Combined drug delivery system based on bioreactor and temperature-sensitive hydrogel and application

Abstract

The invention relates to a combined drug delivery system based on a bioreactor and temperature-sensitive hydrogel, and belongs to the technical field of biomedicine. The invention provides a combined drug delivery system based on a bioreactor and temperature-sensitive hydrogel, the bioreactor is genetically engineered bacteria which selectively colonize in tumor tissues and induce intratumor vascular thrombosis, and the temperature-sensitive hydrogel is prepared from a temperature-sensitive material and is loaded with a hypoxia activation prodrug. The effect of the hypoxia activated prodrug is improved by enhancing the hypoxia environment, and the action time of the drug on a target part is prolonged by the temperature-sensitive hydrogel. A luxCDABE gene is expressed in genetically engineered bacteria, a bioluminescence system is constructed, a bioreactor is subjected to autofluorescence in a normal oxygen environment and is subjected to fluorescence weakening in a hypoxic environment, fluorescence signals are detected through an in-vitro imaging system, the best administration time of the temperature-sensitive hydrogel can be judged in an auxiliary mode, meanwhile, the treatment process is visually monitored, and the treatment effect is good. Good clinical application values and prospects are realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a combined drug delivery system based on a bioreactor and a thermosensitive hydrogel and its application. Background Art

[0002] As early as the 19th century, studies have confirmed that bacteria have a therapeutic effect on tumors. After treatment with Streptococcus pyogenes and Serratia marcescens, researchers observed that the patient's tumor had significantly regressed. This discovery opened the door to bacterial tumor treatment. In recent years, with the advancement of synthetic biology, gene editing technology has been used to genetically modify bacteria to prepare engineered bacteria that can synthesize specific functional proteins such as tumor antigens, cytokines, antibodies, and specific enzymes, and use them for the treatment and diagnosis of tumors. Subsequently, researchers discovered that some bacteria can destroy the tumor vascular system and induce thrombosis, thereby cutting off the nutrition and oxygen supply to the tumor. More importantly, based on the unique hypoxia and immunosuppressive characteristics of the tumor microenvironment, both obligate and facultative anaerobes can specifically colonize in tumor tissues, thus giving bacteria tumor targeting capabilities.

[0003] In the clinical treatment of tumors, chemotherapy has been used as one of the main anti-tumor therapies to date because of its good tumor suppression effect. However, due to the lack of tumor specificity, traditional chemotherapy drugs often cause non-selective killing of normal tissue cells while killing tumor cells. Therefore, tumor patients often experience serious toxic side effects after chemotherapy. Tirapazamine (TPZ) is a hypoxia-activated prodrug that can produce oxidative free radicals under hypoxic conditions, leading to biomacromolecule damage and cell death. In contrast, TPZ exhibits minimal cytotoxicity under normoxic conditions. Compared with normal tissues, because the tumor microenvironment is in a hypoxic state, TPZ can exhibit selective killing characteristics for tumor tissues, thereby showing the potential for tumor targeted therapy and having fewer toxic side effects on normal tissues.

[0004] However, the lack of hypoxia in tumors and the fact that the hypoxic microenvironment is limited to certain areas of tumor tissues have limited the ideal tumor therapeutic effect of TPZ. Therefore, it is urgent to develop a strategy to enhance tumor hypoxia and improve the efficacy of TPZ treatment. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a combined drug delivery system based on a bioreactor and a thermosensitive hydrogel, wherein the bioreactor is a genetically engineered bacterium that selectively colonizes in tumor tissue and induces intratumoral vascular thrombosis, and the thermosensitive hydrogel is prepared from a thermosensitive material and loaded with a hypoxia-activated prodrug. The luxCDABE gene is expressed in the genetically engineered bacteria, and a bioluminescent system is constructed. The bioreactor spontaneously fluoresces in a normoxic environment, and the fluorescence is weakened in an anaerobic environment. The fluorescence signal is detected by an in vitro imaging system, which can assist in determining the optimal administration time of the thermosensitive hydrogel and realize visual monitoring of the treatment process.

[0006] The first object of the present invention is to provide a combined drug delivery system based on a bioreactor and a thermosensitive hydrogel, wherein the bioreactor is a genetically engineered bacterium that selectively colonizes in tumor tissue and induces intratumoral vascular thrombosis, and the thermosensitive hydrogel is prepared from a thermosensitive material and loaded with a hypoxia-activated prodrug.

[0007] Furthermore, the chassis strain of the genetically engineered bacteria is Escherichia coli TOP10.

[0008] Escherichia coli TOP10 is tumor-targeting and can selectively colonize in tumor tissues, induce vascular thrombosis in the tumor, and increase the level of hypoxia in the tumor.

[0009] Furthermore, the genetically engineered bacteria express the luxCDABE gene.

[0010] Furthermore, the expression of the luxCDABE gene is driven by the EM7 promoter.

[0011] In one embodiment of the present invention, the luminescence system was constructed in E. coli TOP10 using pGEN-luxCDABE plasmid.

[0012] Furthermore, the hypoxia-activated prodrug is tirapazamine.

[0013] Furthermore, the temperature-sensitive polymer material is Pluronic F127.

[0014] Furthermore, in the preparation process of the thermosensitive hydrogel, the amount of the thermosensitive polymer material added is affected by the hypoxia-activated prodrug. The gelation temperature of the prepared thermosensitive hydrogel is different depending on the hypoxia-activated prodrug loaded.

[0015] In one embodiment of the present invention, the preparation method of the thermosensitive hydrogel is as follows:

[0016] (1) preparing a tirapazamine solution with a concentration of 1 mg / mL;

[0017] (2) Weigh Pluronic F127 powder;

[0018] (3) Add Pluronic F127 powder to the tirapazamine solution and place on ice with constant stirring until it is completely dissolved.

[0019] Furthermore, the concentration of the Pluronic F127 is 15-18%.

[0020] Preferably, the concentration of Pluronic F127 is 18%.

[0021] The second object of the present invention is to provide the use of the above-mentioned combined drug delivery system in the preparation of tumor therapeutic drugs.

[0022] Furthermore, there is an interval between the administration timings of the bioreactor and the thermosensitive hydrogel, and the interval is 8-24 hours.

[0023] In one embodiment of the present invention, the interval is 24 hours.

[0024] Furthermore, the intensity of the fluorescence signal from the bioreactor can be used to determine the degree of hypoxia in the tumor environment, thereby determining the optimal administration time of the thermosensitive hydrogel.

[0025] Furthermore, the administration method is in situ injection, intravenous injection or oral administration.

[0026] Preferably, the administration is by in situ injection.

[0027] Beneficial effects of the present invention:

[0028] (1) Using genetically engineered bacteria as bioreactors to target tumor cells and enhance their hypoxic environment helps activate hypoxia-activated prodrugs, thereby improving the killing effect on tumor cells;

[0029] (2) Thermosensitive hydrogels are used as drug carriers, which can form gel in situ inside the tumor under the action of body temperature and achieve sustained release of drugs, which helps to maintain the effective concentration of drugs in the tumor site and further improve the therapeutic effect;

[0030] (3) LuxCDABE bioluminescence system was introduced into E. coli TOP10, so that the genetically engineered bacteria TOP10-Lux could emit light autonomously. Since the bioluminescence intensity of TOP10-Lux is related to the number of thrombi in the tumor and the degree of hypoxia in the tumor, the treatment process can be monitored in real time by an in vitro imaging device, thereby realizing the visualization of the treatment process. The optimal administration time of tirapazamine can also be determined according to the intensity of the fluorescence signal, so as to achieve the best efficacy of tirapazamine.

[0031] (4) E. coli TOP10 has low pathogenicity and high biocompatibility, and is a commonly used and safe engineered strain. At the same time, the thermosensitive polymer material Pluronic F127 also has good biocompatibility and safety, ensuring the safety of the system in clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0033] Figure 1 is the pGEN-luxCDABE plasmid map involved in the present invention;

[0034] Figure 2 It is the quantification of the bioluminescent signal of Escherichia coli TOP10 and genetically engineered bacteria TOP10-Lux under normoxic conditions in Example 2 of the present invention;

[0035] Figure 3 It is the quantification of the bioluminescent signal of the genetically engineered bacteria TOP10-Lux under normoxic or anoxic conditions in Example 2 of the present invention;

[0036] Figure 4 The biodistribution of the genetically engineered bacteria TOP10-Lux in Example 3 of the present invention in mice bearing colon cancer on days 3 and 6 after injection;

[0037] Figure 5 It is the quantification of the bioluminescent signal after the genetically engineered bacteria TOP10-Lux in Example 4 of the present invention is incubated with red blood cells of different concentrations;

[0038] Figure 6 The bioluminescent signal quantification of the tumor site of mice at different time points after the genetically engineered bacteria TOP10-Lux in Example 4 of the present invention is injected into the tumor of colon cancer-bearing mice;

[0039] Figure 7 It is the gel temperature test result of F127 hydrogel and F127@TPZ hydrogel at different F127 concentrations (16%, 18%, 20%, 22%, 24%) in Example 5 of the present invention;

[0040] Figure 8 are the rheological test results of the F127 hydrogel and the F127@TPZ hydrogel in Example 5 of the present invention at 4°C and 37°C, wherein a is the modulus change of the hydrogel at 4°C, and b is the modulus change of the hydrogel at 37°C;

[0041] Fig. 9is the test result of the cumulative release of TPZ over time of the F127@TPZ hydrogel at 37°C in Example 6 of the present invention;

[0042] Fig.10 is the test result of cytotoxicity of TPZ on CT26 cells under normoxic and hypoxic conditions in Example 6 of the present invention, wherein a is normoxic conditions and b is hypoxic conditions;

[0043] Fig.11 This is a graph showing the weight change of mice in the treatment of colon cancer-bearing mice with the genetically engineered bacteria TOP10-Lux combined with TPZ in Example 7 of the present invention;

[0044] Fig.12 The results of routine blood examination and blood biochemical test of mice bearing colon cancer treated with the genetically engineered bacteria TOP10-Lux combined with TPZ in Example 7 of the present invention, wherein a is the result of routine blood examination of mice, and b is the result of blood biochemical test of mice;

[0045] Fig.13 This is a graph showing the growth curve of mouse tumors in which the genetically engineered bacteria TOP10-Lux combined with TPZ in Example 7 of the present invention treat colon cancer-bearing mice;

[0046] Fig.14 This is a graph showing the survival time of mice treated with colon cancer tumors by the genetically engineered bacteria TOP10-Lux combined with TPZ in Example 7 of the present invention;

[0047] Fig.15 It is the quantification of bioluminescent signals at the tumor site of mice during the treatment of colon cancer-bearing mice by the genetically engineered bacteria TOP10-Lux combined with TPZ in Example 8 of the present invention. DETAILED DESCRIPTION

[0048] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0049] The pGEN-luxCDABE plasmids capable of autonomous luminescence involved in the following examples were purchased from Addgene, USA. The plasmid map is shown in Figure 1 shown.

[0050] Example 1: Preparation of a genetically engineered bacterial living bioreactor

[0051] 1. Preparation of Competent Bacteria

[0052] (1) Pick a single TOP10 colony and culture it in LB medium at 37°C in a shaking incubator overnight (about 16 hours);

[0053] (2) Transfer 1 mL of overnight cultured bacteria into 100 mL of LB medium and culture at 37°C with shaking (180 rpm) for about 3 hours;

[0054] (3) Preparation of 0.1M CaCl 2 The solution was pre-cooled on ice;

[0055] (4) Pipette 1.5 mL of the cultured bacterial solution into a 2 mL centrifuge tube and cool on ice for 10 minutes;

[0056] (5) Centrifugation at 3000 g for 5 min at 4°C;

[0057] (6) Discard the supernatant and add 100 μL of pre-cooled 0.1 M CaCl 2 Solution, gently pipette up and down to mix the bacterial solution, and place it on ice for 20 minutes;

[0058] (7) Repeat steps (5) and (6);

[0059] (8) The bacterial suspension can be used immediately for transformation or added with a cryoprotectant (15%-20% glycerol) and then ultra-frozen for storage at -80°C.

[0060] 2. Plasmid Transfection

[0061] (1) Pipette 5 μL of 0.2 μg / μL pGEN-luxCDABE plasmid (see the map) Figure 1 As shown) were placed in 40 μL competent TOP10 bacterial solution and placed on ice for 30 minutes;

[0062] (2) Heat shock in a 42°C water bath for 90 seconds and quickly transfer to ice for 5 minutes;

[0063] (3) Add 200 μL of LB culture medium (without resistance) to the competent TOP10 bacterial solution containing the plasmid in a sterile clean bench, then transfer to a culture tube and culture at low speed in a shaker at 37°C for 1 hour;

[0064] (4) 200 μL of bacterial suspension was added to the ampicillin-resistant LB solid culture medium, the suspension was evenly spread with a spreading rod, and the suspension was inverted and cultured in a 37°C constant temperature incubator for 16 hours to obtain the engineered bacteria TOP10-luxCDABE (TOP10-Lux) capable of self-luminescence, which is the genetically engineered bacteria living bioreactor described in the present invention.

[0065] Finally, the above recombinant E. coli TOP10-Lux strain was picked up, added to LB medium containing ampicillin, cultured at 37°C in a shaker for 24 hours, and then stored in 25% sterile glycerol and kept in a -80°C refrigerator for further use.

[0066] Example 2: Characterization of bioluminescence capability of genetically engineered bacteria in vivo bioreactor

[0067] TOP10-Lux bioluminescence ability test: Pick three frozen bacterial suspensions of TOP10 and TOP10-lux and culture them in 20mL LB medium containing ampicillin, and culture them in a shaking incubator at 37℃. After 24 hours, take 1mL of bacterial suspension from each tube and add it to a 24-well plate. Place the well plate in a small animal live imaging instrument to observe bioluminescence. Figure 2 As shown, TOP10 does not have inherent bioluminescence ability, while TOP10-lux can spontaneously produce bioluminescence under normoxic conditions, which confirms that pGEN-luxCDABE was successfully transformed and the obtained TOP10-Lux has bioluminescence ability.

[0068] Detection of the effect of oxygen content on the luminescence ability of TOP10-Lux: Select frozen TOP10-Lux strains and culture them in 6 tubes of 20 mL LB medium containing ampicillin, and culture them in a shaking incubator at 37°C. After 24 hours, take 1 mL of bacterial suspension from 3 tubes and add them to 2 24-well plates. The well plates of the hypoxic treatment group were placed in an anaerobic bag for pretreatment for 1 hour, and the well plates were placed in a small animal live imaging instrument to observe the bioluminescence of each sample well. Figure 3 As shown, the bioluminescence intensity of TOP10-Lux under hypoxia was significantly reduced compared with that in the normoxic group, indicating that TOP10-Lux can be used as an efficient active bioreactor for hypoxia detection.

[0069] Example 3: Study on the biodistribution of genetically engineered bacteria living bioreactor in tumor-bearing mice

[0070] A subcutaneous CT26 colon cancer-bearing mouse model was established by intratumoral injection of TOP10-Lux (5×10 per mouse). 8 CFU), and then monitor the bacterial retention in CT26 tumor-bearing mice. The mice were euthanized before injection and on the 3rd and 6th days after injection, and tumor tissues and organs, including heart, liver, spleen, lung, and kidney, were collected under a sterile environment. They were weighed and homogenized with 1 mL of sterile PBS. After homogenization and serial dilution, the organ and tumor tissue slurry was applied to LB plates containing ampicillin, incubated in an incubator at 37°C, and the number of colonies on each sample culture plate was counted.

[0071] The results are as follows Figure 4As shown in the figure, the genetically engineered bacteria TOP10-Lux selectively enriches and colonizes in tumor tissues, while only a very small amount or even none exists in the main organs of mice. This phenomenon is related to the tumor-targeting properties of TOP10 itself and the immune system of the organism, and also shows the feasibility of TOP10-Lux for tumor treatment.

[0072] Example 4: Identification of hypoxia degree by monitoring TOP10-Lux in a genetically engineered bacterial living bioreactor

[0073] (1) The effect of TOP10-Lux-induced thrombosis on its own luminescence ability

[0074] Since TOP10 can cause rapid and strong blood coagulation in the tumor area, leading to the formation of thrombus, it is speculated that the bioluminescence intensity of TOP10-Lux is also related to thrombus formation. To confirm the above hypothesis, blood was collected from the eyeballs of mice, and then red blood cells were extracted from these blood samples and counted.

[0075] Detection of the effect of thrombus on luminescence: Different concentrations of red blood cells were added to a 96-well plate, and then 5×10 8 CFU of TOP10-Lux was incubated with different concentrations of red blood cells, and the bioluminescent signal was detected using a live imaging system. Figure 5 As shown in the figure, as the red blood cell concentration increases, part of the fluorescence is absorbed, resulting in a gradual decrease in the bioluminescence signal detected by TOP10-Lux, indicating that there is a good relationship between the bioluminescence signal and the red blood cell (thrombus) concentration, suggesting that TOP10-Lux can be used as a living biosensor to monitor thrombus and hypoxia in tumor tissue.

[0076] (2) Evaluation of TOP10-Lux’s ability to monitor tumor hypoxia

[0077] Construction of subcutaneous CT26 colon cancer-bearing mouse model: CT26 mouse colon cancer cells were cultured in 1640 cell culture medium until the exponential growth phase, then digested by blowing with culture medium, centrifuged at 900 rpm, 4°C for 3 minutes to collect cells, resuspended in PBS, and inoculated subcutaneously in Balb / c mice. The mouse tumor volume grew to about 150 mm 3 Experiments were conducted at the same time.

[0078] Imaging monitoring of tumor-bearing mice: TOP10-Lux (5×10 8 CFU / mouse), and the bioluminescence of TOP10-Lux at the tumor site was detected by an in vivo imaging system.

[0079] like Figure 6As shown, over time, the fluorescence signal at the tumor site first decreased, then increased, and finally decreased, and the signal intensity reached a peak at 48 hours. This trend suggests that after the injection of TOP10-Lux at the tumor site, the initial reaction is the slow proliferation of bacteria, which quickly induces thrombosis, resulting in a gradual weakening of the tumor fluorescence signal. Subsequently, the bacteria proliferated logarithmically, resulting in an increase in the tumor fluorescence signal. Ultimately, as TOP10-Lux-induced thrombosis and the degree of hypoxia in the tumor increased, the bioluminescence signal at the tumor site gradually decreased, indicating that TOP10-Lux can be used as an efficient biosensor for detecting tumor hypoxia levels.

[0080] Example 5: Preparation and characterization of thermosensitive injectable hydrogel drug delivery system

[0081] Preparation of F127 hydrogel: Weigh F127 powder and add ddH 2 O and placed on ice with constant stirring until completely dissolved. When preparing F127@TPZ hydrogel, F127 powder was added to TPZ solution (1 mg / mL) and F127@TPZ hydrogel was obtained by the same steps.

[0082] Study on the gelation behavior of hydrogel drug delivery system: Prepare different concentrations of F127 and F127@TPZ solutions on ice, and use the inverted observation method to raise the temperature by 0.5°C each time starting from 0°C. For each concentration, take 0.1mL of sample and introduce it into the EP tube. Invert the sample for 30 seconds and observe the gelation behavior of F127 and F127@TPZ. If there is no flow, the corresponding temperature is the gelation temperature of F127 and F127@TPZ. The test results are as follows Figure 7 As shown in the figure, when the temperature reaches a certain critical value, sol-gel transition occurs in both F127 and F127@TPZ solutions, and the gelation temperature decreases with the increase of F127 concentration. In addition, the addition of TPZ has an effect on the gelation temperature of F127 solution. In order to ensure the injectability of F127@TPZ at room temperature and shorten its gelation time in vivo (37°C), F127@TPZ with an F127 concentration of 18% was selected for heat-triggered in situ gelation (hereinafter referred to as F127 concentration of 18%).

[0083] Rheological properties of hydrogels before and after gelation: The viscoelastic properties of hydrogels were analyzed using frequency sweep mode rheological measurements. Figure 8 As shown in a, at 4°C, the elastic modulus (G') is smaller than the viscous modulus (G"), showing characteristics similar to those of a solution. Figure 8 As shown in b, at 37°C, the elastic modulus (G') is greater than the viscous modulus (G"), showing a gel-like behavior.

[0084] Example 6: Characterization of drug release behavior and in vitro toxicity testing of thermosensitive injectable hydrogel drug delivery system

[0085] Study on the release behavior of TPZ in F127@TPZ: Take 1 mL of 18% F127 solution containing TPZ (1 mg / mL) and add it to a centrifuge tube, and place it in a 37°C water bath. Repeat three samples. Then, slowly add 10 mL of phosphate buffered saline (PBS) to the centrifuge tube and place it in a 37-degree water bath. Pipette 100 μL of supernatant at different time points, measure the absorbance at 460 nm with an enzyme reader, and draw a release efficiency curve. Fig. 9 As shown, TPZ was released continuously in F127 hydrogel.

[0086] TPZ cytotoxicity test: CT26 cells were prepared into a cell suspension and inoculated into a 96-well plate, 200 μL per well. On the next day, a predetermined concentration of TPZ solution was added to the cells in the 96-well plate; for the hypoxia treatment group, this group of cells was placed in an anaerobic bag. After incubation for 24 hours, the old culture medium was discarded, 100 μL of MTT (5 mg / mL) was added to each well, and the culture was continued in a 37°C incubator for 4 hours. Aspirate the MTT, add 200 μL of DMSO to each well, leave at room temperature for 30 minutes, and shake every 5 minutes. Finally, the OD value of each group of sample wells was measured at a wavelength of 570 nm on an enzyme reader, and statistical analysis was performed. Fig.10 As shown in the figure, under hypoxic conditions, as the concentration of TPZ increased, the viability of CT26 cells decreased significantly, indicating that TPZ had a significant cytotoxic effect on hypoxic cells. Under normoxic conditions, there was no significant change in cell viability after TPZ treatment. The results showed that TPZ can only produce cytotoxic effects on cells under hypoxic conditions.

[0087] Example 7: Evaluation of the anti-tumor efficacy of genetically engineered bacteria in vivo bioreactor combined with thermosensitive hydrogel drug delivery system F127@TPZ

[0088] Construction of subcutaneous CT26 colon cancer-bearing mouse model: CT26 mouse colon cancer cells were cultured in 1640 cell culture medium and then digested. Cells were collected by centrifugation at 900 rpm and 4°C for 3 minutes. After resuspending in PBS, they were subcutaneously inoculated into Balb / c mice. The tumor volume of the mice grew to about 150 mm 3 Experiments were conducted at the same time.

[0089] Grouping and administration of tumor-bearing mice: The mice were randomly divided into 5 groups, including the non-administered group, TOP10-Lux group, F127@TPZ group, TOP10-Lux+TPZ group, and TOP10-Lux+F127@TPZ group. When the tumor size reached 150 mm 3 TOP10-Lux was injected intratumorally (5×10 8CFU), and TPZ or F127@TPZ treatment was performed 24 hours later (50 μL was injected intratumorally in each mouse). TPZ or F127@TPZ was injected again on the 6th day.

[0090] In order to evaluate the safety of genetically engineered bacteria, the weight of mice in each group was weighed and recorded every two days, and blood was collected from each group. The blood routine indexes of mice were tested by whole blood, and the biochemical indexes of mice were tested by serum. The results showed that the combined treatment strategy did not cause a significant decrease in the weight of tumor-bearing mice ( Fig.11 ), and all blood-related indicators fluctuated within the normal range ( Fig.12 ). These comprehensive tests confirmed that the genetically engineered bacteria TOP-Lux of the present invention has good biosafety.

[0091] After the first injection of TPZ or F127@TPZ, the tumor volume and body weight of the mice were monitored every two days. The length and width of the tumor were measured with a caliper, and the total volume of the tumor was calculated as (length × width 2 ) / 2. When the tumor load reaches 1500mm 3 At this time, the mice were euthanized and the number of dead mice was recorded to calculate the survival curve of tumor-bearing mice. Fig.13 As shown in the figure, TOP10-Lux+TPZ treatment significantly delayed tumor growth in mice compared with the TOP10-Lux or F127@TPZ groups. It is speculated that this is due to the microthrombi induced by bacterial colonization in the tumor, which enhanced the degree of tumor hypoxia and further activated TPZ. In addition, TOP10-Lux+F127@TPZ treatment showed the strongest tumor inhibitory effect among all treatments, and even led to tumor eradication in one mouse, which was attributed to the sustained release of TPZ by F127 hydrogel. Fig.14 As shown in the figure, compared with the untreated group, the survival time of mice in the TOP10-Lux+F127@TPZ group was also significantly improved, indicating that the combined treatment of TOP10-Lux and F127@TPZ can significantly improve the efficiency of tumor treatment and has better biosafety. The above results comprehensively show that TOP10-Lux combined with the thermosensitive hydrogel drug delivery system F127@TPZ has a good therapeutic effect on mouse tumors.

[0092] Example 8: Evaluation of the monitoring capability of the genetically engineered bacterial living bioreactor TOP10-Lux during treatment of CT26 tumor-bearing mice

[0093] Construction of subcutaneous CT26 colon cancer-bearing mouse model: CT26 mouse colon cancer cells were cultured in 1640 cell culture medium and then digested. Cells were collected by centrifugation at 900 rpm and 4°C for 3 minutes. After resuspending in PBS, they were subcutaneously inoculated into Balb / c mice. The tumor volume of the mice grew to about 150 mm 3 Experiments were conducted at the same time.

[0094] Imaging monitoring of tumor-bearing mice during treatment: TOP10-Lux (5×10 8 CFU / mouse), and 50 μL F127@TPZ was injected intratumorally 24 hours later. During the treatment, the bioluminescence of TOP10-Lux in the tumor site was detected by an in vivo imaging system.

[0095] like Fig.15 As shown in the figure, the bioluminescence intensity at the tumor site first decreased, then increased, and finally decreased again. After intratumoral injection of TOP10-Lux, thrombosis was rapidly triggered in the tumor, resulting in enhanced hypoxia in the tumor and a gradual weakening of the tumor bioluminescence signal. Then, with the rapid growth of bacteria, the bioluminescence intensity at the tumor site gradually increased. Finally, with the massive production of blood clots, hypoxia in the tumor was further aggravated, and the bioluminescence signal of the tumor also decreased again, reflecting the enhanced activation of TPZ. Therefore, these results verify that TOP10-Lux can effectively monitor the treatment process of TPZ.

[0096] In summary, the present invention utilizes the facultative anaerobic Escherichia coli TOP10 strain to selectively colonize the tumor site, induce thrombosis in the tumor area, and enhance hypoxia in the tumor. The anti-tumor prodrug TPZ produces reactive oxygen species in a hypoxic environment, leading to tumor cell death, but has little toxicity to normal cells under normoxic conditions. When TPZ was used in combination with TOP10, we observed an enhanced therapeutic effect on tumors. In order to improve the efficacy of TPZ, we used an injectable thermosensitive hydrogel F127 to load TPZ and promote the sustained release of TPZ at the tumor site. This may have implications for the use of hypoxia-activated prodrugs in tumor treatment.

[0097] In addition, assessing the level of hypoxia within the tumor is a key issue. To address this issue, the present invention introduces the luxCDABE bioluminescence system into TOP10 to achieve autonomous bioluminescence. The present invention confirms that the bioluminescence intensity of TOP10 is negatively correlated with the degree of hypoxia within the tumor. Therefore, this TOP10-Lux allows real-time monitoring of the treatment process by in vitro imaging equipment. We have developed a living bioreactor based on engineered TOP10 bacteria, which can improve the therapeutic effect of anti-tumor prodrugs and monitor the treatment process, providing an effective strategy for bacteria-based tumor-targeted chemotherapy and treatment monitoring. The present invention provides a new research direction for promoting tumor treatment and visualizing the treatment process through genetically engineered bacteria.

[0098] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A combined drug delivery system based on a bioreactor and a thermosensitive hydrogel, characterized in that: The bioreactor is a genetically engineered bacterium that selectively colonizes in tumor tissue and induces thrombosis in tumor blood vessels. The thermosensitive hydrogel is prepared from a thermosensitive material and loaded with a hypoxia-activated prodrug.

2. The combined drug delivery system according to claim 1, characterized in that: The chassis strain of the genetically engineered bacteria is Escherichia coli TOP10.

3. The combined drug delivery system according to claim 1, characterized in that: The genetically engineered bacteria express the luxCDABE gene.

4. The combined drug delivery system according to claim 3, characterized in that: The luxCDABE gene is expressed by the EM7 promoter.

5. The combined drug delivery system according to claim 1, characterized in that: The hypoxia activated prodrug is tirapazamine.

6. The combined drug delivery system according to claim 1, characterized in that: The temperature-sensitive polymer material is Pluronic F127.

7. The combined drug delivery system according to claim 6, characterized in that: The concentration of Pluronic F127 is 15-18%.

8. Use of the combined drug delivery system according to any one of claims 1 to 7 in the preparation of tumor therapeutic drugs.

9. The use according to claim 8, characterized in that: There is an interval between the administration timings of the bioreactor and the thermosensitive hydrogel, and the interval is 8-24 hours.

10. The use according to claim 8, characterized in that: The administration method is in situ injection, intravenous injection or oral administration.

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