CN117599085B - Preparation method and application of bioheterozygous free radical generator based on streptococcus pneumoniae - Google Patents

Abstract

The invention relates to a preparation method and application of a biological heterozygous free radical generator based on streptococcus pneumoniae, which can continuously generate high-activity OH to kill tumors without being limited by tumor hypoxia, and the technical scheme is that the preparation method comprises the following steps: 1) Preparing azide modified streptococcus pneumoniae s.pn-N ₃; 2) Preparing alkynyl modified Fe ₃O₄ nano-particles Fe ₃O₄ -DBCO; 3) Preparing a bioheterozygous free radical generator Fe ₃O₄ @S.pn based on streptococcus pneumoniae; the method of the invention has the advantages of convenient operation, stability and reliability, the prepared tumor biological heterozygote free radical generator has the advantages of high selectivity, lasting efficiency and the like, can break through the limitation of the tumor hypoxia microenvironment to selectively and continuously generate high activity, OH, improves the tumor treatment effect, and is an innovation in tumor treatment medicaments.

Description

Preparation method and application of a biological hybrid free radical generator based on Streptococcus pneumoniae

1. Technical Field

The present invention relates to the field of biomedicine, and in particular to a preparation method and application of a biological hybrid free radical generator based on Streptococcus pneumoniae.

2. Background Technology

Reactive oxygen species (ROS) are highly reactive oxygen-containing bioactive species that can induce cell death by inducing lipid peroxidation or damaging proteins and DNA. Since tumor cells are more sensitive to exogenous ROS and more susceptible to damage by exogenous ROS than normal cells, extensive research efforts have been directed toward the design of ROS-generating systems for tumor therapy. However, although tumor cells have higher hydrogen peroxide (H ₂ O ₂ ) levels (50-100 μM) compared with normal cells, endogenous H ₂ O ₂ is still insufficient to continuously generate large amounts of ROS and achieve satisfactory antitumor effects. Therefore, there is an urgent need to develop a paradigm that can selectively and efficiently generate ROS continuously in tumor tissues.

It is worth noting that a large amount of ROS is produced during bacterial infection, and high concentrations of ROS accumulation can cause serious damage to host cells. In particular, a large amount of H ₂ O ₂ is produced during infection with Streptococcus pneumoniae. Due to the lack of catalase to neutralize H ₂ O ₂ , the H ₂ O ₂ production can accumulate to millimolar, which provides a new idea for the use of Streptococcus pneumoniae in anti-tumor therapy. In addition, the sensing and chemotaxis ability of hypoxic sites and the tumor immunosuppressive microenvironment enable bacteria to selectively and efficiently colonize in tumor tissues. Therefore, the excellent tumor targeting and efficient H ₂ O ₂ production efficiency of Streptococcus pneumoniae are expected to be used for efficient anti-tumor therapy. Based on this, we conducted a preliminary exploration of the tumor killing effect of Streptococcus pneumoniae. However, the tumor treatment effect was poor due to the weak oxidation activity of H ₂ O _2. Therefore, the conversion of H ₂ O ₂ produced by Streptococcus pneumoniae into highly active hydroxyl radicals (·OH) is expected to enhance the anti-tumor effect mediated by Streptococcus pneumoniae.

In recent years, bacteria have been combined with functional nanoparticles, and nanomaterial modification can significantly expand the functions of bacteria. Studies have shown that ferroferric oxide (Fe ₃ O ₄ ) nanoparticles have peroxidase-like activity and can be used as Fenton's reagent to catalyze H ₂ O ₂ to produce ·OH. However, most nanomaterial modifications have harmful effects on bacteria and interfere with the inherent properties of microorganisms, including growth vitality, tropism, and membrane protein expression. As a modification method with mild reaction and high binding rate, bioorthogonal click chemistry achieves coupling through the chemical reaction of complementary groups of two bioorthogonal precursors in the biological system without interfering with the active life activities in the biological system. Therefore, the use of bioorthogonality to modify Fe ₃ O ₄ on Streptococcus pneumoniae is expected to achieve continuous ·OH generation. Therefore, it is imperative to invent a biohybrid system that can continuously produce highly active ·OH to enhance anti-tumor therapy without being restricted by tumor hypoxia.

III. Summary of the invention

In view of the above situation, in order to solve the defects of the prior art, the purpose of the present invention is to provide a preparation method and application of a biological hybrid free radical generator based on Streptococcus pneumoniae, which can continuously produce highly active ·OH and efficiently kill tumors without being restricted by tumor hypoxia.

The technical solution provided by the present invention is to provide a method for preparing a biological hybrid free radical generator based on Streptococcus pneumoniae. First, the surfaces of S.pn (Streptococcus pneumoniae) and Fe ₃ O ₄ are modified with an azide group N ₃ and DBCO (dibenzocyclooctyne) respectively, and a click chemical reaction occurs between N ₃ and the alkynyl group of DBCO to obtain a biological hybrid free radical generator, which specifically comprises the following steps:

1) Preparation of azide-modified Streptococcus pneumoniae S.pn-N ₃

S.pn was cultured for 8-10 hours, and the cells were expanded 10 times with freshly prepared THB medium + 5% fetal bovine serum for 3-5 hours. After the culture was completed, the cells were collected by centrifugation at 6000 g for 5-10 minutes, and resuspended in fresh THB medium containing 4-6 mM HD-DAP (N ₃ )·HCL or fresh THB medium + 5% fetal bovine serum for further 60-80 minutes. The cells were then collected by centrifugation at 6000 g for 5-10 minutes, and the cells were washed twice with PBS buffer to obtain azide-modified Streptococcus pneumoniae S.pn-N ₃ ;

2) Preparation of Fe ₃ O ₄ nanoparticles modified with alkynyl groups Fe ₃ O ₄ -DBCO

10-40 mg DSPE-PEG2000-DBCO (distearoylphosphatidylethanolamine-polyethylene glycol-diphenylcyclooctyne) was dissolved in 2-8 mL chloroform, ultrasonicated for 5-10 min, and then transferred to a dry glass round-bottom flask; subsequently, 5-20 mg oleic acid-coated Fe ₃ O ₄ nanoparticles were dissolved in 1-4 mL chloroform, the Fe ₃ O ₄ solution was added to the DSPE-PEG2000-DBCO solution, ultrasonically mixed for 5-10 min, and then the glass round-bottom flask was rotary evaporated at 55° C. until the solvent was completely evaporated, and 5-20 mL ultrapure water was added and ultrasonicated for 20-30 min to obtain Fe ₃ O ₄ -DBCO nanoparticles;

3) Preparation of a biological hybrid free radical generator Fe ₃ O ₄ @S.pn based on Streptococcus pneumoniae

The S.pn-N ₃ cells prepared in step 1) are resuspended in 3-12 mL of the Fe ₃ O ₄ -DBCO preparation system with a concentration of 50-200 μg/mL prepared in step 2) and incubated for 3-5 hours. Subsequently, 200 μL of the bio-orthogonal bacterial solution is taken, centrifuged at 4000 rpm and 4°C for 3-5 minutes, the supernatant is discarded, the solution is washed twice with PBS and resuspended in 3-12 mL of PBS to obtain a biohybrid free radical generator based on Streptococcus pneumoniae.

The particle size of S.pn-N ₃ in step 1) is 0.5-1 μm, the particle size of Fe ₃ O ₄ -DBCO nanoparticles in step 2) is 10-30 nm, and the particle size of Fe ₃ O ₄ @S.pn in step 3) is 0.5-1 μm.

The THB culture medium formula is: beef powder 10g/L, tryptone 20g/L, glucose 2g/L, sodium bicarbonate 2g/L, sodium chloride 2g/L, disodium hydrogen phosphate 0.4g/L, pH value 7.8±0.1, 25°C.

The application of the biohybrid free radical generator based on Streptococcus pneumoniae in the preparation of anti-tumor drug injection.

The application of the biohybrid free radical generator based on Streptococcus pneumoniae in self-supplying _H2O2 to improve tumor chemokinetics therapeutic _drugs .

The application of the biohybrid free radical generator based on Streptococcus pneumoniae in hypoxic tumor treatment drugs.

The application of the biohybrid free radical generator based on Streptococcus pneumoniae in a drug that utilizes the natural bacterial infection pathway to induce tumor cell death.

The method of the present invention is easy to operate, stable and reliable. The prepared tumor biohybrid free radical generator has the advantages of high selectivity, continuous and high efficiency, etc. In tumor treatment, it can break through the limitation of tumor hypoxic microenvironment and selectively and continuously produce highly active OH, thereby improving the tumor treatment effect. It is an innovation in tumor treatment drugs with huge economic and social benefits.

IV. Description of the drawings

FIG. 1 is a result of investigating the hydrogen peroxide production capacity of Streptococcus pneumoniae of the present invention.

FIG. 2 shows the effect of different oxygen partial pressures on the hydrogen peroxide production capacity of Streptococcus pneumoniae according to the present invention.

FIG3 shows the distribution behavior of Streptococcus pneumoniae in vivo after intravenous injection of the present invention.

FIG. 4 is the experimental result of the present invention showing that Fe ₃ O ₄ catalyzes H ₂ O ₂ to produce hydroxyl radicals.

FIG5 is a transmission electron microscopy characterization result of Streptococcus pneumoniae and the biological hybrid free radical generator of the present invention.

FIG. 6 is the characterization result of the Fe ₃ O ₄ @S.pn biohybrid free radical generator of the present invention by laser confocal microscopy.

FIG. 7 is a result of investigating the continuous generation of hydroxyl radicals by the biohybrid free radical generator of the present invention.

FIG8 is a result of investigating the cytotoxicity of the biohybrid free radical generator of the present invention to 4T1 cells.

V. Specific implementation methods

The specific implementation of the present invention is further described in detail below with reference to the accompanying drawings and examples.

Example 1

In the specific implementation of the present invention, the preparation method comprises the following steps:

1) Preparation of azide-modified Streptococcus pneumoniae S.pn-N ₃

S.pn was cultured for 8 hours, and then expanded 10 times with freshly prepared THB medium + 5% fetal bovine serum for 3 hours. After the culture was completed, the cells were collected by centrifugation at 6000 g for 5 minutes, and resuspended in fresh THB medium/fresh THB medium + 5% fetal bovine serum containing 4 mM HD-DAP(N ₃ )·HCL and continued to be cultured for 60 minutes. Then, the cells were collected by centrifugation at 6000 g for 5 minutes, and the cells were washed twice with PBS buffer to obtain azide-modified Streptococcus pneumoniae S.pn-N ₃ ;

2) Preparation of Fe ₃ O ₄ nanoparticles modified with alkynyl groups Fe ₃ O ₄ -DBCO

10 mg DSPE-PEG2000-DBCO was dissolved in 2 mL chloroform, ultrasonicated for 5 min, and then transferred to a dry glass round-bottom flask; subsequently, 5 mg oleic acid-coated Fe ₃ O ₄ nanoparticles were dissolved in 1 mL chloroform, the Fe ₃ O ₄ solution was added to the DSPE-PEG2000-DBCO solution, ultrasonically mixed for 5 min, the glass round-bottom flask was rotary evaporated at 55 °C until the solvent was completely evaporated, 5 mL ultrapure water was added and ultrasonicated for 20 min to obtain Fe ₃ O ₄ -DBCO nanoparticles;

3) Preparation of a biological hybrid free radical generator Fe ₃ O ₄ @S.pn based on Streptococcus pneumoniae

The S.pn-N ₃ cells prepared in step 1) were resuspended in 3 mL of 50 μg/mL Fe ₃ O ₄ -DBCO preparation system prepared in step 2) and incubated for 3 h. Subsequently, 200 μL of the bio-orthogonal bacterial solution was taken, centrifuged at 4000 rpm, 4°C for 3 min, the supernatant was discarded, the solution was washed twice with PBS and resuspended in 3 mL of PBS to obtain a biohybrid free radical generator based on Streptococcus pneumoniae.

Example 2

In the specific implementation of the present invention, the preparation method comprises the following steps:

1) Preparation of azide-modified Streptococcus pneumoniae S.pn-N ₃

S.pn was cultured for 9 h, and the cells were expanded 10-fold with freshly prepared THB medium + 5% fetal bovine serum for 4 h. After the culture was completed, the cells were collected by centrifugation at 6000 g for 7 min, and resuspended in fresh THB medium/fresh THB medium + 5% fetal bovine serum containing 5 mM HD-DAP(N ₃ )·HCL and continued to be cultured for 70 min. Then, the cells were collected by centrifugation at 6000 g for 7 min, and the cells were washed twice with PBS buffer to obtain azide-modified Streptococcus pneumoniae S.pn-N ₃ ;

2) Preparation of Fe ₃ O ₄ nanoparticles modified with alkynyl groups Fe ₃ O ₄ -DBCO

20 mg of DSPE-PEG2000-DBCO was dissolved in 4 mL of chloroform, ultrasonicated for 7 min, and then transferred to a dry glass round-bottom flask; subsequently, 10 mg of oleic acid-coated Fe ₃ O ₄ nanoparticles were dissolved in 2 mL of chloroform, the Fe ₃ O ₄ solution was added to the DSPE-PEG2000-DBCO solution, ultrasonically mixed for 7 min, the glass round-bottom flask was rotary evaporated at 55 °C until the solvent was completely evaporated, 10 mL of ultrapure water was added and ultrasonicated for 25 min to obtain Fe ₃ O ₄ -DBCO nanoparticles;

3) Preparation of a biological hybrid free radical generator Fe ₃ O ₄ @S.pn based on Streptococcus pneumoniae

The S.pn-N ₃ cells prepared in step 1) were resuspended in 6 mL of 100 μg/mL Fe ₃ O ₄ -DBCO preparation system prepared in step 2) and incubated for 4 h. Subsequently, 200 μL of the bio-orthogonal bacterial solution was taken, centrifuged at 4000 rpm, 4°C for 4 min, the supernatant was discarded, the solution was washed twice with PBS and resuspended in 6 mL of PBS to obtain a biohybrid free radical generator based on Streptococcus pneumoniae.

Example 3

In the specific implementation of the present invention, the preparation method comprises the following steps:

1) Preparation of azide-modified Streptococcus pneumoniae S.pn-N ₃

S.pn was cultured for 10 hours, and then expanded 10-fold with freshly prepared THB medium + 5% fetal bovine serum for 5 hours. After the culture was completed, the cells were collected by centrifugation at 6000 g for 10 minutes, and resuspended in fresh THB medium/fresh THB medium + 5% fetal bovine serum containing 6 mM HD-DAP(N ₃ )·HCL and continued to be cultured for 80 minutes. Then, the cells were collected by centrifugation at 6000 g for 10 minutes, and the cells were washed twice with PBS buffer to obtain azide-modified Streptococcus pneumoniae S.pn-N ₃ ;

2) Preparation of Fe ₃ O ₄ nanoparticles modified with alkynyl groups Fe ₃ O ₄ -DBCO

40 mg of DSPE-PEG2000-DBCO was dissolved in 8 mL of chloroform, ultrasonicated for 10 min, and then transferred to a dry glass round-bottom flask. Subsequently, 20 mg of oleic acid-coated Fe ₃ O ₄ nanoparticles were dissolved in 4 mL of chloroform. The Fe ₃ O ₄ solution was added to the DSPE-PEG2000-DBCO solution, and ultrasonically mixed for 10 min. The glass round-bottom flask was rotary evaporated at 55 °C until the solvent was completely evaporated, and 20 mL of ultrapure water was added and ultrasonicated for 30 min to obtain Fe ₃ O ₄ -DBCO nanoparticles.

3) Preparation of a biological hybrid free radical generator Fe ₃ O ₄ @S.pn based on Streptococcus pneumoniae

The S.pn-N ₃ cells prepared in step 1) were resuspended in 12 mL of 200 μg/mL Fe ₃ O ₄ -DBCO preparation system prepared in step 2) and incubated for 5 h. Subsequently, 200 μL of the bio-orthogonal bacterial solution was taken, centrifuged at 4000 rpm, 4°C for 5 min, the supernatant was discarded, the solution was washed twice with PBS and resuspended in 12 mL of PBS to obtain a biohybrid free radical generator based on Streptococcus pneumoniae.

The preparation method of the present invention is simple, and utilizes the hypoxic tropism of Streptococcus pneumoniae and the immunosuppressive microenvironment of tumor tissue to selectively colonize Streptococcus pneumoniae modified with azide groups in tumor tissue. At the same time, through the in-situ bioorthogonality of the alkyne groups on the surface of Fe ₃ O ₄ -DBCO and the azide tumor on the surface of Streptococcus pneumoniae, Fe ₃ O ₄ in-situ catalyzes the conversion of a large amount of H ₂ O ₂ produced by S.pn into highly active ·OH, significantly improving the anti-tumor therapeutic effect. After repeated tests, consistent results were obtained. The relevant test data are as follows:

1. Characterization of the Biohybrid Free Radical Generator Based on Streptococcus pneumoniae

1. Investigation of the ability of Streptococcus pneumoniae to produce hydrogen peroxide

After overnight culture of Streptococcus pneumoniae for 9 hours, the culture was expanded 10 times with freshly prepared THB medium (+5% fetal bovine serum). After culture for 4 hours, the cells were collected by centrifugation at 6000g for 5 minutes. The cells were divided into two parts, one part was resuspended in fresh THB medium (+5% fetal bovine serum) containing 4mM HD-DAP(N ₃ )·HCL and cultured for 60 minutes, and the other part was resuspended in fresh THB medium (+5% fetal bovine serum). The cells were collected by centrifugation at 6000g for 5 minutes, and the cells were washed twice with PBS buffer. The culture was expanded 10 times with Optimzm medium for 9 hours, and a 3mg/mL ABTS and 0.2mg/mL HRP mixture was prepared. In a 96-well plate, 180μL of bacterial solution was first added, and then 20μL of the mixture was added. After incubation at room temperature in the dark for 20 minutes, the absorbance value was detected at 415nm. The results are shown in Figure 1.

According to the results, it can be seen that the concentration of H ₂ O ₂ produced by Streptococcus pneumoniae reaches 1.2 mM, and azide modification does not affect its H ₂ O ₂ production ability.

2. Investigate the effect of different oxygen partial pressures on the ability of Streptococcus pneumoniae to produce hydrogen peroxide

Pick a single colony of Streptococcus pneumoniae, culture it overnight for 9 hours, expand it 10 times with freshly prepared THB medium (+5% fetal bovine serum), culture it for 4 hours, and collect the cells by centrifugation at 6000g for 5 minutes. Then divide it into two parts, expand it 10 times with Optimzm medium for 9 hours, one of the culture medium is placed in a culture bag together with a microaerobic gas production bag (Mitsubishi, Japan) and placed in an incubator to induce anoxic environment, and the other culture medium is cultured in a normoxic environment. Prepare a mixture of 3mg/mL ABTS and 0.2mg/mL HRP, add 180μL of bacterial solution to a 96-well plate, then add 20μL of the mixture, incubate it at room temperature in the dark for 20 minutes, and detect the absorbance value at 415nm.

As shown in Figure 2, there was no statistical difference in the concentration of H ₂ O ₂ produced by S. pneumoniae under normoxic and anaerobic conditions, indicating that the H ₂ O ₂ production capacity of S. pneumoniae is not limited by hypoxia.

3. Investigate the distribution behavior of Streptococcus pneumoniae after intravenous injection

Pick a monoclonal colony of Streptococcus pneumoniae and place it in 3mL of THB medium containing 5% fetal bovine serum, place it in a 37℃ incubator, and culture it anaerobically overnight. Take the bacterial solution and expand it to a bacterial concentration of 1×10 ⁸ CFU/mL, then inject 100μL into each mouse through the tail vein. After the injection, return the mice to the feeding room for feeding, and detect the weight changes of the mice every day; collect the spleen, liver, kidney, heart, lung, and solid tumor of the tumor-bearing mice on the 1st, 3rd, and 7th day after the injection of S.pn, weigh the weight of each tissue and keep a record; add steel beads for homogenization to the centrifuge tubes of each tissue and organ, and break the tissue by a tissue homogenizer; dilute each tissue homogenate in multiple ratios; take an equal volume of each diluted tissue homogenate onto the TSA blood plate, place it in a 37℃ incubator, and culture it anaerobically overnight. Take out the TSA blood plate and count the number of monoclonal colonies on each plate.

As shown in Figure 3, compared with other tissues, Streptococcus pneumoniae is most distributed in tumor tissues and can reside for more than 7 days.

4. Experiment on the generation of hydroxyl radicals by Fe ₃ O ₄ catalyzing H ₂ O ₂

Electron spin resonance (ESR) technology was used to detect the ability of Fe ₃ O ₄ to catalyze H ₂ O ₂ to generate ·OH, and BrukerEMX ESR spectrum was used for detection at room temperature. 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was used as a spin trap to detect ROS. 20μL DMPO was added to the Fe ₃ O ₄ solution (1.3mM), followed by 20μL H ₂ O ₂ (10M) for immediate detection. H ₂ O _2, Fe ₃ O ₄ +H ₂ O ₂ and Fe ₃ O ₄ -DBCO+H ₂ O ₂ were used as controls. The ESR measurement parameters were set as follows: microwave power 21.8mW, scanning range 150G, field modulation 1G. The results are shown in Figure 4.

The characteristic absorption peaks of 1:2:2:1 for the production of ·OH from H ₂ O ₂ catalyzed by Fe ₃ O ₄ were found, and DBCO modification did not affect the ability of Fe ₃ O ₄ to catalyze the production of ·OH from H ₂ O ₂ .

5. Transmission Electron Microscopy Characterization of Streptococcus Pneumoniae and Biohybrid Free Radical Generators

S.pn and Fe ₃ O ₄ @S.pn biohybrid free radical generator were dissolved in ultrapure water to prepare 0.1 mg/mL solutions respectively, and then 10 μL was dropped on the copper mesh. After the liquid evaporated, this operation was repeated 3 times. The morphology was observed by transmission electron microscopy. The results are shown in FIG5 .

The edges of Fe ₃ O ₄ @S.pn were rough, and particles of about 20 nm were visible on the surface of S. pneumoniae, indicating that Fe ₃ O ₄ was successfully modified on the surface of S.pn by bioorthogonality.

6. Characterization of _{Fe 3} O ₄ @S.pn Biohybrid Free Radical Generator by Laser Confocal Imaging

S.pn was cultured for 9 hours, expanded 10 times with freshly prepared THB medium (+5% fetal bovine serum), cultured for 4 hours, collected by centrifugation at 6000g for 5 minutes, and resuspended in fresh THB medium (+5% fetal bovine serum) containing 4mM HD-DAP(N ₃ )·HCL for 60 minutes, then collected by centrifugation at 6000g for 5 minutes, washed twice with PBS buffer, and resuspended in 3ml 250μg/mL Fe ₃ O ₄ -DBCO-RHB system for 3 hours. 200μL of bio-orthogonal bacterial solution was taken, centrifuged at 4000rpm, 4℃, 3min, the supernatant was discarded, washed once with PBS, resuspended in 180μL PBS, and then 5μL 3.34mM STYO-9 staining solution was added, incubated at 37℃ for 15min, protected from light, and finally imaged by laser confocal microscopy to observe the fluorescence co-localization. The results are shown in Figure 6.

The results showed that S.pn-N ₃ modified by STYO-9 and Fe ₃ O ₄ -DBCO modified by RHB had obvious fluorescence co-localization, indicating that the two were successfully bio-orthogonal.

7. Investigation of continuous generation of hydroxyl radicals by a biological hybrid free radical generator

At 1, 3, 6, 9, 12, and 24 h, 100 μL of equal amounts of non-bioorthogonal S.pn+Fe ₃ O ₄ -DBCO and bioorthogonal hybrid Fe ₃ O ₄ @S.pn were taken and resuspended in PBS (the final concentration of Fe ₃ O ₄ was 1 mg/mL), 40 μL of 10 mM 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was added to a 96-well plate, and ultrapure water was added to 200 μL. The mixture was mixed with a pipette, incubated at room temperature in the dark for 20 min, and detected at 415 nm. The results are shown in Figure 7.

The results showed that the biohybrid free radical generator continuously produced a large amount of free radicals to oxidize ABTS, and its oxidation capacity was stronger during the logarithmic growth phase, and then tended to be flat. Compared with the non-bioorthogonal group, the oxidation capacity of the biohybrid free radical generator was about 3 times that of the non-bioorthogonal group.

8. Investigation of the cytotoxicity of the biohybrid free radical generator to 4T1 cells

4T1 cells were seeded into 6-well plates (3×10 ⁵ /well) and the original culture medium was discarded after culturing for 12 hours. Fresh culture medium containing equal concentrations of S.pn, Fe ₃ O ₄ and Fe ₃ O ₄ @S.pn was added to each well. After each preparation was incubated with cells for 4 hours, the drug-containing culture medium was discarded and the cells were washed three times with PBS. After the cells were placed in the incubator and cultured for another 12 hours, the cells were digested with EDTA-free trypsin and collected by centrifugation. Finally, the cells were dispersed in 0.8 mL of buffer, 8 μL of Annexin V-FITC and 8 μL of PI were added in sequence, and the cells were incubated at room temperature for 10 minutes before detection by flow cytometry, and the data were analyzed using FlowJO V10.

As shown in Figure 8 , the apoptosis rate of 4T1 cells induced by the bio-orthogonal group was doubled compared with that of untreated cells, indicating that the bio-hybrid free radical generator can efficiently induce apoptosis of 4T1 cells.

It can be seen from the above experiments that the present invention has the following beneficial effects compared with the prior art:

(1) The biohybrid free radical generator based on Streptococcus pneumoniae provided by the present invention has good tumor selectivity, can colonize in the tumor site for a long time and continuously produce highly active hydroxyl free radicals, thereby improving the efficiency of anti-tumor treatment;

(2) The in situ generated biological hybrid free radical generator based on Streptococcus pneumoniae provided by the present invention is not limited by hypoxia, breaking through the limitation of traditional free radical therapy on oxygen.

2. Conclusion

The present invention _utilizes the click chemistry reaction between azide and alkyne groups, firstly performs azidoglycosylation modification on the surface of Streptococcus pneumoniae, and then adds DBCO-modified _Fe3O4 with POD enzyme activity for combination, so as to form a biohybrid system based on Streptococcus pneumoniae. The prepared tumor in situ biohybrid free radical generator has the advantages of high selectivity, continuous and high efficiency, etc. In terms of tumor treatment, it can break through the limitation of tumor hypoxic microenvironment and continuously and efficiently generate ·OH in situ of tumor, thereby improving the tumor treatment effect. It is an innovation in tumor treatment drugs, and has huge economic and social benefits.

Claims (9)

1. A method for preparing a biological hybrid free radical generator based on Streptococcus pneumoniae, characterized in that, firstly, the surfaces of S.pn and Fe ₃ O ₄ are modified with azide groups N ₃ and DBCO, respectively, and a click chemical reaction occurs between N ₃ and the alkynyl group of DBCO to obtain a biological hybrid free radical generator, specifically comprising the following steps: 1) Preparation of azide-modified Streptococcus pneumoniae S.pn-N ₃ S.pn was cultured for 8-10 hours, and the culture was expanded 10 times with freshly prepared THB medium + 5% fetal bovine serum for 3-5 hours. After the culture was completed, the cells were collected by centrifugation at 6000g for 5-10 minutes, and resuspended in fresh THB medium containing 4-6mM HD-DAP (N ₃ )·HCL or fresh THB medium + 5% fetal bovine serum for further culturing for 60-80 minutes, and then the cells were collected by centrifugation at 6000g for 5-10 minutes, and the cells were washed twice with PBS buffer to obtain azide-modified Streptococcus pneumoniae S.pn-N ₃ ; 2) Preparation of alkynyl-modified Fe ₃ O ₄ nanoparticles Fe ₃ O ₄ -DBCO 10-40 mg DSPE-PEG2000-DBCO was dissolved in 2-8 mL chloroform, ultrasonicated for 5-10 min, and then transferred to a dry glass round-bottom flask; subsequently, 5-20 mg oleic acid-coated Fe ₃ O ₄ nanoparticles were dissolved in 1-4 mL chloroform, the Fe ₃ O ₄ solution was added to the DSPE-PEG2000-DBCO solution, ultrasonically mixed for 5-10 min, the glass round-bottom flask was rotary evaporated at 55°C until the solvent was completely evaporated, 5-20 mL ultrapure water was added and ultrasonicated for 20-30 min to obtain Fe ₃ O ₄ -DBCO nanoparticles; 3) Preparation of a biological hybrid free radical generator Fe ₃ O ₄ @S.pn based on Streptococcus pneumoniae The S.pn-N ₃ cells prepared in step 1) were resuspended in 3-12 mL of 50-200 μg/mL Fe ₃ O ₄ -DBCO preparation system prepared in step 2) and incubated for 3-5 h. Subsequently, 200 μL of the bio-orthogonal bacterial solution was taken, centrifuged at 4000 rpm, 4°C for 3-5 min, the supernatant was discarded, the solution was washed twice with PBS and resuspended in 3-12 mL PBS to obtain a biohybrid free radical generator based on Streptococcus pneumoniae. 2. The method for preparing a biological hybrid free radical generator based on Streptococcus pneumoniae according to claim 1, characterized in that it comprises the following steps: 1) Preparation of azide-modified Streptococcus pneumoniae S.pn-N ₃ S.pn was cultured for 8 hours, and then expanded 10-fold with freshly prepared THB medium + 5% fetal bovine serum for 3 hours. After the culture was completed, the cells were collected by centrifugation at 6000g for 5 minutes, and resuspended in fresh THB medium containing 4mM HD-DAP (N ₃ )·HCL or fresh THB medium + 5% fetal bovine serum for further 60 minutes. The cells were then collected by centrifugation at 6000g for 5 minutes, and the cells were washed twice with PBS buffer to obtain azide-modified Streptococcus pneumoniae S.pn-N ₃ ; 2) Preparation of alkynyl-modified Fe ₃ O ₄ nanoparticles Fe ₃ O ₄ -DBCO 10 mg of DSPE-PEG2000-DBCO was dissolved in 2 mL of chloroform, ultrasonicated for 5 min, and then transferred to a dry glass round-bottom flask; subsequently, 5 mg of oleic acid-coated Fe ₃ O ₄ nanoparticles were dissolved in 1 mL of chloroform, the Fe ₃ O ₄ solution was added to the DSPE-PEG2000-DBCO solution, ultrasonically mixed for 5 min, and the glass round-bottom flask was rotary evaporated at 55 °C until the solvent was completely evaporated, and 5 mL of ultrapure water was added and ultrasonicated for 20 min to obtain Fe ₃ O ₄ -DBCO nanoparticles; 3) Preparation of a biological hybrid free radical generator Fe ₃ O ₄ @S.pn based on Streptococcus pneumoniae The S.pn-N ₃ cells prepared in step 1) were resuspended in 3 mL of 50 μg/mL Fe ₃ O ₄ -DBCO preparation system prepared in step 2) and incubated for 3 h. Subsequently, 200 μL of the bio-orthogonal bacterial solution was taken, centrifuged at 4000 rpm, 4°C for 3 min, the supernatant was discarded, the solution was washed twice with PBS and resuspended in 3 mL of PBS to obtain a biohybrid free radical generator based on Streptococcus pneumoniae. 3. The method for preparing the biohybrid free radical generator based on Streptococcus pneumoniae according to claim 1, characterized in that it comprises the following steps: 1) Preparation of azide-modified Streptococcus pneumoniae S.pn-N ₃ S.pn was cultured for 9 h, and then expanded 10-fold with freshly prepared THB medium + 5% fetal bovine serum for 4 h. After the culture was completed, the cells were collected by centrifugation at 6000 g for 7 min, and resuspended in fresh THB medium containing 5 mM HD-DAP (N ₃ )·HCL or fresh THB medium + 5% fetal bovine serum and continued to be cultured for 70 min. Then, the cells were collected by centrifugation at 6000 g for 7 min, and the cells were washed twice with PBS buffer to obtain azide-modified Streptococcus pneumoniae S.pn-N ₃ ; 2) Preparation of alkynyl-modified Fe ₃ O ₄ nanoparticles Fe ₃ O ₄ -DBCO 20 mg of DSPE-PEG2000-DBCO was dissolved in 4 mL of chloroform, ultrasonicated for 7 min, and then transferred to a dry glass round-bottom flask; subsequently, 10 mg of oleic acid-coated Fe ₃ O ₄ nanoparticles were dissolved in 2 mL of chloroform, the Fe ₃ O ₄ solution was added to the DSPE-PEG2000-DBCO solution, ultrasonically mixed for 7 min, the glass round-bottom flask was rotary evaporated at 55 °C until the solvent was completely evaporated, 10 mL of ultrapure water was added and ultrasonicated for 25 min to obtain Fe ₃ O ₄ -DBCO nanoparticles; 3) Preparation of a biological hybrid free radical generator Fe ₃ O ₄ @S.pn based on Streptococcus pneumoniae The S.pn-N ₃ cells prepared in step 1) were resuspended in 6 mL of 100 μg/mL Fe ₃ O ₄ -DBCO preparation system prepared in step 2) and incubated for 4 h. Subsequently, 200 μL of the bio-orthogonal bacterial solution was taken, centrifuged at 4000 rpm, 4°C for 4 min, the supernatant was discarded, the solution was washed twice with PBS and resuspended in 6 mL of PBS to obtain a biohybrid free radical generator based on Streptococcus pneumoniae. 4. The method for preparing a biological hybrid free radical generator based on Streptococcus pneumoniae according to claim 1, characterized in that it comprises the following steps: 1) Preparation of azide-modified Streptococcus pneumoniae S.pn-N ₃ S.pn was cultured for 10 hours, and the cells were expanded 10-fold with freshly prepared THB medium + 5% fetal bovine serum for 5 hours. After the culture was completed, the cells were collected by centrifugation at 6000 g for 10 minutes, and resuspended in fresh THB medium containing 6 mM HD-DAP (N ₃ )·HCL or fresh THB medium + 5% fetal bovine serum and continued to be cultured for 80 minutes. Then, the cells were collected by centrifugation at 6000 g for 10 minutes, and the cells were washed twice with PBS buffer to obtain azide-modified Streptococcus pneumoniae S.pn-N ₃ ; 2) Preparation of alkynyl-modified Fe ₃ O ₄ nanoparticles Fe ₃ O ₄ -DBCO 40 mg of DSPE-PEG2000-DBCO was dissolved in 8 mL of chloroform, ultrasonicated for 10 min, and then transferred to a dry glass round-bottom flask. Subsequently, 20 mg of oleic acid-coated Fe ₃ O ₄ nanoparticles were dissolved in 4 mL of chloroform. The Fe ₃ O ₄ solution was added to the DSPE-PEG2000-DBCO solution, and ultrasonically mixed for 10 min. The glass round-bottom flask was rotary evaporated at 55 °C until the solvent was completely evaporated, and 20 mL of ultrapure water was added and ultrasonicated for 30 min to obtain Fe ₃ O ₄ -DBCO nanoparticles. 3) Preparation of a biological hybrid free radical generator Fe ₃ O ₄ @S.pn based on Streptococcus pneumoniae The S.pn-N ₃ cells prepared in step 1) were resuspended in 12 mL of 200 μg/mL Fe ₃ O ₄ -DBCO preparation system prepared in step 2) and incubated for 5 h. Subsequently, 200 μL of the bio-orthogonal bacterial solution was taken, centrifuged at 4000 rpm, 4°C for 5 min, the supernatant was discarded, the solution was washed twice with PBS and resuspended in 12 mL of PBS to obtain a biohybrid free radical generator based on Streptococcus pneumoniae. 5. The method for preparing a biological hybrid free radical generator based on Streptococcus pneumoniae according to claim 1, characterized in that the particle size of S.pn-N ₃ in step 1) is 0.5-1 μm, the particle size of Fe ₃ O ₄ -DBCO nanoparticles in step 2) is 10-30 nm, and the particle size of Fe ₃ O ₄ @S.pn in step 3) is 0.5-1 μm. 6. The method for preparing a biological hybrid free radical generator based on Streptococcus pneumoniae according to claim 1, characterized in that the THB medium formula is: beef powder 10g/L, tryptone 20g/L, glucose 2g/L , sodium bicarbonate 2g/L, sodium chloride 2g/L, disodium hydrogen phosphate 0.4g/L, pH 7.8±0.1, 25°C. 7. Use of the biohybrid free radical generator based on Streptococcus pneumoniae according to any one of claims 1 to 4 in the preparation of anti-tumor drug injections. 8. Use of the biohybrid free radical generator based on Streptococcus pneumoniae according to any one of claims 1 to 4 in the preparation of drugs for treating hypoxic tumors. 9. Use of the biohybrid free radical generator based on Streptococcus pneumoniae according to any one of claims 1 to 4 in the preparation of a drug that utilizes the natural bacterial infection pathway to induce tumor cell death.