CN117482117A - ATP-responsive manganese-based bacterial composite material and preparation method and application thereof - Google Patents

ATP-responsive manganese-based bacterial composite material and preparation method and application thereof Download PDF

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CN117482117A
CN117482117A CN202311413789.9A CN202311413789A CN117482117A CN 117482117 A CN117482117 A CN 117482117A CN 202311413789 A CN202311413789 A CN 202311413789A CN 117482117 A CN117482117 A CN 117482117A
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毛峥伟
杨黄
郭全世
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Zhejiang University ZJU
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Abstract

The invention provides an ATP-responsive manganese-based bacterial composite material, which is a core-shell material with single active bacteria inside and ATP-responsive manganese-based materials outside, wherein the surface of the single active bacteria is modified with phenylboronic acid modified polyethyleneimine polymer (PEI-PBA) molecules; the ATP-responsive manganese-based material is composed of a manganese-containing metal coordination complexSelf-assembling. The invention also provides a preparation method of the ATP-responsive manganese-based bacterial composite material and application of the ATP-responsive manganese-based bacterial composite material in preparation of antitumor drugs. The ATP-responsive manganese-based bacteria composite material can release Mn in a tumor high ATP environment 2+ To improve the activation effect of bacteria on the cGAS-STING channel, realize the selective synergistic activation of the cGAS-STING channel, and have good tumor immunotherapy effect.

Description

ATP-responsive manganese-based bacterial composite material and preparation method and application thereof
Technical Field
The invention belongs to the field of biological materials, and particularly relates to an ATP-responsive manganese-based bacterial composite material, and a preparation method and application thereof.
Technical Field
Immunotherapy, a very promising approach to tumor treatment, is the realization of tumor suppression by activating the human immune system. However, this treatment is only clinically applicable to a few patients, mainly because of the immunosuppressive nature of the tumor microenvironment. Current research indicates that GMP-AMP synthase (cGAS) -interferon gene Stimulator (STING) signaling plays a key role in improving immune microenvironment. First, cGAS in host cells acts as a DNA sensor, recognizing a specific type of DNA and producing the secondary messenger cGAMP; then, STING protein is activated by cGAMP and induces the cells to produce type I interferon (IFN-I), thereby promoting DC cell maturation and activating effector T cells, achieving tumor immunotherapy.
The DNA that activates the cGAS-STING pathway can be classified into exogenous DNA (e.g., bacterial DNA and viral DNA) and endogenous DNA (e.g., damaged DNA of the nucleus and mitochondria) according to the source. Dai et al have used nuclear and/or mitochondrial damaged DNA resulting from chemotherapy, radiation therapy or hyperthermia to activate the cGAS-STING pathway. However, the damaged DNA produced by these methods is dependent on the therapeutic window of anticancer drugs and is difficult to maintain for a long period of time. In contrast, bacteria can continue to secrete extracellular DNA (eDNA) during proliferation and growth to achieve continuous stimulation of the cGAS-STING pathway. In addition, the use of bacteria for anti-tumor therapy has other unique advantages such as intratumoral colonization, space-time controlled distribution and drug carriers for tumors.
However, the activation of cGAS-STING pathway by only bacterial eDNA is not only limited by weak stimulation, but also has a problem of poor selectivity. To address these challenges, it is necessary to explore a composite material that can selectively release cGAS-STING agonists. Divalent manganese ion (Mn) 2+ ) Is a trace element essential for human body. Research shows that Mn 2+ Stimulation of the cGAS-STING pathway by DNA is enhanced by increasing the sensitivity of cGAS to dsDNA recognition and enhancing binding between STING and cGAMP. The extracellular adenosine 5' -triphosphate (ATP) concentration (0.1-0.4 mM) was much higher in tumor tissues than in normal tissues (1-10 nM). And, immunogenic deathTumor cells secrete ATP to further increase extracellular ATP concentration. Thus, ATP is an ideal stimulus for extracellular responsive materials.
Based on the above, the present invention is expected to provide an ATP-responsive manganese-based bacterial composite material capable of fully utilizing the high concentration ATP environment in tumor tissue by releasing Mn 2+ To improve the activating effect of bacteria on the cGAS-STING channel, thereby realizing tumor immunotherapy.
Disclosure of Invention
In order to solve the problems of weak stimulation and low selectivity in the bacterial activation cGAS-STING pathway, the present invention aims to provide a method capable of releasing Mn in a tumor high ATP environment 2+ In the hope of achieving selective synergistic activation of the cGAS-STING pathway.
In order to achieve the above purpose, the invention adopts the following product material design and preparation scheme:
in a first aspect, the invention provides an ATP-responsive manganese-based bacterial composite material, which is a core-shell material with single active bacteria inside and ATP-responsive manganese-based materials outside, wherein the surface of the single active bacteria is modified with phenylboronic acid modified polyethyleneimine polymer (PEI-PBA) molecules; the ATP-responsive manganese-based material is formed by self-assembly of a manganese-containing metal coordination complex.
In the composite material, the single active bacteria are arranged on a nuclear layer, and the ATP-responsive manganese-based material is uniformly distributed on a shell layer to wrap the active bacteria; PEI-PBA molecules are modified on the surface of bacteria through covalent action and electrostatic action, so that on one hand, the adsorption capacity of the bacteria to manganese-containing metal coordination complex ligands is improved, on the other hand, the manganese-containing metal coordination complex is promoted to form a manganese-based material coating on the surface of the bacteria through in-situ self-assembly, and on the other hand, ATP-responsive polymer can provide ATP responsiveness for the composite material. The composite material of the present invention is different from a mixture formed by disordered mixing of bacteria and manganese-containing metal coordination complexes, but is a homogeneous material having a typical core-shell structure.
In the composite material of the invention, the single active bacteria can be any bacteria capable of secreting eDNA, for example, can be various gram-positive bacteria or gram-negative bacteria, and can be specifically selected from bacillus coagulans, staphylococcus aureus, escherichia coli, salmonella and the like.
In the composite material, PEI-PBA is prepared from polyethylenimine and bromomethyl phenylboronic acid. The polyethyleneimine is of a linear, branched or hyperbranched structure, and the molecular weight range is 1-250 kDa.
In the composite material of the invention, the manganese-containing metal coordination complex is based on Mn (OAc) 2 And different benzoic acid-containing ligands are prepared according to different mass ratios. The benzoic acid-containing ligand may be selected from terephthalic acid, nitroterephthalic acid, 4' -biphenyldicarboxylic acid, trimesic acid, 1,3, 5-tris (4-carboxyphenyl) benzene, 5,10,15, 20-tetrakis (4-carboxyphenyl) porphyrin, 1,3, 5-tris (3, 5-m-carboxyphenyl) benzene or 5,5,5,5,5- (1, 3,6, 8-pyrenetetrayl) tetrakis [1, 3-phthalic acid]Any one of them; preferably 5,10,15, 20-tetrakis (4-carboxyphenyl) porphyrin.
In the preferred composite material of the present invention, in order to further enhance the biocompatibility of the composite material, the surface of the ATP-responsive manganese-based material is further modified with a phospholipid polymer conjugate, more preferably a phospholipid-polyethylene glycol (DSPE-PEG) molecule by hydrophobic interaction.
In a second aspect, the invention also provides a method of preparing the ATP-responsive manganese-based bacterial composite of the first aspect, comprising:
1) Mixing active bacteria and PEI-PBA solution, controlling the ratio of the active bacteria to PEI-PBA to be 10 8 cfu is 0.01-1 mg, PEI-PBA molecules are modified on the surface of the active bacteria through self-assembly under the ultrasonic condition to obtain a first solution;
2) Mn (OAc) is added to the first solution obtained in 1) 2 And a benzoic acid-containing ligand and adjusting the pH of the reaction system to 8-9, preferably 8.5, to obtain a second solution; controlling active bacteria, mn (OAc) in said second solution 2 And a ratio of benzoic acid-containing ligand of 10 8 cfu：0.1～10mg：002-2 mg; mn (OAc) under magnetic stirring 2 And forming a manganese-containing metal coordination complex by using a benzoic acid-containing ligand, and self-assembling on the surface of the active bacteria modified with PEI-PBA to form a shell layer, thereby obtaining the ATP-responsive manganese-based bacteria composite material.
In the production method according to the second aspect of the present invention, the active bacterium of 1) may be any eDNA-secreting bacterium, and may be, for example, various gram-positive or gram-negative bacteria, and may be specifically selected from bacillus coagulans, staphylococcus aureus, escherichia coli, salmonella and the like.
In a preferred preparation method according to the second aspect of the present invention, 1) the PEI-PBA is prepared from polyethylenimine and bromomethylphenylboronic acid, wherein the mass ratio of polyethylenimine to bromomethylphenylboronic acid is 1 mg/0.1-5 mg, more preferably 1 mg/0.1-1 mg, still more preferably 1 mg/0.7-1 mg. The polyethyleneimine is of a linear, branched or hyperbranched structure, and the molecular weight range is 1-250 kDa.
In the preparation method according to the second aspect of the present invention, 1) the ratio of active bacteria to PEI-PBA in the first solution affects the potential of the bacterial surface, thereby affecting the self-assembly efficiency of the manganese-based complex. In a preferred preparation method of the invention, the mass ratio of the active bacteria described in 1) to PEI-PBA is 10 8 cfu is 0.01mg to 0.5mg; further preferably 10 8 cfu is 0.05mg to 0.1mg. In this ratio range, PEI-PBA can adjust the surface potential of the active bacteria to be most suitable for self-assembly of manganese-containing metal coordination complex, and can obtain an ATP-responsive manganese-based material coating with uniform distribution on the surface of the bacteria.
In the preparation method according to the second aspect of the present invention, 2) the second solution has PEI-PBA-surface-modified active bacteria and Mn (OAc) 2 The proportion of benzoic acid-containing ligand can influence the thickness of the manganese-based material shell generated by self-assembly, and further influence the exposure speed of bacteria under ATP response. In addition PEI-PBA and Mn (OAc) on the surface of active bacteria 2 The proportion of benzoic acid-containing ligand also has an effect on the ATP responsiveness of the composite. In a certain range, when the PEI-PBA proportion is higher, the composite materialThe ATP responsiveness of the material is improved; however, too high a PEI-PBA ratio will have an adverse effect on the self-assembly of the manganese-based material in step 2). Thus, in a preferred method of preparation of the invention, 2) the active bacteria, mn (OAc) 2 The mass ratio of the benzoic acid-containing ligand is 10 8 cfu is 0.1-5 mg, and cfu is 0.02-1 mg; more preferably 10 8 cfu is 0.5-1 mg, and cfu is 0.1-0.2 mg. In this ratio range, mn (OAc) 2 And the benzoic acid-containing ligand can be self-assembled on the surface of the active bacteria with high efficiency to form the ATP-responsive manganese-based material coating with moderate thickness.
In the preparation method according to the second aspect of the present invention, the benzoic acid-containing ligand of 2) may be selected from any one of terephthalic acid, nitroterephthalic acid, 4' -biphenyldicarboxylic acid, trimesic acid, 1,3, 5-tris (4-carboxyphenyl) benzene, 5,10,15, 20-tetrakis (4-carboxyphenyl) porphyrin, 1,3, 5-tris (3, 5-m-carboxyphenyl) benzene or 5,5,5,5,5- (1, 3,6, 8-pyrenetetrayl) tetrakis [1, 3-phthalic acid ]; preferably 5,10,15, 20-tetrakis (4-carboxyphenyl) porphyrin.
In a preferred preparation method according to the second aspect of the present invention, in order to further enhance the biocompatibility of the composite material, the method for preparing an ATP-responsive manganese-based bacterial composite material further includes a post-treatment step 3), specifically including: the ATP-responsive manganese-based bacterial composite obtained in 2) is collected by centrifugation and to this is added a phospholipid polymer conjugate, preferably DSPE-PEG, to give a post-treatment mixture, which is then incubated for 3-6 hours at room temperature.
In a further preferred preparation method according to the invention, the mass ratio of active bacteria to DSPE-PEG in the aftertreatment mixture according to 3) is 10 8 cfu is 0.01-0.2 mg; more preferably 10 8 cfu:0.01～0.5mg。
In a third aspect, the invention also provides an application of the ATP-responsive manganese-based bacterial composite material in preparing antitumor drugs.
Aiming at the technical background that the cGAS-STING channel is activated only by the bacterial eDNA, the stimulation effect is weak, and the selectivity is poor. The present invention addresses this problem by innovatively designing ATP-responsive manganese-based bacterial complexes. The composite material of the present invention comprises three sectionsThe method comprises the following steps: active bacteria at the nuclear layer, an ATP-responsive polymer (PEI-PBA molecule) at the surface of the active bacteria, and a manganese-based coordination complex at the shell layer. Wherein PEI-PBA molecules can be modified on the surface of bacteria through covalent action and electrostatic action, and the manganese-based coordination complex in situ self-assembly on the surface of bacteria is promoted by improving the adsorption capacity of bacteria to benzoic acid ligands. The shell layer of the composite material of the invention can be gradually degraded and release Mn in ATP environment 2+ Thereby exposing the active bacteria of the nuclear layer. The exposed active bacteria secrete eDNA, which in turn activates the cGAS-STING pathway within Dendritic Cells (DCs). Mn released in the composite material 2+ With the aid of which the sensitivity of cGAS to eDNA is increased and the binding capacity between STING and cGAMP is also enhanced. Thus, the ATP-responsive manganese-based bacterial composite material disclosed by the invention can selectively and synergistically activate the cGAS-STING channel.
Compared with the prior art, the invention has the advantages of wide applicability, sensitive response and good cGAS-STING stimulation effect:
1) In the present invention, the ATP-responsive manganese-based coating is capable of in situ self-assembly on a variety of bacterial surfaces. Thus, the coating has a broad spectrum.
2) In the present invention, the surface coating of ATP-responsive manganese-based bacterial material is capable of degrading in an extracellular low concentration ATP environment (0.1-0.4 mM) of tumor tissue. Thus, the coating has a sensitive responsiveness.
3) In the present invention, the surface coating of ATP-responsive manganese-based bacterial material is releasing Mn 2+ After that, the stimulation of the cGAS-STING pathway in immune cells by the bacteria can be sensitized. Thus, the complex has the effect of synergistically activating the cGAS-STING pathway.
Drawings
FIG. 1 is a TEM image of the ATP-responsive manganese-based bacterial complex of example 1.
FIG. 2 is a TEM image of the ATP-responsive manganese-based bacterial complex of example 2.
FIG. 3 is a TEM image of the ATP-responsive manganese-based bacterial complex of example 3.
FIG. 4 is a TEM image of the ATP-responsive manganese-based bacterial complex of example 4.
FIG. 5 is a TEM image of the ATP-responsive manganese-based bacterial complex of example 5.
FIG. 6 is a TEM image of the ATP-responsive manganese-based bacterial complex of example 6.
FIG. 7 is an SEM photograph of an ATP-responsive manganese-based bacterial complex according to example 7.
FIG. 8 is a TEM image of the ATP-responsive manganese-based bacterial complex of example 7 in different ATP environments.
FIG. 9 shows the Mn of the ATP-responsive manganese-based bacterial complexes of example 7 in different ATP environments 2+ Release rate.
FIG. 10 shows the synergistic activation of cGAS-STING in DC2.4 cells by the ATP-responsive manganese-based bacterial complex of example 7. In FIG. 10, parts A and B show the expression levels of pSTING and pIRF in DC cells under different conditions (including example 7 and comparative example 1, the same applies below); the expression level of IFN beta and ISG genes in DC cells under different conditions is reflected in the parts C and D; parts E and F show the levels of IFN beta and TNF alpha secreted by DC cells under different conditions.
FIG. 11 is an inhibition of B16F10 tumors by ATP-responsive manganese-based bacterial complexes of example 7. Wherein, part A shows the tumor volume change curve of tumor-bearing mice under different condition treatments; part B shows the final tumor mass after the end of the tumor suppression experiment under different conditions.
FIG. 12 is an in vivo immune activation of ATP-responsive manganese-based bacterial complexes in tumor-bearing mice of example 7. Wherein part A shows the content of mature DC cells in lymph nodes under different conditions; part B shows the content of effector T cells in lymph nodes treated under different conditions.
Detailed Description
The technical problems, technical schemes and beneficial effects to be solved by the invention are described in detail below with reference to specific embodiments. The following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any way. It should be noted that several variations and modifications can be made by those skilled in the art without departing from the precursors of the inventive concept. These are all within the scope of the present invention.
The invention relates to an ATP-responsive manganese-based bacterial composite material, which is a core-shell material with single active bacteria inside and ATP-responsive manganese-based materials outside, wherein the surface of the single active bacteria is modified with phenylboronic acid modified polyethyleneimine polymer (PEI-PBA) molecules; the ATP-responsive manganese-based material is formed by self-assembly of a manganese-containing metal coordination complex.
The ATP-responsive manganese-based bacterial composite material of the invention is prepared by the following method:
1) PEI-PBA solution is prepared by reacting PEI with 2-bromomethylphenylboronic acid. The polyethyleneimine is of a linear, branched or hyperbranched structure, and the molecular weight range is 1-250 kDa. The mass ratio of PEI to 2-bromomethyl phenylboronic acid is 1mg: 0.1-1 mg; preferably 1mg, 0.1 to 5mg, more preferably 1mg, 0.1 to 1mg, further preferably 1mg, 0.7 to 1mg.
2) Mixing active bacteria and the PEI-PBA solution under ultrasonic conditions to obtain a first solution. Controlling the ratio of the bacteria to PEI-PBA in the first solution to be 10 8 cfu: 0.01-1 mg; preferably 10 8 cfu is 0.01mg to 0.5mg; further preferably 10 8 cfu is 0.05mg to 0.1mg. The active bacteria may be any bacteria capable of secreting eDNA, for example, may be various gram-positive bacteria or gram-negative bacteria, and may specifically be selected from Bacillus coagulans, staphylococcus aureus, escherichia coli, salmonella and the like.
3) Mn (OAc) is added to the first solution under magnetic stirring 2 And a benzoic acid-containing ligand, and the pH of the reaction system was adjusted to 8.5 using a NaOH solution to obtain a second solution. Controlling the bacteria, mn (OAc) 2 And a ratio of benzoic acid-containing ligand of 10 8 cfu: 0.1-10 mg: 0.02-2 mg; preferably 10 8 cfu is 0.1-5 mg, and cfu is 0.02-1 mg; more preferably 10 8 cfu is 0.5-1 mg, and cfu is 0.1-0.2 mg. The benzoic acid-containing ligand can be selected from terephthalic acid, nitroterephthalic acid, 4' -biphenyl dicarboxylic acid and homobenzeneBenzene tricarboxylic acid, 1,3, 5-tris (4-carboxyphenyl) benzene, 5,10,15, 20-tetrakis (4-carboxyphenyl) porphyrin, 1,3, 5-tris (3, 5-m-carboxyphenyl) benzene or 5,5,5,5,5- (1, 3,6, 8-pyrenetetrayl) tetrakis [1, 3-phthalic acid]Any one of them; preferably 5,10,15, 20-tetrakis (4-carboxyphenyl) porphyrin. The product of the step is the ATP-responsive manganese-based bacterial composite material.
4) After collecting the product in the second solution by centrifugation, DSPE-PEG was added and the ratio of active bacteria to DSPE-PEG was controlled to 10 8 cfu is 0.01-0.2 mg; preferably 10 8 cfu is 0.01-0.5 mg. After incubating for 4 hours at room temperature and centrifugally collecting the product, the ATP-responsive manganese-based bacteria composite material with high biocompatibility can be obtained.
Based on the above method, the present invention provides the following examples, comparative examples and performance tests for explaining the aspects and technical effects of the present invention in detail.
Example 1:
the preparation method of the ATP-responsive manganese-based bacteria composite material comprises the following specific raw materials and steps:
(1) First, 2.0g of Polyethylenimine (PEI) was slowly added to 20 ml of methanol until completely dissolved. Next, 1.68g of bromomethylphenylboronic acid was slowly added to the PEI solution and reacted for 24 hours with stirring at 50 ℃. After the completion of the reaction, the reaction solution was subjected to centrifugation (7000 rpm,5 minutes), and the supernatant was precipitated with diethyl ether. And (3) centrifuging, collecting, repeatedly dissolving and precipitating the supernatant to obtain the pure PEI-PBA.
(2) Under ultrasonic conditions, 1mL of E.coli bacterial liquid (10 8 cfu/mL, pure water) was added to 1mL of PEI-PBA solution (100. Mu.g/mL), and sonicated for 3 minutes.
(3) Under magnetic stirring, 4mL of Mn (oAc) was added to the above mixed solution 2 Solution (250. Mu.g/mL, water) and 20. Mu.L of 5,10,15, 20-tetrakis (4-carboxyphenyl) porphyrin (TCPP) solution (10 mg/mL, DMSO). Subsequently, the pH of the above reaction system was adjusted to 8.5 using NaOH solution, and reacted at room temperature for 30 minutes. The product was collected by centrifugation (5000 rpm,5 minutes) and repeatedly washed 3 times with water. The prepared microorganism composite material is named as E.coli@PDMCTEM images of the samples are shown in FIG. 1.
Example 2:
the preparation and use methods are generally the same as in example 1, except that: adding 0.1mL of PEI-PBA solution in the step (2); in step (3), 0.4mL of Mn (OAc) was added 2 Solution and 2. Mu.L of TCPP solution. TEM pictures of the prepared microorganism composite material are shown in figure 2. As can be seen from FIG. 2, the thickness of the shell layer of the obtained composite material is smaller in comparison with example 1 when the amount of PEI-PBA and the amount of the manganese-based material are reduced.
Example 3:
the preparation and use methods are generally the same as in example 1, except that: adding 10mL of PEI-PBA solution in the step (2); 40mL of Mn (OAc) was added in step (3) 2 Solution and 200. Mu.L of TCPP solution. TEM pictures of the prepared microorganism composite material are shown in figure 3. As can be seen from FIG. 3, the thickness of the shell layer of the obtained composite material is larger in the case of increasing the amounts of PEI-PBA and the manganese-based material as compared with example 1.
Example 4:
the preparation and use methods are generally the same as in example 1, except that: in step (2), bacillus coagulans was used. TEM pictures of the prepared microorganism composite material are shown in figure 4. As can be seen from fig. 4, in comparison with the case of using different types of bacteria in example 1, the manganese-based material was still able to form a uniform shell layer to encapsulate the bacteria by self-assembly.
Example 5:
the preparation and use methods are generally the same as in example 1, except that: staphylococcus aureus is used in step (2). TEM images of the prepared microorganism composite material are shown in figure 5. As can be seen from fig. 5, in comparison with the case of using different types of bacteria in example 1, the manganese-based material was still able to form a uniform shell layer to encapsulate the bacteria by self-assembly.
Example 6:
the preparation and use methods are the same as in general example 1, except that: salmonella is used in step (2). TEM image of the prepared microorganism composite material is shown in figure 6. As can be seen from fig. 6, in comparison with the case of using different types of bacteria in example 1, the manganese-based material was still able to form a uniform shell layer to encapsulate the bacteria by self-assembly.
Example 7:
example 7 is an addition of post-treatment steps to example 1, the post-treatment steps comprising: E.coli@PDMC obtained in step (3) of example 1 (concentration 10 8 cfu/mL, diluted with pure water) and centrifuged (rotation at 5000rpm for 5 minutes) before the product is redispersed in 50. Mu.g/mL DSPE-PEG solution. After the mixture was shaken at room temperature for 4 hours, the centrifugation was again performed (rotation speed: 5000rpm,5 minutes), and the suspension was re-suspended with pure water. The biocompatibility of the product of example 1 was thus improved, and the final product was named E.coli@PDMC-PEG. TEM pictures of the E.coli@PDMC-PEG are shown in FIG. 7.
Comparative example 1:
the preparation and use methods are generally the same as in example 7, except that: in step (2) no bacteria were used and the product obtained was in the form of a precipitate, designated "PDMC-PEG".
Performance test:
in the following, an in vitro responsiveness test, an in vitro cGAS-STING stimulatory capability test, an in vivo antitumor capability test, and an in vivo immunostimulatory capability test were performed for example 7 and comparative example 1, respectively.
In vitro responsiveness test:
detecting a sample:
E.coli@PDMC-PEG prepared in example 7 of the present invention.
The detection method comprises the following steps:
2mL of E.coli@PDMC-PEG (concentration 10 8 cfu/mL) solution was placed in a dialysis bag (MWCO 8-14 kDa). Next, the dialysis bag containing the test sample was immersed in ATP solutions of different concentrations (0, 0.1, 0.4 mM) and treated in a constant temperature water bath at 37 ℃ with shaking at 200 rpm. After 1 hour of treatment, 10. Mu.L of the test sample was taken out of the dialysis bag, and the morphology thereof was observed using a Transmission Electron Microscope (TEM), and the test results are shown in FIG. 8. As can be seen from FIG. 8, E.coli@PDMC-PEG was treated in a solution having an ATP concentration of 0 for 1 hourThe stable structure was maintained, while the E.coli@PDMC-PEG had degraded to a different extent after 1 hour in solutions with ATP concentrations of 0.1mM and 0.4 mM.
2mL of E.coli@PDMC-PEG (concentration 10) 8 cfu/mL) solution was placed in a dialysis bag (MWCO 8-14 kDa). Then, the dialysis bag containing the test sample was immersed in ATP solutions of different concentrations (0, 0.1, 0.4 mM) and treated in a thermostat water bath at 37℃under shaking conditions of 200 rpm. At a preset time point, 1mL of liquid is taken out of the solution outside the dialysis bag, and 1mL of ATP solution of the corresponding concentration is added for replenishment. To the removed liquid was added 1mL of aqua regia and allowed to stand overnight. Subsequently, the above solution was diluted to 10mL using a 2% nitric acid solution, and the content of Mn element therein was detected using an inductively coupled plasma mass spectrometer (ICP-MS), and the cumulative release amount of Mn ions was calculated. The test results are shown in FIG. 9. As can be seen from FIG. 9, E.coli@PDMC-PEG released Mn ions efficiently in the first 1h in ATP solutions of various concentrations, but at different release rates. The release rate of the E.coli@PDMC-PEG in a 0.4mM ATP solution for 1h is close to 80%, which is significantly higher than that in a 0.1mM ATP solution for 1 h. In addition, the release rate of Mn ions of E.coli@PDMC-PEG in 0.1-0.4mM ATP solutions with different concentrations can reach a peak value in the first 1h, which proves that the E.coli@PDMC-PEG prepared in the embodiment 7 of the invention has more sensitive ATP response capability.
In vitro cGAS-STING stimulation capability test:
detecting an object:
e.coli, PDMC-PEG prepared in comparative example 1 and E.coli@PDMC-PEG prepared in example 7
The detection method comprises the following steps:
DC2.4 cells were diluted using RPMI1640 medium containing 10% Fetal Bovine Serum (FBS) and seeded at a concentration of 107 cells/well to the bottom of a 24-well plate. After 12 hours, DC2.4 cells were attached to the bottom of the well plate. Subsequently, the cells were grouped and subjected to different treatments: (1) NC group, (2) ATP group, (3) PDMC-PEG+ATP group, (4) E.coll+ATP group, (5) E.coll@PDMC-PEG group, (6) E.coll@PDMC-PEG+ATP group. In groups (1) and (5), the medium was replaced with fresh RPMI1640 medium;in groups (2), (3), (4), (6), the medium was replaced with fresh RPMI1640 medium containing ATP (0.4 mM); in groups (3), (4), (5) and (6), a Transwell chamber was added to the well plate, and 200. Mu.L of PDMC-PEG prepared in comparative example 1, E.coli and E.coli@PDMC-PEG prepared in example 7 (wherein Mn concentration was 60.5. Mu.g/mL and E.coli concentration was 5X 10) were added to the chambers, respectively 8 cfu/mL). After 8 hours of treatment of DC2.4 cells, the expression level of cGAS-STING pathway associated protein (pstings, pIRF 3) in DC2.4 cells was detected using Western Blotting (WB) method; detecting the expression level of cGAS-STING pathway related genes (ISG and ifnβ genes) in the DC2.4 cells using a real-time fluorescent quantitative PCR (RT-qPCR) method; the expression level of cGAS-STING pathway-related inflammatory factors (ifnβ, tnfα) in DC2.4 cells was detected using an enzyme-linked immunosorbent assay (ELISA) kit, and the test results are shown in fig. 10. As shown in FIG. 10, under various treatment conditions, the E.coli@PDMC-PEG+ATP group DC2.4 intracellular levels of pSTING and pIRF3 proteins, the relative levels of IFN beta and ISG genes, and the extracellular IFN beta and TNF alpha inflammatory factors were significantly higher than those of the other groups. Demonstrating that E.coli@PDMC-PEG prepared in example 7 of the present invention can respond to ATP concentration and effectively stimulate intracellular cGAS-STING pathways.
In vivo antitumor capability test:
detecting an object:
e.coli, PDMC-PEG prepared in comparative example 1 and E.coli@PDMC-PEG prepared in example 7
The detection method comprises the following steps:
all animal experiments were conducted in compliance with the guidelines of the institutional animal care and use committee of Zhejiang university and under the protocol approved by the committee. Six week old female C57 mice were used in this experiment to construct B16F10 tumor models. Specifically, 5×10 5 The individual B16F10 cells were suspended in 50. Mu.L of PBS medium and injected into the hind legs of the mice until the tumor volume increased to 80mm 3 And when the tumor model is left and right, the B16F10 tumor model is successfully established.
After the B16F10 tumor model was established successfully, this time was noted as day 0 and tumor bearing mice were randomly grouped into four groups. On days 0, 2 and 4, the following materials were injected aside four groups of mice, respectively: (1) PB (PB)S, (2) PDMC-PEG prepared in comparative example 1, (3) E.coli and (4) E.coli@PDMC-PEG prepared in example 7 (50. Mu.L, wherein E.coli: 5X 10) 8 cfu/mL, mn:60.5 μg/mL). The body weight of the mice, the length and width of the tumor were recorded every two days, and the tumor volume (length×width 2 /2). On day 5, one mouse per group was sacrificed and the tumor was subsequently removed for hematoxylin-eosin (H&E) Staining and TUNEL staining analysis. On day 10, all mice were sacrificed and tumors were removed for photographing and weighing, and the test results are shown in fig. 11. As shown in fig. 11, there was a significant inter-group difference in tumor volume and weight in mice: after 10 days, the average tumor weight of the mice injected with E.coli@PDMC-PEG prepared in example 7 is less than 0.5g, while the average tumor weights of the mice injected with PDMC-PEG and E.coli are both between 1 and 2 g; in 10 days, the tumor volume of the mice injected with E.coli@PDMC-PEG prepared in example 7 is not obvious, and is always kept at 300mm 3 The average tumor volume of mice injected with PDMC-PEG and E.coli became rapidly larger below, and exceeded 1000mm after 10 days 3 . The E.coli@PDMC-PEG prepared in example 7 is shown to have an ideal in vivo antitumor capability.
In vivo immunostimulant ability test:
detecting an object:
e.coli, PDMC-PEG prepared in comparative example 1 and E.coli@PDMC-PEG prepared in example 7
The detection method comprises the following steps:
after the B16F10 tumor model was established successfully, this time was counted as day 0 and tumor bearing mice were randomly grouped into four groups of three mice each. On day 0, day 2 and day 4, the following materials were injected aside four groups of mice tumors, respectively: (1) PBS, (2) PDMC-PEG prepared in comparative example 1, (3) E.coli and (4) E.coli@PDMC-PEG prepared in example 7 (50. Mu.L, wherein E.coli: 5X 10) 8 cfu/mL, mn:60.5 μg/mL). On the tenth day after injection administration, lymph node tissue was removed and ground and filtered, followed by analysis of immune cells by cell flow (CD 8 + ，CD3 + ，CD86 + ，CD11c + ) The test results are shown in FIG. 12. As shown in FIG. 12, E.coli@PDMC-PEG prepared in example 7 can significantly promote the lymphopoiesis of miceThe level of immune cells in the tissue was significantly higher than in the other groups. The E.coli@PDMC-PEG of the embodiment 7 of the invention has ideal in vivo immunostimulating capability, can effectively activate effector T cells, and is very suitable for tumor immunotherapy.

Claims (9)

1. An ATP-responsive manganese-based bacterial composite, characterized by: the core-shell material is characterized in that the core-shell material is internally provided with single active bacteria and externally provided with ATP-responsive manganese-based materials, and the surface of the single active bacteria is modified with phenylboronic acid modified polyethyleneimine polymer (PEI-PBA) molecules; the ATP-responsive manganese-based material is formed by self-assembly of a manganese-containing metal coordination compound; the manganese-containing metal coordination complex is composed of Mn (OAc) 2 And benzoic acid-containing ligands; the benzoic acid-containing ligand is selected from terephthalic acid, nitroterephthalic acid, 4' -biphenyl dicarboxylic acid, trimesic acid, 1,3, 5-tri (4-carboxyphenyl) benzene, 5,10,15, 20-tetra (4-carboxyphenyl) porphyrin, 1,3, 5-tri (3, 5-m-carboxyphenyl) benzene or 5,5,5,5,5- (1, 3,6, 8-pyrenetetrayl) tetra [1, 3-phthalic acid]Any one of them; preferably 5,10,15, 20-tetrakis (4-carboxyphenyl) porphyrin.
2. The ATP-responsive manganese-based bacterial composite of claim 1, wherein: the surface of the ATP-responsive manganese-based material is further modified with a phospholipid polymer conjugate, more preferably a phospholipid-polyethylene glycol (DSPE-PEG) molecule through hydrophobic interaction.
3. A method of making the ATP-responsive manganese-based bacterial composite of claim 1, comprising:
1) Mixing active bacteria and PEI-PBA solution, controlling the ratio of the active bacteria to PEI-PBA to be 10 8 cfu is 0.01-1 mg, PEI-PBA molecules are modified on the surface of the active bacteria through self-assembly under the ultrasonic condition to obtain a first solution;
2) Mn (OAc) is added to the first solution obtained in 1) 2 And a benzoic acid-containing ligand, and adjusting the pH value of the reaction systemSection 8-9, preferably 8.5, to obtain a second solution; the benzoic acid-containing ligand is selected from terephthalic acid, nitroterephthalic acid, 4' -biphenyl dicarboxylic acid, trimesic acid, 1,3, 5-tri (4-carboxyphenyl) benzene, 5,10,15, 20-tetra (4-carboxyphenyl) porphyrin, 1,3, 5-tri (3, 5-m-carboxyphenyl) benzene or 5,5,5,5,5- (1, 3,6, 8-pyrenetetrayl) tetra [1, 3-phthalic acid]Any one of them; preferably 5,10,15, 20-tetrakis (4-carboxyphenyl) porphyrin; controlling active bacteria, mn (OAc) in said second solution 2 And a ratio of benzoic acid-containing ligand of 10 8 cfu: 0.1-10 mg: 0.02-2 mg; mn (OAc) under magnetic stirring 2 And forming a manganese-containing metal coordination complex by using a benzoic acid-containing ligand, and self-assembling on the surface of the active bacteria modified with PEI-PBA to form a shell layer, thereby obtaining the ATP-responsive manganese-based bacteria composite material.
4. A method as claimed in claim 3, characterized in that: 1) The PEI-PBA is prepared from polyethylenimine and bromomethylbenzoic acid, wherein the mass ratio of the polyethylenimine to the bromomethylbenzoic acid is 1 mg/0.1-5 mg, more preferably 1 mg/0.1-1 mg, still more preferably 1 mg/0.7-1 mg; the polyethyleneimine is of a linear, branched or hyperbranched structure, and the molecular weight range is 1-250 kDa.
5. A method as claimed in claim 3, characterized in that: 1) The mass ratio of the active bacteria to PEI-PBA is 10 8 cfu is 0.01mg to 0.5mg; further preferably 10 8 cfu:0.05mg～0.1mg。
6. A method as claimed in claim 3, characterized in that: 2) The active bacteria, mn (OAc) 2 The mass ratio of the benzoic acid-containing ligand is 10 8 cfu is 0.1-5 mg, and cfu is 0.02-1 mg; more preferably 10 8 cfu:0.5～1mg:0.1～0.2mg。
7. A method of making the ATP-responsive manganese-based bacterial composite of claim 2, wherein: after obtaining the ATP-responsive manganese-based bacterial composite material according to the method of claim 3, a post-treatment step 3) is performed, comprising: the ATP-responsive manganese-based bacterial composite as claimed in claim 3 is collected by centrifugation and phospholipid polymer conjugate, preferably DSPE-PEG, is added thereto to give a post-treatment mixture, which is then incubated at room temperature for 3-6 hours.
8. The method of claim 7, wherein: 3) The mass ratio of the active bacteria to DSPE-PEG in the post-treatment mixture is 10 8 cfu is 0.01-0.2 mg; more preferably 10 8 cfu:0.01～0.5mg。
9. Use of the ATP-responsive manganese-based bacterial composite of any one of claims 1-2 in the manufacture of an anti-tumor medicament.
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