CN114149941B

CN114149941B - Bacteria with surface combined with targeting ligand and application thereof

Info

Publication number: CN114149941B
Application number: CN202111276432.1A
Authority: CN
Inventors: 刘尽尧; 耿忠民
Original assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2021-10-29
Filing date: 2021-10-29
Publication date: 2023-09-19
Anticipated expiration: 2041-10-29
Also published as: CN114149941A

Abstract

The invention belongs to the field of biological medicine, relates to bacteria with surface-bound targeting ligand and application thereof, and in particular relates to bacteria with surface-bound targeting ligand, a preparation method thereof, a pharmaceutical composition containing the bacteria, a drug delivery system and application thereof. Wherein the bacteria are any one or more selected from gram-negative bacteria; the targeting ligand is a nucleic acid aptamer, preferably an aminated nucleic acid aptamer. The nucleic acid aptamer is modified on the surface of bacteria, so that the stability of the nucleic acid aptamer in an in-vivo environment is improved, the specificity and the engraftment capability of the nucleic acid aptamer to a target site are improved, for example, the tumor site and cells are improved, and the immune response in the tumor is highly activated. The bacteria can be used as a drug carrier, and can release the drug after being customized at a target position to improve accumulation and permeation of the drug in tumor tissues, so that the therapeutic effect is maximized.

Description

Bacteria with surface combined with targeting ligand and application thereof

Technical Field

The invention belongs to the field of biological medicine, relates to bacteria with surface-bound targeting ligand and application thereof, and in particular relates to bacteria with surface-bound targeting ligand, a preparation method thereof, a pharmaceutical composition containing the bacteria, a drug delivery system and application thereof.

Background

In recent years, the incidence of malignant tumors has increased year by year, and various treatment methods have been layered endlessly. In particular, with recent advances in the fields of immunology and biotechnology, bacteria have received more attention as immunomodulators and drug delivery vehicles in tumor biotherapy. Bacterial infection can promote the immunogenicity of tumors and further induce effective anti-tumor immune responses. In addition, colonization of tumor tissue by bacteria can trigger activation of innate immune cells and release of pro-inflammatory factors, resulting in destruction of the tumor vasculature and associated thrombosis. The application of bacteria as drug carriers has the greatest advantage that the bacteria are beneficial to colonization at tumor sites due to anaerobic conditions, eutrophication and immunosuppressive microenvironments in the tumor. Disadvantageously, however, bacterial-mediated tumor treatment is always plagued by inefficient tumor growth inhibition and adverse dose-dependent side effects.

Although bacteria have been modified or engineered by chemical or genetic means to address these challenges. For example, in order to reduce systemic toxicity, several bacterial strains including listeria, escherichia coli and clostridium are attenuated by removing critical virulence factors; alternatively, various types of substrates, such as synthetic nanoparticles and therapeutic drugs, are decorated on the bacterial surface for enhancing antitumor activity. However, the modification by chemical and genetic techniques in the prior art still does not improve the tumor specificity of the bacteria after administration and its ability to localize at tumor sites. Therefore, how to improve the tumor targeting of bacteria and thus the safety and anti-tumor efficacy of bacterial therapies remains a technical problem that has not been solved or significantly improved in the art.

Furthermore, prior studies have shown that nucleic acid aptamers, also known as "chemical antibodies", have been widely used as targeting ligands due to their ability to specifically recognize cell surface targets (such as nucleic acids and proteins), whole cells and even tissues. Similar to antibodies, nucleic acid aptamers exhibit many advantageous features, such as high affinity, excellent specificity, and low immunogenicity. In addition, the nucleic acid aptamer may be synthesized in vitro by mature solid state synthesis techniques and may be chemically modified with various functional groups. By virtue of these unique properties, in the prior art, researchers have evaluated the potential of partial therapeutic nucleic acid aptamers in the treatment of cancer in clinical trials. However, aptamer-based drug delivery has focused mainly on the development of aptamer-drug conjugates and aptamer-functionalized nanoparticles, and to date there has been no method or product for actively delivering living cells to tumor sites through specific ligand-receptor interactions by using the aptamer.

Based on this, the inventors of the present application and their team expect to modulate bacterial behavior with the aid of nucleic acid aptamers, enhancing the ability of bacterial tumor localization and thus therapeutic effects of biological therapies.

Disclosure of Invention

Aiming at the blank and the defects in the tumor treatment field of bacteria and targeted nucleic acid aptamer in the prior art, the inventor and team of the application of the invention provide a bacteria with the surface bound with the targeted ligand and application thereof, in particular to a bacteria with the surface bound with the targeted ligand, a preparation method thereof, a pharmaceutical composition containing the bacteria, a drug delivery system and application thereof in preparing tumor treatment drugs.

In a first aspect, the present application provides a bacterium having a surface-bound targeting ligand, the bacterium having a nucleic acid aptamer bound to the surface of the bacterium by a single step amidization process.

Further, the bacterium is any one or more selected from gram-negative bacteria. Optionally, E.coli; optionally salmonella, preferably attenuated salmonella.

Further, the targeting ligand is a nucleic acid aptamer, preferably an aminated nucleic acid aptamer.

In a second aspect, the present application provides a method of preparing a surface-bound ligand-targeted bacterium as described above, comprising: binding of the targeting ligand to the bacterial surface is performed by a single amidation process, wherein the targeting ligand is a nucleic acid aptamer.

Further, the nucleic acid aptamer is an aminated nucleic acid aptamer.

Further, the single-step amidation process, specifically, magnetic stirring at 600rpm at room temperature, adds 0.10-0.02. Mu. Mol/mL of the functionalized aptamer, EDC and NHS to the bacterial suspension and stirs to obtain bacteria with proper surface binding.

Further, the aminated nucleic acid aptamer may be an oligonucleotide strand as shown in SEQ1 or SEQ 2.

Further, the grafting density of the aptamer on the bacterial surface was 0.7X10 ⁵ -5.7×10 ⁵ Nucleic acid aptamer/cell.

In a third aspect, the present application provides a pharmaceutical composition comprising any one of the above surface-bound targeting ligand bacteria and a pharmaceutically acceptable carrier therefor.

Further, in the pharmaceutical composition, the surface-bound targeting ligand has a bacterial content of 1-99.9 wt%. Preferably 10-90 wt%, more preferably 50-80 wt%. Wherein the grafting density of the aptamer is 0.7X10 ⁵ -5.7 ×10 ⁵ Nucleic acid aptamer/cell.

In a fourth aspect, the present application provides a drug delivery system for tumor treatment comprising at least a bacterium having a surface bound targeting ligand of any of the above, the bacterium having the targeting ligand bound to the surface of the bacterium by a single amidation process, wherein the targeting ligand is a nucleic acid aptamer.

Further, the nucleic acid aptamer is an aminated nucleic acid aptamer.

Further, the drug delivery system uses the bacteria with the surface bound targeting ligand as a carrier to load other active drug components. Such as nanobodies, small molecule drugs, and more particularly immunosuppressants.

In a fifth aspect, the present application also provides the use of any one of the above surface-bound targeting ligand bacteria for the preparation of a medicament for the treatment of a tumor.

Further, the nucleic acid aptamer is used for treating different types of tumors according to the selected nucleic acid aptamer. The tumor may be a common solid tumor including lung cancer, stomach cancer, colon cancer, uterine cancer, rectal cancer, throat cancer, breast cancer, etc., preferably stomach cancer, breast cancer.

By amide condensation, the aminated aptamer is combined with the carboxyl of N-acetyl muramic acid on the bacterial cell wall, and the combination stability is good. The nucleic acid aptamer is combined on the surface of bacteria, so that the stability of the nucleic acid aptamer in an in-vivo environment is improved, the specificity and the engraftment capacity of the nucleic acid aptamer to a target site are improved, for example, the tumor site and cells are improved, and the immune response in the tumor is highly activated. The modified bacteria are used as drug carriers, and can release drugs after being customized at target positions to the greatest advantage, so that accumulation and permeation of the drugs in tumor tissues are improved, and the therapeutic effect is maximized.

Advantageous effects

The invention discloses bacteria with the surface combined with a targeting ligand, a preparation method thereof, a pharmaceutical composition containing the bacteria, a drug delivery system using the bacteria and application thereof.

Based on experimental study, the binding process of the aptamer to the surface of the bacteria is an amidation process compatible with cells, and can remarkably promote the positioning of the bacteria at the tumor site of the whole body after the administration. The surface density of the aptamer can be easily adjusted through different feeding ratios. Furthermore, the chemical bond bonding to the bacterial cell wall surface can increase the stability of the aptamer in vivo environment as compared to the aptamer alone. Approximately 2-fold and 4-fold aggregation in tumor tissue occurred at 12 and 60h, respectively, compared to unmodified bacteria. In the 4T1 and H22 tumor-bearing mouse models, attenuated salmonella in combination with the aptamer showed significantly enhanced anti-tumor efficacy and highly activated immune response within the tumor. The examples and experimental examples of the present application demonstrate the particular outstanding advancement of aptamer-bound bacteria in targeting tumors and their colonization within tumors, which shows great potential and important clinical application value in tumor biotherapy.

Drawings

Figure 1 shows the design, preparation and characterization of ApCB. Wherein:

(a) Preparing ApCB by an amide condensation method;

(b) Nucleic acid aptamer-assisted tumor-localized bacteria for enhancing biological therapy;

(c) Typical LSCM images of aptamer-bound bacteria, red and green channels represent aptamer binding to Cy5 and EcN, respectively, producing GFP, scale bar: 10 μm;

(d) Flow cytometry analysis EcN and EcN conjugated to Cy5 labeled and AS 1411;

(e) Percentage of bound bacteria EcN at different feed ratios, wherein error bars represent standard deviation (n=3), data are expressed as mean ± SD;

(f) Quantifying the average number of binding of the aptamer on each bacterium by calculating the difference in fluorescence intensity of the aptamer solution after the reaction, wherein the error bars represent standard deviations (n=3), and the data are expressed as mean ± SD;

(g) LB agar plate count EcN, 2ApCB, 5ApCB, 10ApCB bacterial viability, wherein, prior to counting, the dishes were incubated at 37℃for 24h (n=3), the data were expressed as mean+ -SD, the significance of the differences was assessed using a unpaired two-tailed Student's assay (two-tailed), ns indicating no significant difference;

(h) Kinetics of degradation of bound AS1411 in 90% phosphate buffered serum solution at 37 ℃, wherein error bars represent standard deviation (n=3).

Fig. 2 shows flow cytometry analysis EcN and ApCB by tracking fluorescence intensity of Cy 5-labeled AS 1411.

FIG. 3 shows that ApCB binds to 4T1 cells. Wherein:

(a) Respectively with PBS and EcN at 37deg.C _GFP 、CTL _GFP 、2ApCB _GFP 、5ApCB _GFP And 10ApCB _GFP Representative LSCM image of 4T1 cells after incubation for 2h, where cells were washed with PBS prior to observation, green channel indicated EcN produced GFP, scale bar: 10 μm;

(b) Flow cytometric analysis of co-incubated 4T1 cells.

In fig. 4, (a) after incubation of 293T, H and 4T1 cells with PE-labeled anti-nucleolin antibodies, flow cytometry analysis of cell surface nucleolin; (b) After co-incubation at 37℃for 2h, apCB _GFP Percentage of bound 4T1 cells. Error bars represent standard deviation (n=3). Data are expressed as mean ± SD. The significance of the differences was assessed using the unpaired two-tailed Student's test (two-tailed), giving p-value: 10ApCB vs EcN,0.000004;5ApCB vs EcN,0.0001;2ApCB vs EcN,0.0007; PBS vs. EcN,0.0004.ns indicates no significant difference. * P<0.001，****p<0.0001。

Figure 5 shows the specific binding of ApCB to cancer cells. Wherein:

(a) Representative SEM images of 4T1 cells after incubation with EcN and 5ApCB for 2h at 37 ℃ respectively, bacteria were circled in red and dotted scale: 5 μm;

(b) 293T cells and EcN respectively _GFP And 5ApCB _GFP Flow cytometric analysis after co-incubation for 2h at 37 ℃;

(c) Percentage of 293T cells bound to EcNGFP and 5ApCBGFP after co-incubation at 37 ℃ for 2h, respectively, wherein error bars represent standard deviation (n=3), data are expressed as mean ± SD, the significance of the differences was assessed using unpaired two-tailed Student's test (two-tailed), ns representing no significant difference;

(d) Representative LSCM images of 293T cells incubated with EcNGFP and 5ApCBGFP, respectively, for 2h at 37℃were obtained by washing the cells with PBS prior to observation, green channel indicated EcN for GFP production, scale bar 10. Mu.m.

(e) Intravenous injection 1×10 ⁷ Confocal images of tumor tissue sections 12h after CFU bacteria, where green and blue represent Cyanine 3-labeled anti-e.coli and DAPI stained nuclei, scale bar: 20 μm.

Figure 6 shows the in vivo inflammatory response evaluated. Wherein the blood routine analysis comprises (a) white blood cell count and (b) platelet count. Specifically, cytokine levels in serum are determined using a commercially available enzyme-linked immunosorbent assay (ELISA) kit, comprising (c) IL-6 and (d) IL-10. The tail vein was injected and blood was collected from the orbit 60 hours later. Error bars represent standard deviation (n=4). Data are expressed as mean ± SD. The significance of the differences was assessed using the unpaired two-tailed Student's test (two-tailed), ns indicating no significant difference.

Fig. 7 shows the biodistribution of ApCB in tumor-bearing mice. Wherein:

(a) PBS or 1×10 respectively was injected in the tail vein ⁷ CFU EcN _Lux 、CTL _Lux And 5ApCB _Lux IVIS imaging of 4T1 tumor bearing mice 12, 36, 60h later;

(b) The average intensity of the luminescence signal at 12, 36 and 60h tumor sites after injection, wherein the luminescence intensity (atomic units (a.u.)) was quantified by the Caliper IVIS Lumina II system, and the region of interest (n=4) containing the entire solid tumor was plotted as mean ± SD. The significance of the differences was assessed using the unpaired two-tailed Student's test (two-tailed), giving p-value: 5ApCB vs PBS,0.081 at 60 h; 5ApCB vs EcN,0.0131;5ApCB vs CTL,0.0132; at 36h, 5ApCB vs PBS,0.0370,5ApCB vs EcN 0.0339,5ApCB vs CTL,0.0287; ns, no significant difference.

(c) With PBS or 1X 10 respectively ⁷ After 60h of CFU EcN, CTL and 5ApCB tail intravenous injection, the mice were mainly organ and biodistributed by bacteria in tumor tissue. Bacterial numbers were determined by plate counting and data are expressed as mean ± SD (n=4). The differences were evaluated to be significant using the unpaired two-tailed Student's test (two-tailed)Sex, p-value is given: 5ApCB vs PBS, 0.0012;5ApCB vs EcN,0.0138;5ApCB vs CTL,0.0108; ns: no significant differences were found.

(d) 12h after injection, ecn number of implants in tumor, where error bars represent standard deviation (n=3), data are expressed as mean ± SD. The significance of the differences was assessed using the unpaired two-tailed Student's test (two-tailed), giving p-value: 5ApCB vs PBS,0.0027;5ApCB vs EcN,0.0251;5ApCB vs CTL,0.0390; ns indicates no significant difference. * p <0.05, p <0.01.

Fig. 8 shows the properties of nucleic acid aptamer binding to VNP. Wherein:

(a) The aptamer binds to a typical LSCM image of VNP. Wherein the red and green channels represent the production of GFP by the aptamer binding to Cy5 and VNP, respectively. Scale bar: 10 μm.

(b) Flow cytometry analyzed VNP and VNP bound to Cy 5-labeled AS 1411.

(c) Percentage of bound VNP at different feed ratios. Error bars represent standard deviation (n=3). Data are expressed as mean ± SD.

(d) The average number of aptamer binding per bacteria was quantified by calculating the difference in aptamer solution fluorescence intensity after the reaction. Error bars represent standard deviation (n=3). Data are expressed as mean ± SD.

Fig. 9 shows the in vivo therapeutic effect of ApCB. Wherein:

(a) Tumor relative growth after different treatments (n=5). Data are expressed as mean ± SD. The significance of the differences was assessed using the unpaired two-tailed Student's test (two-tailed), giving p-value: 5ApCB vs PBS,0.000005;5ApCB vs VNP,0.00002.

(b) Representative photographs of mouse tumor tissue at the end of treatment. Scale bar: 1cm.

(c) The change in body weight of the treated mice during each treatment period was observed (n=5), wherein data are expressed as mean ± SD.

(d) Percentage of Ki67 (cd3+ cell gating) inside tumor after treatment (n=4), where data are expressed as mean ± SD; the significance of the differences was assessed using the unpaired two-tailed Student's test (two-tailed), giving p-value: 5ApCB vs PBS, 0.0003;5ApCB vs VNP,0.0411.

(e) Analyzing the number of intra-tumor cd4+ T cells (cd3+ cell gating) after treatment with flow cytometry (n=4) wherein data are expressed as mean ± SD; the significance of the differences was assessed using the unpaired two-tailed Student's test (two-tailed), giving p-value: 5ApCB vs PBS,0.0002;5ApCB vs VNP,0.0088.

(f) Percentage of intra-tumor IFN- γ (gating on cd3+ cells) after treatment (n=4), where data are expressed as mean ± SD; the significance of the differences was assessed using the unpaired two-tailed Student's test (two-tailed), giving p-value: 5ApCB vs PBS,0.00009;5ApCB vs VNP,0.0192.

(g) (h) tumor tissue was stained with TNF- α (green) and TUNEL (green). Blue is DAPI stained nuclei. The ratio ruler: 50 μm.

(i) Representative H & E staining images of tumor sections after different treatments. The necrotic areas are circled with white dashed lines. Scale bar: 200 μm. Wherein p <0.05, p <0.01, p <0.001, p <0.0001.

FIG. 10 shows binding of ApCB to 4T1 cells. Wherein:

(a) Respectively using PBS and VNP _GFP 、CTL _GFP 、2ApCB _GFP 、5ApCB _GFP 、10ApCB _GFP Representative LSCM images of 4T1 cells after incubation at 37 ℃ for 2 h. Cells were washed with PBS prior to observation. The green channel indicates that VNP produces GFP. Scale bar: 15 μm.

(b) Flow cytometric analysis of co-incubated 4T1 cells.

Figure 11 shows the use of ApCB with TLS11a aptamer in H22 tumor models. Wherein:

(a) ApCB was prepared by combining VNP with TLS11a via an amide condensation reaction.

(b) H22 cells were incubated with PBS, VNP, T-5ApCB for 1H at 37 ℃ and then analyzed by flow cytometry.

(c) Digital photographs of mice at day 12 post-treatment. Tumors were marked with red circles. H22 tumor-bearing mice (1X 10 inoculation) ⁶ Cancer cells) were randomly divided into three groups, i.v. PBS and 5 x 10 ⁵ CFU VNP or T-5ApCB when the tumor size reaches about 100mm ³ (defined as day 0).

(d) Relative growth of tumors after different treatments (n=5), where data are expressed as mean ± SD; the significance of the differences was assessed using the unpaired two-tailed Student's test (two-tailed), giving p-values: T-5ApCB vs PBS,0.000002; T-5ApCB vs VNP,0.0052.

(e) H22 tumor-bearing mice were subjected to different treatments for survival (n=7), where data are expressed as mean ± SD. The significance of the differences was assessed using the unpaired two-tailed Student's test (two-tailed), giving p-value: t-5ApCB vs PBS, p < 0.0001; T-5ApCB vs VNP,0.0017.

(f) Change in body weight during treatment, wherein p <0.01, p <0.0001.

Wherein p <0.05, p <0.01, p <0.001, p <0.0001, ns no significant difference (n=5).

Figure 12 shows the percentage of 4T1 cells bound to ApCBGFP after 2h incubation at 37 ℃. The cells were quantitatively analyzed using flow cytometry. Error bars represent standard deviation (n=3). Data are expressed as mean ± SD. The significance of the differences was assessed using the unpaired two-tailed Student's test (two-tailed), giving p-value: 10ApCB vs VNP,0.0158;5ApCB vs VNP,0.0006;2ApCB vs VNP,0.0096; PBS vs VNP,0.0016.* p <0.05, < p <0.01, < p <0.001, ns represents a significant difference.

In FIG. 13, (a) percentage of Ki67, (b) number of CD4+ T cells, (c) percentage of IFN- γ in tumors after various treatments. All cells are cd3+ cell-gated cells.

FIG. 14 shows images of tumor tissue stained with (a) TNF- α and (b) TUNEL after various treatments. Scale bar: 50 μm.

FIG. 15 shows H22 cells and T-5ApCB _GFP Geometric mean fluorescence intensity after co-incubation, PBS and VNP were used as control groups. Error bars represent standard deviation (n=3). Data are expressed as mean ± SD. The significance of the differences was assessed using the unpaired two-tailed Student's test (two-tailed), giving p-value: T-5ApCB vs PBS,0.0004; t (T)-5ApCB vs VNP， 0.0279。*p<0.05，***p<0.001。

FIG. 16 shows a gating strategy for cell sorting. Specifically, a gating strategy for screening CD3+, CD4+, IFN-. Gamma.Ki 67 cells from tumor cells cultured in vivo as shown in FIGS. 9.d-f and 13.

Detailed Description

The technical scheme of the present invention will be further described with reference to the specific embodiments, but the present invention is not limited thereto. Any modification, adjustment or modification, or equivalent replacement method that can be implemented by those skilled in the art to which the present invention pertains will fall within the scope of the claimed invention without departing from the technical idea and technical solution of the present invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

In embodiments of the present application, the nucleic acid aptamer is bound to the bacterial surface by a single amidation process, thereby obtaining a surface-bound targeting ligand bacteria.

Further, the bacterium is any one or more selected from gram-negative bacteria, preferably, escherichia coli or salmonella, more preferably, attenuated salmonella.

In experimental examples, the aminated nucleic acid aptamer is an oligonucleotide chain as shown in SEQ1 or SEQ 2.

SEQ1:5’-GGT GGT GGT GGT TGT GGT GGT GGT GGT TTT TTT TTT TT-NH2-3’

SEQ2:5’-ACA GCA TCC CCA TGT GAA CAA TCG CAT TGT GAT TGT TAC GGT TTC CGC CTC ATG GAC GTG CTG TTT-NH2-3’

Further, the nucleic acid aptamer is an aminated nucleic acid aptamer.

Further, the bacterium is any one or more selected from gram-negative bacteria. In one embodiment, salmonella is selected as the original strain to be engineered, preferably attenuated Salmonella.

In another embodiment, probiotics are selected as engineered primary strains, preferably E.coli, e.g. Nissle 1917 (EcN).

Further, the single step amidation process. Specifically, 0.1mL of amino-functionalized aptamer (0.05. Mu. Mol/mL), 0.55mg of EDC and 0.65mg of NHS were added to the bacterial suspension at room temperature, and after stirring for 3 hours, bacteria with surface-bound nucleic acid aptamer were obtained.

Further, the drug delivery system may also be a delivery system in which the bacteria with the surface bound targeting ligand are used as a carrier to carry other drug components.

Further, the nucleic acid aptamer is used for treating different types of tumors according to the selected nucleic acid aptamer. The tumor may be a common solid tumor including lung cancer, stomach cancer, colon cancer, uterine cancer, rectal cancer, throat cancer, breast cancer, etc., preferably stomach cancer, breast cancer. Specifically, different targeting ligands are selected depending on the type of tumor, more specifically, nucleic acid aptamers of a specific tumor type are selected from existing libraries.

In each example, the aminated aptamer was bound to the carboxyl group of N-acetylmuramic acid on the bacterial cell wall by amide condensation to obtain bacteria with surface-bound aptamer, and specific characteristics and parameters thereof were confirmed by experimental examples.

Example 1: preparation of bacteria with surfaces bound by aptamer

The aminated aptamer binds to the bacterial surface by an amide condensation reaction. Specifically, 1×10 ⁸ The bacteria of each cell were dispersed in 1mL of PBS solution, and 0.1mL of the aminated aptamer (0.05. Mu. Mol/mL), 0.55mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 0.65mg of N-hydroxysuccinimide (NHS) were added simultaneously to the bacterial suspension at room temperature.

EDC is able to activate carboxyl groups during amide condensation, forming unstable O-acylisoureas. The activated carboxyl groups can be stabilized by adding NHS to the reaction mixture, thereby increasing the conversion of amide condensation.

After stirring for 3h, the modified bacteria were isolated by centrifugation, 10000rpm,1min, and washed 3 times with PBS.

Experimental example 1.1:

the aminated aptamer AS1411 was attached to the surface of E.coli Nissle 1917 (EcN) by an amide condensation reaction. Specifically, 1×10 ⁸ EcN cells were dispersed in 1mL PBS solutionTo the bacterial suspension, 0.1mL of the amino-functionalized aptamer (0.05. Mu. Mol/mL), 0.55mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), and 0.65mg of N-hydroxysuccinimide (NHS) were added simultaneously at room temperature. After stirring for 3h, the modified bacteria were isolated by centrifugation (10000 rpm,1 min) and washed 3 times with PBS.

Experimental example 1.2:

the amination aptamer AS1411 is connected to the surface of attenuated Salmonella typhimurium VNP20009 (VNP) by an amide condensation reaction. Specifically, 1×10 ⁸ VNP cells were dispersed in 1mL PBS and 0.1mL of amino-functionalized aptamer (0.05. Mu. Mol/mL), 0.55mg EDC and 0.65mg NHS were added to the bacterial suspension at room temperature. After stirring for 3h, the modified bacteria were isolated by centrifugation (10000 rpm,1 min) and washed 3 times with PBS.

Experimental example 1.3:

the aminated aptamer TLS11a was attached to the surface of attenuated Salmonella typhimurium VNP20009 (VNP) by amide condensation. Specifically, 1×10 ⁸ VNP cells were dispersed in 1mL PBS and 0.1mL of amino-functionalized aptamer (0.05. Mu. Mol/mL), 0.55mg EDC and 0.65mg NHS were added to the bacterial suspension at room temperature. After stirring for 3h, the modified bacteria were isolated by centrifugation (10000 rpm,1 min) and washed 3 times with PBS.

Materials and methods

1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) was purchased from Adamas-beta (Shanghai, china). RPMI 1640 medium, antibiotic/antifungal solution, and phosphate buffered saline (1 XPBS) were all supplied by Sigma-Aldrich (USA).

The following nucleic acid aptamers and oligonucleotide strands were synthesized and supplied by Tsingke synthesis biotechnology limited (china, shanghai).

AS1411(5'GGT GGT GGT GGT TGT GGT GGT GGT GGT TTT TTT TTT TT-NH2-3’)

TLS11a(5'ACA GCA TCC CCA TGT GAA CAA TCG CAT TGT GAT TGT TAC GGT TTC CGC CTC ATG GAC GTG CTG TTT-NH ₂ -3’)

CTL(5’-CCT CCT CCT CCT TCT CCT CCT CCT CCT TTT TTT TTT TT-NH ₂ -3’)

EcN and Salmonella typhimurium are both purchased from the China general microbiological culture collection center GMCC.

Both the 4T1 breast cancer cell line and the 293T cell line were obtained from the American type biological culture Collection (ATCC), CRL-2539; CRL-3216). H22 hepatoma cell lines were purchased from the BeNa culture Collection (BNCC) (category: BNCC 338327). Both cell lines were cultured in Dulbecco's modified eagle's medium (Sigma, USA) supplemented with 10% (v/v) inactivated Fetal Bovine Serum (FBS) (Sigma, USA) and 1% (v/v) antibiotic/antifungal solution (Sigma, america), 37℃in a 5% CO2 incubator. Mycoplasma assessment is performed periodically on the cell culture environment to ensure that there is no mycoplasma contamination.

All reagents, such as plasmid pBBR1MCS2-Tac-GFP, pMD18-luxCDABE, were purchased from domestic suppliers.

BALB/c female mice (6-8 weeks, 18-20 g) were supplied by Jegsi laboratory animal Co., ltd (Shanghai, china). All mice were kept under dark/light cycle and SPF (ambient temperature (25 ℃) and humidity (55%) conditions for 12/12h animal study protocol was approved by the institutional animal Care and use Committee of Shanghai university of transportation medical college when tumor size exceeded 2000mm ³ When this time, the mice were euthanized. Tumor size/burden did not exceed the maximum.

I. Experiment and detection

Photoluminescence (PL) spectra were measured on a FluoMax-4 fluorescence spectrometer. Confocal laser scanning microscopy (CLSM, leica, SP 8) was used to characterize the cell fluorescence images. Flow cytometry (Beckman CytoFlex) quantitatively detects cellular fluorescence. In vivo imaging of mice was done by the in vivo imaging system (Caliper LifeSciences, USA). Morphological observations were performed on a Scanning Electron Microscope (SEM) (Sirion 200, usa).

Experiment 1: bacterial growth

EcN carrying pBBR1MCS2-Tac-GFP or pMD18-luxCDABE was cultured overnight at 37℃in 10mL LB liquid medium supplemented with 50. Mu.g/mL kanamycin.

Salmonella typhimurium was cultured in Tryptone Soy Broth (TSB) medium at 37℃and after overnight culture, 1:50 (v/v) dilution to fresh LB liquid medium was continued at 37℃for 2-3h. Bacteria were collected, centrifuged at 6000 Xg for 10min, and resuspended in ice-cold PBS to obtain bacterial suspension. After dilution of the bacterial suspension obtained, it was incubated on LB agar plates at 37℃overnight, colony Forming Units (CFU) were counted, and the bacteria were determined and counted.

Experiment 2: preparation of ApCB

The aminated aptamer binds to the bacterial surface by an amide condensation reaction. Specifically, 1×10 ⁸ The bacteria of each cell were dispersed in 1mL of PBS solution, and 0.1mL of the aminated aptamer (0.05. Mu. Mol/mL), 0.55mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 0.65mg of N-hydroxysuccinimide (NHS) were added simultaneously to the bacterial suspension at room temperature. Specifically, the bacteria and nucleic acid aptamers selected are described in experimental examples 1.1-1.3 above.

Experiment 3: the average number of binding of the appropriate ligand per bacterial surface was calculated.

First, several Cy 5-labeled AS1411 solutions were prepared at a certain concentration. Then, fluorescence intensity of the Cy 5-labeled AS1411 concentration was plotted using photoluminescence spectrum having an excitation wavelength of 647nm and an emission wavelength of 670nm, and a standard curve was established. After determining the regression equation for the standard curve, the concentration of free Cy 5-labeled AS1411 after the reaction can be calculated. The number of binding ligands on the surface of each bacterial cell was calculated according to the following formula.

N _avg ＝((c ₀ v-c _a v)×NA)/N _a

Wherein Navg represents average number/bacterial cell, c ₀ Represents the concentration of Cy 5-labeled AS1411 released before the reaction, c _a Indicating the free form after the reactionCy 5-labeled AS1411, v is the volume in which the reaction occurs, NA is the Avgalileo constant 6.02X10 ²³ ，N _a Is the total number of bacterial cells.

Experiment 4: detection of stability of bound bacteria in serum

To investigate the serum stability of bacteria surface-bound with appropriate ligands, 1X 10 of Cy5-labelled AS1411 was surface-bound ⁹ CFU ApCB was suspended in 5ml PBS containing 10% fetal bovine serum at 37 ℃ and time points 0, 1, 4, 8, 12, 24 and 48h were predetermined. At the end of each time point, 100 μl was removed from each sample and centrifuged. After rinsing with PBS, the final samples were resuspended in 100. Mu.L of PBS and placed in quartz tubes. The fluorescence emission intensity of each sample was measured at room temperature under 647nm excitation light and 670nm emission light. And calculating the concentration of the bound proper ligand by using a regression equation obtained by the standard curve.

Experiment 5: cell absorption

At 2X 10 per confocal dish ⁵ Density of individual cells were inoculated in 1ml of RPMI-1640 medium containing 10% fetal bovine serum and antibiotics. 4T1 cells were cultured in 5% CO2 at 37℃for 24h. The stock medium was then replaced with fresh 1640 medium, treated with equal volumes of EcN or ApCB for 2h at 37 ℃, washed 3 times with pbs and the cells were observed with LSCM.

To quantify cell uptake behavior, cells were digested and collected for flow cytometry analysis.

To directly observe the binding of bacteria to cancer cells, the co-incubated cells were fixed with 4% glutaraldehyde in 0.5mL of PBS, 4 ℃,40min. Then, dehydration was performed in a series of ethanol/water solutions, increasing the ethanol content from 30% to 100%. Finally, the relevant ethanol was removed from the cells by freeze-drying.

Experiment 6: construction of subcutaneous tumor model

This experiment was carried out under SPF (specific pathogen-free, SPF) conditions on female BALB/C mice (6-8 weeks) kept for 6 d. For the 4T1 and H22 tumor models, 1X 10 injections were subcutaneously injected into the right hind limb of each BALB/C mouse ⁶ Cells, 100 μl serum-free 1640 medium. By calipersTumor volume per mouse was measured and the formula was calculated: (Width) ² X length x 0.5.

Experiment 7: binding of bacteria to cancer cells in vivo

Preparation of a 4T1 tumor-bearing mouse model, 1X 10 right hind limb injection per Balb/c nude mouse ⁶ Cells, 100 μl serum-free 1640 medium. The vaccinated mice were randomly split into PBS, ecN (1X 10) ⁷ CFU) and 5ApCB (1×10) ⁷ CFU) 3 groups. 100 μl each was taken and gastric lavaged via tail vein. For bacterial staining, mice were euthanized on day 12 post injection to harvest tumor tissue. Samples were fixed in 4% paraformaldehyde fixative, then transferred to sucrose-containing PBS, soaked overnight at 4deg.C, cut into slides, and FITC-labeled anti-E.coli antibody (FITC-labeled anti-Escherichia coli antibody) was added to PBS containing 0.1% normal serum and incubated in the dark for 1-2h at room temperature. Nuclei were stained with 4', 6-diamino-2-phenylindole (DAPI). After 3 washes in PBS, the specimens were fixed imaged.

Experiment 8: biodistribution of living beings

To study the biological distribution of EcN and 5ApCB in vivo, we will contain 1X 10 ⁷ The tail vein of the 4T1 tumor mouse was injected with 0.1mL of physiological saline of EcN or 5ApCB of CFU. Each group had 4 mice. All mice were imaged by the in vivo imaging system at the indicated time points and then harvested tissue was sacrificed. Homogenizing liver, spleen, lung, heart, kidney and tumor in a glass homogenizer. Equal amounts of each homogenate were serially diluted with LB, 50. Mu.L of each dilution was plated on LB agar plates containing antibiotics, incubated overnight at 37℃and counted.

Experiment 9: tumor imaging

Preparation of a 4T1 tumor-bearing mouse model, 1X 10 right hind limb injection per Balb/c nude mouse ⁶ Cells, 100 μl serum-free 1640 medium. Tumor inoculation of mice to about 100cm ³ Post random grouping (4 per group). To detect the accumulation of EcN and 5ApCB in tumors, we will be 1×10 ⁷ EcN or 5ApCB, which CFU expresses LuxCDABE, was injected into tumor bearing mice. Fluorescence imaging was performed at 12, 48, 60h after injection, respectively. All mice were imaged by an in vivo imaging system and at a predetermined timeTime points were sacrificed for further analysis. All images were analyzed using the Caliper IVIS Lumina II system. Within each group, regions of interest (ROIs) of the tumor tissue are defined. The average intensity of the tumor ROI in each frame of image is calculated and the background intensity within the ROI is subtracted.

Experiment 10: bacterial-mediated cancer treatment and histological analysis

The tumor size reached about 100cm ³ Mice of (E) were divided into PBS, VNP (5X 10) ⁵ CFU)、5ApCB(5×10 ⁵ CFU) 3 groups (5 per group). The tail vein was administered at 100. Mu.L each. Tumor size was measured every other day with digital calipers. For histological analysis, tumors were taken, fixed with 4% paraformaldehyde, paraffin embedded, hematoxylin Eosin (HE) stained. Each group of tumor tissues was collected simultaneously for TUNEL and TNF- α immunofluorescence staining analysis.

Experiment 11: flow cytometry to detect immune responses

Tumor tissue was harvested, treated with 1mg/mL collagenase I (Gibco) for 1h, and ground with the rubber end of a syringe. Cells were filtered through a nylon mesh filter and washed with PBS. The cells were further stained with the corresponding fluorescently labeled antibodies. To analyze the subpopulations of T cells in the induced tumors, single cell suspensions prepared from these specimens were examined using flow cytometry. The following primary antibodies were used: perCP-cyanide5.5-labeled anti-CD3 (BioLegend, catalog: 100328), APC-labeled anti-CD4 (BioLegend, catalog: 100412), PE-Cy7-labeled anti-IFN-gamma (BioLegend, catalog: 505826), and FITC-labeled anti-Ki67 (eBioscience, catalog: 11-5698-82). The dilution factor of the antibody was 1:200. Data analysis was performed using CytoFLEX and analysis was performed using cytexert (Beckman Coulter, USA) and FlowJo (TreeStar, USA) software.

Statistical analysis and reproducibility

All data are expressed as mean ± Standard Deviation (SD) of at least three independent experiments. Statistical analysis was performed using Prism 8.0 (GraphPad, USA). Comparison between the two groups was performed using the unpaired two-tailed Student's test (two-tailed). Fig. 1.C, 3.A, 5.a, 5.D-e, 7.B-d and fig. 8.A, 10.A, 13.A-b, n=3 independent experiments. In all events, a statistical difference is considered if p <0.05 (< p 0.05, < p <0.01, < p <0.001, and < p < 0.0001).

II, results of experiment and detection

1. ApCB and characterization of the preparation

The aminated AS1411 was attached to the carboxyl group of the n-acetyl muramic acid by an amide condensation process (FIG. 2). Because n-glycolyl muramic acid is widely present in the cell wall of gram-negative bacteria, it has higher reactivity than other carboxyl groups of glutamic acid or aspartic acid residues.

ApCB was prepared by incubating EcN with AS1411 in Phosphate Buffered Saline (PBS) for 3h at room temperature with the aid of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) and the resulting bacteria were purified by simple centrifugation. For measurement by Laser Scanning Confocal Microscopy (LSCM) and flow cytometry, apCB was prepared using AS1411 labeled with fluorescent dye Cyanine5 (Cy 5) at the 5' end of the sequence. AS shown in fig. 1.C, LSCM images showed that EcN expressing Green Fluorescent Protein (GFP) was effectively modified by AS1411 (red) under experimental conditions. Flow cytometry further demonstrated that AS1411 was successfully modified on the bacterial cell surface. The average fluorescence intensity of ApCB was about 10 times that of EcN (fig. 2).

Taking a certain number of 1×10 ⁸ EcN of CFU, labeled AS1411 with Cy5 of 0, 2, 5 and 10nmol, respectively (specifically EcN _Cy5 ,2ApCB _Cy5 ,5ApCB _Cy5 And 10ApCB _Cy5 ) And (5) incubating. The resulting bacteria were then analyzed by flow cytometry. AS a result, the fluorescence signal was enhanced and the concentration of AS1411 was increased, clearly indicating a higher intensity of the diversion of the distinct continuous curve (fig. 1.D and e). The number of grafted oligonucleotide aptamers per cell was counted and the detection of EcN bound to different concentrations of Cy 5-collimated AS1411 was performed using a fluorescence spectrophotometer. As shown in FIG. 1.f, the binding amount of AS1411 to each cell surface of 2, 5 and 10ApCBCy5 was calculated to be 0.7X10, respectively ⁵ 、2.8×10 ⁵ And 5.7X10 ⁵ And an aptamer.

To investigate whether the preparation process and the aptamer had no toxic side effects on bacterial viability, we used plate counting to evaluate the growth of ApCB. EcN, 2ApCB, 5ApCB, 10ApCB were serially diluted and plated onto Luria Bertani (LB) agar plates. After 24h incubation at 37 ℃, the activity of ApCB was determined by recording the number of colonies. As can be seen from figure 1.g, there was no significant difference in colony numbers CFU for 2ApCB, 5ApCB, 10ApCB compared to EcN, indicating that bacterial viability was unaffected after binding the appropriate ligand.

Furthermore, serum tolerance of aptamer-based therapeutic drugs is critical for in vivo applications. Based on existing theory and experimental data. It is speculated that binding of a suitable ligand to the bacterial surface may greatly increase the steric hindrance of AS1411 by enzymatic hydrolysis of the nuclease.

To demonstrate this conclusion, we monitored the stability of the aptamer bound to EcN by tracking the fluorescence intensity of Cy 5-labeled AS 1411. ApCBCy5 was analyzed by flow cytometry after incubation of ApCBCy5 with 10% fetal bovine serum in PBS at 37℃to a predetermined time point. According to the inventors team, it has previously been reported that more than 60% of free AS1411 rapidly degrades after 48h incubation (j.am. Chem. Soc.2020,142,2, 937-944). However, it is notable that under the same experimental conditions, AS1411 bound to the EcN surface, its stability was greatly improved, and more than 70% of the bound aptamer remained intact at the same time point (fig. 1. H).

Increased binding of ApCB to cancer cells

CTL is a scrambled oligonucleotide of the same number of bases AS1411 but without secondary structure, and serves AS a control. To study the role of AS1411 in surface-bound AS1411 in EcN invading cancer cells, we evaluated the use of nucleolin-overexpressing 4T1 cells in PBS, ecN, respectively _GFP 、CTL _GFP 、2ApCB _GFP 、5ApCB _GFP And 10ApCB _GFP Is shown (FIG. 4. A). LSCM imaging showed that AS1411 binding to the cell surface enhanced EcN binding to 4T1 cells (fig. 3. A). Based on confocal imaging results and flow cytometry images, AS1411 grafting density was from 0.5X10 ⁵ -2.8×10 ⁵ Cell, binding efficiency increases with increasing grafting density. The grafting density was further increased to 5.5X10 ⁵ SubsequentlyBinding efficiency began to drop (figure 3.b).

Under the same culture conditions, ecN is respectively combined with PBS and EcN _GFP 、CTL _GFP 、2ApCB _GFP 、5ApCB _GFP And 10ApCB _GFP After 2h incubation, the ratio of 4T1 cells to EcN bound was about 0.2%, 5.5%, 4.5%, 15%, 50%, 30%, respectively (fig. 4. B). These results indicate that aptamer modification can promote bacterial interactions with targeted cancer cells through specific ligand-receptor recognition, and that this depends on grafting density.

To visually observe binding of ApCB to cancer cells, 4T1 cells and 5ApCB cells after co-incubation with EcN were observed with scanning electron error copy (SEM), respectively (fig. 5.a). Representative SEM images showed that more bacteria were attached to the 4T1 cell surface 5ApCB compared to EcN, consistent with flow cytometry analysis results.

Flow cytometric analysis showed that 293T cells were differentiated from EcN _GFP and 5ApCB _GFP There was no significant difference in fluorescence intensity after binding (FIGS. 5.B and c). Meanwhile, LSCM images showed that 5ApCBGFP binds 293T cells with similar efficiency as EcNGFP. There was limited bacterial attachment on the surface of 293T cells (fig. 5. D).

In addition, to further evaluate the binding efficiency of ApCB to cancer cells in vivo, we established a 4T1 tumor model by intravenously administering PBS and 1×10 respectively to the tumor-bearing mice obtained ⁷ EcN of CFU or 5ApCB. At 12h post injection, tumor tissue was taken and stained with FITC-labeled anti-Escherichia coli and 4', 6-diamino-2-phenylindole (DAPI) (FIG. 5.e).

The tumor targeting ability of ApCB was determined by tumor imaging using a 4T1 tumor-bearing mouse model. According to the in vitro experimental results, 5ApCB with the highest binding efficiency was used to evaluate tumor aggregation. To achieve visual tracking, luxCDABE engineered 5ApCBLux was injected into tumor bearing mice via tail vein, while PBS, CTL _Lux And EcN surface-free aptamer _Lux As a control group. At the indicated time points after injection, the dosed mice were observed with an In Vivo Imaging System (IVIS).

As shown in FIG. 7As indicated in a, the mean luminescence signal of the tumor obtained from ivis increases with increasing time intervals from 12 to 60 hours after administration. Importantly, at each time point, the average intensity of the luminescence signal of the tumor site of the mice injected with 5ApCBLux was significantly higher than that of the mice injected with PBS and CTL respectively _Lux And EcN _Lux Is shown (FIG. 7. B). In particular, the relative signal intensity is CTL 60h after injection _Lux And EcN _Lux 8 times the group.

To quantify tumor colonization capacity, bacterial biodistribution was examined at the end point of animal studies. After euthanizing the mice, heart, kidney, liver, lung, spleen and tumor tissues were collected, homogenized, and plated on LB agar plates for bacterial enumeration. The number of bacteria in the tumor tissue of the 5ApCB injected mice sections was calculated to be 4 times that of EcN and CTL groups (fig. 7. C).

The distribution of bacteria in tumor tissue is unusual compared to major organs, with about 99.9% of bacteria colonizing the tumor interior. The massive aggregation of EcN within tumor tissue is associated with their preference for hypoxia, eutrophication and immunosuppressive microenvironment of the tumor. To demonstrate this hypothesis, we quantified the localization of EcN within 4T1 tumors in a short time after injection using plate counting. As shown in FIG. 7.D, the number of bacteria localized in tumor tissue reached 1.75X10 at 12h post-5 ApCB administration ⁷ CFU/g tissue was 2-fold compared to EcN without binding or CTL modification. That is, surface conjugation does provide a greater opportunity to localize tumor sites via ligand-receptor interactions between aptamer-modified bacteria and tumor cells.

In addition, the potential side effects of 5ApCB were assessed by blood routine and cytokine detection. The values of White Blood Cells (WBC) and Platelets (PLT) of mice injected with a dose of 5ApCB were similar to those of the PBS group with interleukin-6 (IL-6) and interleukin (IL-10) 60h after injection of PBS, indicating satisfactory compatibility (FIG. 6).

3. Evaluation of ApCB anti-cancer efficacy

Compared to EcN, due to the modification of AS1411, a comparable increase in VNP can be caused during binding of VNP to 4T1 cells (fig. 7, 9 and 1 2). Using the 4T1 mouse subcutaneous tumor model, the tumor-bearing mice obtained were randomly divided into three groups (n=5) and then received different treatments, including PBS, VNP and 5ApCB, respectively. It has been reported that a single intravenous VNP dose of 1X 10 ⁴ -1×10 ⁶ CFU/mice were tolerizing (see Oncol Res.2001;12 (11-12): 501-8 for details). Thus, each mouse in the VNP and 5ApCB groups was injected 5X 10 intravenously on day 0 ⁵ CFU bacteria. Studies that concisely evaluate tumor volume tumor inhibition showed that VNP and 5ApCB could inhibit tumor growth compared to PBS group (figures 9.a and b). As shown in fig. 9, changes in mice body weight record every other day after treatment revealed limited side effects of bacteria at the experimental dose.

To reveal the bacterially mediated immune response, activation of T cells in tumor tissue was analyzed at the end of treatment. The infiltration of CD3+ T cells in the tumor was detected using the non-histone Ki67 on the nucleus as a marker. As shown in fig. 9.d, 13.A and 16, cd3+ T cell proliferation was significantly increased in 5ApCB treated mice compared to PBS and VNP groups. Furthermore, 5ApCB treatment significantly increased the percentage of cd3+cd4+ T cells (fig. 9.e, fig. 13.B and fig. 16), suggesting that aptamer-modified bacteria may be more effective in promoting tumor infiltration of immune effector cells.

Further, the inventors also examined the percentage of tumor cytokines (e.g., interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha)) that were primarily responsible for modulating cellular immune responses the results indicated that this percentage was significantly increased in 5ApCB treated mice (FIGS. 9.f and g, FIGS. 13.C and 14. A.) the increased levels of TNF-alpha and IFN-gamma in tumor tissue indicated that 5ApCB treatment activated anti-tumor immune responses.

Taken together, inhibition of tumor growth may be due to aptamer-mediated enhancement of tumor targeting ability of bacteria and activation of bacterial-associated anti-tumor immune responses.

Versatility of ApCB

Similarly, TLS11a based ApCB (T-ApCB) was obtained by co-incubation with VNP (fig. 11. A). The details of the preparation of T-ApCB are briefly described herein. Specifically, 1×10 ⁸ VNP of CFU was incubated with 5nmol of TLS11a to prepare T-5ApCB.

Flow cytometric analysis showed that, with T-5ApCB _GFP The fluorescence intensity of co-incubated H22 cells was higher than that of H22 cells co-incubated with VNPGFP, indicating an increased affinity of TLS11 a-bound bacteria for binding to cancer cells (fig. 11.B and fig. 15).

Next, we evaluated the anti-tumor efficacy of the T-5ApCB thus produced in H22 tumor-bearing mice. Day 0 single intravenous injection of PBS, 5×10 ⁵ VNP or T-5ApCB of CFU (tumor size about 100 mm) ³ ) Tumor size and body weight of treated mice were recorded.

As shown in FIGS. 11.C and d, the T-5ApCB group has a significant inhibitory effect on tumor growth compared to the PBS group and the VNP group, since the binding of the aptamer to its surface increases the ability of the bacteria to localize within the tumor. Survival of the different groups was further verified that treatment of H22 tumor-bearing mice with T-5ApCB significantly prolonged survival, with nearly 70% of treated mice successfully surviving within 36 days (fig. 11. E). While neither PBS nor VNP mice survived under the same conditions. Furthermore, changes in body weight during treatment showed that mice had slightly decreased in weight in the group injected with bacteria, but quickly recovered to the same level as in the PBS group (fig. 11. F). Slight fluctuations in body weight indicate that 5ApCB has limited side effects at the experimental dose.

In summary, in the present application, the binding of the aptamer to the surface of the bacterium is a cell-compatible amidation process, which can significantly promote localization of the bacterium at the tumor site after systemic administration. The surface density of the aptamer can be easily adjusted through different feeding ratios, and the coupling can improve the stability of the anchoring aptamer. Average binding per cell of 2.8X10 ⁵ Optimally for each aptamer, the aptamer-coupled bacterium enhances its specificity for tumor cells in vitro, wherein when averaged each cell node2.8X10 of ⁵ The specificity is optimal when the aptamer is used. Approximately 2-fold and 4-fold aggregation was produced in tumor tissue at 12 and 60h, respectively, compared to unmodified bacteria. In the 4T1 and H22 tumor-bearing mouse models, the aptamer-bound attenuated salmonella showed significantly enhanced anti-tumor efficacy, as well as a highly activated immune response within the tumor. This work demonstrates how the behavior of the modified bacteria is mediated by surface coupling and supports the potential of aptamer-conjugated bacteria in targeting within tumors and enhancing tumor biotherapy.

The above are only specific examples of the present invention and experimental examples for confirming the inventive concept of the present application, but the present invention is not limited thereto. All modifications which may be directly derived or suggested by the teachings of this invention will be considered to be included within the scope of this invention by those skilled in the art.

Claims

1. A bacterium having a surface-bound targeting ligand, wherein the bacterium is obtained by binding a nucleic acid aptamer to the surface of the bacterium through a single amidation process, and wherein the bacterium is any one or more selected from the group consisting of gram-negative bacteria.

2. The bacterium of claim 1, wherein the nucleic acid aptamer is an aminated nucleic acid aptamer.

3. The bacterium of claim 2, wherein said aptamer has a grafting density of 0.7x10 ⁵ -5.7×10 ⁵ Nucleic acid aptamer/cell.

4. A method of preparing a bacterium having a surface-bound targeting ligand as described above, comprising: binding a targeting ligand to the surface of a bacterium by a single amidation process, wherein the bacterium is any one or more selected from gram-negative bacteria and the targeting ligand is a nucleic acid aptamer.

5. The method of claim 4, wherein the nucleic acid aptamer is an aminated nucleic acid aptamer.

6. The method of claim 4, wherein the aminated nucleic acid aptamer is an oligonucleotide strand as set forth in SEQ ID No.1 or SEQ ID No. 2.

7. The method of claim 4, wherein the aptamer has a grafting density of 0.7X10 ⁵ -5.7×10 ⁵ Nucleic acid aptamer/cell.

8. A pharmaceutical composition comprising the surface-bound ligand-targeting bacterium of any one of claims 1-3 and a pharmaceutically acceptable carrier thereof.

9. A drug delivery system for tumour therapy, characterized in that said system comprises at least a surface-bound targeting ligand bacterium according to any of the preceding claims 1-3, wherein the targeting ligand is a nucleic acid aptamer.

10. The drug delivery system of claim 9, wherein the drug delivery system is loaded with additional active drug components using the surface-targeting ligand-binding bacteria as a carrier.

11. Use of a surface-bound ligand-targeting bacterium according to any one of claims 1-3 for the manufacture of a medicament for the treatment of a tumor.

12. The use according to claim 11, for the treatment of different types of tumors, depending on the aptamer selected, wherein said tumors are common solid tumors, including lung cancer, stomach cancer, colon cancer, uterine cancer, rectal cancer, laryngeal cancer, breast cancer, etc.,

13. The use according to claim 12, wherein the tumor is gastric cancer, breast cancer.