Engineering living body photo-thermal hydrogel as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, and discloses engineering living body photo-thermal hydrogel as well as a preparation method and application thereof. The engineering living body photo-thermal hydrogel comprises a sodium alginate hydrogel matrix loaded with polypyrrole nanoparticles, and gene engineering modified escherichia coli entrapped in the sodium alginate hydrogel matrix, wherein the gene engineering modified escherichia coli expresses lactate oxidase or glucose oxidase. According to the engineered living body photo-thermal hydrogel provided by the invention, temperature-controlled engineered escherichia coli expressing GOX / LOX is combined with the photo-thermal hydrogel, a triple synergistic anti-tumor effect is triggered through 1064 nm laser, tumors are directly killed through photo-thermal heating, glucose / lactic acid is consumed in real time through GOX / LOX, energy metabolism is blocked, and a pentose phosphate pathway is inhibited; immunogenic cells are induced to die through a metabolite ROS, and whole-body anti-tumor immunity is activated.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an engineered in vivo photothermal hydrogel, its preparation method, and its application. Background Technology

[0002] Photothermal hydrogels are innovative biomedical materials combining photothermal therapy and hydrogel technology. Photothermal therapy utilizes the localized high temperatures generated by a photothermal agent absorbing near-infrared laser light to kill tumor cells. Within the temperature range of 40-50°C, the high temperature disrupts cell membranes, inducing irreversible cell necrosis with minimal damage to normal tissues. Near-infrared lasers, due to their strong tissue penetration and low absorption characteristics, are ideal light sources for initiating photothermal therapy. Hydrogels, as cross-linked polymers with a three-dimensional network structure, possess high water absorption and biocompatibility, mimicking the natural extracellular matrix to support cell migration, tissue regeneration, and wound healing. Combining photothermal therapy with hydrogels to form photothermal hydrogels offers new possibilities for tumor treatment; this material retains the excellent biocompatibility and drug release capabilities of hydrogels while possessing the precision and efficiency of photothermal therapy.

[0003] In synergistic treatment strategies, photothermal gels can regulate drug release through the photothermal effect, precisely targeting tumor sites, improving treatment efficiency, and reducing toxic side effects. However, traditional photothermal therapy has poor targeting, traditional photosensitizers are prone to off-target effects leading to toxic side effects, and photothermal therapy alone cannot regulate the tumor microenvironment, easily inducing drug resistance and recurrence. It also lacks intervention methods for tumor cell energy metabolism (such as glucose and lactate). Summary of the Invention

[0004] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, one objective of the present invention is to provide an engineered in vivo photothermal hydrogel.

[0005] The second objective of this invention is to provide a method for preparing the engineered living photothermal hydrogel.

[0006] The third objective of this invention is to provide applications of the engineered living photothermal hydrogel.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A first aspect of the present invention provides an engineered in vivo photothermal gel, comprising:

[0009] A sodium alginate hydrogel matrix loaded with polypyrrole nanoparticles, and genetically engineered Escherichia coli encapsulated therein; wherein the genetically engineered Escherichia coli expresses lactate oxidase (GOX) or glucose oxidase (LOX).

[0010] In some embodiments of the present invention, the engineered live photothermal hydrogel contains both genetically engineered Escherichia coli expressing lactate oxidase and genetically engineered Escherichia coli expressing glucose oxidase.

[0011] In some embodiments of the present invention, the sodium alginate hydrogel matrix loaded with polypyrrole nanoparticles has a porous network structure with a pore size of less than 10 μm.

[0012] In some preferred embodiments of the present invention, the sodium alginate hydrogel matrix loaded with polypyrrole nanoparticles has a porous network structure with a pore size of 0.1-5 μm.

[0013] In some embodiments of the present invention, the loading amount of polypyrrole nanoparticles in the sodium alginate hydrogel matrix loaded with polypyrrole nanoparticles is 0.5wt%-5wt%.

[0014] In some embodiments of the present invention, the polypyrrole nanoparticles are prepared by a method comprising the following steps:

[0015] Potassium permanganate (KMnO4) was dissolved in water, and pyrrole monomer was added to obtain polypyrrole nanoparticles. The product was collected, washed with pure water and ethanol, and then freeze-dried for storage.

[0016] In some embodiments of the present invention, the loading capacity of the genetically engineered Escherichia coli is 10. 6 -10 10 CFU / mL.

[0017] In some embodiments of the present invention, the genetically engineered Escherichia coli is prepared by a method comprising the following steps:

[0018] The plasmid encoding lactate oxidase or glucose oxidase was transformed into Escherichia coli, followed by resistance screening and single-clone preservation to obtain the genetically engineered Escherichia coli.

[0019] In some embodiments of the present invention, the preparation of genetically engineered Escherichia coli includes plasmid transformation using a heat shock method.

[0020] In some embodiments of the present invention, the preparation of genetically engineered Escherichia coli includes resistance screening using at least one of ampicillin, carbenicillin, kanamycin, and chloramphenicol.

[0021] In some embodiments of the present invention, the preparation of genetically engineered Escherichia coli specifically includes the following steps:

[0022] After inoculating *E. coli* in LB liquid medium and culturing overnight, the cells were transferred to fresh LB medium and cultured to the logarithmic growth phase. The cells were collected by centrifugation, washed with pre-cooled CaCl2 solution, and resuspended to obtain competent cells. Plasmids encoding lactate oxidase or glucose oxidase were added to the competent cells, and the cells were heat-shocked at 42°C after an ice bath at 4°C. The resulting bacterial culture was then added to LB medium for recovery and culture. The cells were collected by centrifugation, spread on LB plates containing ampicillin, and cultured overnight. Single colonies were then picked for preservation to obtain genetically engineered *E. coli*.

[0023] A second aspect of the present invention provides a method for preparing the engineered living photothermal hydrogel described in the first aspect of the present invention, comprising the following steps:

[0024] Genetically engineered Escherichia coli was resuspended in sodium alginate solution, and polypyrrole nanoparticles and a crosslinking agent were added to obtain the engineered living photothermal hydrogel.

[0025] In some embodiments of the present invention, the concentration of the sodium alginate solution is 0.5wt%-5wt%.

[0026] In some preferred embodiments of the present invention, the concentration of the sodium alginate solution is 2wt%-4wt%.

[0027] In some embodiments of the present invention, the density of the genetically engineered Escherichia coli is OD. 600 =0.3-1.5.

[0028] In some preferred embodiments of the present invention, the density of the genetically engineered Escherichia coli is OD. 600 =0.5-1.0.

[0029] In some embodiments of the present invention, the amount of the crosslinking agent is 20-50 mmol / L.

[0030] In some preferred embodiments of the present invention, the amount of the crosslinking agent is 20-40 mmol / L.

[0031] In some embodiments of the present invention, the crosslinking agent includes manganese chloride (MnCl2).

[0032] The third aspect of the present invention provides the application of the engineered in vivo photothermal hydrogel described in the first aspect of the present invention in the preparation of tumor therapeutic drugs.

[0033] The basic principles of this invention are explained as follows:

[0034] Engineered bacteria refer to microbial systems that have been selectively modified using modern biotechnology such as gene recombination and metabolic regulation. Their high targeting, multifunctionality, and long-lasting effects make them a promising platform for tumor treatment. When photothermal gels are combined with engineered bacteria therapy, their potential is further amplified. By embedding engineered bacteria into photothermal gels, the activity of the bacterial community and drug release can be precisely controlled using the photothermal effect, while simultaneously improving the tumor microenvironment, activating the immune response, and forming a powerful synergistic therapeutic modality.

[0035] This invention uses a sodium alginate hydrogel matrix to provide a three-dimensional network structure, encapsulating engineered bacteria and a photothermal agent:

[0036] 1) When preparing engineered living photothermal gels using polypyrrole nanoparticles (Ppy NPs) as photothermal agents and MnCl2 as crosslinking agents, the effect of polypyrrole nanoparticles on sodium alginate-MnCl2... 2+ The cross-linked network exhibits minimal interference, and polypyrrole (Ppy), as a conjugated polymer, possesses a π-π* conjugated system in its main chain. The band gap (Eg) between the valence band (HOMO) and conduction band (LUMO) is approximately 1.17 eV, corresponding to a photon energy of 1064 nm (1.16 eV). This means that the 1064 nm photon energy perfectly matches the electronic transition band gap of Ppy, resulting in the highest absorption efficiency. When irradiated with a 1064 nm near-infrared laser, the 1064 nm photon is captured by the π electrons of Ppy. The electron transitions from the HOMO to the LUMO. The excited-state electron releases energy through internal conversion (electron relaxation from a high vibrational energy level to a low energy level) and electron-phonon coupling (electron energy transfer to molecular lattice vibration), generating localized heat that directly kills tumor cells, achieving photothermal therapy. Furthermore, it triggers the expression of engineered bacterial enzymes through thermal triggering.

[0037] 2) Genetically engineered E. coli expressing lactate oxidase or glucose oxidase can be temperature-controlled to express lactate oxidase / glucose oxidase. First, lactate oxidase can degrade lactate and relieve immunosuppression, while glucose oxidase consumes glucose and cuts off the energy supply, thereby achieving the purpose of metabolic intervention. The catalysis of glucose and lactate can also affect the pentose phosphate pathway of tumor cells, reducing the supply of substances required for tumor cell proliferation. In addition, genetically engineered E. coli expressing lactate oxidase or glucose oxidase can increase the level of ROS at the cellular level and induce immunogenic cell death (ICD) through ROS, thereby enhancing the anti-tumor immune response.

[0038] 3) Encapsulating genetically engineered Escherichia coli in a sodium alginate hydrogel matrix loaded with polypyrrole nanoparticles can limit bacterial spread through the hydrogel and reduce off-target toxicity through the engineered bacteria's natural tumor colonization ability.

[0039] Compared with the prior art, the beneficial effects of the present invention are:

[0040] The engineered in vivo photothermal hydrogel provided by this invention combines temperature-controlled engineered E. coli expressing lactate oxidase or glucose oxidase with the photothermal hydrogel. Triggered by a 1064 nm laser, it exhibits a triple synergistic anti-tumor effect: directly killing tumors through photothermal heating; consuming glucose / lactic acid in real time via GOX / LOX, blocking energy metabolism and inhibiting the pentose phosphate pathway; and inducing immunogenic cell death through the metabolite ROS, thus activating systemic anti-tumor immunity. The hydrogel encapsulates photosensitizers, restricting their movement and enabling precise delivery to tumor tissue via injection. Furthermore, the engineered bacteria used possess natural tumor-targeting capabilities, sensing and colonizing tumors, reducing off-target rates and toxic side effects. This overcomes the bottlenecks of drug resistance and off-target toxicity in traditional therapies and demonstrates good biosafety. Attached Figure Description

[0041] Figure 1 The protein expression of engineered E. coli in Examples 1 and 2;

[0042] Figure 2 Here is a SEM image of the photothermal hydrogel in Comparative Example 1;

[0043] Figure 3 Here is a SEM image of the engineered in vivo photothermal hydrogel in Example 3;

[0044] Figure 4 The results are for the cytotoxicity test of engineered in vivo photothermal hydrogels;

[0045] Figure 5 Characterization results of ROS enhancement at the cellular level in tumor cells induced by engineered in vivo photothermal hydrogel;

[0046] Figure 6 Characterization results of ICD induced in tumor cells by engineered in vivo photothermal hydrogel;

[0047] Figure 7 To reduce the concentration of tumor cell-related substrates through the catalytic action of engineered in vivo photothermal hydrogels;

[0048] Figure 8 To pass NADP + The change in the / NADPH ratio characterizes the level of inhibition of the pentose phosphate pathway in tumor cells by engineered in vivo photothermal hydrogels;

[0049] Figure 9 Results of in vivo tumor treatment using engineered in vivo photothermal hydrogels;

[0050] Figure 10 The change in body weight of mice in each group during the treatment process. Detailed Implementation

[0051] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0052] Example 1

[0053] This embodiment prepares an engineered living photothermal hydrogel, and the steps are as follows:

[0054] S1. *E. coli* (BL21(DE3)) was inoculated into LB liquid medium and cultured overnight. The culture was then transferred to fresh LB medium and cultured until the logarithmic growth phase. The cells were collected by centrifugation, washed once with pre-cooled CaCl2 solution, and resuspended to obtain competent cells. A plasmid encoding lactate oxidase was added to the competent cells, and the cells were incubated at 4°C for 30 min on ice followed by heat shock at 42°C for 90 s to allow the bacterial cells to absorb the plasmid. The resulting bacterial culture was added to LB medium and incubated at 37°C for 1 h. The cells were collected by centrifugation, plated on LB agar plates containing ampicillin, and cultured overnight at 37°C. Single colonies were then picked for preservation to obtain engineered *E. coli* expressing lactate oxidase. These cells were then incubated at 37°C in a constant temperature environment, and their OD value was measured. 600 Value, when growing to OD 600 When the concentration is approximately 0.8, it is used for the preparation of engineered living photothermal hydrogels;

[0055] S2. The engineered E. coli expressing lactate oxidase was resuspended in sodium alginate solution, polypyrrole nanoparticles were added and mixed thoroughly, then MnCl2 was added and mixed thoroughly to obtain an engineered live photothermal gel, denoted as LE, wherein the loading capacity of the engineered E. coli expressing lactate oxidase was 10. 8 The concentration of the sodium alginate solution was 3 wt%, the loading of polypyrrole nanoparticles in the sodium alginate hydrogel matrix was 2 wt%, and the amount of MnCl2 used was 30 mmol / L.

[0056] Example 2

[0057] This embodiment prepares an engineered living photothermal hydrogel, and the steps are as follows:

[0058] S1. *E. coli* (BL21(DE3)) was inoculated into LB liquid medium and cultured overnight. The culture was then transferred to fresh LB medium and cultured until the logarithmic growth phase. The cells were collected by centrifugation, washed once with pre-cooled CaCl2 solution, and resuspended to obtain competent cells. A plasmid encoding glucose oxidase was added to the competent cells, and the cells were incubated at 4°C for 30 min on ice followed by heat shock at 42°C for 90 s to allow the bacterial cells to absorb the plasmid. The resulting bacterial culture was added to LB medium and incubated at 37°C for 1 h. The cells were collected by centrifugation, plated on LB agar plates containing ampicillin, and cultured overnight at 37°C. Single colonies were then picked for preservation to obtain engineered *E. coli* expressing glucose oxidase. These were then incubated at 37°C in a culture medium, and their OD value was measured. 600 Value, when growing to OD 600 When the concentration is approximately 0.8, it is used for the preparation of engineered living photothermal hydrogels;

[0059] S2. The engineered E. coli expressing glucose oxidase was resuspended in sodium alginate solution, polypyrrole nanoparticles were added and mixed thoroughly, then MnCl2 was added and mixed thoroughly to obtain an engineered live photothermal gel, denoted as GE, in which the loading capacity of the engineered E. coli expressing glucose oxidase was 10. 8 The concentration of the sodium alginate solution was 3 wt%, the loading of polypyrrole nanoparticles in the sodium alginate hydrogel matrix was 2 wt%, and the amount of MnCl2 used was 30 mmol / L.

[0060] Example 3

[0061] This embodiment prepares an engineered living photothermal hydrogel, and the steps are as follows:

[0062] Engineered *E. coli* expressing lactate oxidase were prepared according to the method in Example 1, and engineered *E. coli* expressing glucose oxidase were prepared according to the method in Example 2. Both engineered *E. coli* strains were resuspended in sodium alginate solution, polypyrrole nanoparticles were added and mixed thoroughly, and then MnCl2 was added and mixed completely to obtain an engineered live photothermal hydrogel, denoted as LGE. The total loading capacity of the two engineered *E. coli* strains was 10. 8 The concentration of CFU / mL was 1:1, the concentration of sodium alginate solution was 3wt%, the loading of polypyrrole nanoparticles in sodium alginate hydrogel matrix was 2wt%, and the amount of MnCl2 used was 30 mmol / L.

[0063] Comparative Example 1

[0064] In this comparative example, a photothermal gel was prepared using the following steps:

[0065] Polypyrrole nanoparticles were added to sodium alginate solution and mixed evenly. Then MnCl2 was added and mixed thoroughly to obtain photothermal hydrogel, denoted as pH.

[0066] Comparative Example 2

[0067] In this comparative example, a photothermal gel encapsulating bacteria was prepared using the following steps:

[0068] Escherichia coli (E. coli BL21(DE3)) was inoculated into LB liquid medium and cultured overnight, then transferred to fresh LB medium and cultured until OD. 600 ≈0.8, resuspended in sodium alginate solution, polypyrrole nanoparticles were added and mixed evenly, then MnCl2 was added and mixed thoroughly to obtain a photothermal hydrogel encapsulating bacteria, denoted as BPH, in which the total encapsulation capacity of Escherichia coli was 10. 8 The concentration of the sodium alginate solution was 3 wt%, the loading of polypyrrole nanoparticles in the sodium alginate hydrogel matrix was 2 wt%, and the amount of MnCl2 used was 30 mmol / L.

[0069] Characterization and performance testing

[0070] 1. Protein expression assay of engineered E. coli:

[0071] The protein expression of the engineered E. coli in Examples 1 and 2 was verified by Western blotting. Figure 1 The figures show the protein expression of engineered E. coli in Examples 1 and 2, where... Figure 1 The left-hand image shows the protein expression of engineered E. coli expressing lactate oxidase (LOX) in Example 1. Figure 1 The right-hand side of the figure shows the protein expression of engineered E. coli expressing glucose oxidase (GOX) in Example 2. Figure 1 It can be seen that after 20 minutes of induction, the bands corresponding to the two enzymes can be clearly seen, proving that the two target proteins, GOX and LOX, were successfully expressed by the corresponding engineered E. coli.

[0072] 2. SEM characterization of the photothermal gel in Comparative Example 1 and the engineered living photothermal gel in Example 3:

[0073] Figure 2 The image shows a SEM image of the photothermal hydrogel in Comparative Example 1. Figure 2 It can be seen that the photothermal hydrogel in Comparative Example 1 exhibits a network porous structure with a pore size of less than 10 μm.

[0074] Figure 3 This is a SEM image of the engineered living photothermal hydrogel from Example 3. Figure 3As can be seen, the engineered live photothermal hydrogel in Example 3 maintained the network porous structure of the photothermal hydrogel with a pore size of less than 10 μm, and the engineered Escherichia coli was encapsulated in the network structure, indicating that the engineered live photothermal hydrogel was successfully prepared.

[0075] 3. Cytotoxicity test:

[0076] Tumor cells were seeded into 96-well plates and cultured overnight. PBS buffer (denoted as Ctrl), engineered in vivo photothermal gels (LE, GE, LGE) prepared in Examples 1-3, photothermal gel (PH) prepared in Comparative Example 1, and bacterial-encapsulated photothermal gel (BPH) prepared in Comparative Example 2 were then added. Irradiation with a 1064 nm laser (0.5 w / cm²) was applied. 2 In two cases, with and without laser irradiation, the samples were cultured for 4 h, then aspirated and washed with PBS, cultured for another 24 h, thiazolyl blue (MTT) solution was added, and the samples were incubated in the dark for 4 h. The supernatant was aspirated and dimethyl sulfoxide (DMSO) was added. The absorbance at 570 nm of each well was measured.

[0077] Figure 4 The cytotoxicity test results for engineered in vivo photothermal hydrogels were obtained from... Figure 4 It can be seen that under laser irradiation, the photothermal gel (PH) prepared in Comparative Example 1 can induce some toxicity through its own photothermal effect; the proportion of tumor cell death caused by encapsulating unengineered Escherichia coli (BPH) is not significantly changed; the proportion of tumor cell death caused by encapsulating genetically engineered Escherichia coli expressing lactate oxidase or glucose oxidase (LE, GE) and irradiating with laser can significantly increase the proportion of tumor cell death; when simultaneously encapsulating Escherichia coli expressing lactate oxidase / glucose oxidase (LGE), the two synergistically lead to the highest level of tumor cell death.

[0078] 4. Validation of in vitro antitumor mechanism

[0079] 1) The ability of PBS buffer (denoted as Ctrl), engineered in vivo photothermal gels (LE, GE, LGE) prepared in Examples 1-3, photothermal gel (PH) prepared in Comparative Example 1, and photothermal gel (BPH) containing bacteria prepared in Comparative Example 2 to induce ROS generation was characterized using the ROS probe DCFH-DA:

[0080] Figure 5 The results characterize the increase in ROS at the cellular level induced by engineered in vivo photothermal gels in tumor cells. Here, "Laser" refers to the irradiation of the corresponding photothermal gel with a 1064 nm laser (0.5 w / cm²). 2 (20 min), by Figure 5It can be seen that when irradiated with a 1064nm laser, the engineered in vivo photothermal gels prepared in Examples 1-3 can all induce an increase in ROS at the cellular level in tumor cells. Among them, the induction effect of the engineered in vivo photothermal gel in Example 3 is significantly better than that in Examples 1 and 2, indicating that the engineered Escherichia coli expressing lactate oxidase or glucose oxidase can be significantly induced to increase ROS levels when the photothermal gel is loaded with both engineered Escherichia coli. The highest level of cellular ROS can be obtained when both engineered Escherichia coli are loaded at the same time.

[0081] 2) The ability of PBS buffer (denoted as Ctrl), engineered in vivo photothermal gels (LE, GE, LGE) prepared in Examples 1-3, photothermal gel (PH) prepared in Comparative Example 1, and photothermal gel (BPH) containing bacteria prepared in Comparative Example 2 to induce ICD in tumor cells was characterized by analyzing immunogenic cell death (ICD) markers. The three markers were high-mobility group box 1 (HMGB1), calreticulin (CRT), and adenosine triphosphate (ATP).

[0082] Figure 6 The characterization results of ICD induced in tumor cells by engineered in vivo photothermal hydrogel are as follows: Figure 6 (a) shows the level characterization results of the biomarker high-mobility group box 1 (HMGB1). Figure 6 (b) shows the results of characterizing the level of the biomarker calreticulin (CRT). Figure 6 (c) shows the level characterization results of the biomarker adenosine triphosphate (ATP). Laser refers to the irradiation of the corresponding photothermal gel with a 1064 nm laser (0.5 w / cm²). 2 (20 min), by Figure 6 It can be seen that the engineered in vivo photothermal gels prepared in Examples 1-3 can all induce an increase in HMGB1, CRT, and ATP levels. Among them, the induction effect of the engineered in vivo photothermal gel in Example 3 is significantly better than that in Examples 1 and 2. ROS can induce ICD in tumor cells. The characterization results of the engineered in vivo photothermal gel inducing ICD in tumor cells are consistent with... Figure 5 The consistency between the medium phase and the medium phase indicates that the engineered in vivo photothermal hydrogel provided by the present invention can enhance the anti-tumor immune response by increasing the ROS level and inducing the ICD effect.

[0083] 3) Characterize lactate and glucose levels at the cellular level, and through NADP... +The / NADPH ratio characterizes the inhibitory levels of the pentose phosphate pathway in tumor cells by PBS buffer (denoted as Ctrl), engineered in vivo photothermal gels (LE, GE, LGE) prepared in Examples 1-3, photothermal gel (PH) prepared in Comparative Example 1, and photothermal gel (BPH) containing bacteria prepared in Comparative Example 2.

[0084] The substrates for the LOX and GOX catalytic reactions are lactate and glucose, respectively, which are important metabolic substrates in the tumor microenvironment. Therefore, the levels of these two substrates were characterized. Figure 7 To reduce the concentration of tumor cell-related substrates through the catalytic action of engineered in vivo photothermal hydrogels, among which, Figure 7 In (a), the catalytic effect of engineered in vivo photothermal hydrogels reduces the concentration of lactic acid. Figure 7 (b) shows the catalytic effect of engineered in vivo photothermal gels in reducing glucose concentration. "Laser" refers to the irradiation of the corresponding photothermal gel with a 1064 nm laser (0.5 W / cm²). 2 (20 min), by Figure 7 It can be seen that the engineered live photothermal gel LGE prepared in Example 3 can minimize the levels of lactic acid and glucose at the cellular level, indicating that the engineered live photothermal gel provided by the present invention can specifically consume lactic acid / glucose and inhibit energy supply.

[0085] The catalysis of glucose and lactate simultaneously affects the pentose phosphate pathway in tumor cells, which provides substances necessary for tumor cell proliferation and is also a major source of reduced nicotinamide adenine dinucleotide phosphate (NADPH). Due to the interaction between NADPH and NADP... + They can be interconverted, therefore, by characterizing NADP + The ratio of EBPH to NADPH was used to study the effect of EBPH on the pentose phosphate pathway in tumor cells. Figure 8 To pass NADP + The change in the NADPH ratio characterizes the inhibitory level of engineered in vivo photothermal gels on the pentose phosphate pathway in tumor cells. Here, "Laser" refers to the irradiation of the corresponding photothermal gel with a 1064 nm laser (0.5 W / cm²). 2 (20 min), by Figure 8 It can be seen that the engineered live photothermal hydrogel LGE prepared in Example 3 can effectively reduce NADP under external laser irradiation. + The ratio of pentose phosphate to NADPH indicates that the pentose phosphate pathway is inhibited.

[0086] 5. In vivo tumor treatment

[0087] Tumor-bearing mice were randomly divided into 7 groups and treated with PBS buffer, engineered in vivo photothermal gels (LE, GE, LGE) prepared in Examples 1-3, photothermal gel (PH) prepared in Comparative Example 1 with / without laser irradiation, and photothermal gel (BPH) samples containing bacteria prepared in Comparative Example 2. Changes in tumor size and body weight were recorded.

[0088] Figure 9 This refers to the in vivo tumor treatment results of engineered in vivo photothermal gels, where "Laser" refers to the irradiation of the corresponding photothermal gel with a 1064 nm laser (0.5 w / cm²). 2 (20 min), 1-PBS buffer, 2-pH, 3-BPH, 4-pH, Laser, 5-LE, Laser, 6-GE, Laser, 7-LGE, Laser, from Figure 9 It is known that photothermal hydrogels can inhibit tumor development to a certain extent after laser irradiation (PH, Laser), and the expression of GOX or LOX further inhibits tumor growth. When both enzymes are expressed simultaneously, tumor regression can be induced.

[0089] Figure 10 The change in body weight of mice in each group during treatment is shown. Laser refers to the treatment administered with a 1064nm laser (0.5 w / cm²) to the corresponding photothermal gel. 2 (20 min), 1-PBS buffer, 2-pH, 3-BPH, 4-pH, Laser, 5-LE, Laser, 6-GE, Laser, 7-LGE, Laser, from Figure 10 It can be seen that the body weight of mice in each group showed a slight upward trend throughout the treatment process, indicating that the engineered in vivo photothermal hydrogel provided by the present invention has good biocompatibility.

Claims

1. An engineered living photothermal gel, characterized in that, include: A sodium alginate hydrogel matrix loaded with polypyrrole nanoparticles, and genetically engineered Escherichia coli encapsulated therein; wherein the genetically engineered Escherichia coli expresses lactate oxidase or glucose oxidase.

2. The engineered living photothermal hydrogel according to claim 1, characterized in that, The sodium alginate hydrogel matrix loaded with polypyrrole nanoparticles has a porous network structure with a pore size of less than 10 μm.

3. The engineered living photothermal hydrogel according to claim 2, characterized in that, In the sodium alginate hydrogel matrix loaded with polypyrrole nanoparticles, the loading amount of polypyrrole nanoparticles is 0.5wt%-5wt%.

4. The engineered living photothermal hydrogel according to claim 1, characterized in that, The genetically engineered E. coli had an encapsulation capacity of 10. 6 -10 10 CFU / mL.

5. The engineered living photothermal hydrogel according to claim 4, characterized in that, The genetically engineered Escherichia coli was prepared by a method comprising the following steps: The plasmid encoding lactate oxidase or glucose oxidase was transformed into Escherichia coli, followed by resistance screening and single-clone preservation to obtain the genetically engineered Escherichia coli.

6. The engineered living photothermal hydrogel according to claim 5, characterized in that, The genetically engineered Escherichia coli was obtained, and the plasmid transformation included the use of heat shock.

7. The method for preparing the engineered living photothermal hydrogel according to any one of claims 1-6, characterized in that, Includes the following steps: Genetically engineered Escherichia coli was resuspended in sodium alginate solution, and polypyrrole nanoparticles and a crosslinking agent were added to obtain the engineered living photothermal hydrogel.

8. The preparation method according to claim 7, characterized in that, The concentration of the sodium alginate solution is 0.5wt%-5wt%; The density of the genetically engineered E. coli was OD. 600 =0.3-1.

5.

9. The preparation method according to claim 7, characterized in that, The amount of the crosslinking agent used is 20-50 mmol / L.

10. The use of the engineered in vivo photothermal hydrogel according to any one of claims 1-6 in the preparation of tumor therapeutic drugs.

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