Engineered bacteria for in situ synthesis of melanin in tumors and applications thereof

Abstract

The application belongs to the technical field of biological medicine, and specifically discloses an engineered bacterium for in-situ synthesis of melanin for tumors and application thereof. The engineered bacterium is obtained by transforming a temperature-controlled expression vector containing a gene encoding tyrosinase into a bacterium. The bacterium can accumulate and proliferate in a tumor microenvironment after entering a living organism. The engineered bacterium can realize controllable synthesis of melanin in-situ for tumors. The engineered bacterium constructed in the application can be used for mass production of melanin. After entering a living organism, the bacterium can target and colonize in a tumor microenvironment. Then, melanin synthesis can be started by near-infrared laser irradiation, controllable amplification of drugs at a tumor site and photothermal combined immunotherapy can be realized, and the killing effect and safety on tumors can be improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and more specifically, relates to an engineered bacterium for in situ synthesis of melanin in tumors and its application. Background Technology

[0002] Cancer, as one of the most serious diseases threatening human life and health, remains a formidable challenge for countless clinical and basic researchers. The characteristics of most solid tumors, such as hypoxia, dense extracellular matrix, abnormal vascularity, and lack of lymphatic drainage, along with tumor heterogeneity and the immunosuppressive microenvironment of tumor tissue, further complicate the development of cancer treatment methods. Researchers recognize the excessive toxicity and limited efficacy of traditional chemotherapy and radiotherapy due to their dependence on tumor cell sensitivity, creating an urgent need for safer and more effective cancer treatments. This has led researchers to focus on tumor bacterial therapy.

[0003] Tumor bacterial therapy is a cancer treatment strategy developed by researchers based on the characteristic that bacteria preferentially accumulate and proliferate in solid tumors. Through surface modification or genetic engineering, bacteria are transformed into low-toxicity tumor-targeting carriers to deliver various anti-tumor drugs, thereby achieving targeted anti-tumor effects. Although using bacteria as tumor-targeting carriers can improve the targeted accumulation of anti-tumor drugs and increase drug concentration at the tumor site to some extent, the proliferation of drug-loaded bacteria in tumor tissue cannot simultaneously increase the amount of drug loaded. As the bacteria are cleared by the body's immune system, the drug is gradually lost. To address this issue, researchers use synthetic biology techniques to customize gene expression products, designing engineered bacteria to express cytotoxic proteins or immunomodulatory factors to inhibit tumors. External inducers are used to control expression, allowing bacteria to proliferate in the hypoxic regions deep within the tumor while continuously expressing therapeutic molecules, achieving local drug amplification and further enhancing the therapeutic effect.

[0004] However, most bacterial therapies encounter difficulties in the clinical trial stage, with single bacterial treatments proving ineffective and failing to achieve a balance between efficacy and safety. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide an engineered bacterium for in situ synthesis of melanin in tumors and its application, aiming to solve the problems of low efficacy and safety of single-bacterial therapy.

[0006] To achieve the above objectives, the present invention provides an engineered bacterium for in situ melanin synthesis in tumors. The engineered bacterium is obtained by transforming a temperature-controlled expression vector containing a gene encoding tyrosinase into bacteria. The bacteria are capable of accumulating and proliferating in the tumor microenvironment after entering the organism. The engineered bacteria can achieve controlled melanin synthesis in situ in tumors.

[0007] Preferably, the gene encoding tyrosinase has undergone codon optimization, and its nucleotide sequence is shown in SEQ ID No. 1 of the sequence listing.

[0008] Preferably, the bacteria is Escherichia coli Nissle 1917, and the temperature-controlled expression vector can initiate the expression of the gene encoding tyrosinase under near-infrared laser irradiation.

[0009] According to another aspect of the present invention, the use of the above-mentioned engineered bacteria in the preparation of an antitumor drug, wherein the antitumor drug comprises the engineered bacteria is provided.

[0010] Preferably, the antitumor drug further includes the ability to release Cu in the tumor microenvironment. 2+ Copper nanoparticles with tyrosine.

[0011] Preferably, the preparation method of the copper nanoparticles includes the following steps: adding a mixture of Cys-Tyr dipeptide aqueous solution and NaOH solution dropwise to CuSO4 solution while stirring continuously; centrifuging after the addition is completed, removing the supernatant, dissolving the precipitate with ethanol, and freeze-drying under vacuum to obtain copper nanoparticles CuNPs solid.

[0012] According to another aspect of the present invention, a method for producing melanin is provided, comprising the following steps: fermenting and culturing the above-mentioned engineered bacteria, and adding Cu to the fermentation broth. 2+ With L-tyrosine, melanin was synthesized by induction culture at temperatures above 42°C.

[0013] Preferably, the induction culture at a temperature above 42°C is a constant temperature induction culture at 45°C for at least 24 hours.

[0014] Preferably, the induction culture at a temperature above 42°C is performed using a power density of 1.5 W / cm². 2 The fermentation broth was irradiated with an 808nm laser for at least 15 minutes and then cultured.

[0015] Preferably, oxygen is introduced into the fermentation broth during the induction culture.

[0016] In summary, the technical solutions conceived by this invention have the following beneficial effects compared with the prior art:

[0017] (1) This invention constructs an engineered bacterium capable of temperature-controlled expression of tyrosinase (TYR). Tyrosinase (monophenol monooxygenase EC1.14.18.1) is a copper-containing enzyme involved in melanin synthesis. Utilizing molecular oxygen, this enzyme catalyzes the hydroxylation of L-tyrosine to L-DOPA (cresolase activity), followed by oxidation to dopaquinone (catecholase activity), and then polymerization to form melanin through a series of non-enzymatic redox reactions. Melanin, as a photothermal therapy agent with high photothermal conversion efficiency, good biocompatibility, and safety, has shown good tumor-suppressing effects in in vivo tumor treatment. The engineered bacteria constructed using this invention can target and deliver melanin to tumor tissue, enhancing the killing effect on tumor cells. Simultaneously, using a temperature-controlled expression vector, the engineered bacteria can be spatiotemporally controlled to colonize the tumor microenvironment before efficiently expressing tyrosinase and synthesizing melanin, thereby enhancing the targeted tumor treatment effect. The bacteria combined with photothermal therapy can, on the one hand, induce immunogenic death of tumor cells, exposing tumor antigens and synergistically enhancing the immunotherapeutic effect of the bacteria; on the other hand, the strong thermal effect can eradicate bacteria in tumor tissue while killing tumor cells, reducing the potential post-treatment toxicity caused by excessive bacterial proliferation in tumor tissue.

[0018] (2) The present invention optimizes the codons of the gene encoding tyrosinase on the expression vector to make it easier to express in Escherichia coli host, improve translation efficiency, and thus improve the expression level of tyrosinase.

[0019] (3) *Escherichia coli* Nissle 1917 (EcN) is a Gram-negative bacterium with significant probiotic characteristics. EcN is non-pathogenic and is easily cleared from the body by serum. Currently, EcN has been authorized as a drug for treating diseases. This invention constructs engineered *Escherichia coli* Nissle 1917, which has high biosafety. Furthermore, EcN possesses strong tumor targeting and intratumoral colonization capabilities, effectively inducing an anti-tumor immune response in the body, making it a promising anti-tumor bacterial drug. The temperature-controlled expression vector in this invention's engineered bacteria can initiate expression under near-infrared laser irradiation. Compared to isothermal induction, this significantly shortens the induction time and provides greater operability for anti-tumor treatment.

[0020] (4) This invention provides a drug comprising engineered bacteria capable of temperature-controlled expression of tyrosinase. These engineered bacteria can proliferate in tumor tissue while simultaneously expressing a large amount of tyrosinase, utilizing Cu nanoparticles (CuNPs) that are present in vivo or provided to them at specific sites. 2+The substrate tyrosine is further oxidized to synthesize melanin, a biophotothermal agent, achieving continuous accumulation and amplification of melanin in the tumor site for use in combined photothermal and immunotherapy for tumors. This invention uses non-pathogenic engineered bacteria as a carrier, which can be controlled by external light to initiate the synthesis of biocompatible melanin as a therapeutic drug, exhibiting high biosafety and promising potential application in the clinical treatment of tumors.

[0021] (5) Melanin is a class of complex and diverse biopolymer pigments widely found in animals, plants, and microorganisms, possessing many different functions, including protecting humans and animals from ultraviolet damage, antibiotic function, thermoregulation, free radical damage, and involvement in some nervous system functions. This invention provides a method for producing melanin using engineered bacteria through fermentation. The engineered bacteria express tyrosinase under high-temperature induction, then convert L-tyrosine into melanin, which is secreted extracellularly. This method is low-cost, simple, and yields high output. Furthermore, the melanin produced by microorganisms is non-toxic, harmless, and has good stability.

[0022] (6) This invention investigated the optimized conditions for melanin synthesis by engineered bacteria. It was found that a temperature rise effect similar to that of constant temperature induction for 24 hours could be achieved after 15 minutes of photothermal induction. Furthermore, the photothermal temperature rise effect of the engineered bacteria increased with the increase of photothermal induction time, indicating that photothermal induction has a higher melanin production efficiency. In addition, the study found that increasing the oxygen content in the fermentation broth of engineered bacteria can greatly increase the melanin production. Attached Figure Description

[0023] Figure 1 The pBV220-VS1-6×His plasmid map constructed in Example 1 of this invention.

[0024] Figure 2 These are TEM images of EcN(A) and Mel@EcN-T(B) in Embodiment 2 of the present invention.

[0025] Figure 3 The Fourier transform infrared spectrum of the melanin synthesized by EcN-T in Example 2 of this invention.

[0026] Figure 4 The photothermal heating curves of Mel@EcN-T under different induction conditions in Example 3 of the present invention are shown.

[0027] Figure 5 The results of Western blotting detection of Histag protein in bacteria under different induction conditions in Example 3 of this invention are shown.

[0028] Figure 6 The photothermal heating curves of Mel@EcN-T under different ventilation conditions in Embodiment 3 of the present invention are shown.

[0029] Figure 7 The photothermal heating curves of different concentrations of Mel@EcN-T in Example 4 of this invention are shown.

[0030] Figure 8 OD in Embodiment 4 of the present invention 600 Photothermal heating curves of Mel@EcN-T with a power density of 0.8 under near-infrared light irradiation at different power densities.

[0031] Figure 9 The photothermal stability curve of Mel@EcN-T in Embodiment 4 of the present invention is shown.

[0032] Figure 10 The photothermal conversion efficiency of Mel@EcN-T in Embodiment 4 of the present invention is given.

[0033] Figure 11 Different concentrations of Mel@EcN-T in Example 5 of this invention were subjected to 808nm laser irradiation (1.5W·cm). -2 Cytotoxicity of H22 cells under (5 min) or without laser irradiation.

[0034] Figure 12 In Example 5 of this invention, H22 cells were co-incubated with different concentrations of Mel@EcN-T and then irradiated with an 808nm laser (1.5W·cm). -2 Fluorescence imaging (5 min) or without laser irradiation was performed, with live cells stained green by Calcein-AM and dead cells stained red by PI.

[0035] Figure 13 This is a TEM image of CuNPs prepared in Example 6 of the present invention.

[0036] Figure 14 This is a hydrodynamic size distribution diagram of CuNPs in Embodiment 6 of the present invention.

[0037] Figure 15 This is an X-ray photoelectron spectroscopy analysis diagram of CuNPs in Example 6 of the present invention.

[0038] Figure 16 This is an X-ray diffraction image of CuNPs in Embodiment 6 of the present invention.

[0039] Figure 17 In Example 6 of this invention, CuNPs were used in an in vitro simulated tumor microenvironment. 2+ Release status.

[0040] Figure 18 Fluorescence imaging (A) and semi-quantitative analysis (B) of mouse tumor tissues at different time points after tail vein injection of EcN and EcN-T solutions in Example 7 of this invention.

[0041] Figure 19 The results of plating and counting of major tissues from mice sacrificed at different time points after tail vein injection of EcN-T solution in Example 7 of this invention.

[0042] Figure 20 The Histag protein Western Blot results of tumor grinding samples from different treatment groups (PBS group, EcN-T group, EcN-T+CuNPs group, EcN-T+Laser group, EcN-T+CuNPs+Laser group) in Example 8 of the present invention are used to characterize the expression of tyrosinase in tumor tissues.

[0043] Figure 21 The relative content of melanin in tumor grinding samples from different treatment groups (PBS group, EcN-T+CuNPs group, EcN-T+CuNPs+Laser group, EcN-T+CuNPs+HBO group, EcN-T+CuNPs+Laser+HBO group) in Example 8 of the present invention is based on the content of the PBS group.

[0044] Figure 22 This is a flowchart illustrating the drug administration process of the EcN-T in vivo photothermal combined with immunotherapy strategy in Example 9 of the present invention.

[0045] Figure 23 In Example 9 of this invention, H22 tumor-bearing mice were treated with different methods and then irradiated with an 808nm laser (1.5W·cm). -2 The temperature rise curve of tumor tissue at 5 min.

[0046] Figure 24 The tumor growth curves of H22 tumor-bearing mice after different treatments in Example 9 of this invention are shown.

[0047] Figure 25 The tumor weight of H22 tumor-bearing mice after different treatments in Example 9 of this invention.

[0048] Figure 26 The images show tumor images of H22 tumor-bearing mice after different treatments in Example 9 of this invention.

[0049] Figure 27 The images show H&E staining of tumor tissues from H22 tumor-bearing mice after different treatments in Example 9 of this invention. Detailed Implementation

[0050] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0052] Example 1: Construction of engineered Escherichia coli EcN-T

[0053] The specific process for constructing engineered Escherichia coli EcN-T provided in this embodiment is as follows:

[0054] Using the pBV220 vector containing the bacteriophage temperature-sensitive gene cI857 as a backbone, an ampicillin resistance gene was introduced. The tyrosinase gene (VS1) from Bacillus megaterium was linked with a 6×His tag sequence and cloned into the pBV220 vector, resulting in the recombinant plasmid pBV220-VS1-6×His. Its plasmid map is shown below. Figure 1 As shown in the figure. In this invention, the VS1 gene underwent codon optimization to facilitate its expression in *E. coli* hosts, resulting in the VS1 gene shown in SEQ ID No. 1 of the sequence listing. The plasmid pBV220-VS1-6×His was then introduced into *E. coli* Nissle 1917 (EcN) via electroporation. The electroporation conditions were: voltage 2.5 kV, capacitance 25 μF, and electroporation time 4 ms, using a solution containing 100 μg / mL... -1 Positive transformants were screened using LB agar plates, and single colonies grown on the plates were cultured in liquid medium. Plasmids were extracted and digested for verification. The results showed that the engineered *E. coli* EcN / pBV220-VS1-6×His strain was successfully constructed. Intracellular protein expression analysis of the original and engineered strains (EcN-T) revealed that the tyrosinase gene VS1 was successfully expressed in the engineered strain.

[0055] In a concentration of 100 μg·mL -1 Single colonies of engineered E. coli were picked from Amp's LB agar plates and diluted to 100 μg / mL. - 1 The engineered E. coli EcN-T was obtained by culturing the bacteria in LB liquid medium of Amp and allowing the medium to become turbid after a period of growth. It can be induced to highly express tyrosinase at temperatures above 42°C.

[0056] Example 2: Production of melanin using engineered Escherichia coli EcN-T fermentation culture

[0057] A constant temperature of 45℃ was selected as the condition for inducing tyrosinase expression. 0.2 mM copper sulfate (CuSO4) and 40 mg·L⁻¹ were added to the EcN-T bacterial culture that had reached the plateau phase. -1Tyrosine (Tyr) was added, and then the sample was transferred to a 45℃ constant temperature shaker for 24 h to obtain melanin-containing bacteria Mel@EcN-T. The morphological characteristics of Mel@EcN-T were observed using transmission electron microscopy (TEM). Using EcN as a control, a sample diluted to a certain concentration was dropped onto a carbon support film copper grid. After the sample dried, it was loaded onto an HT7700 transmission electron microscope and observed at 100 kV. The results are as follows. Figure 2 As shown in the figure. TEM observations revealed that Mel@EcN-T maintained an ellipsoidal bacterial structure similar to wild-type EcN, with no significant change in size. Furthermore, the contrast between the inside and outside of Mel@EcN-T was significantly enhanced, confirming the large-scale synthesis of melanin-like substances within the bacteria.

[0058] Extraction and purification of melanin produced by engineered bacteria: The bacterial fermentation broth cultured for 24 hours was centrifuged at 7500g for 15 min to remove bacteria and debris. The pH of the supernatant was then adjusted to 2.0 with 6mol / L HCl, allowed to stand for 4 hours, and then centrifuged at 9000g for 15 min to collect the precipitate, obtaining crude melanin extract. This extract was then sequentially extracted with organic solvents such as chloroform, ethyl acetate, and ethanol. The resulting solid was washed four times with distilled water and centrifuged at 9000g for 15 min to obtain purified melanin. The purified melanin was lyophilized and stored at -20℃.

[0059] Validation of melanin produced by engineered bacteria: Based on reported characterization methods for microbial melanin, the purified melanin was validated using Fourier Transform Infrared Spectroscopy (FTIR). Figure 3 As can be seen, this melanin spectrum exhibits obvious characteristics of eumelanin. The results of this embodiment demonstrate that EcN-T can synthesize large quantities of melanin under isothermal induction conditions at 45℃.

[0060] Example 3: Optimization of Melanin Induction Conditions for Mel@EcN-T Production

[0061] This embodiment investigated the amount of melanin synthesis under different induction conditions and times, and under different ventilation conditions.

[0062] (1) Optimization of induction conditions and time

[0063] Besides constant temperature induction at 45℃, laser irradiation can also achieve a similar induction effect. Adding 0.2 mM CuSO4 and 40 mg·L⁻¹ to EcN / pBV220-VS1-6×His bacterial culture that has reached the plateau phase... -1 After Tyr, a power density of 1.5 W / cm² was used. 2Mel@EcN-T samples with varying melanin content were obtained by irradiating the bacterial culture with an 808nm laser for different durations. The melanin content of each group was evaluated using a photothermal heating experiment. Mel@EcN-T samples were divided into five groups based on different induction conditions: 24h induction at 45℃ (H-24h), 5min (L-5m), 10min (L-10m), 15min (L-15m), and 20min (L-20m) irradiation with an 808nm laser (maintaining the bacterial culture temperature at 45℃). After induction, using LB as a control, 1mL of each sample was irradiated with an 808nm near-infrared laser for 10min at a power density of 1.5W·cm². -2 Simultaneously, a FLIR infrared thermal imager was used to monitor the photothermal temperature rise in each group. Figure 4 It can be seen that as the photothermal induction time increases, the photothermal heating effect of Mel@EcN-T also increases. After 15 minutes of photothermal induction, it can achieve a heating effect similar to that of 24 hours of isothermal induction, indicating that the melanin production of the two is close at this time. This also shows that photothermal induction has a higher melanin production efficiency.

[0064] In addition, to evaluate the tyrosinase content expressed by the engineered strain EcN-T under different induction conditions, Western blotting was used to detect the expression of TYR-Histag in EcN-T cells that were uninduced, induced at constant temperature for 15 min (H-15m), induced by photothermal radiation for 15 min (L-15m), and induced at constant temperature for 24 h (H-24h). Histag expression in wild-type EcN cells was also detected. The results are as follows: Figure 5 As shown, the Histag protein band in EcN-T samples induced by photothermal radiation for 15 min showed little difference from that in EcN-T samples induced by isothermal radiation for 24 h, both remaining at a high level. However, the intensity of the protein band in the 15 min isothermal-induced sample was significantly lower, only slightly higher than the background expression of uninduced EcN-T. This indicates that EcN-T samples induced by photothermal radiation for the same duration showed significantly higher tyrosinase expression levels than those induced by isothermal radiation, and that 15 min of photothermal radiation could achieve the same expression effect as 24 h of isothermal radiation.

[0065] Both the photothermal heating and Western blotting results in this embodiment show that photothermal induction can significantly shorten the induction time of EcN-T, making it possible to induce tyrosinase expression in mouse tumors through photothermal induction. Therefore, 15 min of photothermal induction was chosen as the induction condition for subsequent in vivo photothermal therapy strategies.

[0066] (2) Optimization of ventilation conditions

[0067] During the induction of EcN-T at a constant temperature of 45℃, oxygen (O2 group), nitrogen (N2 group), and no treatment (Normal group) were introduced into the culture flasks. After 24h of induction culture, the photothermal temperature rise of different groups was detected in the same way as in (1), and the melanin content was evaluated.

[0068] from Figure 6 It can be seen that with the increase of O2 content, the amount of melanin synthesized by EcN-T also gradually increases. This indicates that the increase of O2 content can significantly promote the synthesis of melanin by EcN-T.

[0069] Example 4: Detection of the in vitro photothermal properties of Mel@EcN-T

[0070] To evaluate the in vitro photothermal properties of Mel@EcN-T, 1 mL of OD was taken. 600 Mel@EcN-T bacterial cultures of 0.1, 0.2, 0.4, and 0.8 g were placed in 1.5 mL centrifuge tubes, with an equal volume of LB as a control. The power density was 1.5 W·cm³. -2 The cells were irradiated with an 808nm near-infrared laser for 10 minutes, and the photothermal temperature rise in each group was monitored. Additionally, 4 mL of OD was taken... 600 Mel@EcN-T bacterial suspension with a concentration of 0.8 was divided into 4 groups, 1 mL per group, and the power density was adjusted to 0.5, 0.75, 1, and 1.5 W·cm⁻¹, respectively. -2 Using LB as a control, the group was irradiated with an 808nm near-infrared laser for 10 minutes, and the photothermal temperature rise of each group was monitored.

[0071] By measuring different concentrations (in OD) 600 Reflecting bacterial concentration) and the photothermal heating curves of Mel@EcN-T under different light power densities ( Figure 7 and Figure 8 The results showed that under 808 nm near-infrared light irradiation, the temperature of Mel@EcN-T bacterial culture increased significantly, and the temperature rise increased continuously with increasing concentration and light power density. When the light power density was 1.5 W·cm⁻¹... -2 At that time, in OD 600 When the values ​​were 0.1, 0.2, 0.4, and 0.8, the temperature increases were 15.1, 21, 29, and 40.4 °C, respectively. Under the same conditions, the temperature of LB only increased by 3.6 °C. This indicates that the increase in bacterial culture temperature was mainly due to the photothermal effect of Mel@EcN-T, which is used to synthesize melanin.

[0072] To verify the photothermal stability of Mel@EcN-T for synthesizing melanin, 1 mL of Mel@EcN-T bacterial culture was used and subjected to a 1.5 W·cm⁻¹ near-infrared laser at 808 nm. -2Irradiate at a certain power for 10 minutes, allow to cool, then irradiate again for 10 minutes, repeating this process 5 times, monitoring and recording the temperature changes. Figure 9 As shown, the temperature rise of the bacterial solution remained basically consistent with each irradiation, demonstrating extremely good photothermal stability.

[0073] To determine the photothermal conversion efficiency of Mel@EcN-T for melanin synthesis, the absorbance of the Mel@EcN-T bacterial culture at 808 nm was first measured using a UV-vis spectrophotometer. Then, 0.5 mL of the sample was placed in a 1.5 mL centrifuge tube and irradiated with an 808 nm near-infrared laser at a power density of 1.5 W·cm³. -2 The temperature of the bacterial solution was monitored using an infrared thermal imager, recorded every 30 seconds, until the temperature of the bacterial solution stabilized and remained relatively stable. The laser was then turned off, and the bacterial solution was allowed to cool naturally to room temperature. The temperature change of the bacterial solution during this process was monitored, recorded every 30 seconds. After data collection, the photothermal conversion efficiency of Mel@EcN-T was calculated according to the method reported in the literature (W.Feng et al, AdvMater, 2019, 31(5):1805919), and it was found that its photothermal conversion efficiency was as high as 58.6% (e.g., ...). Figure 10 As shown in the figure, it is far superior to that of ordinary inorganic photothermal nanoparticles.

[0074] Through a series of photothermal performance evaluations in this embodiment, the excellent in vitro photothermal performance of Mel@EcN-T of the present invention has been confirmed, making it an ideal photothermal agent for tumor photothermal therapy.

[0075] Example 5: Detection of the photothermal cytotoxicity of Mel@EcN-T against tumor cells in vitro

[0076] (1) CCK-8 cytotoxicity assay

[0077] To evaluate the photothermal cytotoxicity of Mel@EcN-T cells, bacteria (upper chamber) and H22 hepatocellular carcinoma cells (lower chamber) were separated using 0.4 μm Transwell chambers, and the specific detection was performed using a CCK-8 assay kit. H22 cells in logarithmic growth phase were counted, centrifuged, resuspended in fresh culture medium, and seeded at 2.0 × 10⁶ cells / well in each well of a 24-well plate. 5 1 H22 cells were added to the upper chamber of the Transwell apparatus in each experimental group, maintaining a total volume of 1 mL. OD was added to the upper chamber of the Transwell apparatus in each experimental group. 600 300 μL of Mel@EcN-T bacterial suspension (0.125, 0.25, 0.5) was added to each well, while an equal volume of RPMI-1640 medium was added to the upper chamber of the control group. Four parallel wells were set up for each group. The non-illuminated groups were then incubated in a 37℃ CO2 incubator for 6 hours, while the illuminated groups were pre-incubated for 2 hours before being incubated with 1.5 W·cm⁻¹. -2After irradiation with 808nm near-infrared laser for 5 min, cells were incubated for 4 h. After incubation, cells were aspirated, centrifuged at 200g for 5 min to remove supernatant, washed twice with PBS, and resuspended in 1 mL of culture medium. 100 μL of each group was added to a 96-well plate, with 10 μL of CCK-8 reagent added to each well. Cells were incubated at 37℃ until the control group solution turned orange-yellow. The A content of each group's solution was detected using a multi-functional microplate reader. 450 Cell viability was calculated according to the instructions of the CCK-8 assay kit.

[0078] like Figure 11 As shown, at 1.5 W·cm -2 Under 808nm laser irradiation, due to the excellent photothermal heating properties of Mel@EcN-T, co-incubation of H22 cells with different concentrations of Mel@EcN-T followed by photothermal treatment all resulted in a certain degree of inhibition of H22 cell growth. Furthermore, the inhibition of cell growth became stronger with increasing Mel@EcN-T concentration. When the OD of Mel@EcN-T... 600 At a concentration of 0.5, bacterial photothermal effects reduced cell survival to 43.9%, significantly lower than the Control group. In contrast, the cell survival rate in the non-photothermal group with the same Mel@EcN-T concentration was 93.8%, showing no significant difference from the Control group. These results demonstrate that the Mel@EcN-T of this invention can effectively kill tumor cells through photothermal action, and does not cause significant cell damage under photothermal conditions, exhibiting good biocompatibility.

[0079] (2) Calcein-AM / PI live / dead cell staining assay

[0080] The activity of H22 cells before and after photothermal treatment was evaluated using the Calein-AM / PI live / dead cell staining kit. The photothermal killing experiment of H22 cells with Mel@EcN-T was performed as described in (1). After treatment, H22 cells in each group were centrifuged to remove the supernatant, washed twice with PBS, and then incubated with the prepared Calein-AM and PI mixed staining solution at 37°C for 15 min. The dye was removed by centrifugation, and the cells were washed twice and resuspended in PBS. The cells were then dropped into clean confocal dishes and observed under an FV3000 laser confocal microscope. Live and dead cells were stained with green fluorescence (Calcein-AM) and red fluorescence (PI), respectively.

[0081] To more intuitively observe the photothermal killing effect of Mel@EcN-T on tumor cells, this example used Calcein-AM and propidium iodide (PI) to perform live / dead staining on H22 cells incubated with different concentrations of Mel@EcN-T and subjected to photothermal treatment, and studied the ratio of live to dead cells. Figure 12 As shown, H22 cells in the Control group and the Control+Laser group exhibited extremely high viable cell ratios, indicating that irradiation with 808nm laser alone did not significantly affect the activity of H22 cells. No significant increase in dead cells was also observed in the non-illuminated Mel@EcN-T group, demonstrating that Mel@EcN-T itself has no significant dark toxicity. Simultaneous treatment with Mel@EcN-T and 808nm laser irradiation showed a significant concentration-dependent photothermal killing effect on H22 cells, with the OD of Mel@EcN-T increasing as the concentration of dead cells increased. 600 At a concentration of 0.5, photothermal activity can cause significant damage to H22 cells.

[0082] The above results collectively demonstrate that the Mel@EcN-T of this invention has a significant photothermal killing effect on tumor cells in vitro, making it an excellent photothermal therapy drug that helps advance the research on the photothermal anti-tumor effect of the EcN-T treatment strategy in animals.

[0083] Example 6: Preparation and Characterization of CuNPs

[0084] (1) Preparation of CuNPs

[0085] EcN-T requires Cu to synthesize melanin under photothermal induction. 2+ With the involvement of Tyr, this embodiment constructs a method for releasing Cu at the tumor site. 2+ Copper nanoparticles (CuNPs) with Cys-Tyr were prepared. The specific preparation method is as follows: Prepare 10 mL of a 2.5 mM CuSO4 solution, then prepare 5 mL each of a 10 mM Cys-Tyr dipeptide aqueous solution and a NaOH solution. Mix the two solutions and add the mixture dropwise to the CuSO4 solution using a micro-injection pump while continuously stirring. React for 5 minutes. After the addition of the mixture is complete, stop the reaction and centrifuge at 10,000 rpm for 6 minutes to obtain CuNPs. Dissolve the supernatant in ethanol and freeze-dry using a vacuum freeze dryer to obtain solid CuNPs.

[0086] (2) Morphological and structural characterization of CuNPs

[0087] The CuNPs collected by centrifugation in (1) were dissolved in ethanol, and the particle size distribution of CuNPs was detected after equilibration at room temperature for 2 min using a nanoparticle size analyzer.

[0088] The morphological characteristics of CuNPs were observed using transmission electron microscopy. CuNPs samples diluted to a certain concentration were dropped onto a copper grid of a carbon support film and allowed to stand at room temperature until the film dried. The samples were then loaded onto an HT7700 transmission electron microscope and observed at 100 kV.

[0089] CuNPs were observed using TEM, such as Figure 13 As shown, CuNPs exhibit a structure where a Cu core is coordinated with a Cys-Tyr dipeptide, and the dipeptides are cross-linked. The structure is relatively stable, with an average Cu core size of approximately 80 nm. Further dynamic light scattering (DLS) measurements revealed an average hydrodynamic diameter of 222.3 ± 1.4 nm. Figure 14 ).

[0090] The valence state of copper ions in CuNPs solid powder was analyzed using an AXIS SUPRA+ X-ray photoelectron spectroscopy (XPS) instrument. X-ray photoelectron spectroscopy results ( Figure 15 The results show that copper ions in CuNPs exist in a divalent form, which can be used to release Cu from within the tumor. 2+ It participates in the synthesis of tyrosinase from EcN-T.

[0091] The X-ray diffraction peaks of CuNPs solid powder were detected using an X'pert3 powder X-ray diffractometer (XRD), with scanning angles ranging from 10° to 70°. Analysis of the crystal structure information of CuNPs revealed that CuNPs have an amorphous structure (e.g., ...). Figure 16 As shown in the figure, the position of the diffraction peak is consistent with that of the Cys-Tyr dipeptide, which proves that copper nanoparticles successfully loaded Cys-Tyr.

[0092] (3) Evaluation of CuNPs Cu 2+ Release ability

[0093] A tumor redox microenvironment was simulated using PBS with a GSH concentration of 1 mM, an H2O2 concentration of 0.1 mM, and a pH of 6.5. CuNPs solution was placed in a dialysis bag and released in a 37°C constant-temperature shaker. 5 mL of the outer layer of the release medium was collected at time points of 5, 10, 15, 20, 25, 30, 60, 120, 240, and 720 min. The Cu content was determined using an inductively coupled plasma atomic emission spectrometer (ICP). 2+ The CuNPs in the in vitro simulated tumor microenvironment were calculated. 2+ Changes in release over time.

[0094] CuNPs Cu 2+ Release result ( Figure 17 This indicates that in PBS with a GSH concentration of 1 mM, an H2O2 concentration of 0.1 mM, and a pH of 6.5, CuNPs can rapidly release Cu. 2+Within 30 minutes, 49.5% of the CuNPs can be released, and within 1 hour, 73.2% can be released, demonstrating the excellent Cu content of CuNPs in an in vitro simulated tumor microenvironment. 2+ Release capabilities.

[0095] Example 7: Assessment of the accumulation and proliferation of EcN-T in different tissues of mice.

[0096] (1) In vivo fluorescence imaging of small animals

[0097] To investigate the tumor-targeting ability of EcN-T, EcN was used as a control. Bacterial cells were labeled with IR-780 fluorescent dye. Three male H22 tumor-bearing BALB / c mice were injected intravenously with the same dose of EcN and EcN-T. The mice were anesthetized and placed in the IVIS Lumina XR small animal in vivo imaging system at time points of 0, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, and 144 h to observe the distribution of IR-780 fluorescence in tumor tissue and perform fluorescence quantification.

[0098] like Figure 18 As shown, the accumulation of EcN and EcN-T in tumor tissue can be directly observed in the fluorescence imaging results of mice, proving that EcN-T retains the good tumor targeting of bacteria. Moreover, the accumulation of EcN-T in the tumor reaches its peak 72 hours after injection and remains at a high level thereafter. This provides an important reference for the selection of the time point for in vivo photothermal therapy.

[0099] (2) Bacterial smear count

[0100] To investigate the tissue distribution and proliferative capacity of EcN-T in tumors, approximately 10 μL of EcN-T was injected via the tail vein into H22 tumor-bearing male BALB / c mice (n=3 per group). 6 EcN-T mice with CFU were sacrificed at 1, 2, 3, 7, and 14 days. Major organs such as heart, liver, spleen, lung, and kidney, as well as subcutaneous tumors, were collected, washed with PBS, and weighed. The samples were then ground using a tissue homogenizer and diluted to appropriate fold gradients. 100 μL of the sample was added to the surface of a pre-pouring solid culture medium containing Amp, spread evenly with a spreader, and incubated at 37°C for 24 hours. The number of single colonies (CFU) growing on each group of plates was then counted.

[0101] The results are as follows Figure 19 As shown, EcN-T, after intravenous injection, can reach major organs in the body, and the concentration of EcN-T in each organ decreases over time, reflecting the body's immune system's clearance of bacteria. Conversely, the concentration of EcN-T in tumors gradually increases in the first 3 days, far exceeding the initial injection dose of 10. 6CFU was present, and the bacteria could maintain a high concentration for more than 10 days after reaching their peak. This indicates that EcN-T can escape immune response in the tumor immunosuppressive microenvironment and then proliferate in large numbers in the hypoxic region of the tumor.

[0102] The above results indicate that EcN-T has extremely strong tumor targeting and accumulation capabilities and can proliferate in large quantities locally in tumors. The bacterial population density reaches its peak around the 3rd day after intravenous injection, which is conducive to the next step of the treatment strategy of amplifying melanin drugs inside the tumor.

[0103] Example 8: Assessment of intratumoral tyrosinase and melanin production in EcN-T tumors

[0104] (1) Western Blot Analysis

[0105] Mice were divided into four groups according to different treatments during EcN-T therapy: PBS group, EcN-T group, EcNT+CuNPs group, EcN-T+Laser group, and EcN-T+CuNPs+Laser group. After treatment, mice were sacrificed and subcutaneous tumors were collected. The tumors were cleaned in PBS, weighed, and then thoroughly ground using a tissue homogenizer. The samples were diluted with PBS to 20 mg / mL according to tumor mass. -1 Tumor tissue suspension was prepared. 20 μL of tumor tissue suspension was placed in a 1.5 mL centrifuge tube, and an equal volume of RIPA lysis buffer containing 1 mM PMSF was added. The mixture was lysed on ice for 30 min, then 10 μL of 5× Loading Buffer was added, and the solution was boiled at 100 °C for 10 min until clear. This yielded the experimental samples for subsequent Western blotting. The content of Tyrosinase-Histag fusion protein in each group of tumor tissues was detected using anti-Histag antibody.

[0106] Due to the synthesis of tyrosinase and photothermal induction and Cu 2+ Therefore, these two variables are relevant, and the experiment is conducted by controlling for their relevance. For example... Figure 20 As shown, the tyrosinase content in tumor tissues of the group simultaneously treated with CuNPs and the photothermally induced EcN-T+CuNPs+Laser group was significantly higher than that of other groups, while injection of CuNPs only (EcN-T+CuNPs group) and photothermal induction only (EcN-T+Laser group) only caused a slight increase in EcN-T tyrosinase expression. These results indicate that when both CuNPs and photothermal induction conditions are met, EcN-T cells enriched in the tumor can express tyrosinase in situ in large quantities.

[0107] (2) Melanin detection

[0108] Mice were divided into four groups based on different treatments during EcN-T therapy: PBS group, EcN-T+CuNPs group, EcN-T+CuNPs+HBO group, EcN-T+CuNPs+Laser group, and EcN-T+CuNPs+Laser+HBO group. After treatment, mice were sacrificed and subcutaneous tumors were collected. The tumors were weighed, ground, and diluted to obtain the same concentration (20 mg / mL). -1 Tumor tissue suspensions were collected. The tumor tissue suspensions were treated overnight at 60°C with 1M NaOH. After centrifugation, the supernatant of each group of solutions was aliquoted into 96-well plates, 100 μL per group, with 4 sub-wells. The A value of each group of samples was detected using a UV-Vis spectrophotometer. 492 The relative content of melanin composed of each combination was evaluated.

[0109] Melanin was isolated from tumors in different treatment groups and detected by colorimetry. Figure 21 As shown, the melanin content in tumor tissues of the EcN-T+CuNPs+Laser+HBO group, which was simultaneously treated with photothermal induction and HBO, was significantly increased. 492 The absorbance was 3.6 times that of the PBS group, while the absorbance of other groups was relatively low, with no significant difference compared to the PBS group. These results indicate that photothermal induction and HBO treatment of tumors in the presence of CuNPs can significantly improve the efficiency of EcN-T in situ melanin synthesis.

[0110] Example 9: Evaluation of the EcN-T photothermal combined with immunosuppressive effect on tumor suppression

[0111] (1) Mouse tumor photothermal heating experiment

[0112] according to Figure 22 The drug administration procedure shown was used to conduct tumor suppression experiments in mice: approximately 10 mg / L of the drug was administered intravenously to each H22 tumor-bearing BALB / c male mouse (n=3 per group). 6 CFU EcN-T was administered intratumorally to mice at a concentration of 1 mg / kg body weight 3 days after the bacteria had completed tumor-targeted enrichment and multiplication. -1 CuNPs can release Cu within the tumor. 2+ Mice were treated with Cys-Tyr, followed by a first laser irradiation for 15 minutes. Afterward, EcN-T cells in the tumor region expressed a large amount of tyrosinase. Twenty-four hours later, hyperbaric oxygen (HBO) treatment for 1.5 hours increased local O2 levels in the tumor. During this process, Tyr cells reacted with O2 under the catalysis of tyrosinase to synthesize large amounts of melanin. After another 24 hours, once a certain amount of melanin had accumulated, mice in each group underwent a second laser irradiation of the subcutaneous tumor (1.5 W / cm²). -2Irradiation with an 808nm laser for 10 minutes (i.e., combined bacterial and photothermal therapy, PTT) resulted in photothermal conversion of melanin. Mice were divided into five groups based on different treatments during EcN-T therapy: PBS+Laser+HBO+PTT group, EcN-T+CuNPs+PTT group, EcN-T+CuNPs+Laser+PTT group, EcN-T+CuNPs+HBO+PTT group, and EcN-T+CuNPs+Laser+HBO+PTT group. Tumor temperature changes were monitored minute by minute using a FLIR infrared thermal imager, and tumor photothermal temperature rise curves were plotted accordingly.

[0113] like Figure 23 As shown, with increasing irradiation time, the tumor tissue in the EcN-T+CuNPs+Laser+HBO+PTT group exhibited a significant temperature increase, reaching 22.3℃ after 10 minutes of laser irradiation, with the final tumor temperature exceeding 55℃, effectively causing photothermal killing of tumor cells. In contrast, the temperature in the control group (PBS+Laser+HBO+PTT) only increased to 43℃, indicating that laser irradiation at this power density is safe and reliable for normal tissues. Furthermore, the EcN-T+CuNPs+PTT, EcN-T+CuNPs+Laser+PTT, and EcN-T+CuNPs+HBO+PTT groups also showed varying degrees of temperature increase, demonstrating that even without photothermal induction and HBO treatment, EcN-T can still produce a certain amount of melanin at the tumor site based on its background expression. The above results show that the photothermal effect of mouse tumor tissue treated with EcN-T was significantly enhanced, reflecting the large-scale synthesis of melanin in the tumor area, proving that the EcN-T photothermal therapy strategy of the present invention is effective in achieving local melanin drug amplification in tumors.

[0114] (2) Evaluation of the in vivo tumor-suppressing effect of EcN-T photothermal therapy strategy

[0115] H22 tumor-bearing male BALB / c mice were randomly divided into eight groups according to different treatments: PBS group, PBS+Laser+HBO+PTT group, EcN-T+CuNPs group, EcN-T+CuNPs+PTT group, EcNT+CuNPs+Laser+HBO group, EcN-T+CuNPs+Laser+PTT group, EcN-T+CuNPs+HBO+PTT group, and EcN-T+CuNPs+Laser+HBO+PTT group. On the first day, each H22 tumor-bearing mouse (n=6 per group) was injected with approximately 10 6 CFU EcN-T, intratumoral injection concentration on day 4: 1 mg / kg -1CuNPs were induced by laser irradiation for 15 minutes after 2 hours, followed by HBO treatment for 1.5 hours on the fifth day. On the sixth day, each group of tumors was treated with 808nm laser irradiation for 10 minutes, and then cultured in mice for 14 days. During the treatment period, the mice were weighed at fixed times every day, and the long and short diameters of the tumors were monitored with calipers. The formula "Volume = 1 / 2 × Long Diameter × (Short Diameter)" was used. 2 "Tumor volume was calculated, and tumor inhibition curves were plotted to evaluate the photothermal therapy effect of EcN-T. After treatment, mice were sacrificed, tumors were collected, photographed, and tumor weight was recorded. The tumors were then fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with H&E. The H&E-stained sections of the tumors were observed under an optical microscope to examine the tumor tissue damage and apoptosis status in each group."

[0116] like Figure 24 As shown, compared with the PBS group, the tumor growth in the PBS+Laser+HBO+PTT group was not significantly inhibited, and the tumor volume even increased. This indicates that simple laser irradiation and HBO treatment not only cannot inhibit tumors, but may also lead to tumor deterioration due to the pro-angiogenic effect of HBO. Similarly, the tumor growth trends in the EcN-T+CuNPs group and the EcN-T+CuNPs+PTT group also demonstrate that EcN-T, CuNPs, and laser irradiation alone have little effect on tumor growth. The results of the EcN-T+CuNPs+Laser+HBO group show that photothermal induction and HBO treatment can promote melanin production in hypoxic areas of the tumor, but cannot significantly inhibit tumor growth. However, the EcN-T+CuNPs+Laser+HBO+PTT group, which underwent a second photothermal treatment after the bacteria completed melanin synthesis, achieved good tumor treatment results. The tumors of 5 out of 6 tumor-bearing mice were completely cleared, with a tumor inhibition rate of 99.3%. Furthermore, the tumor inhibition rates of the EcN-T+CuNPs+Laser+PTT group and the EcN-T+CuNPs+HBO+PTT group were 64.6% and 59.1%, respectively, indicating that EcN-T can still synthesize a small amount of melanin even without photothermal induction or HBO treatment, thus achieving a certain photothermal therapeutic effect.

[0117] Figure 25 and Figure 26 The results of the tumor weight data and tumor images are consistent with the results of the tumor growth curves mentioned above. Figure 27H&E staining results of tumor tissues showed that almost no tumor cells were observed in the EcN-T+CuNPs+Laser+HBO+PTT group, while the tumor cells in the EcN-T+CuNPs+Laser+PTT and EcN-T+CuNPs+HBO+PTT groups were also quite sparse, with significantly lower numbers than in other groups. These results collectively indicate that the EcN-T photothermal combined with immunotherapy strategy can effectively kill tumor cells through photothermal action, inducing photothermal ablation of H22 subcutaneous tumors, and even achieving the effect of eradicating the primary tumor.

[0118] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of engineered bacteria for in situ melanin synthesis in tumors in the preparation of antitumor drugs, characterized in that: The antitumor drug includes engineered bacteria, as well as bacteria capable of releasing Cu in the tumor microenvironment. 2+ Copper nanoparticles with tyrosine; The engineered bacteria are obtained by transforming a temperature-controlled expression vector containing a gene encoding tyrosinase into bacteria; the bacteria are bacteria that can accumulate and proliferate in the tumor microenvironment after entering the organism; the nucleotide sequence of the gene encoding tyrosine is shown in SEQ ID No. 1; the temperature-controlled expression vector can initiate the expression of the gene encoding tyrosinase under near-infrared laser irradiation. The preparation method of the copper nanoparticles includes the following steps: a mixture of Cys-Tyr dipeptide aqueous solution and NaOH solution is added dropwise to CuSO4 solution while stirring continuously. After the addition is completed, the mixture is centrifuged, the supernatant is removed, the precipitate is dissolved in ethanol, and the copper nanoparticles are obtained by vacuum freeze-drying. After colonizing the tumor microenvironment, the engineered bacteria can initiate the expression of tyrosinase under near-infrared laser irradiation and form melanin through the copper nanoparticles via oxidative polymerization. In other words, the engineered bacteria can achieve controllable melanin synthesis in situ within the tumor.

2. The application according to claim 1, characterized in that: The bacteria in question is Escherichia coli Nissle 1917.

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