Preparation and application of bacterial-loaded cationized photosensitizer complex for tumor photothermal / photodynamic therapy

Abstract

The application provides a bacteria-light sensitizer compound and a preparation method and application thereof, and relates to the technical field of nanomedicine. The bacteria-light sensitizer compound is composed of a light sensitizer and bacteria, and the bacteria and the light sensitizer are combined through electrostatic adsorption. The light sensitizer is a cationized near-infrared cyanine light sensitizer, and can be combined with the surface of the negatively charged bacteria. The compound aims to solve the problem that the existing bacteria treatment has poor tumor treatment effect. The bacteria-light sensitizer compound provided by the application can electrostatically adsorb a photothermal / photodynamic light sensitizer on the surface of the bacteria, thereby improving the safety and targeting of the bacteria in the body, and realizing the combined application of the bacteria treatment and the photothermal / photodynamic treatment, so that the effectiveness of the tumor treatment is significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to the biomedical field of a bacterial-photosensitizer complex for anti-tumor treatment. Specifically, it relates to the design of a cationized near-infrared cyanine photosensitizer, the preparation method of the complex, and its application in anti-tumor bacterial-photothermal / photodynamic combined therapy. Background Technology

[0002] Cancer remains one of the leading causes of death worldwide, posing a major threat to human health. [1] Despite significant advancements in oncology research for diagnosis and treatment, numerous challenges remain, including the complexity of early cancer diagnosis, the high heterogeneity of tumors, drug resistance, and the toxic side effects of treatment. [2-3] Against this backdrop, the core objective that researchers are constantly pursuing is to ensure therapeutic efficacy while avoiding collateral damage to healthy tissues.

[0003] In recent years, research has shown that bacterial-mediated antitumor therapy has once again become a research hotspot due to its unique mechanism. The history of this therapy can be traced back to 1891, when William Coley first used "Coley toxin," prepared from Streptococcus pyogenes and Serratia marcescens, to treat patients with inoperable cancers and observed positive therapeutic effects, bringing bacterial therapy into the field of cancer treatment research. [4] In recent years, with the development of modern immunology and synthetic biology technologies, the molecular mechanisms of bacterial-mediated tumor therapy and its clinical application potential have been elucidated in greater depth, and the unique advantages of this therapy have once again attracted widespread attention.

[0004] Bacteria naturally possess tumor-targeting capabilities, surviving in the hypoxic, low-pH tumor microenvironment. This allows them not only to induce host immune activation but also to act as delivery vectors for anti-tumor drugs and genes, eliminating tumor cells through multiple mechanisms. [5] In addition, bacteria can exert anti-tumor effects by secreting cytotoxins or directly interfering with tumor cell signaling pathways. [6] Furthermore, the application of gene editing technology allows researchers to reduce the virulence of wild-type bacteria and, through synthetic biology strategies, endow them with non-inherent functions, such as tumor microenvironment sensing, controlled expression, targeted delivery, and biosynthesis, thereby further enhancing their potential for bacterial therapy. [7-8] .

[0005] Single-dose bacterial therapy has shown limited tumor-suppressive effects in clinical applications, and is accompanied by dose-dependent toxicity issues, limiting its effectiveness in preventing tumor metastasis and achieving complete eradication. [9]Given current advancements in medical technology, combining bacterial-mediated biotherapy with traditional treatment strategies (such as chemotherapy, photothermal therapy, radiotherapy, immunotherapy, and bacterial-mediated prodrug activation therapy) has yielded significant results in research and clinical applications. [10-14] The combined treatment approach demonstrates three significant advantages: First, the synergistic effect of different treatment methods can significantly enhance the anti-tumor efficacy; second, utilizing bacteria with tumor-targeting properties can reduce systemic toxicity and treatment-related side effects; and finally, this strategy can effectively activate the body's systemic immune response, thereby inhibiting tumor metastasis and reducing the risk of recurrence.

[0006] Phototherapy is a local treatment strategy that utilizes light of specific wavelengths to act on photosensitive materials, thereby triggering a series of biological effects. Photothermal therapy (PTT) relies on photothermal agents (such as noble metal nanomaterials, carbon-based materials, and organic dyes) absorbing near-infrared (NIR) light and efficiently converting it into heat energy, thus inducing an increase in local tissue temperature, leading to tumor cell apoptosis or necrosis. Photodynamic therapy (PDT), on the other hand, utilizes photosensitizers to generate reactive oxygen species (ROS) under the excitation of specific wavelengths of light, inducing cell damage and immune responses. Phototherapy is minimally invasive, highly targeted, and controllable, enabling precise local treatment while reducing systemic toxicity. Although photosensitizers have high photothermal conversion efficiency, traditional photosensitizers generally suffer from low water solubility, poor pharmacokinetic properties, or potentially high toxicity. To overcome these limitations, various functional materials are widely used in the delivery of photothermal agents to improve their water solubility, circulating half-life, and biocompatibility.

[16] However, although nanoformulations can be used to load and deliver photosensitizers, their effective enrichment at tumor sites remains a challenge. Studies have shown that the overall enrichment rate of photosensitizers delivered via intravenous injection at tumor sites is less than 1.4%, making it difficult to achieve an effective therapeutic dose. Even with local injection strategies, the diffusion capacity of photosensitizers in distal tumor tissues is still limited due to factors such as high interstitial fluid pressure within the tumor and the barrier effect of the dense extracellular matrix, leading to uneven heating of the tumor tissue and ultimately treatment failure.

[16] .

[0007] The combined treatment of bacterial infections and phototherapy, as an innovative treatment strategy, has shown broad application potential in the treatment of a variety of diseases. [17-18]This strategy combines the advantages of both bacterial therapy and phototherapy, primarily in the following aspects: First, the combined treatment significantly enhances the targeting of the treatment. Bacteria can serve as highly efficient delivery carriers, specifically accumulating at the lesion site, especially in hypoxic tumor areas where traditional therapies are difficult to effectively target.

[19] This characteristic allows phototherapy to precisely target tumor tissue, enhancing the local effect of treatment while reducing damage to surrounding normal tissues, thereby improving the specificity and effectiveness of the treatment. Secondly, combined therapy can enhance the overall therapeutic effect. Phototherapy directly induces tumor cell death through local high temperatures, causing tumor cell lysis and releasing tumor-associated antigens (TAAs), thus activating the body's immune system. This immune activation effect not only helps to eliminate residual tumor cells but also promotes the establishment of anti-tumor immune responses, improving the long-term efficacy of treatment. Finally, combined therapy can effectively reduce the risk of drug resistance. Phototherapy kills tumor cells through physical high-temperature effects, rather than relying on the biological activity of chemical drugs, and therefore does not directly induce drug resistance in bacteria or tumor cells. This feature is particularly crucial for long-term treatment or diseases prone to relapse, helping to maintain the continuity and stability of treatment and improving its clinical application value. Summary of the Invention

[0008] This invention aims to provide an immunotherapy strategy based on bacterial-loaded cationic photosensitizers for the combination therapy of solid tumors. This strategy combines bacterial therapy with photothermal / photodynamic therapy (PTT / PDT) to improve the deep delivery efficiency of the photosensitizer in tumor tissue, thereby overcoming the safety issues and limited therapeutic effects of bacterial therapy alone. Specifically, this invention utilizes electrostatic interactions to load the newly synthesized photosensitizer Cy-BnNM-NH2 into attenuated Salmonella VNP20009, constructing a novel bacterial-mediated PTT / PDT combination therapy system to enhance tumor targeting and promote anti-tumor immune responses. This strategy features a simple and high-yield preparation process suitable for industrial production. The resulting bacterial-photosensitizer complex exhibits excellent biocompatibility, efficient photothermal conversion capability, strong reactive oxygen species (ROS) generation capacity, and precise tumor targeting characteristics, providing a new solution for the efficient treatment of solid tumors.

[0009] To achieve the above objectives, the first aspect of the present invention provides a cationic photosensitizer Cy-BnNM-NH2, the chemical structure of which is shown in formula (1).

[0010]

[0011] Secondly, the present invention also provides a method for preparing the above-mentioned probe, comprising the following steps:

[0012]

[0013] Thirdly, this invention provides a schematic diagram of the VNP20009@Cy-BnNM-NH2 complex in adjuvant tumor phototherapy and enhanced immunotherapy.

[0014] Fourthly, this invention provides the photothermal and photodynamic properties of VNP20009@Cy-BnNM-NH2.

[0015] Fifthly, the present invention provides detailed characterization data for the VNP20009@Cy-BnNM-NH2 complex.

[0016] In a sixth aspect, the present invention evaluated the phototoxicity and dark toxicity of the VNP20009@Cy-BnNM-NH2 complex.

[0017] In a seventh aspect, the present invention demonstrates the results of in vivo antitumor experiments on the VNP20009@Cy-BnNM-NH2 complex. Attached Figure Description

[0018] Figure 1 This describes the process and mechanism of the VNP20009@Cy-BnNM-NH2 complex in assisting tumor phototherapy and enhancing immunotherapy.

[0019] Figure 2 This indicates the photothermal properties of Cy-BnNM-NH2.

[0020] Figure 3 This represents the photodynamic properties of Cy-BnNM-NH2.

[0021] Figure 4 The binding characterization results of VNP20009@Cy-BnNM-NH2 are shown.

[0022] Figure 5 This indicates the phototoxicity and dark toxicity assessment of the VNP20009@Cy-BnNM-NH2 complex.

[0023] Figure 6 This indicates the phototoxicity of the VNP20009@Cy-BnNM-NH2 complex at the cellular level.

[0024] Figure 7 This indicates that the VNP20009@Cy-BnNM-NH2 complex produces reactive oxygen species at the cellular level.

[0025] Figure 8 This indicates the in vivo tumor-suppressive ability of the VNP20009@Cy-BnNM-NH2 complex. Detailed Implementation

[0026] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0027] In a first aspect, the present invention provides a cationized photosensitizer Cy-BnNM-NH2 with the structure shown in formula (1);

[0028]

[0029] Example 1: Preparation steps of photosensitizer Cy-BnNM-NH2

[0030]

[0031] Preparation Example 1, Synthesis of Compound 2

[0032] 1,6-Dibromohexane (5.4 g, 22 mmol) was dissolved in 5 mL of acetone, and the mixture was heated to 50 °C. Compound 1 (1 g, 7.4 mmol) was dissolved in 6 mL of acetone, and then slowly added dropwise to the reaction flask through a dropping funnel. After the addition was complete, the reaction was carried out under argon protection for 16 h. After the reaction was completed, the white solid formed was washed with acetone, filtered, and collected. The solid was dissolved in a small amount of methanol and then recrystallized in acetone to give compound 2 in 93% (2.0 g) yield.

[0033] Preparation Example 2, Synthesis of Compound 3:

[0034] 2,3,3-Trimethyl-3H-indole (0.62 g, 3.3 mol) and compound 2 (1 g, 3.3 mol) were dissolved in 20 mL of toluene, and the mixture was heated under reflux for 24 hours. After cooling to room temperature, the purple solid was collected by filtration. The solid was washed three times with toluene, and compound 3 was purified by silica gel column chromatography (12% MeOH / DCM) in 36% (455 mg) yield.

[0035] Preparation Example 3, Synthesis of Compound 5:

[0036] Compound 4 (91.2 mg, 0.528 mmol) was dissolved in acetic anhydride (10 mL), and sodium acetate (86.6 g, 1.05 mmol) was added. Compound 3 (400 mg, 1.05 mmol) was slowly added, and the mixture was stirred at 40 °C for 4 h under argon protection. After the reaction was complete, the reaction liquid was poured into 60 mL of diethyl ether, and the precipitated green color was collected.

[0037] Solid. Compound 5 was purified by silica gel column chromatography (8% MeOH / DCM) in 45.6% (214 mg).

[0038] Preparation Example 4, Synthesis of compound Cy-BnNM-NH2:

[0039] Compound 1,1-dimethylethyl (4-hydroxyphenyl)carbamate (52.7 mg, 0.252 mmol) was dissolved in DMF (2 mL), and potassium carbonate (46.4 mg, 0.336 mmol) was added. The reaction mixture was stirred for 30 minutes at room temperature under argon protection, and compound 5 (150 mg, 0.168 mmol) was added. The mixture was heated to 80 °C and stirred for 4 hours. After the reaction was complete, the reaction mixture was diluted with 15 mL of DCM and washed five times with water. The organic layer was dried over anhydrous sodium sulfate and concentrated. After drying, trifluoroacetic acid (1.2 mL, 16.8 mmol) was slowly added dropwise to the concentrate under argon protection and an ice bath. The reaction was carried out at room temperature for 15 minutes, followed by distillation under reduced pressure using DCM. The concentrate was dissolved in a small amount of methanol, and 10 times its volume of ethyl acetate was added. Recrystallization yielded a green solid, Cy-BnNM-NH2, in a yield of 43% (68 mg).

[0040] Pharmacological experiments

[0041] Experimental Example 1: Photothermal Properties of Cy-BnNM-NH2

[0042] Different concentrations of Cy-BnNM-NH2 were prepared and irradiated with laser for 10 min (785 nm, 1.2 W / cm²). 2 During the process, an infrared thermal imager was used to record temperature changes, and the heating-cooling curves were further analyzed. Combined with the relevant time constant τs, the photothermal conversion efficiency of Cy-BnNM-NH2 was determined. Furthermore, to evaluate the photothermal stability of Cy-BnNM-NH2, heating-cooling curves were generated under four irradiation cycles; the results are shown below. Figure 2 It can be seen that as the concentration of photosensitizer increases, the temperature also increases; 50 μM photosensitizer can raise the temperature by 26 °C; the photothermal conversion efficiency of 50 μM photosensitizer is 54.9%; and this photosensitizer has good photostability.

[0043] Experimental Example 2: Photodynamic Properties of Cy-BnNM-NH2

[0044] To evaluate the ability of Cy-BnNM-NH2 to generate reactive oxygen species (ROS) in solution, we used the ROS fluorescent probe DCFH-DA (2,7-dichlorofluorescein diacetate) for detection. First, under light-protected conditions, 10 mM NaOH was added, and DCFH-DA (1 mM) was hydrolyzed at room temperature to generate DCFH. Then, Tris buffer (pH 7.4) was added to terminate the deacetylation process. The DCFH solution was further diluted to 40 μM with Tris buffer and stored frozen. Subsequently, Cy-BnNM-NH2 was diluted to 10 μM with the above DCFH solution in a 96-well plate. Then, a laser (λ) was used to detect the ROS generation. ex =785nm, 1.2W / cm 2 Irradiation for 30 seconds, then fluorescence intensity (λ) is recorded every 5 seconds using a microplate reader. ex =480nm, λ em =525nm), n=3, results are shown in Figure 3 (a).

[0045] For the detection of superoxide anion (O2·-), the indicator diethyldihydrogen ion (DHE, 0.82 mg) was dissolved in 1.3 mL of dimethyl sulfoxide to obtain a 2 mM diethyldihydrogen ion DMSO solution. The diethyldihydrogen ion indicator solution should be stored in a light-protected refrigerator at 4°C for later use. When using, the diethyldihydrogen ion solution was added to Tris buffer (20 mM, pH 7.4) to prepare a 20 μM diethyldihydrogen ion Tris solution. Cy-BnNM-NH2 was diluted to 10 μM with the above DHE-containing Tris buffer solution and added to a 96-well plate. The laser (λ) was used for detection. ex =785nm, 1.2W / cm 2 Irradiation was performed for 5 seconds, with fluorescence intensity recorded every 5 seconds using a microplate reader. This process was repeated 6 times, for a total irradiation time of 30 seconds. The excitation wavelength was 535 nm, and the emission wavelength was 610 nm. Results are shown below. Figure 3 (b)

[0046] 1,3-Diphenylisobenzofuran (DPBF) was used as a singlet oxygen indicator. The probe and DPBF solution were mixed in DMSO, yielding absorbances of approximately 0.2 and 1.0 at 415 nm, respectively. The solution was then placed in a 96-well plate and analyzed using a laser (785 nm, 1.2 W / cm²). 2 Irradiation was performed, and the DPBF absorbance was immediately measured. ICG was selected as the reference compound (Φ in DMSO). Δ ICG = 7.7%

[20] ), calculate Φ according to the following equation Δ probe = Φ ΔICG × (kprobe / kICG) × (FICG / Fprobe), where k is the slope of the absorbance curve of DPBF at 415 nm over time, and F = 1-10. -O.D (OD is the optical density at 785 nm). See the results below. Figure 3 (c) In the presence of Cy-BnNM-NH2 and ICG, the absorption intensity of DPBF decreased by 37% and 79% respectively within 3 min. ICG (ΦΔ=7.7%), Cy-BnNM-NH2... 1 O2 quantum yield (ΦΔ=2.74%).

[0047] Experimental Example 3: Binding Characterization of the VNP20009@Cy-BnNM-NH2 Complex

[0048] VNP20009 binds to Cy-BnNM-NH2 via electrostatic adsorption. The change in zeta potential confirmed the electrostatic interaction between the photosensitizer and VNP20009. Results are shown in [Figure number missing]. Figure 4 (a) Cy-BnNM-NH2 changed the bacterial zeta potential from -32 mV to -15 mV. Furthermore, to further understand the effect of this binding on the optical properties of the material, the changes in absorption spectra before and after binding were measured; the results are shown in […]. Figure 5 (b) Finally, to quantitatively evaluate the binding efficiency of Cy-BnNM-NH2 to VNP20009, we used ultra-high performance liquid chromatography (UHPLC) for precise determination. The results are shown in [Figure number missing]. Figure 4 (c) The results show that the probe has a binding rate of 88.5% with the 50 μM photosensitive material.

[0049] Experimental Example 4: Phototoxicity and dark toxicity of the VNP20009@Cy-BnNM-NH2 complex

[0050] 1. Phototoxicity

[0051] 4T1 cell suspension was prepared at a ratio of 4 × 10⁴ cells per well. 3 200 μL of cells were seeded into 96-well plates, with three parallel wells for cell control. Cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. The culture medium was discarded, and VNP20009@Cy-BnNM-NH2 PBS solution was added to the 96-well plates, along with 100 μL of blank control solution per well. Cells were incubated at 37°C in a 5% CO2 incubator for 1 hour. A 785 nm laser lamp at 1.2 W / cm² was then used. 2Irradiate at the specified intensity for 5 minutes. Discard the culture medium, add 110 μL / well of 10% CCK8 DMEM culture medium, and incubate at 37°C for 30 minutes. Then, measure the absorbance of each well at 450 nm using a Tecan Spark 10M multi-functional microplate reader. Calculate cell viability: Cell viability (%) = (A - A0) / (As - A0) × 100%. A represents the absorbance of the experimental group, As represents the absorbance of the blank control group, and A0 represents the absorbance of the cell-free culture medium group. Results are shown below. Figure 5 (a) The results show that VNP20009@Cy-BnNM-NH2 has significant phototoxicity.

[0052] 2. Dark Poison

[0053] 4T1 cell suspension was prepared at a ratio of 4 × 10⁴ cells per well. 3 Two hundred and ten cells (200 μL) were seeded into 96-well plates, with three parallel wells for each group serving as cell control wells. The plates were incubated at 37°C in a 5% CO2 incubator for 24 hours. The culture medium was discarded, and Cy-BnNM-NH2 solutions (0-100 μM) prepared in DMEM medium were added to the wells, along with 100 μL of blank control solution per well. The plates were incubated at 37°C in a 5% CO2 incubator for 24 hours. The culture medium was then discarded, and 110 μL of 10% CCK8 DMEM medium was added to each well. After incubation at 37°C for 30 minutes, the absorbance at 450 nm was measured using a Tecan Spark 10M multi-functional microplate reader. Cell viability was calculated as: Cell viability (%) = (A-A0) / (As-A0) × 100%. A represents the absorbance of the experimental group, As represents the absorbance of the blank control group, and A0 represents the absorbance of the cell-free culture medium group. Each experiment was repeated three times. Results are shown below. Figure 5 (b) The results showed that Cy-BnNM-NH2 had no obvious dark toxicity.

[0054] Experimental Example 5: Cellular Phototoxicity of VNP20009@Cy-BnNM-NH2 Complex

[0055] 4T1 cells (300 μL, 4 × 10⁻⁶) were added. 4 VNP20009@Cy-BnNM-NH2 and 1 μg / mL PIPBS solution were seeded in 8-well coverslips for laser confocal fluorescence imaging (Thermo Fisher, Nunc LAB-Tek TMII). After incubation at 37°C and 5% CO2 for 24 hours, the old culture medium was removed, and the cells were washed twice with PBS buffer. VNP20009@Cy-BnNM-NH2 and 1 μg / mL PIPBS solution were added to each well, and the cells were incubated for another hour. After 1 hour, the cells were then incubated using a 785nm laser at 1.2 W / cm².2 Cells were irradiated at a specific intensity for 5 minutes. After washing three times with PBS, 20 μg / mL Hoechst 33324 and phenol red-free medium were added, and the cells were incubated at 37°C in a 5% CO2 incubator for 30 minutes before laser confocal fluorescence imaging. All images were acquired using a Leica TCS SP8 X laser confocal scanning microscope (63x oil immersion). PI excitation wavelength was 488 nm, and fluorescence emission collection wavelength was 600-650 nm; Hoechst 33324 excitation wavelength was 405 nm, and fluorescence emission collection wavelength was 440-490 nm. Results are shown below. Figure 6 .

[0056] Experiment Example 6: Experiment on ROS and Superoxide Anion Production at the Cellular Level using VNP20009@Cy-BnNM-NH2

[0057] 4T1 cells (300 μL, 4 × 10⁻⁶) were added. 4 VNP20009@Cy-BnNM-NH2 and 40 μM DCFH-DAPBS solutions were seeded in 8-well coverslips (Thermo Fisher, Nunc LAB-Tek TMII) for laser confocal fluorescence imaging. After 24 hours of incubation at 37°C and 5% CO2, the old culture medium was removed, and the cells were washed twice with PBS buffer. VNP20009@Cy-BnNM-NH2 and 40 μM DCFH-DAPBS solution were added to each well, and the cells were incubated for another hour. After 1 hour, the cells were seeded using a laser (785 nm, 1.2 W / cm²). 2 Irradiation for 5 minutes. After washing cells three times with PBS, 20 μg / mL Hoechst 33324 and phenol red-free medium were added, and the cells were incubated at 37°C in a 5% CO2 incubator for 30 minutes before laser confocal fluorescence imaging. Images were acquired using a Leica TCS SP8 X laser confocal scanning microscope (63x oil immersion). The excitation wavelength of DCFH-DA was 488 nm, and the fluorescence emission collection wavelength was 495-550 nm; the excitation wavelength of Hoechst 33324 was 405 nm, and the fluorescence emission collection wavelength was 440-490 nm. Results are shown below. Figure 7 (a).

[0058] 4T1 cells (300 μL, 4 × 10⁻⁶) were added. 4VNP20009@Cy-BnNM-NH2 and 20 μM DHE PBS solution were seeded in 8-well coverslips (Thermo Fisher, Nunc LAB-Tek TMII) for laser confocal fluorescence imaging. After 24 hours of incubation at 37°C and 5% CO2, the old culture medium was removed, and the cells were washed twice with PBS buffer. VNP20009@Cy-BnNM-NH2 and 20 μM DHE PBS solution were added to each well, and the cells were incubated for another hour. After 1 hour, the cells were seeded using a laser (785 nm, 1.2 W / cm²). 2 Irradiation for 5 minutes. After washing cells three times with PBS, 20 μg / mL Hoechst 33324 and phenol red-free medium were added, and the cells were incubated at 37°C in a 5% CO2 incubator for 30 minutes before laser confocal fluorescence imaging. Images were acquired using a Leica TCS SP8 X laser confocal scanning microscope (63x oil immersion). DHE excitation wavelength was 535 nm, and fluorescence emission collection wavelength was 600-650 nm; Hoechst 33324 excitation wavelength was 405 nm, and fluorescence emission collection wavelength was 440-490 nm. Results are shown below. Figure 7 (b)

[0059] Experiment Example 7: In vivo verification of the tumor-suppressive and immune-activating capabilities of VNP20009@Cy-BnNM-NH2

[0060] First, we established a 4T1 tumor model using BALB / c mice. When the tumor size grew to approximately 100 mm... 3 Mice were randomly divided into groups and treated with PBS (Group I), VNP20009 (Group II), VNP20009+ light (Group III), VNP20009@Cy-BnNM-NH2 (Group IV), and VNP20009@Cy-BnNM-NH2+ light (Group V). Each group received the drug via tail vein injection. Twenty-four hours after injection, mice were treated with a 785nm laser (1.2W / cm²). 2 Irradiate the tumor for 5 minutes. Record the body weight and tumor volume of each mouse every 2 days. Figure 8 As shown in (a), (b), (c), and (d), compared to group I mice, group V mice showed a significant slowdown in tumor growth during treatment. Figure 8 (e)), and the tumor size was significantly reduced ( Figure 8 (h, i). Furthermore, during the overall treatment process, the mice's body weight did not change significantly. Figure 8 (f)). H&E staining of tumor tissue sections after day 14 of treatment in different experimental groups confirmed that VNP20009@Cy-BnNM-NH2 could induce more B16 tumor cell death under light activation. Figure 8 (k)) to demonstrate the ability of VNP20009@Cy-BnNM-NH2 bacteria in combination with photothermal therapy for tumors at the in vivo level.

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Claims

1. A bacteria-photosensitizer complex, characterized in that, It includes a photosensitizer and bacteria, wherein the bacteria and the photosensitizer are connected by electrostatic adsorption; preferably, the bacteria and the photosensitizer are combined by electrostatic adsorption.

2. The bacterial-photosensitizer complex according to claim 1, characterized in that, The photosensitizer is a cationic near-infrared cyanine photosensitizer, and the photosensitizer has the structure shown in formula (1):

3. The bacteria-photosensitizer complex according to claim 2, characterized in that, The preparation method of the cationic near-infrared cyanine photosensitizer includes condensing the compound shown in formula (2) with the Vilsmeier intermediate in an organic solvent to generate a tricationic heptamethylcyanine photosensitizer; 4. The method for preparing a bacterial-photosensitizer complex according to claim 1, characterized in that, The process includes the following steps: mixing bacteria and photosensitizer, so that the bacteria and photosensitizer bind together through electrostatic adsorption, thus obtaining a bacteria-photosensitizer complex.

5. The use of the bacterial-photosensitizer complex according to claim 1 in the preparation of antitumor drugs.

6. The application according to claim 5, characterized in that, The combined therapy using the complex exhibited a mild photothermal therapeutic effect, which helps reduce damage to normal tissues and can induce immunogenic cell death, thereby improving the efficacy of immunotherapy.

Images