof, belonging to the technical field of biological medicines. The compound of the invention is obtained by taking a bacterial cell membrane as a drug carrier and carrying a DNA tetrahedron. The invention is based on DNA tetrahedron, takes the bacterial cell membrane as a drug carrier, utilizes the natural chemotaxis of the bacterial cell membrane to the bacterial tumor biomembrane, transports the DNA tetrahedron drug to the bacterial biomembrane specifically gathered around the tumor, and releases the drug at the same time, so that the DNA tetrahedron drug can be accurately applied to the tumor cells. The bacterial cell membrane of the present invention is preferably a Streptococcus mutans cell membrane. The invention not only has good tumor biomembrane chemotaxis, greatly enhances the infiltration of the drug to solid tumors, but also has the function of enhancing immunity because the bacterial carrier can influence immune cells in a tumor microenvironment, and finally enhances the inhibition of the drug to the tumor growth.

Description

Bacterial cell membrane/DNA tetrahedral compound and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a bacterial cell membrane/DNA tetrahedral compound and a preparation method and application thereof.

Background

A large number of researches show that the self-assembled and synthesized DNA tetrahedron can promote the regeneration of various stem cells (fat stem cells, bone marrow stem cells, neural stem cells and the like) as a novel nucleic acid nano-drug, and the anti-inflammatory and antioxidant functions of the DNA tetrahedron can also be applied to the treatment of various diseases. In addition, the three-dimensional tetrahedral structure has excellent biosafety and flexible editability and can be taken up by most cells through an endocytic pathway. Thus, DNA tetrahedrons are often used as drug carriers, on which various nucleic acid sequences (aptamers, siRNA, microRNA, etc.) and chemotherapeutic drugs (paclitaxel, doxorubicin, etc.) are incubated, chemically linked, or otherwise loaded, and carried in large numbers into cells to exert their respective effects.

The patent application with the publication number of CN109663134B discloses that DNA tetrahedron is adopted to carry 5-fluorouracil and AS1411, the anticancer effect of the DNA tetrahedron is remarkably stronger than that of the DNA tetrahedron carried by the 5-fluorouracil, and the safety of the DNA tetrahedron is better. 5-fluorouracil is the most common uracil antimetabolite, and is converted into fluorouracil deoxynucleotide (F-dUMP) after entering the body, and the fluorouracil deoxynucleotide is covalently bound with the active center of thymidylate synthase to inhibit the enzymatic activity of thymidylate synthase, so that deoxynucleotide deficiency affects the DNA synthesis. In addition, fluorouracil (FUMP) can be incorporated as a pseudo-metabolite into DNA and RNA to affect cellular function and produce cytotoxicity. AS1411 is an oligonucleotide sequence rich in guanine and composed of 26 bases, can form a G-quadruplex, can be specifically combined with nucleolin highly expressed on the nuclear membrane of tumor cells to play a role of a nucleic acid aptamer, and can form a complex with nucleolin to cause nucleolin redistribution, inhibit ribosome biosynthesis and induce apoptosis of tumor cells. The AS1411 and 5-fluorouracil modified DNA tetrahedron has tumor cell targeting and killing properties, firstly, the AS1411 targets tumor cells, and the 5-fluorouracil is carried into the tumor cells and released by the DNA tetrahedron, so that the synthesis of DNA and RNA of the tumor cells is influenced, and the killing effect of the tumor cells is exerted.

New studies have shown that microorganisms are present in tumor cells and immune cells, suggesting that these microorganisms may influence the state of the tumor microenvironment. The microorganisms of tumor tissues are obviously different from those of normal tissues, and certain microorganisms are specifically gathered in the tumor tissues, so that the tumor tissues become strong therapeutic targets.

Tumors are closely related to the tumor microenvironment, and can influence the microenvironment by releasing cell signaling molecules, promote angiogenesis and induce immune tolerance, while immune cells in the microenvironment can influence the growth and development of cancer cells. Tumor microenvironment refers to the surrounding microenvironment in which tumor cells reside, including surrounding blood vessels, immune cells, fibroblasts, myeloid-derived inflammatory cells, various signaling molecules, and extracellular matrix. The tumor microenvironment usually presents an overall anoxic and acidic environment, a great amount of apoptosis of tumor and surrounding tissue cells can cause inflammatory infiltration and inflammatory factor secretion, and the occurrence and development of the tumor can also trigger corresponding immune response. T cells are used as a main group of cellular immunity and play an important role in killing tumor immune cells. In the tumor microenvironment, the CD8 positive T subtype in the T cells can kill the tumor cells.

The DNA tetrahedron jointly modified by AS1411 and 5-fluorouracil is a novel DNA nano-drug, and has tumor cell targeting and killing properties: firstly, the DNA aptamer AS1411 is used for targeting tumor cells, and the DNA tetrahedral drug transport carrier carries 5-fluorouracil into the tumor cells and releases the 5-fluorouracil, so that the synthesis of DNA and RNA of the tumor cells is influenced, and the killing effect of the tumor cells is exerted. When the compound is applied in vitro, the compound has a relatively ideal killing effect on tumor cells, but when the compound is applied in vivo, the infiltration of the medicine on tumors is poor, the concentration of the medicine accumulated in tumor tissues is low, and the growth inhibition effect on solid tumors is poor. Meanwhile, the DNA tetrahedron modified by AS1411 and 5-fluorouracil together cannot influence immune cells in a tumor microenvironment, and plays a role in enhancing immunity.

Disclosure of Invention

Although 5-fluorouracil and AS1411 modified DNA tetrahedron have ideal tumor cytotoxicity in vitro, when applied to in vivo tumors, the treatment effect on solid tumors is poor due to poor tumor infiltration of the drug. In order to solve the above problems, the present invention provides a bacterial cell membrane/DNA tetrahedral complex and a preparation method and use thereof. The invention aims at the problems of poor tumor infiltration and unsatisfactory in-vivo application effect of the DNA tetrahedral medicament, takes the bacterial cell membrane as a carrier, carries the tetrahedral medicament to tumor tissues by virtue of the natural chemotaxis of the bacterial cell membrane on bacterial biological membranes gathered outside tumors, and accurately acts on tumor cells to improve the medicament curative effect of the tetrahedral medicament in vivo.

The invention provides a bacterial cell membrane/DNA tetrahedron compound, which is obtained by loading DNA tetrahedron by using bacterial cell membrane as a drug carrier.

Further, the compound is obtained by mixing and incubating the bacteria and the DNA tetrahedron.

Further, the bacterial concentration during the incubation is 10 ⁸ ～10 ¹⁰ CFU/ml; and/or the concentration of the DNA tetrahedron is 500-4000 nM.

Further, the bacterial concentration during the incubation is 10 ⁹ CFU/ml; and/or, the DNThe concentration of A tetrahedra was 2000nM.

Further, the temperature of the incubation is 37 ℃; and/or the incubation time is 10-30 min;

preferably, the incubation time is 15min.

Further, the incubation is followed by purification, comprising the steps of: centrifuging, collecting precipitate, fixing glutaraldehyde solution, washing, and centrifuging again;

preferably, the centrifugation condition is 8000-10000 rpm centrifugation for 5-10 min;

and/or, the concentration of the glutaraldehyde solution is 2-5%;

and/or the fixed time is 10-30 min.

Further, the air conditioner is provided with a fan,

the bacteria are streptococcus mutans;

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 respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 5; 5' -end of the nucleotide sequence shown in SEQ ID NO.3 is connected with 5-fluorouracil.

Further, the method for synthesizing the DNA tetrahedron comprises the following steps: adding the four DNA single strands into a TM buffer solution, maintaining the temperature at 95 ℃ for 10min, and cooling to 4 ℃ for more than 30min to obtain the DNA single strand;

preferably, the four single DNA strands are an equimolar ratio of the four single DNA strands.

The present invention also provides a method for preparing the above-mentioned bacterial cell membrane/DNA tetrahedral complex, which comprises the steps of:

(1) Adjusting the bacteria concentration to 10 ⁸ ～10 ¹⁰ CFU/ml；

(2) Adjusting the concentration of the DNA tetrahedron to 500-4000 nM;

(3) Mixing the two solutions, and incubating;

preferably, in step (1), the concentration of bacteria is adjusted to 10 ⁹ CFU/ml；

And/or, in step (2), adjusting the tetrahedral concentration of the DNA to 2000nM;

and/or, in the step (3), the incubation temperature is 37 ℃; and/or the incubation time is 10-30 min;

and/or, in the step (3), the incubation further comprises purification, and the steps are as follows: centrifuging, collecting the precipitate, fixing the glutaraldehyde solution, washing and centrifuging again;

more preferably, in step (3), the incubation time is 15min;

and/or in the step (3), the centrifugation condition is 8000-10000 rpm centrifugation for 5-10 min;

and/or, the concentration of the glutaraldehyde solution is 2-5%;

and/or the fixed time is 10-30 min.

The invention also provides the application of the bacterial cell membrane/DNA tetrahedral complex in preparing an anti-tumor medicament;

preferably, the neoplasm is human oral squamous carcinoma.

The invention also provides an anti-tumor drug which is prepared by taking the bacterial cell membrane/DNA tetrahedral compound as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

Compared with the prior art, the invention has the beneficial effects that:

the invention is based on DNA tetrahedral nano-drug, takes the bacterial cell membrane as the drug carrier, utilizes the natural chemotaxis of the bacterial cell membrane to the bacterial tumor biomembrane, transports the DNA tetrahedral nano-drug to the bacterial biomembrane specifically gathered around the tumor, and releases the drug at the same time, so that the DNA tetrahedral nano-drug can be accurately applied to the tumor cells. The bacterial cell membrane of the present invention is preferably a Streptococcus mutans cell membrane. The invention not only has good tumor biomembrane chemotaxis, greatly enhances the infiltration of the drug to solid tumors, but also has the function of enhancing immunity because the bacterial carrier can influence immune cells in a tumor microenvironment, and finally enhances the inhibition of the drug to the tumor growth.

It will be apparent that various other modifications, substitutions and alterations can be made in the present invention without departing from the basic technical concept of the invention as described above, according to the common technical knowledge and common practice in the field.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 shows the identification results of DNA nano-drugs carried by the cell membrane of Streptococcus mutans: a, selecting the result of the optimal reaction time for a fluorescence quantitative method; b is the result of observing the co-localization condition of the streptococcus mutans and the DNA nano-drug by immunofluorescence staining; c selecting the working concentration result of AT5 for flow cytometry; d is the Zeta potential results of AT5, simple bacteria (S.m) and S.m-AT 5; e is the characterization result of the transmission electron microscope and the immune electron microscope on the material form; f is the characterization of the dynamic light scattering on the material size.

FIG. 2 shows the efficacy and safety results of DNA nano-drugs carried by the cell membrane of Streptococcus mutans: a is the result of detecting the metabolic time of different materials in a mouse by live imaging of a small animal; b is the distribution of the colony culture observation material in vivo; c is a statistical chart of the distribution condition of the colony culture observation material in the body; d is the hemolytic result of the material detected by the hemolytic experiment; e is a statistical chart of hemolytic results.

FIG. 3 is the result of in vitro experiments demonstrating the uptake of the drug of the present invention in tumor cells: a is the tumor biological membrane chemotaxis of immunofluorescence detection S.m-AT5 and the release result of AT 5; b is a Transwell model for in vitro studies; c is the uptake of S.m-AT5 by human oral squamous carcinoma cells (SCC 25) observed by immunofluorescence.

FIG. 4 shows the result of cytotoxicity test of DNA nano-drug carried by the cell membrane of Streptococcus mutans: a is a Transwell model for in vitro studies; b is a CCK-8 experiment detection result; c is the statistical result of the cell cycle detected by flow cytometry; d is the statistical result of detecting the apoptosis by the flow cytometry; e is the result of detecting the protein expression of the cell by the immunoblotting method; f, g is a statistical histogram of protein expression results of cells detected by an immunoblotting method; h, i is the protein expression level of the immunofluorescence staining detection cell.

FIG. 5 shows the results of flow cytometry: a is a cell cycle result detected by flow cytometry; b is the result of detecting cell apoptosis by flow cytometry.

FIG. 6 shows the results of animal experiments with the drug of the present invention: a is a schematic diagram of a tumor-bearing animal treatment model; b is the result of tumor infiltration of the immunofluorescence observation drug; c is a solid tumor photographing measurement result; d is the tumor volume calculation; e is the survival rate result of the mouse; f is H & E staining result; g is TUNEL staining result.

Fig. 7 shows the tumor microenvironment study results of the inventive drug: a is the result of sorting mature dendritic cells by flow cytometry; b is a histogram of flow cytometry sorting mature dendritic cells; c is the result of CD11c expression observed by immunofluorescence staining; d is flow cytometry sorting of mature dendritic cells with antigen presenting capacity; e is a statistical chart of the flow cytometry sorting of mature dendritic cells with antigen presenting capacity; f is flow cytometry sorting of tumor-infiltrating CD3 and CD8 cells; g is a statistical plot of flow cytometry sorting of tumor infiltrating CD3 and CD8 cells; h is the result of CD4 and CD8 expression of tumor infiltration observed by immunofluorescence staining; i is the result of CD4 and CD8 expression in lymph nodes infiltrated by the tumor observed by immunofluorescence staining; j is the ratio of the effective memory T cells (Tem) and the central memory T cells (Tcm) in the peripheral blood of the mouse sorted by flow cytometry; k is a proportion statistical chart of effective memory T cells (Tem) and central memory T cells (Tcm) in the peripheral blood of the mouse sorted by flow cytometry; l is the infiltration of neutrophils in the tumor observed by immunofluorescence staining.

Detailed Description

The raw materials and equipment used in the embodiment of the present invention are known products and obtained by purchasing commercially available products.

Example 1 preparation of bacterial cell Membrane/DNA tetrahedral complexes of the invention

1. Preparation of 5-fluorouracil and AS1411 modified DNA tetrahedron

The preparation method is as described in patent No. CN109663134B, and comprises the following steps:

preparing four single chains of a DNA tetrahedron, namely S1, S2, S3 and S4-AS1411; wherein, 5' end of S3 chain is connected with 5-fluorouracil (5-FU); S4-AS1411 is the 5' end of the S4 strand linked to AS 1411. The sequences of single strands S1, S2, S3, S4-AS1411 and AS1411 are shown in Table 1, with the sequence of AS1411 in the lower case.

TABLE 1 sequence listing

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Four 1. Mu.l single strands S1, S2, S3 and S4-AS1411 each at a concentration of 1M were added to 96. Mu.l MgCl-containing solution ₂ And Tris and pH 8.0 (Tris-base 0.605g, mgCl) ₂ .6H ₂ O5.075 g in 50ml ddH ₂ O), the whole 100 microliter system was heated to 95 ℃ for 10 minutes and then cooled to 4 ℃ for 30 minutes, synthesizing 5-fluorouracil and AS1411 modified DNA tetrahedron (DNA nanopharmaceutical, AT 5) in a volume of 100 microliter AT a concentration of 1000 nM.

Cy 5-labeled DNA tetrahedral drugs can be prepared by linking Cy5 to the 5' end of the S1 chain, labeling S1-Cy5, and replacing S1 with S1-Cy5 according to the above-mentioned method.

2. Synthesis of Streptococcus mutans membrane/DNA tetrahedral complex

Streptococcus mutans in logarithmic growth phase was collected and adjusted to a concentration of 10 ⁹ CFU/mL, simultaneously concentrating and re-suspending the DNA nano-drug (AT5) prepared in the step 1 to 2000nM by using DPBS, combining 1mL of streptococcus mutans and 1mL of AT5 in an incubator AT 37 ℃ for 15min, centrifuging AT 10000rpm for 5min, discarding supernatant, collecting precipitate, fixing with 2% glutaraldehyde solution for 30min, washing with DPBS three times, and centrifuging again to obtain the DNA nano-drug (S.m-AT5) carried by the streptococcus mutans cell membrane with the concentration of 2000nM.

3. Identification

(1) AT5 was incubated with streptococcus mutans as described above, with varying incubation times. And selecting the optimal incubation time of the DNA nano-drug and the streptococcus mutans by a fluorescence quantitative method. By calculating the DNA concentration of the supernatant in the reaction system at different time points, the binding rate of the DNA drug at different reaction time is calculated and compared with 2000nM of the original DNA.

The results are shown in FIG. 1 a: AT 15min, up to about 57.5% of AT5 was bound to the cell membrane of S.mutans, and therefore 15min was used as the optimal binding time.

(2) And (3) observing the co-localization condition of the streptococcus mutans and the DNA nano-drug by adopting immunofluorescence staining: and (3) labeling AT5 with Cy5 (Cy 5 labels S1 chain, and then Cy5 labeled AT5 is prepared according to the method), simultaneously staining the streptococcus mutans with live bacteria by SYTO 9, incubating AT5 and the streptococcus mutans according to the method, and observing the combination condition of the bacteria and the drugs when the two are incubated for 15min, 1h and 4 h.

The results are shown in FIG. 1 b: AT 15min, the greatest co-localization of AT5 with S.mutans was observed, consistent with the results in FIG. 1 a.

(3) Flow cytometry to select working concentration of AT 5: cy 5-labeled AT5 was prepared in a concentration gradient of 500, 1000, 2000, 4000nM, and complexes were prepared as described above and the binding ratio to the cell membrane of Streptococcus mutans was determined, respectively.

The results are shown in FIG. 1 c: when AT5 was concentrated to 2000nM, it bound bacteria up to 82.8%, so 2000nM was chosen as the working concentration.

(4) The zeta potential of AT5, S.simplex mutans (S.m), and S.m-AT5 was measured.

The results are shown in FIG. 1 d: AT5, as a DNA material, exhibits a negative potential, streptococcus mutans, as a bacterium, also exhibits a negative charge, whereas s.m-AT5, a weak negative charge, represents a combination of the two.

(5) Transmission electron microscope and immune electron microscope characterize material morphology

The results are shown in FIG. 1 e: the shape of AT5 is approximately triangular under a transmission electron microscope, the particle size is within 20nm, the shapes of bacteria (S.m) and S.m-AT5 are similar, and the particle size is about 1000 nm.

(6) Characterization of material size by dynamic light scattering

The results are shown in FIG. 1 f: in accordance with the results of transmission electron microscopy, the particle size of AT5 was 19.15nm, while the particle sizes of bacteria (S.m) and S.m-AT5 were 1125.09nm and 1132.18nm, respectively.

4. Effectiveness and safety

(1) The metabolic time of different materials (Cy 5-labeled AT5 and Cy 5-labeled s.m-AT 5) in vivo in mice was examined using live small animal imaging: after local injection of the Cy 5-labeled drug (balb/c male tumor bearing mice, locally injected at 500nM concentration under the buccal mucosa of mouse buccal horn), the mice were placed into a mouse in vivo imager.

The results are shown in FIG. 2 a: AT5 is rapidly metabolized to the liver within 4 hours of local injection and is discharged out of the body after 12 hours; after 12 hours of local injection, s.m-AT5 was partially metabolized to the liver and still partially retained in the tumor.

(2) Colony culture observation of material distribution in vivo: using balb/c male tumor-bearing mice, injecting the drug locally under the buccal mucosa of the mouse mouth at the concentration of 500nM for 5 times after completing the drug injection (wherein the CTRL group is injected with physiological saline), collecting the heart, the liver, the spleen, the lung and the kidney and the tumor of the mice and preparing homogenate on the 17 th day of the treatment course, mixing the homogenate with a bacterial solid culture medium, culturing the bacteria on a bacterial agar plate for 24h, counting the bacterial colony number and counting.

The results are shown in FIGS. 2b and 2 c: the local injection causes most of the materials to be gathered in tumor tissues, more materials are located in the kidney, and other organs are fewer, so that the biological safety of the medicine to other organs is indicated.

(3) Hemolysis assay the hemolysis of the test material: the quantitative colorimetric method is used to determine the hemoglobin (TBH) in whole blood and the content of free hemoglobin (PFH) released into plasma after blood contacts nanoparticles (Cy 5-labeled AT5 and Cy 5-labeled S.m-AT 5). Hemoglobin and its inducer, besides thiohemoglobin, is oxidized by ferricyanide to methemoglobin in strong base, and then the methemoglobin reacts with cyanide (Drabkin's solution) to form cyanmethemoglobin.

The results are shown in FIGS. 2d and 2 e: in contrast to the Positive Control (PC), both AT5 and S.m-AT5 exhibit essentially no haemolysis.

The advantageous effects of the present invention are demonstrated by specific test examples below.

Test example 1 in vitro experiments demonstrate the uptake of the complexes of the invention in tumor cells

The preparation methods of AT5 and s.m-AT5 used in this test example were the same as in example 1.

(1) Immunofluorescence assay for tumor biofilm chemotaxis of s.m-AT5 and release of AT 5: the bacteria were marked with Alexa Fluor 647 for biofilm exopolysaccharide, AMCA for AT5 and SYTO 9 for bacteria. S.m-AT5 co-labeled with AMCA and SYTO 9 was added to the biofilm and scanned under a confocal microscope to observe the progressive release of chemotaxis to the biofilm and AT5 from 2h,6h to 12h,24h, S.m-AT 5.

The results are shown in FIG. 3 a: it can be seen that green fluorescence representing S.m-AT5 is gathered to the surface of the tumor biological membrane, and blue fluorescence representing AT5 is gradually increased, which means that AT5 is gradually released, and the release amount is gradually increased from 2h to 24 h.

(2) Figure 3b is a Transwell model for in vitro studies.

(3) Immunofluorescence observations of s.m-AT5 uptake by human oral squamous carcinoma cells (SCC 25): culturing a tumor biomembrane in an upper chamber of a Transwell, seeding cells in a lower-layer pore plate of the Transwell, adding Cy 5-labeled S.m-AT5 into the upper-layer biomembrane after 24h, observing the drug intake condition of the cells in the lower-layer pore plate under a confocal microscope, and obtaining the S.m-AT5 intake condition of SCC25 cells after 6h,18h and 30 h.

The results are shown in FIG. 3 c: it can be seen that from 6h to 30h, the increased amount of red fluorescence demonstrates a gradual increase in S.m-AT5 uptake by the cells, up to 30 h.

The experiment result shows that the compound has the characteristics of chemotaxis to bacterial biofilms around tumors, gradual release of the drug and further increase of drug intake of tumor cells.

Test example 2 in vitro cell test of the Compound of the present invention

The procedure for the preparation of AT5 and S.m-AT5 used in this test example was the same as in example 1.

(1) The CCK-8 experiment detects the toxic effect of the drug on tumor cells at different time points: also by Transwell model, the upper chamber is the tumor biofilm with post-added drug and the lower is the tumor cells (SCC 25). And treating the cells with the drugs for 24h, 48h and 72h respectively, adding a CCK-8 detection solution into the cells on the lower layer, incubating for 1-4h, and measuring the absorbance of each hole at the wavelength of 450nm by using an enzyme-labeling instrument.

The results are shown in FIG. 4 b: statistics shows that S.m-AT5 has the most obvious drug treatment effect when SCC25 cells are in 72 hours.

(2) Flow cytometry detection of cell cycle: after the cells were treated with the drug for 72h, the cells in the lower chamber were collected, pre-cooled ethanol was fixed at-20 ℃ for 1h, then resuspended with 100. Mu.l RNase A, incubated at 37 ℃ for 20min, after centrifugation, 400. Mu.l PI was incubated at 4 ℃ for 20min in the dark, and the cell cycle at 488nm was detected by flow cytometry.

The results are shown in FIGS. 4c and 5 a: after the detection on the computer, a figure 5a is obtained, and a figure 4c is obtained through statistics, it can be seen that after the treatment of S.m-AT5, the cells in the S phase representing the cell proliferation state are obviously reduced, which shows the cytotoxicity of S.m-AT 5.

(3) Detecting apoptosis by flow cytometry: after the cells are treated by the drug for 72h, the cells are collected, 5 mul of Annexin V and PI are mixed with binding buffer, and the cells are stained in a dark place for 15min and then are detected on a machine.

The results are shown in FIGS. 4d and 5 b: according to the figure 5b obtained after the on-machine detection, the statistics of the figure 4d show that two quadrants (LR + UR) representing the early apoptosis and the late apoptosis of the cell are the most occupied in the S.m-AT5 group, and the promotion effect of the S.m-AT5 on the apoptosis is proved again

(4) Immunoblotting to detect protein expression in cells: after 48h of drug treatment, cells were collected, total protein was extracted, protein was separated by 15% SDS-PAGE electrophoresis, and after blocking of the membrane, the cells were incubated overnight with beta-actin, annti-caspase-3 (ab 32351) and anti-cleared caspase-3 (# 9661S), the next day secondary antibody was incubated for one hour, and exposed after TBST washing.

The results are shown in FIGS. 4e, 4f and 4 g: from the band analysis results, it can be seen that the S.m-AT5 group represents the lowest expression of caspase-3 of the apoptotic pathway, while the corresponding cleared caspase-3 is the highest expression, demonstrating the pro-apoptotic effect of S.m-AT 5.

(5) Immunofluorescence staining to detect protein expression levels of cells: after the cells are treated by the medicine for 48 hours, culture solution is discarded, paraformaldehyde is used for fixing for 25min,0.5 percent Triton X-100 is used for punching, and goat serum is sealed and incubated overnight at 4 ℃ for the first time; after one hour of rewarming the next day, the secondary antibody is incubated, the cell nucleus is stained with DAPI, and the image is observed and collected by using a laser confocal machine after glycerol mounting.

The results are shown in FIGS. 4h and 4 i: as can be seen, the S.m-AT5 group represents that the caspase-3 fluorescence of the apoptotic pathway is the weakest, while the corresponding cleared caspase-3 fluorescence is the strongest, which proves the apoptosis-promoting effect of S.m-AT 5.

The experimental results show that the compound has stronger cytotoxicity on tumor cells.

Test example 3 animal test of the Compound of the present invention

(1) Schematic representation of tumor-bearing animal treatment model: mouse oral squamous carcinoma cell (SCC 7) is planted under mouse buccal mucosa, and when the tumor volume is increased to 1cm ³ Local injections of drug (100 μ l/dose at 500nM, injected around the tumor) were started every 4 days for 5 times, and were collected after 20 days.

(2) Immunofluorescence tumor infiltration of the drug: isolated tumors, sections, and DAPI staining were collected after 24h peritumoral local injection with syto 9-stained s.m, cy 5-labeled AT5, and syto 9, cy5 co-labeled s.m-AT5, respectively, and whole time protected from light.

The results are shown in FIG. 6 b: the red fluorescence is gathered AT the tumor edge, which means that the AT5 infiltrates the tumor in a shallow range and the accumulation in the tumor is little. The green fluorescence is deposited on the surface of the tumor body in a large amount, which means that the interior infiltration of simple bacteria is less, and the red and green fluorescence of the S.m-AT5 group appears in the tumor, which indicates that the interior infiltration condition of AT5 is better.

(3) Solid tumor photographing measurement

The results are shown in FIG. 6 c: comparison shows that the tumor size of the S.m-AT5 drug treatment group is obviously smaller than that of other drug treatment groups, namely AT5 times.

(4) Calculating the tumor volume: according to the length and width of the tumor measured in the last step, the tumor volume = length x width is calculated ² xπ/6.

The results are shown in FIG. 6 d: the tumor volume of the s.m-AT5 treated group showed a slight increase with the treatment time. The tumor growth was evident in the AT5 treatment group, and the tumor volume showed an obvious increase in all the other treatment groups.

(5) Survival rate of mice: the time to death of each group of mice was recorded until the last surviving mouse remained.

The results are shown in FIG. 6 e: by day 75, half of the S.m-AT5 mice survived, whereas the AT5 group survived 28.6% by day 75, and only half of the other treated mice survived within days 18-30.

(6) H & E staining: after the tumor was fixed, embedded and sectioned, hematoxylin & eosin staining was performed, and the image was taken under a light microscope.

The results are shown in FIG. 6 f: significant squamous carcinoma keratinocytes were observed in the normal saline, simple tetrahedron and 5-fluorouracil-treated groups, significant nuclear heterogeneity and reduced heterogeneity in the AT5 group were observed in the bacteria-only group, while significant tissue necrosis and lymphocyte infiltration were observed in the S.m-AT5 group.

(7) TUNEL staining: paraffin sections are dewaxed, dehydrated and treated by proteinase K, and then the TUNEL detection solution is added for observation under a fluorescence microscope.

The results are shown in FIG. 6 g: FITC, which represents DNA fragmentation, was visualized in the AT5 and S.m-AT5 treated groups, and green fluorescence was greater in S.m-AT 5.

The experimental results show that the compound can be applied to the body of a mouse, can greatly and deeply infiltrate tumor tissues, plays a role in inhibiting the growth of tumors, and has good safety when being used.

Test example 4 study of tumor microenvironment of the Complex of the present invention

The preparation methods of AT5 and s.m-AT5 used in this test example were the same as in example 1. After treating mice according to the method described in test example 3, the following study was performed.

(1) Flow cytometry sorting of mature dendritic cells: tumor-infiltrated lymph nodes from mice in each treatment group were collected, ground and screened, and isolated lymphocytes were stained with CD45, CD11c, CD86, MHC II flow antibodies.

The results are shown in FIGS. 7a and 7 b: FIG. 7a is obtained from the above and it is statistically shown that the ratio CD45+/CD11c + representing mature dendritic cells is highest in the S.m-AT5 treated group, indicating the activation of tumor-infiltrating dendritic cells by S.m-AT 5.

(2) Immunofluorescence staining for CD11c expression: and (3) taking the tumor-infiltrated lymph nodes of the mice of each treatment group, fixing, embedding, and staining sections.

The results are shown in FIG. 7 c: it can be seen that CD11c is the highest in the S.m-AT5 treatment group, as concluded above.

(3) Flow cytometry sorting of mature dendritic cells with antigen presenting capacity

The results are shown in FIGS. 7d and 7 e: FIG. 7d shows the results of the in-vitro detection, and statistics indicate that the ratio of CD86+/MHC II, which indicates the antigen presenting ability of the mature dendritic cells, is the highest in the S.m-AT5 treated group, further illustrating the activation of tumor-infiltrating dendritic cells by S.m-AT 5.

(4) Flow cytometry sorting of tumor-infiltrating CD3 and CD8 cells: tumors from each treatment group were collected, digested, ground, differentially centrifuged, and infiltrated lymphocytes were extracted and stained with CD3, CD8 flow antibody.

The results are shown in FIGS. 7f and 7 g: CD3+ T cells represent all immune cells infiltrated by the tumor, and are obviously increased in simple bacteria and S.m-AT5 groups, which indicates that the existence of the bacteria plays an immune activation role on the immune cells in the tumor tissues. CD8+ T cells represent cytotoxic T cells, i.e., T cells that exert tumor cell killing, with the highest ratio in the s.m-AT5 group, indicating the immune killing effect of s.m-AT 5.

(5) Immunofluorescence staining for CD4 and CD8 expression of tumor infiltration: tumors were collected from mice in each treatment group and after sectioning the sections were stained with CD4, CD8 antibodies.

The results are shown in FIG. 7 h: CD4 and CD8, which represent memory T and cytotoxic T cells, were expressed in the highest amounts in the s.m-AT5 treated group, demonstrating immune activation of the tumor itself after s.m-AT5 drug treatment.

(6) Immunofluorescence staining to observe CD4 and CD8 expression in tumor-infiltrated lymph nodes: collecting tumor-infiltrated lymph nodes of mice in each treatment group, staining the sections with CD4 and CD8 antibodies after sectioning,

the results are shown in FIG. 7 i: CD4 and CD8 have the highest expression in the S.m-AT5 treatment group, which proves the immune activation of the tumor after S.m-AT5 treatment.

(7) Flow cytometry sorting the ratio of effector memory T cells (Tem) and central memory T cells (Tcm) in peripheral blood of mice: the eyedrop method comprises taking peripheral blood from mice, lysing erythrocytes, and staining lymphocytes separated by using flow antibodies of CD3, CD8, CD44 and CD 62L.

The results are shown in FIGS. 7j and 7 k: tem (CD 44+, CD 62L-) located in peripheral blood is stimulated to rapidly secrete virulence factors to play a toxic role, and Tcm (CD 44+, CD62L +) is converted into Tem to play a toxic role. After the detection on the computer, the Tem is obviously increased after the S.m-AT5 treatment, and the corresponding Tcm is reduced, which shows the activation effect of the S.m-AT5 on immune cells in peripheral blood.

(8) The infiltration condition of the neutrophils in the tumor is observed by immunofluorescence staining: tumors were collected from each treatment group and sections were stained with CD11b, ly6G antibody.

The results are shown in FIG. 7 l: when bacteria invade, neutrophils rapidly take phagocytosis as the first line of defense. The expression of proteins (CD 11b and Ly 6G) representing neutrophils was significantly greater in the bacteria alone and in the S.m-AT5 treated group, indicating the activation of neutrophils by S.m-AT 5.

The experimental results show that the compound plays a role in activating the tumor infiltration and the immunity in the tumor microenvironment.

In conclusion, the DNA tetrahedral nano-drug is based on the DNA tetrahedral nano-drug, takes the bacterial cell membrane as the drug carrier, utilizes the natural chemotaxis of the bacterial cell membrane to the bacterial tumor biological membrane, transports the DNA tetrahedral nano-drug to the bacterial biological membrane specifically gathered around the tumor, and releases the drug at the same time, so that the DNA tetrahedral nano-drug can be accurately applied to the tumor cells. The bacterial cell membrane of the present invention is preferably a Streptococcus mutans cell membrane. The invention not only has good tumor biomembrane chemotaxis, greatly enhances the infiltration of the drug to solid tumors, but also has the function of enhancing immunity because the bacterial carrier can influence immune cells in a tumor microenvironment, and finally enhances the inhibition of the drug to the tumor growth.

Claims

1. A bacterial cell membrane/DNA tetrahedral complex characterized by: it is a compound obtained by taking bacterial cell membranes as drug carriers and carrying DNA tetrahedrons.

2. The bacterial cell membrane/DNA tetrahedral complex of claim 1, wherein: the compound is obtained by mixing and incubating bacteria and DNA tetrahedron.

3. The bacterial cell membrane/DNA tetrahedral complex of claim 2, wherein: the bacterial concentration during incubation is 10 ⁸ ～10 ¹⁰ CFU/ml; and/or the concentration of the DNA tetrahedron is 500-4000 nM.

4. The bacterial cell membrane/DNA tetrahedral complex of claim 3, characterized in that: the bacterial concentration during incubation is 10 ⁹ CFU/ml; and/or the concentration of the DNA tetrahedra is 2000nM.

5. The bacterial cell membrane/DNA tetrahedral complex of claim 2, wherein: the incubation temperature was 37 ℃; and/or the incubation time is 10-30 min;

preferably, the incubation time is 15min.

6. The bacterial cell membrane/DNA tetrahedral complex of claim 2, wherein: the method also comprises purification after the incubation, and comprises the following steps: centrifuging, collecting the precipitate, fixing the glutaraldehyde solution, washing and centrifuging again;

and/or, the concentration of the glutaraldehyde solution is 2-5%;

and/or the fixed time is 10-30 min.

7. The bacterial cell membrane/DNA tetrahedral complex of any one of claims 1-4, wherein:

the bacteria are streptococcus mutans;

8. The bacterial cell membrane/DNA tetrahedral complex of claim 7, characterized by: the synthetic method of the DNA tetrahedron comprises the following steps: adding the four DNA single strands into TM buffer solution, maintaining at 95 deg.C for 10min, cooling to 4 deg.C, and maintaining for more than 30min to obtain the final product;

9. A method for preparing a bacterial cell membrane/DNA tetrahedral complex of any one of claims 1 to 8, wherein: the method comprises the following steps:

(1) Adjusting the bacteria concentration to 10 ⁸ ～10 ¹⁰ CFU/ml；

(2) Adjusting the concentration of the DNA tetrahedron to 500-4000 nM;

(3) Mixing the two solutions, and incubating;

and/or, in the step (3), the incubation further comprises purification, and the steps are as follows: centrifuging, collecting precipitate, fixing glutaraldehyde solution, washing, and centrifuging again;

more preferably, in step (3), the incubation time is 15min;

and/or, the concentration of the glutaraldehyde solution is 2-5%;

and/or the fixed time is 10-30 min.

10. Use of a bacterial cell membrane/DNA tetrahedral complex of any one of claims 1 to 8 in the preparation of an anti-tumor medicament;

preferably, the neoplasm is human oral squamous carcinoma.

11. An antitumor agent characterized by: it is prepared by using the bacterial cell membrane/DNA tetrahedron compound of any one of claims 1-8 as active component and adding pharmaceutically acceptable auxiliary materials.