A snps-loaded engineered escherichia coli dual-responsive delivery system, and a preparation method and application thereof

Abstract

The application discloses a kind of SNPs loaded engineered escherichia coli dual-response delivery systems and its preparation method and application, the preparation method includes the engineered escherichia coli of overexpressing Flt3L and TIM-3 scFv is co-incubated with NHS-PEG-CHO, obtain aldehyde functionalized escherichia coli;After activation SNPs is reacted with TFA-hydrazine, obtain hydrazine functionalized SNPs;Aldehyde functionalized escherichia coli and hydrazine functionalized SNPs are reacted;The delivery system of the application can rely on SNPs under laser stimulation Photo-thermal effect, photodynamic effect and generate a large number of ROS, efficiently induce tumor cells to occur immunogenic cell death (ICD), and by overexpressing Flt3L and TIM-3 scFv, activate DC cell and block TIM-3 path, restore DC, T cell function, realize the efficient synergy of photodynamic therapy+photothermal therapy+immunotherapy, and then reach the effective treatment of colorectal cancer.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an engineered Escherichia coli dual-response delivery system loaded with SNPs, its preparation method, and its application. Background Technology

[0002] Photodynamic therapy (PDT) is an FDA-approved local tumor therapy that activates photosensitizers accumulated at the tumor site through specific wavelengths of light, triggering a photochemical reaction and generating reactive oxygen species (ROS) in a spatiotemporally controlled manner, thereby selectively killing tumor cells. A deeper anti-tumor potential of PDT lies in its ability to induce immunogenic cell death (ICD), accompanied by the release of tumor-associated antigens (TAAs) and damage-associated molecular patterns (DAMPs) such as adenosine triphosphate (ATP), high-mobility group box 1 (HMGB1), and heat shock proteins (HSPs). These signals collectively constitute a powerful "danger signal" combination, efficiently recruiting and activating dendritic cells (DCs), promoting their phagocytosis, processing, and cross-presentation of tumor antigens, thereby in situ initiating specific CD8+ activation. + T cell response.

[0003] However, clinically used photosensitizers (such as hematoporphyrin derivatives) suffer from problems such as short excitation wavelengths (insufficient penetration depth), persistent skin phototoxicity, and limited ICD induction efficiency, affecting efficacy and safety. Therefore, improving the performance of photosensitizers and increasing targeted delivery are crucial. In contrast, small organic molecule photosensitizers, due to their good biocompatibility and tunable molecular structure, have become a more promising research direction. Although PDT monotherapy can directly kill tumor cells, incomplete elimination of cancer cells and insufficient long-term immune activation can easily lead to tumor residue and long-term tumor recurrence. Therefore, combining the immune-initiating effect of PDT with immune-modulating strategies targeting key immune checkpoints to construct a synergistic treatment system holds promise for achieving a powerful transformation from local killing to systemic immunity. Summary of the Invention

[0004] The purpose of this invention is to provide an engineered E. coli dual-response delivery system loaded with SNPs, its preparation method, and its application. The engineered E. coli dual-response delivery system loaded with SNPs prepared by this invention can achieve photodynamic and photothermal effects under laser irradiation through the loaded SNPs. It can also activate DC cells by overexpressing Flt3L in E. coli and block the TIM-3 pathway by overexpressing TIM-3 scFv, thereby restoring the function of DC and T cells. Under the synergistic effect, it achieves highly efficient synergy of photodynamic therapy, photothermal therapy, and immunotherapy, thereby significantly improving the efficacy of tumor treatment.

[0005] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: The first aspect of this invention provides a method for preparing an engineered E. coli dual-response delivery system loaded with SNPs, the method comprising the following steps: (a) Engineered Escherichia coli overexpressing Flt3L and TIM-3 scFv were co-incubated with NHS-PEG-CHO to obtain aldehyde-functionalized Escherichia coli; (b) Under ultrasonic conditions, a tetrahydrofuran mixed solution of SKCN and DSPE-PEG-COOH and an aqueous solution of tetrahydrofuran were mixed and ultrasonically treated. The organic solvent was then removed by rotary evaporation. The mixture was then concentrated by membrane filtration, ultrafiltration, and drying to obtain SNPs. The SNPs were activated by EDC / NHS. The activated SNPs were then reacted with TFA-acylhydrazine to obtain acylhydrazine-functionalized SNPs. (c) Under acidic conditions, aldehyde-functionalized Escherichia coli and hydrazide-functionalized SNPs are reacted, centrifuged, and washed to obtain the engineered Escherichia coli dual-response delivery system loaded with SNPs.

[0006] A second aspect of the present invention provides an engineered Escherichia coli dual-response delivery system loaded with SNPs prepared by the above-described method.

[0007] The third aspect of the present invention provides the application of the engineered Escherichia coli dual-response delivery system loaded with SNPs prepared by the above-described method in the preparation of antitumor products.

[0008] Compared with the prior art, the beneficial effects of the present invention include at least the following: The engineered E. coli dual-response delivery system loaded with SNPs prepared in this invention can achieve photodynamic and photothermal effects under laser irradiation through the loaded SNPs. It can also activate DC cells by overexpressing Flt3L in E. coli and block the TIM-3 pathway by overexpressing TIM-3 scFv, thereby restoring the function of DC and T cells. Under the synergistic effect, it can achieve highly efficient synergy of photodynamic, photothermal and immunotherapy, thereby achieving effective treatment of colorectal cancer. Attached Figure Description

[0009] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0010] Figure 1 This is a map of the pBV220-Flt3L-HA plasmid in an embodiment of the present invention; Figure 2 This is a map of the pBAD-TIM-3 scFv-3xflag plasmid in an embodiment of the present invention; Figure 3This is the verification result of temperature-induced Flt3L secretion in the experimental examples of this invention; Figure 4 This is the verification result of L-arabinose-induced TIM-3 scFv secretion in the experimental examples of this invention; Figure 5 This serves as a verification result for temperature-induced Flt3L secretion and L-arabinose-induced TIM-3 scFv secretion in the experimental examples of this invention; Figure 6 The temperature rise of SNPs at different concentrations after laser irradiation in the experimental examples of this invention; Figure 7 The experimental examples of this invention show the temperature rise of SNPs after different laser intensities and irradiation times; Figure 8 The results of the photothermal cycling stability experiment of SNPs in the experimental examples of this invention; Figure 9 This illustrates the degradation of DPBF under different SNP concentrations and time periods in the experimental examples of this invention. Figure 10 The degradation of DPBF under different irradiation powers and times is shown in the experimental examples of this invention. Figure 11 This illustrates the degradation of ABDA at different SNP concentrations and times in the experimental examples of this invention. Figure 12 The degradation of ABDA under different irradiation powers and times is shown in the experimental examples of this invention; Figure 13 This illustrates cell survival at different SNP concentrations in the experimental examples of this invention. Figure 14 This illustrates cell survival at different FT@Ec concentrations in the experimental examples of this invention. Figure 15 This describes the cell survival under different treatment groups in the experimental examples of this invention; Figure 16 The results of Calcein-AM / PI fluorescence staining under different treatment groups in the experimental examples of this invention; Figure 17 These are the detection results of the DCFH-DA fluorescent probe under different treatment groups in the experimental examples of this invention; Figure 18 These are the flow cytometry results of intracellular reactive oxygen species under different treatment groups in the experimental examples of this invention; Figure 19 These are the flow cytometry results of apoptosis under different treatment groups in the experimental examples of this invention; Figure 20 These are the results of γ-H2AX immunofluorescence staining under different treatment groups in the experimental examples of this invention; Figure 21These are the CRT confocal microscope results under different treatment groups in the experimental examples of this invention; Figure 22 These are the confocal microscope results of HMGB1 under different treatment groups in the experimental examples of this invention; Figure 23 These are gross images of tumors under different treatment groups in the experimental examples of this invention; Figure 24 These are the pathological analysis results of various organ tissues under different treatment groups in the experimental examples of this invention. Detailed Implementation

[0011] The embodiments of the technical solution of the present invention will be described in detail below with reference to the examples. The following embodiments are only used to illustrate the technical solution of the present invention more clearly, and are therefore only examples, and should not be used to limit the scope of protection of the present invention.

[0012] It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0013] This invention provides a method for preparing an engineered E. coli dual-response delivery system loaded with SNPs, the method comprising the following steps: (a) Engineered Escherichia coli overexpressing Flt3L and TIM-3 scFv were co-incubated with NHS-PEG-CHO to obtain aldehyde-functionalized Escherichia coli; (b) Under ultrasonic conditions, a tetrahydrofuran mixed solution of SKCN and DSPE-PEG-COOH and an aqueous solution of tetrahydrofuran were mixed and ultrasonically treated. The organic solvent was then removed by rotary evaporation. The mixture was then concentrated by membrane filtration, ultrafiltration, and drying to obtain SNPs. The SNPs were activated by EDC / NHS. The activated SNPs were then reacted with TFA-acylhydrazide to obtain acylhydrazide-functionalized SNPs. (c) Under acidic conditions, aldehyde-functionalized Escherichia coli and hydrazide-functionalized SNPs are reacted, centrifuged, and washed to obtain the engineered Escherichia coli dual-response delivery system loaded with SNPs.

[0014] In one embodiment, in step (a), the engineered E. coli overexpressing Flt3L and TIM-3 scFv is obtained by co-transforming the recombinant plasmid expressing Flt3L and the recombinant plasmid expressing TIM-3 scFv into E. coli and then screening them.

[0015] In one embodiment, in step (a), the incubation time is 1.5-3 hours, and the temperature is room temperature; when engineered E. coli OD overexpressing Flt3L and TIM-3 scFv 600When the concentration is 0.5~1.0, the final concentration of NHS-PEG-CHO added is 1~10 mg / mL.

[0016] In this invention, SKCN is an existing product, and its specific structural formula is as follows: The documented literature is "Quinoidal Semiconductor Nanoparticles for NIR-II Photoacoustic Imaging and Photoimmunotherapy of Cancer" (Web of Science; https: / / doi.org / 10.1002 / adma.202415189). In one embodiment, in step (b), the mass ratio of SKCN to DSPE-PEG-COOH in the tetrahydrofuran mixed solution of SKCN and DSPE-PEG-COOH is 1:(30~50), wherein the concentration of SKCN is 0.1~1.0 mg / mL; the volume ratio of the tetrahydrofuran mixed solution of SKCN and DSPE-PEG-COOH to the tetrahydrofuran aqueous solution is 1:(8~12); and the volume concentration of the tetrahydrofuran aqueous solution is 8%~12%. Ultrasonic treatment for 15-25 minutes, ultrasonic power of 150-250W, frequency of 30-50Hz; The membrane used for membrane filtration concentration has a pore size of 0.45 μm; the ultrafiltration membrane used for ultrafiltration concentration has a molecular weight cutoff of 90~110 KD.

[0017] In one embodiment, in step (b), the reaction time is 2.5 to 4 hours and the temperature is room temperature; the molar ratio of activated SNPs to TFA-hydrazide is 1: (8 to 10).

[0018] In one embodiment, in step (c), the pH value of the acidic conditions is 5.0 to 6.0.

[0019] In one embodiment, in step (c), when aldehyde-functionalized E. coli OD 600 When the concentration is 0.5~1.0, the final concentration of hydrazide-functionalized SNPs added is 50~300 μg / mL.

[0020] Another embodiment of the present invention provides an engineered E. coli dual-response delivery system loaded with SNPs prepared by the above-described method.

[0021] Another embodiment of the present invention provides the application of the engineered E. coli dual-response delivery system loaded with SNPs prepared by the above preparation method in the preparation of antitumor products.

[0022] In one embodiment, the tumor is colorectal cancer.

[0023] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0024] Example 1 This embodiment describes a method for preparing an engineered E. coli dual-response delivery system loaded with SNPs (denoted as FT@Ec-SNPs), the preparation method comprising the following steps: 1. Construction of engineered E. coli overexpressing Flt3L and TIM-3 scFv: Two recombinant plasmids were used: pBV220-Flt3L-HA (expressing mouse Flt3L with HA tag, ampicillin resistance, as shown in the figure). Figure 1 As shown), pBAD-TIM-3 scFv-3xflag (expresses a TIM-3 single-chain antibody with a 3×Flag tag, chloramphenicol resistance, chromatogram as shown) Figure 2 (As shown in the image); all plasmids were purchased from Guangzhou Editgene Technology.

[0025] The plasmids were transformed into *E. coli* (*E. coli* Nissle 1917 (Ec)) using the standard heat shock method. The specific steps were as follows: 1 μg of plasmid DNA was added to 100 μL of *E. coli*. After heat shock at 42°C and recovery culture on LB medium, the transformed bacteria were plated onto LB agar plates containing the corresponding antibiotics: pBV220-Flt3L-HA was treated with 100 μg / mL ampicillin, and pBAD-TIM-3 scFv-3xflag was treated with 34 μg / mL chloramphenicol. After overnight incubation, single colonies were picked and inoculated into LB medium containing the corresponding antibiotics; after overnight incubation, the bacterial culture was mixed with glycerol and stored at -80°C for later use.

[0026] For strains co-expressing two plasmids, pBV220-Flt3L-HA and pBAD-TIM-3 scFv-3xflag were co-transformed into competent cells, and transformants were screened on double-antibody LB plates containing ampicillin (100 μg / mL) and chloramphenicol (34 μg / mL).

[0027] The resulting engineered strains were named F@Ec (pBV220-Flt3L-HA), T@Ec (pBAD-TIM-3 scFv-3xflag), and FT@Ec (expressing two plasmids).

[0028] 2. According to OD 600At a concentration of 0.6% for engineered E. coli overexpressing Flt3L and TIM-3 scFv, the final concentration of NHS-PEG-CHO added was 5 mg / mL. The engineered E. coli overexpressing Flt3L and TIM-3 scFv was incubated with NHS-PEG-CHO in PBS at room temperature for 2 h to perform aldehyde modification. The mixture was dialyzed against ultrapure water (MWCO 100 kDa) to obtain aldehyde-functionalized E. coli. 3. Dissolve SKCN (0.5 mg) and DSPE-PEG2000-COOH (20 mg) separately in 0.5 mL of tetrahydrofuran (THF). After sonicating the two solutions in a 37°C water bath for 5 min, mix them and immediately add them to 10 mL of tetrahydrofuran aqueous solution (10% v / v). Sonicate for another 15 min (200 W, 40 Hz) to promote nanoparticle formation. Then remove THF by rotary evaporation. Filter the resulting dispersion through a 0.45 μm membrane and purify it by three centrifugal ultrafiltrations (Millipore, ultrafiltration membrane molecular weight cutoff MWCO 100 kDa). Collect the final concentrate, dry it, and obtain SNPs. Activate the SNPs in MES buffer using EDC / NHS. Then add TFA-hydrazide at a molar ratio of 1:9 for the activated SNPs and stir the reaction at room temperature for 3 h. Remove unreacted reagents by dialysis with ultrapure water for 24 h to obtain hydrazide-functionalized SNPs. 4. According to OD 600 At a concentration of 0.6 for aldehyde-functionalized E. coli, the final concentration of hydrazide-functionalized SNPs was 150 µg / mL. The aldehyde-functionalized E. coli and hydrazide-functionalized SNPs were mixed in sodium acetate buffer (pH 5.5) and stirred at room temperature for 4 h to form pH-sensitive hydrazone bonds. The resulting E. coli-SNP conjugate (denoted as FT@Ec-SNPs) was collected by centrifugation and filtration, washed three times with PBS, and resuspended in PBS for later use.

[0029] Experimental Example I. Experimental Methods: 1. Validation of protein expression in engineered strains To verify protein expression, strains F@Ec, T@Ec, and FT@Ec were cultured in LB medium containing the corresponding antibiotics. Cultured until OD... 600 Once the concentration reaches 0.5-0.6, protein expression is induced under optimized conditions.

[0030] F@Ec: Induction was performed at 42℃ for different times (5, 10, 15, 30 min), followed by continued culturing at 37℃. The culture supernatant was collected, and the level of secreted Flt3L-HA was detected by Western blot using an anti-HA antibody.

[0031] T@Ec: Different concentrations of L-arabinose (0.02%, 0.1%, 0.15%, 0.2%) were added, and protein expression was induced at 37°C. The culture supernatant after induction was collected, and the secretion level of TIM-3 scFv was detected by Western blot using an anti-Flag antibody to determine the optimal arabinose concentration.

[0032] FT@Ec: Sequential induction was employed: culture was first incubated at 42℃ for different times, and the culture supernatant was collected. The secreted Flt3 L-HA level was detected by Western blot using an anti-HA antibody. Subsequently, different concentrations of L-arabinose were added, and the culture was continued at 37℃ for 12 h. The culture supernatant after induction was collected, and the secretion level of TIM-3 scFv was detected by Western blot using an anti-Flag antibody.

[0033] 2. Photothermal properties and stability of SNPs: The photothermal properties of SNPs were evaluated using an infrared thermal imaging camera under 1064 nm laser irradiation.

[0034] Concentration dependence: SNP solutions of different concentrations (0, 25, 50 and 100 μg / mL) were exposed to 1.0 W / cm² laser irradiation for 12 min, and temperature changes were recorded every 3 min.

[0035] Power dependence: The SNPs solution (50 μg / mL) was irradiated with the same wavelength laser at different power densities (0, 0.5, 1.0 and 1.5 W / cm²), and the temperature was recorded every 3 min for 12 min.

[0036] Photothermal stability: The SNP solution (50 μg / mL) was subjected to three consecutive on-off cycles of irradiation under a 1064 nm laser at 1.5 W / cm², and temperature changes were recorded to generate heating and cooling curves. The photothermal conversion efficiency was calculated according to previously reported methods.

[0037] 3. ROS generation capacity of SNPs: The reactive oxygen species (ROS) generation capacity of SNPs was evaluated using DPBF and ABDA as chemical probes under 1064 nm laser irradiation. All experiments were conducted in the dark.

[0038] DPBF assay: DPBF was prepared to a concentration of 2 mg / mL (230 μM). Mixtures of DPBF with different concentrations of SNPs (0, 25, 50, and 100 μg / mL) were prepared at 1 W / cm². 2 The absorbance at 420 nm was recorded every 5 min under a 1064 nm laser for 20 min, and the decay kinetics curve of DPBF was plotted. Furthermore, a mixture of DPBF and SNPs (50 μg / mL) was subjected to different laser power densities (0, 0.5, 1, and 2 W / cm²). 2 Irradiation with a 1064 nm laser was performed, and the absorbance at 420 nm was recorded every 5 min for 20 min.

[0039] ABDA assay: ABDA was prepared to a concentration of 2 mg / mL (160 μM). ABDA was then mixed with different concentrations of SNPs (0, 25, 50, and 100 μg / mL) at 1 W / cm². 2 The sample was irradiated with a 1064 nm laser, and the absorption peak at 400 nm was measured every 5 min for 20 min to plot the degradation curve. Furthermore, a mixture of ABDA and SNPs (50 μg / mL) was subjected to different laser power densities (0, 0.5, 1, and 2 W / cm²). 2 Irradiation with a 1064 nm laser was performed, and the absorbance at 400 nm was recorded every 5 min for 20 min.

[0040] 4. In vitro cytotoxicity test: CCK-8 assay: CT26 cells were inoculated at a concentration of 1 × 10⁻⁶. 4 Cells were seeded at a density in 96-well plates and cultured for 12 h. Cells were then treated with different concentrations of SNPs (0, 25, 50, 100, and 150 μg / mL) for 24 h. Cells were divided into the following groups to assess their therapeutic potential under different conditions: control group, SNPs group, L group (laser irradiation alone), FT@Ec group, SNPs + L group, and FT@Ec-SNPs + L group. Cell viability was assessed by measuring absorbance at 450 nm using the CCK-8 assay after treatment. All experiments were repeated three times, and viability is expressed as a percentage of the control group.

[0041] 5. Cell viability and cytotoxicity assays: The effects of different treatments on cell viability and cytotoxicity were detected using the Calcein AM / PI assay kit. CT26 cells were seeded in 12-well plates (1×10⁶ cells / wells). 5Cells were cultured at 12 h per well. Cells were then co-incubated for 12 h with designated treatments (control group, SNPs group, L group, FT@Ec group, SNPs + L group, and FT@Ec-SNPs + L group). The SNPs concentration in all SNPs-containing groups was 50 μg / mL, and the FT@Ec concentration in all FT@Ec-containing groups was 2 × 10⁻⁶. 7 The concentration of FT@Ec-SNPs (CFU / mL) was determined by converting the SNP loading to an equivalent SNP concentration. Groups requiring light irradiation used a 1064 nm laser at 1.0 W / cm². 2 Irradiate at a power density of 1000 m / s for 5 min. After treatment, stain with Calcein AM and propidium iodide (PI) according to the kit instructions. Images are acquired using a fluorescence microscope to identify live cells (green) and dead cells (red). Each experiment is repeated three times.

[0042] 6. Intracellular ROS detection: Intracellular ROS levels were assessed using the DCFH-DA probe and observed using confocal laser scanning microscopy (CLSM). CT26 cells (1×10⁻⁶) were... 5 Cells were cultured in 12-well plates for 12 h, then treated with designated treatments (control group, SNPs group, L group, FT@Ec group, SNPs + L group, and FT@Ec-SNPs + L group) for 12 h. The SNPs concentration in all SNP-containing groups was 50 μg / mL, and the FT@Ec concentration in all FT@Ec-containing groups was 2 × 10⁻⁶. 7 The concentration of FT@Ec-SNPs (CFU / mL) was determined by converting the SNP loading to an equivalent SNP concentration. Groups requiring laser irradiation used a 1064 nm laser at 1.0 W / cm². 2 Irradiation for 5 min. After treatment, cells were washed and incubated with 0.2 μM DCFH-DA at 37°C for 30 min. After the final wash, cells were immediately analyzed by CLSM and flow cytometry. All experiments were repeated three times.

[0043] 7. Intracellular DNA damage detection: Intracellular DNA damage was assessed by γ-H2AX immunofluorescence staining. CT26 cells were seeded in confocal culture dishes (2×10⁻⁶). 5 Cells were cultured in dishes (cells / plate) for 12 h. Cells were then treated for 12 h with different methods: control group, SNPs group, L group, FT@Ec group, SNPs + L group, and FT@Ec-SNPs + L group. The SNPs concentration in all SNP-containing groups was 50 μg / mL, and the FT@Ec concentration in all FT@Ec-containing groups was 2 × 10⁻⁶. 7The concentration of FT@Ec-SNPs (CFU / mL) was determined by converting the SNP loading to an equivalent SNP concentration. Groups requiring light irradiation used a 1064 nm laser at 1.0 W / cm². 2 Irradiate for 5 min. After treatment, collect cells, wash, fix, and incubate with anti-γ-H2AX antibody for immunofluorescence staining. Observe stained samples under CLSM. All experiments were repeated three times.

[0044] 8. Apoptosis detection: Apoptosis in CT26 cells was assessed using the Annexin V-FITC / PI apoptosis detection kit. Cells were seeded in 12-well plates (1×10⁶ cells / wells). 5 Cells were cultured at 12 h per well. Cells were then treated for 12 h with different methods: control group, SNPs group, L group, FT@Ec group, SNPs + L group, and FT@Ec-SNPs + L group. The SNPs concentration in all SNPs-containing groups was 50 μg / mL, and the FT@Ec concentration in all FT@Ec-containing groups was 2 × 10⁻⁶. 7 The concentration of FT@Ec-SNPs (CFU / mL) was determined by converting the SNP loading to an equivalent SNP concentration. Groups requiring light irradiation used a 1064 nm laser at 1.0 W / cm². 2 Irradiate for 5 min. After irradiation, cells are incubated for 2 h, then collected and stained with Annexin V and PI according to the manufacturer's instructions. Apoptotic and necrotic cell populations are then distinguished by flow cytometry.

[0045] 9. Intracellular assessment of immunogenic cell death: CLSM was used to detect cell surface CRT exposure and HMGB1 release. CT26 cells were seeded in confocal culture dishes (2 × 10⁻⁶). 5 Cells were cultured in dishes for 12 h. Then, the cells were treated with different methods for 12 h: control group, SNPs group, L group, FT@Ec group, SNPs + L group, and FT@Ec-SNPs + L group. The SNPs concentration in all SNPs-containing groups was 50 μg / mL, and the FT@Ec concentration in all FT@Ec-containing groups was 2 × 10⁻⁶. 7 The concentration of FT@Ec-SNPs (CFU / mL) was determined by converting the SNP loading to an equivalent SNP concentration. Groups requiring light irradiation used a 1064 nm laser at 1.0 W / cm². 2 Irradiate for 5 min. After irradiation, cells are incubated for 2 h, then fixed, co-stained with anti-CRT and anti-HMGB1 antibodies, and imaged under CLSM.

[0046] 10. Evaluation of the efficacy of anti-tumor treatment: Establishment of an orthotopic colorectal cancer model: An orthotopic colorectal cancer model was established in 8-week-old female BALB / c mice. Mice were anesthetized with isoflurane, and the rectum was exposed by incision along the midline of the abdomen. CT26-Luc cells (5 × 10⁻⁶) were then introduced. 6 (Cells / mouse) were slowly injected into the rectal wall. Tumor-forming mice were randomly divided into four groups (n=5): control group, SNPs group, FT@Ec group, and FT@Ec-SNPs +L group. The SNPs concentration in the SNPs group was 3 mg / kg, and the FT@Ec concentration in the FT@Ec group was 2 × 10⁻⁶. 7 The concentration of CFU / mL and FT@Ec-SNPs was determined by converting the SNP loading to an equivalent SNP concentration (3 mg / kg). Treatment began on the same day (referred to as day 0). All formulations were administered intratumorally on days 0, 3, and 6. Six hours after each injection, mice were irradiated with a 1064 nm laser (1.0 W / cm²). 2 Mice were sacrificed on day 15, and primary colorectal tumors and major organs were collected. The collected tissues were fixed and subjected to H&E staining for histological analysis.

[0047] II. Experimental Results 1. Construction and protein expression validation of engineered bacterial systems: 1.1 Validation of temperature-induced Flt3L secretion: To verify the protein secretion capacity of engineered E. coli, Western blot results showed that Flt3L was successfully expressed in engineered E. coli after heat shock treatment at 42℃, and the expression gradually increased over time. Figure 3 This indicates that the temperature-induced system can effectively initiate Flt3L protein expression; 1.2 Verification of arabinose-induced TIM-3 scFv secretion: Western blot results showed that engineered *E. coli* successfully secreted TIM-3 scFv under L-arabinose induction; the expression level of the target protein increased in a concentration-dependent manner with increasing arabinose concentration, indicating that L-arabinose can effectively initiate TIM-3 scFv expression. Figure 4 ).

[0048] 1.3 Validation of secretion by co-expressing bacteria: Western blot results are as follows Figure 5 As shown, engineered E. coli successfully secreted TIM-3 scFv under L-arabinose induction; with increasing arabinose concentration, the expression level of the target protein increased in a concentration-dependent manner. In addition, after heat shock treatment at 42℃, engineered E. coli successfully expressed Flt3L, and the expression gradually increased over time.

[0049] 2. Photothermal properties and reactive oxygen species generation capacity of SNP nanoparticles: 2.1 Photothermal performance evaluation: The photothermal properties of SNPs under 1064 nm laser irradiation were evaluated. Infrared thermal imaging results showed that the temperature rise of the SNP solution was concentration-dependent: at 1.0 W / cm², the temperature rise was at a concentration of 1064 nm. 2 After laser irradiation, the temperatures of the 25, 50, and 100 μg / mL SNPs solutions showed a significant increasing trend, while the control group (0 μg / mL) only showed a slight temperature increase. Figure 6 Power-dependent experiments showed that at a concentration of 100 μg / mL SNPs, as the laser power density increased from 0.5 W / cm², the power dependence increased. 2 As the concentration increased to 1.5 W / cm², the solution temperature exhibited a significant gradient increase. Figure 7 Photothermal cycling stability experiments showed that after three consecutive heating-cooling cycles, the maximum temperature of SNPs did not decrease significantly, indicating that they have good photothermal stability. Figure 8 ).

[0050] 2.2 Assessment of reactive oxygen species generation capacity: The reactive oxygen species (ROS) generation capacity of SNPs was assessed using DPBF and ABDA probes. DPBF assay results showed that SNPs accelerated DPBF degradation in a concentration-dependent manner under 1064 nm laser irradiation. Figure 9 Power-dependent experiments show that as laser power density increases, the ROS generation efficiency significantly improves. Figure 10 The ABDA probe detection results are consistent with the DPBF trend. Figure 11 , Figure 12 This further confirms that SNPs can efficiently generate ROS under 1064 nm laser irradiation.

[0051] 3. In vitro cytotoxicity and therapeutic mechanism: 3.1 Cell viability assay (CCK-8): The in vitro biocompatibility and antitumor therapeutic efficacy of SNPs were evaluated using the CCK-8 assay. First, to verify the biocompatibility of SNPs, different concentrations of SNPs (0, 25, 50, 100, and 150 μg / mL) were co-incubated with CT26 cells for 24 h. Figure 13 As shown, even at the highest concentration (100 μg / mL) without laser irradiation, SNPs did not significantly affect cell viability, and cell survival remained at around 90%, indicating that SNPs have good biocompatibility. Similarly, co-incubating CT26 cells with different concentrations of FT@Ec showed that cell viability remained at a high level at all tested concentrations. Figure 14 This demonstrated that the engineered bacteria themselves had no significant toxicity to tumor cells. Subsequently, the antitumor effects of each treatment group were evaluated. Cells were divided into the following six groups: control group, SNPs group, L group, FT@Ec group, SNPs + L group, and FT@Ec-SNPs + L group. Figure 15 As shown, cell viability in the control group, SNPs group, L group, and FT@Ec group did not show significant changes, indicating that SNPs alone, laser irradiation alone, or engineered bacteria alone could not effectively kill tumor cells. In contrast, cell viability in the SNPs + L group and the FT@Ec-SNPs + L group was significantly reduced, demonstrating the anti-tumor effect of photothermal therapy.

[0052] 3.2 Live / Dead Cell Staining (Calcein-AM / PI): Calcein-AM / PI fluorescence staining results were consistent with the CCK-8 trend. In the control group, SNPs group, L group, and FT@Ec group, the vast majority of cells showed green fluorescence (live cells), with no significant red fluorescence observed, indicating that SNPs alone, laser irradiation alone, or engineered bacteria alone could not effectively induce tumor cell death. In the SNPs + L group and the FT@Ec-SNPs + L group, the number of red fluorescent cells significantly increased, while the number of green fluorescent cells significantly decreased. Figure 16 This further confirms that the synergistic effect of engineered bacteria and photothermal therapy has a highly efficient tumor cell killing ability.

[0053] 3.3 Intracellular reactive oxygen species detection (DCFH-DA): The results of DCFH-DA fluorescent probe detection showed that ( Figure 17 ), streaming data analysis results as follows Figure 18 As shown, by Figures 17-18 It was found that the intracellular ROS levels were extremely low in the control group, SNPs group, L group, and FT@Ec group. The intracellular green fluorescence was significantly enhanced in the SNPs + L group and the FT@Ec-SNPs + L group, indicating that engineered bacteria combined with laser irradiation can efficiently induce intracellular ROS production.

[0054] 3.4 Apoptosis Detection (Annexin V-FITC / PI): Flow cytometry results showed ( Figure 19 The apoptosis rates in the control group, SNPs group, L group, and FT@Ec group were all low. However, the apoptosis rates in the SNPs + L group and the FT@Ec-SNPs + L group were significantly higher, confirming that the synergistic effect of engineered bacteria and photothermal therapy can effectively induce tumor cell apoptosis.

[0055] 3.5 DNA damage detection (γ-H2AX): Immunofluorescence staining results showed ( Figure 20 In the control group, SNPs group, L group, and FT@Ec group, γ-H2AX focal points (DNA damage markers) were not obvious. The SNPs + L group and the FT@Ec-SNPs + L group showed significant γ-H2AX positive signals, indicating that engineered bacteria combined with laser irradiation caused severe DNA damage.

[0056] 3.6 Immunogenic cell death (ICD) assessment: CRT exposure: Confocal microscopy results showed ( Figure 21 In the control group, SNPs group, L group, and FT@Ec group, CRT expression on the cell surface was extremely low. Significant CRT membrane translocation (green fluorescence) was observed in the SNPs + L group and the FT@Ec-SNPs + L group, indicating that the synergistic effect effectively induced CRT exposure.

[0057] HMGB1 release: Confocal microscopy results show ( Figure 22 In the control group, SNPs group, L group, and FT@Ec group, HMGB1 was mainly located in the cell nucleus. The nuclear fluorescence signal was significantly reduced in the SNPs + L group and the FT@Ec-SNPs + L group, confirming that the combined treatment successfully induced the release of HMGB1.

[0058] 4. In vivo anti-tumor efficacy evaluation: 4.1 Treatment efficacy of in situ colorectal cancer model: A CT26-Luc orthotopic colorectal cancer model was established to evaluate the in vivo antitumor effects of different treatment regimens.

[0059] Gross images of tumors from different anatomical groups are shown. Figure 23 The tumors in the control group were larger, while those in the FT@Ec-SNPs+L group were significantly smaller, with almost complete tumor regression in mice. These results indicate that FT@Ec-SNPs+L combined treatment can significantly inhibit colorectal cancer tumor growth. Histopathological analysis was performed on the heart, liver, spleen, lungs, and kidneys of mice after nanomedicine administration. Compared with the blank control group, no significant histopathological damage was observed in any of the major organs in the drug-treated groups. Figure 24 The above results indicate that FT@Ec-SNPs+L exhibits good in vivo biocompatibility, low systemic tissue and organ toxicity, and has the potential for safe in vivo drug delivery.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A method for preparing an engineered E. coli dual-response delivery system loaded with SNPs, characterized in that, The preparation method includes the following steps: (a) Engineered E. coli overexpressing Flt3L and TIM-3 scFv were co-incubated with NHS-PEG-CHO to obtain aldehyde-functionalized E. coli; (b) Under ultrasonic conditions, a tetrahydrofuran mixed solution of SKCN and DSPE-PEG-COOH and an aqueous solution of tetrahydrofuran were mixed and ultrasonically treated. The organic solvent was then removed by rotary evaporation. The mixture was then concentrated by membrane filtration, ultrafiltration, and drying to obtain SNPs. The SNPs were activated by EDC / NHS. The activated SNPs were then reacted with TFA-acylhydrazide to obtain acylhydrazide-functionalized SNPs. (c) Under acidic conditions, aldehyde-functionalized Escherichia coli and hydrazide-functionalized SNPs are reacted, centrifuged, and washed to obtain the engineered Escherichia coli dual-response delivery system loaded with SNPs.

2. The preparation method according to claim 1, characterized in that, In step (a), the engineered E. coli overexpressing Flt3L and TIM-3scFv is obtained by co-transforming the recombinant plasmid expressing Flt3L and the recombinant plasmid expressing TIM-3scFv into E. coli and then screening them.

3. The preparation method according to claim 1, characterized in that, In step (a), the incubation time is 1.5-3 hours, and the temperature is room temperature; when engineered E. coli overexpressing Flt3L and TIM-3 scFv OD 600 When the concentration is 0.5~1.0, the final concentration of NHS-PEG-CHO added is 1~10 mg / mL.

4. The preparation method according to claim 1, characterized in that, In step (b), the mass ratio of SKCN to DSPE-PEG-COOH in the tetrahydrofuran mixed solution of SKCN and DSPE-PEG-COOH is 1:(30~50), wherein the concentration of SKCN is 0.1~1.0 mg / mL; the volume ratio of the tetrahydrofuran mixed solution of SKCN and DSPE-PEG-COOH to the tetrahydrofuran aqueous solution is 1:(8~12); and the volume concentration of the tetrahydrofuran aqueous solution is 8%~12%. Ultrasonic treatment for 15-25 minutes, ultrasonic power of 150-250W, frequency of 30-50Hz; The membrane used for membrane filtration concentration has a pore size of 0.45 μm; the ultrafiltration membrane used for ultrafiltration concentration has a molecular weight cutoff of 90~110 KD.

5. The preparation method according to claim 1, characterized in that, In step (b), the reaction time is 2.5-4 hours and the temperature is room temperature; the molar ratio of activated SNPs to TFA-hydrazide is 1:(8-10).

6. The preparation method according to claim 1, characterized in that, In step (c), the pH value of the acidic conditions is 5.0~6.

0.

7. The preparation method according to claim 1, characterized in that, In step (c), when aldehyde-functionalized E. coli OD 600 When the concentration is 0.5~1.0, the final concentration of hydrazide-functionalized SNPs added is 50~300 μg / mL.

8. The engineered E. coli dual-response delivery system loaded with SNPs prepared by the method of any one of claims 1 to 7.

9. The application of the engineered Escherichia coli dual-response delivery system loaded with SNPs prepared by the preparation method of any one of claims 1 to 7 in the preparation of antitumor products.

10. The application according to claim 9, characterized in that, The tumor is colorectal cancer.

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