Preparation of Environmentally Responsive PDA&DOX@LAC Live Drug Carrier for synergistic tumor therapy

Preparation of Environmentally Responsive PDA&DOX@LAC Live Drug Carrier for synergistic tumor therapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Preparation of Environmentally Responsive PDA&DOX@LAC Live Drug Carrier for synergistic tumor therapy Lu Liu, Xuefen Zhao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4251041/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Jul, 2024 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract The development of intelligent, environmentally responsive, and biocompatible photothermal system holds significant importance for the photothermal combined therapy of tumors. In this study, inspired by lactobacillus, we prepared a biomimetic nanoplatform, PDA&DOX@LAC, for tumor photothermal-chemotherapy by integrating the chemotherapeutic drug doxorubicin (DOX) with dopamine through oxidative polymerization to form polydopamine on the surface of LAC. The PDA&DOX@LAC nanoplatform not only achieves precise and controlled release of DOX based on the slightly acidic microenvironment of tumor tissues, but also exhibits enzyme-like properties to alleviate tumor hypoxia. Under near-infrared light irradiation, it effectively induces photothermal ablation of tumor cells, enhances cellular uptake of DOX with increasing temperature, and thus efficiently inhibits tumor cell growth. Moreover, in vivo experiments further confirmed that photothermal therapy combined with PDA&DOX@LAC induces tumor cells apoptosis, releases tumor-associated antigens, and is engulfed by dendritic cells, activating cytotoxic T lymphocytes, thereby effectively suppressing tumor growth and prolonging the survival period of 4T1 tumor-bearing mice. Therefore, the PDA&DOX@LAC nanoplatform holds immense potential in precise tumor targeting and photothermal combined therapy, providing valuable insights and theoretical foundations for the development of novel tumor treatment strategies based on endogenous substances within the body. Biological sciences/Cancer Biological sciences/Drug discovery Biological sciences/Immunology Physical sciences/Materials science dopamine combination therapy environmental response precise release Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Tumor remains one of the principal threats to human health, with chemotherapy still being the primary clinical approach for its treatment. While this method effectively suppresses tumor growth, it brings about significant toxic side effects on normal tissues and the immune system. 1–2 Therefore, there is a pressing need to develop safer and more effective treatment modalities for tumor. With the deeper understanding of tumor biology, researchers have observed that tumor tissues lack sufficient blood supply, rendering them unable to dissipate heat by regulating blood flow and velocity. 3–4 Consequently, tumor tissues become natural reservoirs for heat accumulation. In light of the monumental development of photothermal therapy for tumors, there is a need to mitigate the damage caused by singular photothermal therapy to surrounding normal cells and tissues. As such, researchers have taken a different approach, utilizing near-infrared light (NIR) to induce localized heat generation within tumor tissues, thereby triggering apoptosis in tumor cells. 5 Despite the advantages of this method such as low invasiveness, high selectivity, and temporal-spatial control, the effective penetration of NIR is limited, making it challenging to eradicate deep-seated tumor cells and hindering its clinical application. 6 Therefore, there is an urgent need for safer and more effective methods for treating tumors. With the widespread development and application of photosensitizers and photothermal materials, the photothermal-chemotherapy combination therapy effectively overcomes the shortcoming of individual treatments. Especially, environmentally responsive nanoplatform prepared based on tumor microenvironment not only achieve precise and controllable release of chemotherapeutic drugs in tumor tissues but also effectively improve the tumor tissue microenvironment, thereby achieving a cascade effect in tumor treatment. 7–8 Currently, nanoplatform extensively developed and utilized primarily consist of exogenous inorganic and organic compounds. However, due to their non-degradability and suboptimal biological metabolism, these carriers inevitably bring unnecessary toxic side effects to normal tissues and organs, hindering the clinical implementation of photothermal-chemotherapy combination therapy. 9–10 Thanks to the development of photothermal nano-materials based on endogenous substances can effectively reduce the body's immune response, minimizing damage to normal tissues and better meeting the requirements for future clinical translation. Currently, widely developed and utilized endogenous photothermal materials mainly including dopamine, serotonin, and adrenaline. 11–13 Among them, dopamine, as one of the important neurotransmitters in the body, plays a crucial role in regulating the development of the nervous system and maintaining pleasure and happiness in humans. Furthermore, as a small molecule catecholamine in the human body, dopamine can undergo oxidative polymerization in an alkaline environment, forming size-controllable polydopamine nanoparticles (PDA NAs). 14 The prepared PDA NAs not only exhibit superior photothermal performance in the near-infrared region but also their unique conjugated molecular structure and special adhesion properties make them highly suitable for loading chemotherapeutic drugs. 15 More importantly, the surface of PDA NAs contains abundant amino and hydroxyl groups, which facilitate surface modification to impart more biological properties, enabling tumor combination therapy based on photothermal effects. 16 Although the biocompatibility of photothermal nanoplatform have been effectively addressed, similar to most photothermal nanoparticles, polydopamine nanoparticles cannot achieve targeted delivery. Moreover, the central region of tumor tissues is severely hypoxic, resulting in the ineffective killing of tumor cells in the central region of tumor tissues by most anti-tumor drugs and photothermal nanoparticles. To sum up, in this study, Lactobacillus (LAC) was used as living drug carrier and integrates the chemotherapeutic drug doxorubicin (DOX) with dopamine through oxidative polymerization to form polydopamine on the surface of LAC, thereby preparing an environmentally responsive PDA&DOX@LAC biomimetic therapeutic platform for photothermal-chemotherapy combined treatment of tumors. As illustrated in Scheme 1a , the one-step preparation process of the PDA&DOX@LAC nanoplatform is gentle, with negligible impact on the biological properties of LAC. Upon intravenous injection into mice, the prepared PDA&DOX@LAC nanoplatform utilizes the anaerobic property of LAC to deliver the anti-tumor drug DOX to tumor tissues and achieves precise and controlled release of DOX based on its protonation propensity under acidic conditions. While preparing the PDA&DOX@LAC nanoplatform, doped Mn ions display peroxidase-like properties, which enable them to react with the high concentration of hydrogen peroxide in tumor tissues, leading to the production of oxygen. This process effectively alleviates the hypoxic condition within the tumor tissues. Upon exposed under NIR irradiation, the PDA&DOX@LAC nanoplatform converts light energy into heat and generates a large amount of reactive oxygen species, inducing apoptosis of tumor cells and releasing tumor-associated antigens. These antigens are taken up by dendritic cells, stimulating infiltration of cytotoxic T cells, which in turn kill tumor cells and inhibit tumor growth (Scheme 1b ). Therefore, the PDA&DOX@LAC nanoplatform has been developed in a simpler and more effective manner, serving not only as an efficient live drug carrier for chemotherapy with tumor tissue-targeted delivery, but also exhibiting peroxidase-like activity to effectively improve the tumor hypoxic microenvironment, providing a theoretical basis and design concept for the development of novel tumor treatment platforms in the future. Materials and Reagents Dopamine, reduced glutathione (GSH), hydrogen peroxide (H 2 O 2 ), 4’,6-diamidino-2-phenylindole (DAPI), doxorubicin (DOX), [Ru(dpp)3]Cl2 (RDPP), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), 1,3-diphenylisobenzofuran (DPBF), cell counting kit-8 (CCK-8), MRS broth, and sodium hydroxide (NaOH) were procured from Aladdin (Shanghai, China). Dulbecco’s modified eagle medium (DMEM), RPMI Medium Modified (RPMI-1640), fetal bovine serum (FBS), phosphate-buffered saline (PBS), and streptomycin and penicillin were obtained from Gibco (Shanghai, China). All chemicals were utilized as received without additional purification. Deionized water was used throughout the experiments. Apparatus and Procedures Transmission electron microscopy (TEM) imaging was conducted using a JEOL 2100 transmission electron microscope. Particle size and ζ-potential measurements were performed using a NanoBrook Omni (Brookhaven, USA). Fourier transform infrared (FTIR) spectra were obtained with a Nicolet Nexus 470 spectrometer (USA). X-ray photoelectron spectroscopy (XPS) analysis of PDA&DOX@Rha was carried out using an XPS spectrometer (Thermo Kalpha). Near-infrared light at 808 nm was delivered using a fiber-coupled NIR laser (MDL-N-808 nm-10W, Beijing Laserwave OptoElectronics Technology Co., Ltd., Beijing, China). An infrared thermal camera (HT-19, Guangzhou, China) was employed to monitor temperature changes and capture infrared thermal images. The preparation of PDA&DOX@LAC The synthesis procedure of the PDA&DOX@LAC nanoplatform followed established protocols with slight adaptations. 17–18 Initially, 1×10 9 Lactobacillus cells were dispersed in 10 mL of pH 8.0 PBS buffer. Subsequently, dopamine (1 mg/mL), doxorubicin (1 mg/mL), and 0.5 mL of KMnO 4 (1 mg/mL) were introduced into the solution according to the prescribed method. The mixture was then stirred at room temperature for 30 min, followed by collection of the precipitate via centrifugation (2000 rpm, 5 min) and subsequent washing with PBS for three times. Finally, the samples were freeze-dried under vacuum conditions. The obtainted PDA&DOX@LAC nanoplatform was stored at 4 ℃ for future applications. The release behavior of DOX from PDA&DOX@LAC To assess the release kinetics of DOX from the PDA&DOX@LAC nanoplatform under various conditions. 19 1×10 7 CFU of PDA&DOX@LAC was separately introduced into (a) PBS (pH = 7.4), (b) GSH (10 mM) and H 2 O 2 (30 mM) in PBS (pH 5.5), and (c) a combination of GSH and H 2 O 2 (pH 5.5, 10 mM GSH, and 30 µM H 2 O 2 ). Subsequently, at predetermined time intervals, samples were withdrawn from each mixture, and the absorbance of DOX at 490 nm was measured using UV-vis spectroscopy to characterize the release profile of DOX. The measurement of photothermal effects PDA&DOX@LAC was resuspended in 200 µL PBS and irradiated with 808 nm NIR (1.5, 1.75, and 2.0 W/cm 2 ) at different powers for different time intervals at room temperature. PDA&DOX@LAC was configured at 100 µg/mL and 200 µg/mL (the number of LACs was approximately 5×10 6 and 1×10 7 ) and temperature changes were recorded at any time using an infrared thermal imaging instrument. The photothermal conversion efficiency (η) of PDA&DOX@LAC was calculated by the following equation: 20–21 θ = (T - T min )/(T max - T min ) ( 1 ) hs = m*C p /ζs ( 2 ) η = hs (ΔT max,mix − ΔT max,H2O )/I(1 − 10 − A808 ) ( 3 ) θ defined the dimensionless driving force temperature, m was 2×10 − 4 kg, Cp was 4.2×10 3 J/(kg·℃), ζ is the slope of T and -Lnθ, ΔTmax,mix is the maximum temperature change of PDA&DOX@LAC, ΔTmax,H 2 O is the maximum temperature change of water. I is the laser power. Where A808 is the absorbance of PDA&DOX@LAC at the wavelength of 808 nm in aqueous solution. Cell culture 4T1 cells (murine breast cancer cells), GES-1 cells (human gastric mucosa epithelial cells), and LAC (Lactobacillus) were procured from SUNNCELL Biological Technology Co., Ltd. The cells were cultured in RPMI Medium Modified (RPMI-1640) supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin. Cell cultures were maintained at 37°C in a 5% CO 2 atmosphere and were approved for use by the Northern Jiangsu People’s Hospital. The evaluation of O 2 generation ability from the PDA&DOX@LAC The oxygen generation capability of PDA&DOX@LAC nanoplatform was assessed by dissolved oxygen meter (JPBJ-608, INESL, Shanghai, China). 22 Initially, 10 mL of PDA&DOX@LAC dispersion solution (200 µg/mL) was added to a 20 mL beaker. Subsequently, H 2 O 2 (30 µM) was introduced into the PDA&DOX@LAC dispersion solution. The dissolved oxygen meter probe was then inserted into the dispersion of PDA&DOX@LAC and H 2 O 2 mix dispersion solution to monitor real-time changes in oxygen concentration. PBS solution, H 2 O 2 solution, and PDA&DOX@LAC dispersion served as control experiments. [Ru(dpp) 3 ]Cl 2 (RDPP) serves as a fluorescent probe for dissolved oxygen detection, where its fluorescence signal is quenched in the presence of oxygen. 23 Thus, 2 mg of PDA&DOX@LAC nanoplatform was added to 20 mL PBS solution containing 30 µM H 2 O 2 and 10 µM RDPP. Fluorescence measurements were taken at predetermined time points by fluorescence spectrometer to detect oxygen concentration in the dispersion of PDA&DOX@LAC. Additionally, 10 µM RDPP fluorescent dye was separately added to 20 mL PBS solution, PBS solution containing H 2 O 2 (30 µM H 2 O 2 ), and PDA&DOX@LAC dispersion (200 µg/mL) as control groups to analyze oxygen concentration in the above solutions. 4T1 cells were co-incubated with PDA&DOX@LAC, and the oxygen generation capability within the cells was assessed by observing the intensity of RDPP fluorescence signals using fluorescence microscopy. The specific steps were as follows: Initially, 4T1 cells (1×10 7 ) were seeded into cell culture dishes and co-incubated with 10 µM RDPP for 4 hours. Then, H 2 O 2 (30 µM), PDA&DOX@LAC (200 µg/mL), and DA&DOX@LAC (200 µg/mL) + H 2 O 2 (30 µM) were separately added to the aforementioned culture, followed by 4 h incubation period and multiple washes with PBS. Finally, fluorescence imaging of 4T1 cells in each treatment group was observed using fluorescence microscopy. 24 The detection of extracellular and intracellular ROS 1,3-diphenylisobenzofuran (DPBF) serves as a chemical probe for monitoring extracellular reactive oxygen species (ROS), exhibiting decreased absorbance when exposed to ROS environments. 25–26 Initially, PDA&DOX@LAC (200 µg/ml) was added to a mixture of DPBF (1 mM) and H 2 O 2 (30 µM), followed by irradiation under 808 nm NIR for 10 minutes. UV-vis spectroscopy was then utilized to detect the UV-visible absorption peaks of DPBF at predetermined time points. DPBF in H 2 O 2 solution (30 µM), dispersed solution of PDA&DOX@LAC (200 µg/ml) + H 2 O 2 (30 µM), PDA&DOX@LAC (200 µg/ml) + NIR, and UV-visible absorption peaks in PDA&DOX@LAC served as control groups. Additionally, the intracellular levels of ROS were assessed using the cell-permeable probe 2',7'-dichlorofluorescein diacetate (DCFH-DA). 27–28 DCFH-DA does not exhibit any fluorescence signal under normal conditions; however, in the presence of abundant ROS within cells, DCFH-DA is rapidly oxidized to yield green fluorescence of 2,7-dichlorofluorescein (DCF). To further validate whether co-incubation of PDA&DOX@LAC with 4T1 cells could induce ROS generation under 808 nm NIR irradiation, 4T1 cells (1×10 7 ) were seeded into 6-well plates and subjected to different treatments (H 2 O 2 , PDA&DOX@LAC + H 2 O 2 , PDA&DOX@LAC + NIR, and PDA&DOX@LAC + NIR + H 2 O 2 ). After 6 h, the cells were exposed under 2.0 W/cm 2 NIR irradiation for 10 minutes, followed by removal of the culture medium and replacement with fresh medium containing DCFH-DA. After 30 min, the medium was discarded, and the cells were washed three times with PBS before fluorescence signals within 4T1 cells were observed using fluorescence microscopy. The temperature regulates cell membrane permeability Different concentrations of PDA&DOX@LAC (100 µg/ml and 200 µg/ml) were co-incubated with 4T1 cells and placed in a cell culture incubator (37°C, 5% CO 2 ). After 6 h, they were subjected to NIR irradiation at different power densities (1.5 and 2.0 W/cm 2 ) for 10 minutes, followed by 3 h incubation period. Subsequently, the culture medium was discarded, and the cells were washed three times with PBS. Then, 1 mL of 4% paraformaldehyde was added to each cell culture dish for fixation for 15 minutes. After discarding the fixative, 1 mL of DAPI staining solution was added to each dish and gently shaken on a horizontal shaker. After 15 min, the DAPI staining solution was removed, and the cells were washed three times with PBS. A small amount of PBS was then added, and the fluorescence intensity of 4T1 cells and GES-1 cells was observed using confocal fluorescence microscopy. 29–30 In vitro cytotoxicity The cytotoxicity of PDA&DOX@LAC combined with NIR was evaluated using the CCK-8 assay. 1×10 4 4T1 cells and GES-1 cells were seeded into 96-well plates and incubated for 12 hours. Subsequently, different concentrations of PDA&DOX@LAC, DOX, PDA, and PDA&DOX nanoparticles were added to each well and co-incubated with the cells for 6 h, followed by irradiation under 808 nm NIR (2.0 W/cm 2 , 10 min). Untreated cells were used as a control group. After 24 h, the cells were washed several times with PBS, and then 10 µl of CCK-8 reagent was added to each well and further incubated for 3 h. The absorbance values (OD) of each well in the 96-well plate were measured using a microplate reader at an excitation wavelength of 450 nm. 31–32 Animal welfare In this study, the animal experiment was conducted in accordance with the guidelines outlined in the Animal Management Rules of the ARRIVE guidelines. Healthy female C57BL/6 mice, aged 6 to 8 weeks, were procured from the Model Animal Genetics Research Center of Yangzhou University (Yangzhou, People’s Republic of China). Every effort was made to minimize animal suffering and reduce the number of animals used in the study. The construction and treatment of 4T1 mouse subcutaneous xenografts model Male Balb/c mice were subcutaneously injected with 0.2 mL of 4T1 cells (5×10 7 ) in the groin area to establish the 4T1 subcutaneous tumor model. 31–32 When the tumor volume reached approximately 100 mm 3 , the 4T1 tumor-bearing mice were randomly divided into 6 groups (n = 6/group): PBS, PBS + NIR, DOX (4 mg/kg), PDA (4 mg/kg) + NIR, PDA&DOX (4 mg/kg) + NIR, and PDA&DOX@LAC + NIR groups. The aforementioned drugs were administered intravenously via the tail vein to the tumor-bearing mice. After 24 h post-injection, NIR irradiation at a power density of 2.0 W/cm 2 was applied for 10 minutes. Injections were administered every 3 days, with real-time monitoring of each mouse's body weight and tumor volume. Tumor volume was calculated using the formula: 0.5 × width 2 × length. Subsequently, tumor tissues and major organs (heart, liver, spleen, lungs, and kidneys) were fixed in 4% formalin buffer and embedded in paraffin. Specimens underwent hematoxylin-eosin (H&E) staining and Terminal Deoxynucleotidyl Transferase Dutp Nick End Labeling (TUNEL) staining. Histological analysis After 15 d, all mice were euthanized using cervical dislocation, and the major organs (heart, liver, spleen, lungs, and kidneys) along with tumor tissues of the 4T1 tumor-bearing mice were collected for histological analysis. The collected tissues were fixed in 4% formalin, followed by embedding in paraffin. Tissue sections were prepared and stained with H&E staining. Tumor tissues were also subjected to TUNEL staining. The prepared tissue sections were placed on a slide scanner to capture tissue images, which were then evaluated by experienced pathologists. 33–34 The detection of mouse blood samples Before treatment, blood tests were conducted on 4T1 tumor-bearing mice in the PDA&DOX@LAC + NIR group. Subsequently, PDA&DOX (4 mg/kg) was injected intravenously via the tail vein into the 4T1 tumor-bearing mice. Blood samples were collected from the mice on days 1, 7, and 14 of treatment to evaluate hematological and biochemical parameters, assessing the biocompatibility of PDA&DOX@LAC. 35 Tumor dendritic cells and infiltrating T cells analysis After treatment, tumor tissues were collected from each mouse in every treatment group. These tissues were minced and digested using Dulbecco's Modified Eagle's Medium (DMEM) supplemented with hyaluronidase (100 µg/mL), DNase I (100 µg/mL), 10% fetal bovine serum (FBS), and collagenase type IV (1 mg/mL) under continuous agitation (200 rpm) in an incubator at 37°C. Following digestion, the cells were filtered through a nylon mesh (500 mesh), then centrifuged at 500 g for 5 minutes. The resulting cell pellet was subjected to red blood cell lysis and purified using a 40% Percoll (GE) solution. Dendritic cells isolated from the extracted cell population were stained with anti-mouse CD11c, CD80, and CD86 antibodies for flow cytometric analysis. Additionally, cytotoxic T lymphocytes present in the single-cell suspension obtained from 4T1 xenograft tumors were stained with anti-mouse CD3, CD4, and CD8a antibodies for subsequent flow cytometric analysis. 36–37 Analysis of tumor dendritic cells and infiltrating T cells After treatment, tumor tissues were collected from each treatment group of each mouse, and continuously shaken in an oven at 37℃ (200 rpm, 40 minutes). Then, they were chopped and digested with Dulbecco modified Dulbecco medium (DMEM) containing hyaluronidase (100 µg/mL), deoxyribonuclease I(100µg/mL), 10% fetal bovine serum (FBS) and collagenase IV (1 mg/mL). Subsequently, the digested cells were filtered with a nylon mesh (500 mesh), collected by centrifugation at 500 g for 5 minutes, and then purified by using erythrocyte lysis and 40% Percoll (GE) solution. The extracted dendritic cells were collected and stained with anti-mouse CD11c, anti-mouse CD80 and anti-mouse CD86 for flow cytometry analysis. In addition, cytotoxic T lymphocytes in single cell suspension were collected from 4T1 xenograft tumors and stained with anti-mouse CD3, anti-mouse CD4 and anti-mouse CD8a for flow cytometry analysis. 36–37 Statistical analysis The experimental data were analyzed by SPSS (17.0) software and OriginPro (2017). All data were presented by mean ± standard deviation. Student’s t-test was used to analyze Two-group comparison and the data from more than three groups were compared and analyzed by one-way analysis of variance (ANOVA). The p value of < 0.05 was considered statistically significant. ‘*** ’ means p < 0.001, ‘**’ means p < 0.01, and ‘*’ means p < 0.05. Results and discussion The preparation and characterization of PDA&DOX@LAC nanosystem Since the pioneering work of W. Busch and W. Coley utilizing bacteria in tumor therapy, bacterial-mediated tumor treatment has been extensively explored and utilized. 38–39 With advancements in biotechnology, materials science, and immunology, bacteria have been genetically engineered to serve as specific immunotherapeutic agents, capable of ameliorating the tumor immunosuppressive microenvironment and enhancing anti-tumor immune responses. 40–41 However, the safety concerns associated with genetically engineered bacteria, including the risk of gene transfer and mutation, have hindered their widespread application as therapeutic vectors. 42 In recent years, inspired by advances in materials science, researchers have employed methods such as covalent coupling, supramolecular interactions, and physical encapsulation to modify bacteria, effectively enhancing the accumulation and retention of therapeutic drugs at tumor sites and imparting additional biological functions. 43 Motivated by these strategies, this study utilized the oxidative polymerization property of dopamine in alkaline environments to coat chemotherapeutic drug DOX onto the surface of LAC bacteria. TEM images demonstrated the successful modification of LAC surface with dopamine and DOX at concentrations of 1 mg/mL, resulting in the formation of uniform nanoparticles with a core-shell structure, termed PDA&DOX@LAC nanosystem (Fig. 1 a). Dynamic light scattering results indicated an increase in average particle size from 1803 ± 158 nm to 2586 ± 312 nm upon PDA loading (Fig. 1 b). Additionally, electrochemical potential observations revealed a shift in potential from − 3.89 ± 0.69 mV (LAC) to -10.2 ± 1.26 mV (PDA@LAC) after PDA coating, and further elevation to -5.89 ± 0.93 mV upon successful loading of DOX onto the surface of PDA&DOX@LAC nanosystem (Fig. 1 c). To further confirm the successful loading of DOX into the PDA&DOX@LAC nanocarrier, UV/Vis spectroscopic results shown a significant increase in absorbance in the suspension of PDA&DOX@LAC nanosystem, with the absorption peak position matching that of DOX (Fig. 1 d). Furthermore, fourier transform infrared (FTIR) spectroscopy results revealed similar vibrational characteristic peaks at 3327 cm − 1 , 2977 cm − 1 , and 1590 cm − 1 for DOX, PDA, and PDA&DOX@LAC nanosystem, corresponding to -OH, C-H, and C = O stretching vibrations, respectively. Moreover, DOX and PDA&DOX@LAC nanosystem exhibited similar absorption peaks between 900–1500 cm − 1 , confirming the successful preparation of the PDA&DOX@LAC nanosystem (Fig. 1 e). Subsequently, the biocompatibility of the PDA&DOX@LAC nanosystem during the preparation process was further validated. Colony counting showed that the survival rate of LAC after encapsulation remained around 94.5% (Fig. 1 f and 1 g). Finally, the impact of the PDA shell on bacterial growth was evaluated through optical density (OD) measurements, revealing that the PDA coating effectively delayed LAC growth without affecting its viability (Fig. 1 h). In conclusion, we successfully prepared a core-shell structured PDA&DOX@LAC nanosystem. In order to evaluate the environmentally responsive release performance of the PDA&DOX@LAC nanosystem, we conducted X-ray photoelectron spectroscopy (XPS) analysis. The results revealed that the PDA&DOX@LAC nanosystem was composed of Na, Mn, O, N, and C elements, with manganese ions primarily existing in the forms of Mn 3+ and Mn 4+ (Fig. 2 c and 2 e). Subsequently, the PDA&DOX@LAC nanosystem was immersed in five different simulated solutions: PBS, pH = 5.5, 30 µM H 2 O 2 , 10 mM GSH, and a simulated tumor microenvironment (TME) (pH = 5.5, 30 µM H 2 O 2 , 10 mM GSH). As depicted in Fig. 2 a, only approximately 15% of DOX was released in the normal physiological environment (PBS), mainly due to the flow of the solution causing DOX release from the surface of the PDA&DOX@LAC nanosystem. However, in the H 2 O 2 solution, around 22.3% of DOX was released from the surface of the PDA&DOX@LAC nanosystem. This release was primarily attributed to the accumulation of H 2 O 2 on the surface of the PDA&DOX@LAC nanosystem, where the manganese ions present in the nanosystem underwent Fenton reaction with H 2 O 2 , weakening the interaction between DOX and the PDA&DOX@LAC nanosystem and resulting in DOX release. When the PDA&DOX@LAC nanosystem was immersed in the GSH solution, nearly 60% of DOX was released. This significant release was attributed to the redox reaction between GSH and the PDA&DOX@LAC nanosystem. Although the elemental composition of PDA&DOX@LAC remained unchanged, a large amount of Mn 3+ and Mn 4+ was reduced to Mn 2+ , causing structural changes in the PDA&DOX@LAC nanosystem and subsequently leading to DOX releasing (Fig. 2 d and 2 f). However, when the PDA&DOX@LAC nanosystem was immersed in an acidic environment, the release rate approached 100% after 24 hours. Fourier-transform infrared spectroscopy (FTIR) spectra results shown that while the absorption peak positions of PDA&DOX@LAC and PDA&DOX@LAC + TME were the same, the absorption peak of DOX between 900–1500 cm − 1 in PDA&DOX@LAC + TME was weakened (Fig. 2 b). This weakening was mainly attributed to the protonation of DOX in the acidic environment, leading to its release from the surface of the PDA&DOX@LAC nanosystem. 44–45 When the PDA&DOX@LAC nanosystem was immersed in an in vitro simulated TME, nearly 100% of DOX was released within 18 hours. The rapid release of DOX was attributed to the combined effects of multiple factors. Thus, the prepared PDA&DOX@LAC nanosystem exhibited environmentally responsive release performance. Enzyme-like property of PDA&DOX@LAC nanosystem Due to the inclusion of Mn ions in the PDA&DOX@LAC nanosystem, which possess catalase-like activity, they can react with the high concentration of H 2 O 2 in tumor tissues, generating oxygen and hydroxyl radicals. This process effectively alleviates the hypoxic microenvironment of tumor tissues and the hydroxyl radicals produced further damage tumor cells. 46–47 In order to investigate the oxygen-generating capacity of the PDA&DOX@LAC nanosystem in response to H 2 O 2 , the nanosystem was immersed in five different simulated solutions: PBS, pH = 5.5, 30 µM H 2 O 2 , 10 mM GSH, and a simulated tumor microenvironment (TME) (pH = 5.5, 30 µM H 2 O 2 , 10 mM GSH). The oxygen content in each solution was monitored in real-time using a dissolved oxygen meter. Over time, as the PDA&DOX@LAC nanosystem was immersed in the H 2 O 2 solution and TME, the oxygen content gradually increased. After 26 min, the oxygen content in these two solutions reached 9.87 mg/mL and 9.93 mg/mL, respectively, while the oxygen content in the other solutions remained relatively unchanged (Fig. 3 a). Photographs were taken of each reaction system, revealing a greater number of bubbles when the PDA&DOX@LAC nanosystem was immersed in the solution containing H 2 O 2 (Fig. 3 b). RDPP, serving as a typical oxygen fluorescence probe, is oxidized in the presence of oxygen, leading to quenching of its fluorescence signal. As shown in Figs. 3 c − 3f, when RDPP was immersed in a mixture of PDA&DOX@LAC and 30 µM H 2 O 2 solution, the fluorescence signal of RDPP gradually weakened. However, in the solution containing RDPP, H 2 O 2 , and PDA&DOX@LAC, there was no significant change in the RDPP fluorescence signal. This result indicates that the dispersion of PDA&DOX@LAC in the H 2 O 2 solution generated oxygen, causing the oxidation of RDPP and subsequent reduction in its fluorescence signal. In an in vitro simulated high-concentration H 2 O 2 environment, PDA&DOX@LAC generated a significant amount of oxygen, prompting further evaluation of intracellular oxygen production. Subsequently, PDA&DOX@LAC was co-incubated with 4T1 cells to assess intratumoral oxygen generation. As shown in Fig. 3 g, when 4T1 cells were co-incubated with PDA&DOX@LAC, the RDPP fluorescence signal exhibited noticeable quenching under fluorescence microscopy. Conversely, when 4T1 cells were co-incubated with PDA&DOX@LAC and H 2 O 2 , the red fluorescence signal was barely detected. In comparison, strong red fluorescence signals were observed when 4T1 cells were solely incubated with RDPP or co-incubated with H 2 O 2 . These results suggest that PDA&DOX@LAC possesses catalase-like properties, capable of generating oxygen through its reaction with H 2 O 2 . Photothermal Properties of PDA&DOX@LAC nanosystem The bacterial suspension of the PDA&DOX@LAC nanosystem exhibits absorbance in the near-infrared (NIR) region, and dopamine nanoparticles demonstrate excellent photothermal conversion properties. This prompted us to further evaluate the photothermal conversion performance of PDA&DOX@LAC. Therefore, bacterial suspensions of different concentrations of PDA&DOX@LAC nanosystem (100 µg/mL and 200 µg/mL) were subjected to irradiation under different power densities of NIR light (1.5 W/cm2, 1.75 W/cm 2 , and 2.0 W/cm 2 ) for 10 minutes. As shown in Figs. 4 a and 4 b, the photothermal conversion performance of the PDA&DOX@LAC nanosystem is positively correlated with time, concentration, and power density. When the concentration of PDA&DOX@LAC nanosystem was 200 µg/mL and irradiated with 2.0 W/cm 2 NIR light for 10 min, the temperature of the PDA&DOX@LAC suspension could increase to 53.2 ℃, with minimal change compared to the temperature of PDA nanoparticles (55.9 ℃) under the same conditions. Furthermore, the photothermal conversion efficiency of the PDA&DOX@LAC nanosystem remained at 35.6% (Fig. 4 c and 4 d). Thus, PDA&DOX@LAC exhibits superior photothermal conversion performance suitable for photothermal therapy of tumors. To further validate the photothermal stability of the PDA&DOX@LAC nanosystem for repeated irradiation, the 200 µg/mL PDA&DOX@LAC suspension was subjected to 2.0 W/cm 2 NIR irradiation for 10 min, followed by NIR excitation shutdown for 10 min. This cycle was repeated four times, and temperature changes were recorded using an infrared thermal imaging instrument (recorded every 2 min). As shown in Fig. 4 e, after four cycles of irradiation, the temperature of the PDA&DOX@LAC suspension remained at 54.1°C, with a slight increase in temperature observed after each cycle, attributed to water evaporation caused by temperature rise. Therefore, the PDA&DOX@LAC nanoplatform prepared in this study demonstrates superior photothermal conversion performance and can withstand multiple repeated irradiation. Generation of ROS inside and outside PDA-DOX @ LAC cells stimulated by near infrared spectroscopy The enzyme-like property and photothermal response property of PDA&DOX@LAC nanosystem prompted us to further explore its ability to generate ROS under the stimulation of near-infrared light for photodynamic therapy of tumors. As a typical extracellular active oxygen probe, DPBF can be oxidized to an oxidation product (O-dibenzoylbenzene) in the presence of ROS, resulting in the decrease of its UV absorption values. To explore whether PDA&DOX@LAC could produce ROS under NIR irradiation, DPBF was respectively immersed in solutions of H 2 O 2 , PDA&DOX@LAC + H 2 O 2 , PDA&DOX@LAC + NIR, and PDA&DOX@LAC + H 2 O 2 + NIR (Fig. 5 a- 5 d). Compared with the H 2 O 2 group in the control group, the UV absorption values of DPBF in other groups decreased to some extent over time. It was mainly attributed to the Fenton reaction between PDA&DOX@LAC and H 2 O 2 , which generated hydroxyl radicals and caused the decrease of absorption value of DPBF. PDA&DOX@LAC produced a large amount of heat after NIR irradiation, which was then transferred to the oxygen in the surrounding tissue. The molecular oxygen transited to the ground state oxygen, causing the decrease of UV absorption values DPBF. However, after H 2 O 2 was added into PDA&DOX@LAC solution and placed in the NIR for 10 minutes, the UV absorption values of DPBF exhibited a more significant decrease, indicating that Fenton reaction occurred after PDA&DOX@LAC was dispersed in H 2 O 2 solution, resulting in the generation of hydroxyl free radicals and a large amount of oxygen. After NIR irradiation, more ROS generated. PDA&DOX@LAC produced large amounts of ROS after extracellular stimulation with H 2 O 2 and NIR, which prompted us to further evaluate the production of ROS in cells. DCFH-DA, as a cell permeability dye, has no fluorescence itself. Once it enters cells, it is easily hydrolyzed by cell esterase into DCFH, which is then rapidly oxidized by ROS to DCF with a strong green fluorescence product. Therefore, DCFH-DA was co-incubated with 4T1 cells to test whether PDA&DOX@LAC could produce ROS in 4T1 cells. As shown in Fig. 5 e, 4T1 cells were randomly divided into 6 groups as control groups, PDA&DOX@LAC + NIR, NIR Only, PDA&DOX@LAC + H 2 O 2 , H 2 O 2 , and PDA&DOX@LAC + H 2 O 2 + NIR. It is found by fluorescence microscope that the green fluorescence signal could be clearly observed when the 4T1 cells were incubated with PDA&DOX@LAC and irradiated with NIR for 10 minutes. In addition, after adding 30 µM H 2 O 2 into the 4T1 cell culture medium and placing it under NIR irradiation for 10 min, the green fluorescence signal was the strongest compared with the control group. The main reason is that H 2 O 2 reacts with PDA&DOX@LAC to produce more oxygen and a large amount of 1 O 2 is produced after NIR irradiation, which leads to the enhancement of green fluorescence signal. However, in other treatment groups like the H 2 O 2 group and the PDA&DOX@LAC group, the green fluorescence signal could hardly be detected, mainly due to the small production of ROS in 4T1 cells, which is consistent with that result of in vitro DPBF detection of ROS. Therefore, PDA&DOX@LAC could produce a large amount of ROS under the excitation of NIR when there was sufficient oxygen around the tissue. Thus PDA&DOX@LAC can be used as an ideal material for PDT treatment. The killing effect of PDA&DOX@LAC on cells PDA&DOX@LAC has superior environmental response performance and the research shows that with the increase of temperature, it can effectively enhance the permeability of cell membrane, and thus enhance the uptake of drugs by cells and enhance the killing performance of cells. 29–30 Therefore, we incubated 200 µg/mL PDA&DOX@LAC with 4T1 cells and GES-1 cells for 12 h respectively, and then treated with NIR(1.5, 1.75 and 2.0 W/cm 2 ) with different powers. The results of confocal microscopy shown that strong fluorescence signals could be detected in 4T1 cells and the fluorescence signal of DOX was also becoming stronger with the gradual increase of NIR power. It was mainly because the simulated tumor microenvironment constructed by 4T1 cells triggers the release of DOX from the surface of PDA&DOX@LAC. Subsequently, NIR causes the temperature to rise, which increases the permeability of 4T1 cells and promotes the uptake of DOX by 4T1 cells. (Fig. 6 a). However, the fluorescence signal of DOX in GES-1 cells did not become stronger with the increase in NIR power (Fig. 6 b). It is mainly due to the fact that the normal tissue microenvironment of GES-1 cells can't release DOX from the surface of PDA&DOX@LAC, which leads to that GES-1 cells can't absorb DOX and its fluorescence signal doesn't get stronger with the increase of NIR power. Thanks to the effective environmental responsiveness of drug release exhibited by PDA&DOX@LAC, coupled with the efficient drug absorption capability of tumor cells, we have delved deeper into its cytotoxic effects on tumor cells. Specifically, the viability of both GES-1 and 4T1 cells was assessed through CCK-8 experiments to gauge the anti-tumor efficacy of the chemo-photothermal synergistic therapy mediated by PDA&DOX@LAC. As depicted in Figs. 6 c and 6 d, the escalation in PDA&DOX@LAC concentration corresponded to a gradual decline in the cell viability of both GES-1 and 4T1 cells. Upon exposure to NIR (10 min, 2.0 W/cm 2 ) under identical concentrations and conditions, the viability of 4T1 cells notably plummeted compared to that of GES-1 cells. Specifically, at a concentration of 200 µg/mL of PDA&DOX@LAC, the cell viability of GES-1 cells remained approximately at 50%, whereas that of 4T1 cells dipped below 20%. These findings underscore the potent cytotoxicity of the chemo-photothermal synergistic therapy mediated by PDA&DOX@LAC against tumor cells, primarily attributed to the effective tumor microenvironment-driven DOX release and subsequent enhancement of 4T1 cell permeability facilitated by photothermal therapy, thereby augmenting DOX absorption. In addition, when DOX and PDA&DOX@LAC were in the same concentration, the killing effect of DOX on GES-1 cells was significantly higher than that of PDA&DOX@LAC. Furthermore, when DOX and PDA&DOX@LAC were administered at equivalent concentrations, the cytotoxic efficacy of DOX against GES-1 cells significantly surpassed that of PDA&DOX@LAC. Hence, PDA&DOX@LAC achieves a multifaceted approach encompassing chemotherapy, photothermal therapy, and photodynamic therapy, bolstered by NIR irradiation, effectively eradicating tumor cells while mitigating DOX-induced damage to normal cells. Antitumor Effect of PDA&DOX@LAC in Vivo Based on the excellent photothermal conversion performance of the PDA&DOX@LAC nanosystem in vitro , we further evaluated its aggregation performance in tumor tissues of 4T1 tumor-bearing mice to examine its anti-tumor effect in vivo . To begin with PBS, PDA nanoparticles, PDA&DOX, and PDA&DOX@LAC were injected into mice with 4T1 xenografts through their tail vein. After 24 h, the 4T1 tumor-bearing mice in each group were irradiated with NIR (2.0W/cm 2 ) for 10 min, and the changes in tumor tissue temperature in each group were recorded by infrared thermal imaging (Fig. 7 a and Fig. 7 b). When 4T1 tumor-bearing mice were treated with PDA-DOX@LAC, the temperature of tumor tissue increased to 54.3 ℃. After PDA and PDA-DOX treatment, the temperature of tumor tissue only increased to 46.5℃ and 46.1 ℃. To sum up, the PDA&DOX@LAC nanosystem increased the targeted delivery of PDA&DOX@LAC to tumor tissues with the help of the anaerobic feature of LAC. Then we further evaluated PDA&DOX@LAC nanosystem anti-tumor performance. When the 4T1 xenografts grew to ~ 100 mm 3 , 4T1 tumor-bearing mice were randomly divided into six groups (n = 6/group) and treated with PBS, PBS + NIR, PDA + NIR, DOX, PDA&DOX + NIR and PDA&DOX@LAC + NIR respectively. The changes in tumor tissues and body weight of these mice were recorded every day. Figure 7 c shown that synergistic treatment of PDA&DOX@LAC + NIR effectively inhibits the growth of xenografts in mice. After 15 d of treatment, the average size of tumor tissues was only 135mm 3 in the PDA&DOX@LAC + NIR group, compared to 405.6 mm 3 , 442.6 mm 3 , and 387.6 mm 3 for the PDA + NIR, DOX, and PDA&DOX + NIR groups, respectively. In addition, NIR irradiation alone did not significantly inhibit the growth of xenografts in mice (Fig. 7 d and Fig. 7 e). It was found that the body weight of the mice decreased rapidly after treatment with DOX, and they even became depressed (Fig. 7 f). In groups of PDA + NIR, PDA&DOX + NIR, and PDA&DOX@LAC + NIR, only a slight change in the body weight of tumor-bearing mice can be observed and the life cycle of the mice was longer than that in the DOX treatment group. In particular, after 40 days of PDA&DOX@LAC + NIR synergistic therapy, the survival rate of mice in this group remained around 30%, while mice in all the other treatment groups had already died (Fig. 7 g). In a word, PDA&DOX@LAC + NIR synergistic treatment of 4T1 tumor-bearing mice could not only effectively inhibit the growth of xenografts in mice, but also greatly reduce the toxic and side effects of DOX and extend their life. Analysis of Antitumor Mechanism of PDA&DOX@LAC in Vivo Given that PDA&DOX@LAC + NIR synergistic therapy effectively inhibits the growth of mouse xenografts, we further analyzed its anti-tumor mechanism. After 15 d of treatment, the tumor tissues of mice in each treatment group were collected for TUNEL and H&E staining, and the necrosis and apoptosis of tumor cells in the tumor tissues were analyzed. As shown in Fig. 8 a, no obvious necrosis and apoptosis of tumor cells were observed in either the PBS or PBS + NIR treatment groups when compared with the PDA + NIR, DOX, PDA&DOX + NIR, and PDA&DOX@LAC + NIR treatment groups. H&E staining results indicated that chemo-photothermal synergistic therapy of PDA&DOX@LAC was highly destructive to tumor cells, which is featured by vacuolation, karyopycnosis, and karyolysis. In addition, TUNEL staining showed that a green fluorescence signal was only detected on the surface of tumor tissue in PDA + NIR and PDA&DOX + NIR treatment groups. while the green fluorescence signal of the tumor tissue was weaker after DOX treatment. However, after chemo-photothermal synergistic therapy of PDA&DOX@LAC + NIR group, a large number of green fluorescence signals could be found. To conclude, the PDA&DOX@LAC nanosystem could effectively transport chemotherapeutic drugs and photothermal carriers to the deep part of tumor tissue to release DOX and kill tumor cells in the deep part of tumor tissue under LAC's ability to easily survive in anoxic and hypoxic environments. And then, apoptotic tumor cells release tumor-associated antigens, thereby promoting the immune response of the body. Therefore, tumor cells from each treatment group were collected to analyze the immune cells in tumor tissues, which helps to explore the immune regulation in the nanosystem PDA&DOX@LAC chemo-photothermal synergistic therapy. As shown in Fig. 8 b, the proportion of dendritic cells (PDA&DOX@LAC) in the PDA&DOX@LAC treatment group exposed to NIR was 39.7%, which was higher than that in other treatment groups (PBS, PBS + NIR, PDA + NIR, DOX, and PDA&DOX + NIR). That results from the efficacy of PDA&DOX@LAC in killing tumor cells and promoting the release of tumor-related antigens after PDA&DOX@LAC chemo-photothermal synergistic therapy, thereby increasing dendritic cells’ uptake of it, eventually leading to the maturation of dendritic cells. However, cytotoxic T lymphocytes (CD8 + T cells) playing an important role in the anti-tumor immune response by being activated by tumor-derived antigens and then directly killing tumor cells. Therefore, the expression of CD8 + T cells in tumor tissue was analyzed. The percentages of CD8 + T cells in the PBS, PBS + NIR, PDA + NIR, DOX, and PDA&DOX + NIR treatment groups were 8.38%, 8.4%, 9.91%, 10.4%, and 14.6%, respectively (Fig. 8 c). However, chemo-photothermal synergistic therapy of PDA&DOX@LAC resulted in the highest percentage of CD8 + T cells among all groups, which was 18.4%. These results indicated that the PDA&DOX@LAC nanosystem, as a potential in situ vaccine, could significantly activate the immune system, promote DC cell maturation and CD8 + T cell activation, and then kill tumor cells and inhibit the growth of tumors. PDA&DOX@LAC Biocompatibility Chemo-photothermal synergistic therapy of the PDA&DOX@LAC nanosystem exhibits superior anti-tumor effect, so it is used for the treatment of mouse xenografts and future clinical application, which requires low toxicity or even non-toxicity in vivo . So the biocompatibility should be analyzed through animal experiments. The biocompatibility of PDA&DOX@LAC in vivo was analyzed by H&E staining during the treatment of tumor-bearing mice. After the treatment, the H&E staining was performed on the main organs (heart, liver, spleen, lung, and kidney) of mice in each treatment group to compare with the control group. It was found that when the mice were injected with PDA&DOX@LAC nanosystem for 15 days, no significant inflammation or tissue damage could be found in the pathological sections. Moreover, the PDA&DOX@LAC nanosystem could effectively alleviate the toxic and side effects of DOX in the tumor treatment process, and especially reduce the damage of DOX to heart tissues (Fig. 9 ). Then, blood samples of the PDA&DOX@LAC + NIR treatment group were collected for routine blood tests and serum biochemical indicators tests. As shown in Fig. 10 , blood was drawn from 4T1 tumor-bearing mice on days 0, 1, 7, and 14 of PDA&DOX@LAC + NIR treatment, respectively. The results shown that there were no obvious changes in routine and biochemical blood test results during the PDA&DOX@LAC + NIR treatment. The above results indicated that PDA&DOX@LAC exhibits excellent biocompatibility as a drug carrier during tumor treatment. Conclusion In conclusion, the investigations detailed above have led to the development of a live bacterial drug carrier endowed with enzyme-like attributes, tailored for synergistic therapy targeting tumors. The PDA&DOX@LAC nanosystem employed exhibits notable advantages: ( 1 ) mitigating the toxicities and adverse effects of DOX on normal tissues in vivo during tumor intervention; ( 2 ) targeted delivery to tumor sites and effective tumor cell eradication facilitated by LAC's adaptability within anoxic environments; ( 3 ) harnessing the catalase property of the PDA&DOX@LAC nanosystem to ameliorate tumor tissue hypoxia, thereby augmenting oxygen availability for photodynamic therapy; ( 4 ) enabling the synergistic photothermal therapy of PTT and PDT via NIR laser activation in a single procedure; ( 5 ) fostering the generation of tumor-associated antigens and eliciting a robust anti-tumor immune response through the chemo-photothermal synergistic therapy mediated by the PDA&DOX@LAC nanosystem. Consequently, outcomes derived from both in vivo and in vitro treatments underscore the innovative and efficacious role of the formulated PDA&DOX@LAC nanosystem in advancing the landscape of tumor synergistic therapy platforms, thus furnishing a theoretical foundation and design paradigm for future iterations of tumor treatment modalities. Declarations Ethics approval and consent to participate All experimental procedures were approved by the Institutional Animal Care and Use Committee of Northern Jiangsu People’s Hospital (no. UJS-IACUC-AP-20190314002). Consent for publication Each coauthor has read the manuscript and approves its submission. This work is being submitted exclusively to your journal. Data Availability The datasets supporting the results of this article are included within the article. Declarations The authors declared that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This research was supported by Natural Science Foundation of Jiangsu Province (SBK2020022937). Authors' contributions Lu Liu are responsible for the experimental operations, data collection and analysis. Xuefen Zhao are responsible for the experimental design and writing of the manuscript. All authors read and approved the final manuscript. Acknowledgements The acknowledged are included within the article. References Yanji Chu, Xiao-Qi Xu, Yapei Wang. Ultradeep Photothermal Therapy Strategies. Ultradeep Photothermal Therapy Strategies. J. Phys. Chem. Lett., 2022. 13: 9564-9572. Zhenglin He, Yihan Wang, Liang Han, et al. The mechanism and application of traditional Chinese medicine extracts in the treatment of lung cancer and other lung-related diseases. Front Pharmacol., 2023. 14: 1330518. Xingshu Li, Jonathan F Lovell, Juyoung Yoon, et al. Clinical development and potential of photothermal and photodynamic therapies for cancer. Nat. Rev. Clin. Oncol., 2020. 17: 657-674. 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Tumor homing-penetrating and nanoenzyme-augmented 2D phototheranostics against hypoxic solid tumors. Acta Biomater., 2022. 150: 391-401. Lei Chen, Shashi Ranjan Tiwari, Yingqi Zhang, et al. Facile Synthesis of Hollow MnO 2 Nanoparticles for Reactive Oxygen Species Scavenging in Osteoarthritis. ACS Biomater. Sci. Eng., 2021. 7: 1686-1692. Scheme Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Scheme1.png Scheme 1. The preparation of PDA&DOX@LAC nanoplatform for tumor synergistic therapy. (a) Construction of a PDA&DOX@LAC nanoplatform. (b) Proposed action mechanism of PDA&DOX@LAC nanoplatform in a mouse tumor model. Cite Share Download PDF Status: Published Journal Publication published 10 Jul, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 29 May, 2024 Reviews received at journal 21 May, 2024 Reviews received at journal 14 May, 2024 Reviews received at journal 06 May, 2024 Reviewers agreed at journal 06 May, 2024 Reviewers agreed at journal 06 May, 2024 Reviewers agreed at journal 06 May, 2024 Reviewers invited by journal 06 May, 2024 Editor assigned by journal 01 May, 2024 Editor invited by journal 30 Apr, 2024 Submission checks completed at journal 30 Apr, 2024 First submitted to journal 11 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4251041","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":299414394,"identity":"0800590c-450b-4d9c-b0ab-9438eef699cf","order_by":0,"name":"Lu Liu","email":"","orcid":"","institution":"The Affiliated Huai’an Hospital of Xuzhou Medical University and The Second People’s Hospital of Huai’an","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Liu","suffix":""},{"id":299414399,"identity":"d4911771-1a7f-4526-9eba-ea1dbfa3f0c8","order_by":1,"name":"Xuefen Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIie3PoWoDQRCA4QkHd2Zo7ARC+gqjrqILEX2RXQJTE1G5MkdhYwK1hbzMpQsXsw9wImJjohsX2atuYa+uYj+xan52BiDL/qGyarafmhUuj6+HqK1KJ3foO4hW5hC6Fccg6WRBIpMYPhT063p2dn7EYhjqaFyLk11bWx08TPeb1C27BzbuhEW1kV7bZ6BTm/yFybgLlth2vQ6PwKQTCa2/E484vC/GFWMSERpOQKJVCcY9jUjQe9ZWkLErhlaQ+kRyv22a843Vkqu36/Vm1WL6nkh+wD/OZ1mWZb/5Amx2ScjyTNjGAAAAAElFTkSuQmCC","orcid":"","institution":"Northern Jiangsu People’s Hospital","correspondingAuthor":true,"prefix":"","firstName":"Xuefen","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2024-04-11 08:08:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4251041/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4251041/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-66966-2","type":"published","date":"2024-07-10T13:07:14+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":56016884,"identity":"16221dda-1c1a-4a52-90bb-30b87ebf8d01","added_by":"auto","created_at":"2024-05-07 15:22:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":369889,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of PDA\u0026amp;DOX@LAC nanosystem. (a) Representative TEM images of LAC and PDA\u0026amp;DOX@LAC. Scale bar: 500 nm. (b) Size distribution and Zeta potential of LAC, PDA@LAC and PDA\u0026amp;DOX@LAC. (D) UV/Vis spectra of DOX, LAC, PDA and PDA\u0026amp;DOX@LAC. (E) FTIR spectra of DOX, PDA and PDA\u0026amp;DOX@LAC. (f) Bacteria clones and bacterial viability of LAC and PDA\u0026amp;DOX@LAC after incubation for 48 h at 37 °C. (g) The corresponding number of viable bacteria of LAC and PDA\u0026amp;DOX@LAC. (h) The corresponding OD value of LAC and PDA\u0026amp;DOX@LAC at different time point.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4251041/v1/0695443f3f9ac4c0e75d31e4.png"},{"id":56018214,"identity":"d0405001-5f82-48bc-87e6-94f689f22729","added_by":"auto","created_at":"2024-05-07 15:38:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":88158,"visible":true,"origin":"","legend":"\u003cp\u003e(a) In vitro profiles of DOX release from PDA\u0026amp;DOX@LAC nanosystem under conditions of PBS, pH = 5.5, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, GSH and simulated TME. (b) FTIR spectra of PDA\u0026amp;DOX@LAC and PDA\u0026amp;DOX@LAC immersed into simulated TME. XPS of (c) PDA\u0026amp;DOX@LAC nanosystem and (c) PDA\u0026amp; DOX@LAC nanosystem immersed into simulated TME. (e) Narrow x-ray photoelectron spectroscopy scan spectra of Mn2p in (e) PDA\u0026amp;DOX@LAC nanosystem and (f) PDA\u0026amp;DOX@LAC nanosystem immersed into simulated TME.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4251041/v1/84d2e3e4d160b23e3737ba83.png"},{"id":56016883,"identity":"3066b977-cf6b-4a75-bf49-9bb3320c8a85","added_by":"auto","created_at":"2024-05-07 15:22:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":395805,"visible":true,"origin":"","legend":"\u003cp\u003e(a) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-triggered O\u003csub\u003e2\u003c/sub\u003e generation in different solutions. (b) Photograph of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-triggered O\u003csub\u003e2\u003c/sub\u003e generation in different solutions. (PDA\u0026amp; DOX@LAC nanosystem immersed into 1: PBS, 2: pH = 5.5, 3: H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 4：GSH, 5：TME ). (c) Fluorescence spectra of RDPP in PBS (c), H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (d), PDA\u0026amp;DOX@LAC nanosystem (e) and (f) PDA\u0026amp;DOX@LAC nanosystem + H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. (g) CLSM images of RDPP in 4T1 cells without any treatments (1), and treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (2), PDA\u0026amp;DOX@LAC nanosystem and PDA\u0026amp;DOX@LAC nanosystem + H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (4). Scale bars: 300 μm\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4251041/v1/bb2ede7e5fa62018c334c1a4.png"},{"id":56016885,"identity":"5067e873-304a-417b-9118-e6a07e05d6b0","added_by":"auto","created_at":"2024-05-07 15:22:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":564529,"visible":true,"origin":"","legend":"\u003cp\u003ePhotothermal Conversion Performance of PDA\u0026amp;DOX@LAC nanosystem.\u003c/p\u003e\n\u003cp\u003e(a) Infrared thermal image shown the temperature distribution of PBS, PDA\u0026amp;DOX@LAC, and PDA suspensions. (b) Temperature changes observed in PBS, PDA\u0026amp;DOX@LAC, and PDA suspensions. (c) Photothermal response of the PDA\u0026amp;DOX@LAC dispersion solution over 600 s of irradiation followed by the cessation of irradiation. (d) Linear relationship between time and -lnθ during the cooling stage. (e) Temperature changes in the 200 μg/mL PDA\u0026amp;DOX@LAC dispersion solution under irradiation with an 808 nm laser (2.0 W/cm\u003csup\u003e2\u003c/sup\u003e) for four on/off cycles. Legend: 1: PBS, 2: PDA\u0026amp;DOX@LAC (100 μg/mL, 2.0 W/cm\u003csup\u003e2\u003c/sup\u003e), 3: PDA\u0026amp;DOX@LAC (200 μg/mL, 1.5 W/cm\u003csup\u003e2\u003c/sup\u003e), 4: PDA\u0026amp;DOX@LAC (200 μg/mL, 1.75 W/cm\u003csup\u003e2\u003c/sup\u003e), 5: PDA\u0026amp;DOX@LAC (200 μg/mL, 2.0 W/cm\u003csup\u003e2\u003c/sup\u003e), 6: PDA (200 μg/mL, 2.0 W/cm\u003csup\u003e2\u003c/sup\u003e).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4251041/v1/2af8bc3f7121f1ae113e2080.png"},{"id":56017676,"identity":"a3912fe6-9891-4b4c-95f6-387779d8120e","added_by":"auto","created_at":"2024-05-07 15:30:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":114767,"visible":true,"origin":"","legend":"\u003cp\u003eUV–vis absorption of DPBF in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (a), PDA\u0026amp;DOX@LAC + H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (b), PDA\u0026amp;DOX@LAC + NIR (c) and PDA\u0026amp;DOX@LAC + NIR (d) at different time points. (e) CLSM images of DCF in 4T1 cells without any treatments (Control),\u0026nbsp;PDA\u0026amp;DOX@LAC + NIR, NIR only, PDA\u0026amp;DOX@LAC+ H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and PDA\u0026amp;DOX@LAC + H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003e+ NIR (6). Scale bars: 400 μm\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4251041/v1/d848439584a1ff27928ddc16.png"},{"id":56017674,"identity":"2a9be49a-4b03-4b66-973a-c380011faba4","added_by":"auto","created_at":"2024-05-07 15:30:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":272873,"visible":true,"origin":"","legend":"\u003cp\u003eCLSM images of (a) 4T1 and (b) GES-1 cells incubated with PDA\u0026amp;DOX@LAC for 12 h and exposed under different power of NIR (1: 1.5 W/cm\u003csup\u003e2\u003c/sup\u003e,\u0026nbsp; 1.75 W/cm\u003csup\u003e2\u003c/sup\u003e and 2.0 W/cm\u003csup\u003e2\u003c/sup\u003e). Scale bars: 30 μm. Viability of (c) 4T1 cells and (d) GES-1 cells upon treatment with PBS, DOX, PDA + NIR, PDA\u0026amp;DOX + NIR and DA\u0026amp;DOX@LAC + NIR.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4251041/v1/77172ca572f63acfe79e26a1.png"},{"id":56016891,"identity":"14dfbe85-ad9b-493a-8da3-7f67c89acb28","added_by":"auto","created_at":"2024-05-07 15:22:44","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":461863,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Infrared thermal images of mice at varied time points of different treatments under NIR irradiation with the power of 2.0 W/cm\u003csup\u003e2\u003c/sup\u003e for 10 min. (b) Temperature change curves at tumor sites of mice in different groups upon NIR irradiation. (c) Average tumor growth curves of 4T1 tumor-bearing mice after 15 d of different treatments (n= 6/group). (d) Photographs of the tumors and (e) average weights of tumor tissues on day 15 after the last treatment. (f) Body weight of mice in different groups during treatment. (g) The survival curves of mice in the different treatment groups. 1: PBS, 2: PBS + NIR, 3: PDA + NIR, 4: DOX, 5: PDA\u0026amp;DOX + NIR, 6: PDA\u0026amp;DOX@LAC + NIR.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4251041/v1/4ddda22059310fd18e297f69.png"},{"id":56016888,"identity":"cac696e7-699e-4373-a4df-85071fe2bcb6","added_by":"auto","created_at":"2024-05-07 15:22:43","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":509716,"visible":true,"origin":"","legend":"\u003cp\u003e(a) H\u0026amp;E staining (up) and TUNEL staining (down)\u0026nbsp;of tumor slices collected from 4T1 tumor-bearing mice after different treatments. Scale bars: 100 μm. (b) Flow cytometry plots of (b) DC cells maturation and (c) CD3\u003csup\u003e+\u003c/sup\u003e/CD8a\u003csup\u003e+ \u003c/sup\u003eT cells extracted from tumor tissues after different treatments. 1: PBS, 2: PBS + NIR, 3: PDA + NIR, 4: DOX, 5: PDA\u0026amp;DOX + NIR, 6: PDA\u0026amp;DOX@LAC + NIR.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4251041/v1/35ba31840cbe54ccb1bd02cf.png"},{"id":56016890,"identity":"64132373-0503-4ef7-87f0-a43ef2679a82","added_by":"auto","created_at":"2024-05-07 15:22:44","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1095756,"visible":true,"origin":"","legend":"\u003cp\u003eH\u0026amp;E staining of various organs collected from 4T1 tumor-bearing mice after different treatments of PBS, PBS + NIR, DOX, PDA + NIR, PDA\u0026amp;DOX + NIR and PDA\u0026amp;DOX@LAC + NIR for 15 days. Scale bars: 100 μm.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4251041/v1/7403984d3d4ecf6186913823.png"},{"id":56016887,"identity":"624cad22-5838-4287-9e24-f48230498fe8","added_by":"auto","created_at":"2024-05-07 15:22:43","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":51919,"visible":true,"origin":"","legend":"\u003cp\u003eThe blood tests (a-h) and the serum biochemistry index (i-l) of the mice on the 0 st, 1st, 7th, and 14th day after the treatment of PDA\u0026amp;DOX@LAC.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-4251041/v1/329a23a56ba4cbd32d92f087.png"},{"id":60222254,"identity":"580a9d24-ea77-4cfb-aff3-8221b543c325","added_by":"auto","created_at":"2024-07-13 13:07:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5352777,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4251041/v1/c126996f-dd0d-483b-86ac-8acca1a92273.pdf"},{"id":56017673,"identity":"27e6252b-1b17-4ba7-a029-5e311be182ab","added_by":"auto","created_at":"2024-05-07 15:30:43","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":272689,"visible":true,"origin":"","legend":"\u003cp\u003eScheme 1. The preparation of PDA\u0026amp;DOX@LAC nanoplatform for tumor synergistic therapy. (a) Construction of a PDA\u0026amp;DOX@LAC nanoplatform. (b) Proposed action mechanism of PDA\u0026amp;DOX@LAC nanoplatform in a mouse tumor model.\u003c/p\u003e","description":"","filename":"Scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-4251041/v1/b19fb435a5433887145c7302.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Preparation of Environmentally Responsive PDA\u0026DOX@LAC Live Drug Carrier for synergistic tumor therapy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTumor remains one of the principal threats to human health, with chemotherapy still being the primary clinical approach for its treatment. While this method effectively suppresses tumor growth, it brings about significant toxic side effects on normal tissues and the immune system.\u003csup\u003e1\u0026ndash;2\u003c/sup\u003e Therefore, there is a pressing need to develop safer and more effective treatment modalities for tumor.\u003c/p\u003e \u003cp\u003eWith the deeper understanding of tumor biology, researchers have observed that tumor tissues lack sufficient blood supply, rendering them unable to dissipate heat by regulating blood flow and velocity.\u003csup\u003e3\u0026ndash;4\u003c/sup\u003e Consequently, tumor tissues become natural reservoirs for heat accumulation. In light of the monumental development of photothermal therapy for tumors, there is a need to mitigate the damage caused by singular photothermal therapy to surrounding normal cells and tissues. As such, researchers have taken a different approach, utilizing near-infrared light (NIR) to induce localized heat generation within tumor tissues, thereby triggering apoptosis in tumor cells.\u003csup\u003e5\u003c/sup\u003e Despite the advantages of this method such as low invasiveness, high selectivity, and temporal-spatial control, the effective penetration of NIR is limited, making it challenging to eradicate deep-seated tumor cells and hindering its clinical application.\u003csup\u003e6\u003c/sup\u003e Therefore, there is an urgent need for safer and more effective methods for treating tumors.\u003c/p\u003e \u003cp\u003eWith the widespread development and application of photosensitizers and photothermal materials, the photothermal-chemotherapy combination therapy effectively overcomes the shortcoming of individual treatments. Especially, environmentally responsive nanoplatform prepared based on tumor microenvironment not only achieve precise and controllable release of chemotherapeutic drugs in tumor tissues but also effectively improve the tumor tissue microenvironment, thereby achieving a cascade effect in tumor treatment.\u003csup\u003e7\u0026ndash;8\u003c/sup\u003e Currently, nanoplatform extensively developed and utilized primarily consist of exogenous inorganic and organic compounds. However, due to their non-degradability and suboptimal biological metabolism, these carriers inevitably bring unnecessary toxic side effects to normal tissues and organs, hindering the clinical implementation of photothermal-chemotherapy combination therapy.\u003csup\u003e9\u0026ndash;10\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThanks to the development of photothermal nano-materials based on endogenous substances can effectively reduce the body's immune response, minimizing damage to normal tissues and better meeting the requirements for future clinical translation. Currently, widely developed and utilized endogenous photothermal materials mainly including dopamine, serotonin, and adrenaline.\u003csup\u003e11\u0026ndash;13\u003c/sup\u003e Among them, dopamine, as one of the important neurotransmitters in the body, plays a crucial role in regulating the development of the nervous system and maintaining pleasure and happiness in humans. Furthermore, as a small molecule catecholamine in the human body, dopamine can undergo oxidative polymerization in an alkaline environment, forming size-controllable polydopamine nanoparticles (PDA NAs).\u003csup\u003e14\u003c/sup\u003e The prepared PDA NAs not only exhibit superior photothermal performance in the near-infrared region but also their unique conjugated molecular structure and special adhesion properties make them highly suitable for loading chemotherapeutic drugs.\u003csup\u003e15\u003c/sup\u003e More importantly, the surface of PDA NAs contains abundant amino and hydroxyl groups, which facilitate surface modification to impart more biological properties, enabling tumor combination therapy based on photothermal effects.\u003csup\u003e16\u003c/sup\u003e Although the biocompatibility of photothermal nanoplatform have been effectively addressed, similar to most photothermal nanoparticles, polydopamine nanoparticles cannot achieve targeted delivery. Moreover, the central region of tumor tissues is severely hypoxic, resulting in the ineffective killing of tumor cells in the central region of tumor tissues by most anti-tumor drugs and photothermal nanoparticles.\u003c/p\u003e \u003cp\u003eTo sum up, in this study, Lactobacillus (LAC) was used as living drug carrier and integrates the chemotherapeutic drug doxorubicin (DOX) with dopamine through oxidative polymerization to form polydopamine on the surface of LAC, thereby preparing an environmentally responsive PDA\u0026amp;DOX@LAC biomimetic therapeutic platform for photothermal-chemotherapy combined treatment of tumors. As illustrated in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1a\u003c/span\u003e, the one-step preparation process of the PDA\u0026amp;DOX@LAC nanoplatform is gentle, with negligible impact on the biological properties of LAC. Upon intravenous injection into mice, the prepared PDA\u0026amp;DOX@LAC nanoplatform utilizes the anaerobic property of LAC to deliver the anti-tumor drug DOX to tumor tissues and achieves precise and controlled release of DOX based on its protonation propensity under acidic conditions. While preparing the PDA\u0026amp;DOX@LAC nanoplatform, doped Mn ions display peroxidase-like properties, which enable them to react with the high concentration of hydrogen peroxide in tumor tissues, leading to the production of oxygen. This process effectively alleviates the hypoxic condition within the tumor tissues. Upon exposed under NIR irradiation, the PDA\u0026amp;DOX@LAC nanoplatform converts light energy into heat and generates a large amount of reactive oxygen species, inducing apoptosis of tumor cells and releasing tumor-associated antigens. These antigens are taken up by dendritic cells, stimulating infiltration of cytotoxic T cells, which in turn kill tumor cells and inhibit tumor growth (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1b\u003c/span\u003e). Therefore, the PDA\u0026amp;DOX@LAC nanoplatform has been developed in a simpler and more effective manner, serving not only as an efficient live drug carrier for chemotherapy with tumor tissue-targeted delivery, but also exhibiting peroxidase-like activity to effectively improve the tumor hypoxic microenvironment, providing a theoretical basis and design concept for the development of novel tumor treatment platforms in the future.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and Reagents","content":"\u003cp\u003eDopamine, reduced glutathione (GSH), hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), 4\u0026rsquo;,6-diamidino-2-phenylindole (DAPI), doxorubicin (DOX), [Ru(dpp)3]Cl2 (RDPP), 2\u0026prime;,7\u0026prime;-dichlorofluorescein diacetate (DCFH-DA), 1,3-diphenylisobenzofuran (DPBF), cell counting kit-8 (CCK-8), MRS broth, and sodium hydroxide (NaOH) were procured from Aladdin (Shanghai, China). Dulbecco\u0026rsquo;s modified eagle medium (DMEM), RPMI Medium Modified (RPMI-1640), fetal bovine serum (FBS), phosphate-buffered saline (PBS), and streptomycin and penicillin were obtained from Gibco (Shanghai, China). All chemicals were utilized as received without additional purification. Deionized water was used throughout the experiments.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eApparatus and Procedures\u003c/h2\u003e \u003cp\u003eTransmission electron microscopy (TEM) imaging was conducted using a JEOL 2100 transmission electron microscope. Particle size and ζ-potential measurements were performed using a NanoBrook Omni (Brookhaven, USA). Fourier transform infrared (FTIR) spectra were obtained with a Nicolet Nexus 470 spectrometer (USA). X-ray photoelectron spectroscopy (XPS) analysis of PDA\u0026amp;DOX@Rha was carried out using an XPS spectrometer (Thermo Kalpha). Near-infrared light at 808 nm was delivered using a fiber-coupled NIR laser (MDL-N-808 nm-10W, Beijing Laserwave OptoElectronics Technology Co., Ltd., Beijing, China). An infrared thermal camera (HT-19, Guangzhou, China) was employed to monitor temperature changes and capture infrared thermal images.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eThe preparation of PDA\u0026amp;DOX@LAC\u003c/h2\u003e \u003cp\u003eThe synthesis procedure of the PDA\u0026amp;DOX@LAC nanoplatform followed established protocols with slight adaptations.\u003csup\u003e17\u0026ndash;18\u003c/sup\u003e Initially, 1\u0026times;10\u003csup\u003e9\u003c/sup\u003e Lactobacillus cells were dispersed in 10 mL of pH 8.0 PBS buffer. Subsequently, dopamine (1 mg/mL), doxorubicin (1 mg/mL), and 0.5 mL of KMnO\u003csub\u003e4\u003c/sub\u003e (1 mg/mL) were introduced into the solution according to the prescribed method. The mixture was then stirred at room temperature for 30 min, followed by collection of the precipitate via centrifugation (2000 rpm, 5 min) and subsequent washing with PBS for three times. Finally, the samples were freeze-dried under vacuum conditions. The obtainted PDA\u0026amp;DOX@LAC nanoplatform was stored at 4 ℃ for future applications.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eThe release behavior of DOX from PDA\u0026amp;DOX@LAC\u003c/h2\u003e \u003cp\u003eTo assess the release kinetics of DOX from the PDA\u0026amp;DOX@LAC nanoplatform under various conditions.\u003csup\u003e19\u003c/sup\u003e 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e CFU of PDA\u0026amp;DOX@LAC was separately introduced into (a) PBS (pH\u0026thinsp;=\u0026thinsp;7.4), (b) GSH (10 mM) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (30 mM) in PBS (pH 5.5), and (c) a combination of GSH and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (pH 5.5, 10 mM GSH, and 30 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e). Subsequently, at predetermined time intervals, samples were withdrawn from each mixture, and the absorbance of DOX at 490 nm was measured using UV-vis spectroscopy to characterize the release profile of DOX.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eThe measurement of photothermal effects\u003c/h2\u003e \u003cp\u003ePDA\u0026amp;DOX@LAC was resuspended in 200 \u0026micro;L PBS and irradiated with 808 nm NIR (1.5, 1.75, and 2.0 W/cm\u003csup\u003e2\u003c/sup\u003e) at different powers for different time intervals at room temperature. PDA\u0026amp;DOX@LAC was configured at 100 \u0026micro;g/mL and 200 \u0026micro;g/mL (the number of LACs was approximately 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e and 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e) and temperature changes were recorded at any time using an infrared thermal imaging instrument. The photothermal conversion efficiency (η) of PDA\u0026amp;DOX@LAC was calculated by the following equation:\u003csup\u003e20\u0026ndash;21\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eθ = (T - T\u003csub\u003emin\u003c/sub\u003e)/(T\u003csub\u003emax\u003c/sub\u003e - T\u003csub\u003emin\u003c/sub\u003e) (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e \u003cp\u003ehs\u0026thinsp;=\u0026thinsp;m*C\u003csub\u003ep\u003c/sub\u003e/ζs (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eη\u0026thinsp;=\u0026thinsp;hs (ΔT\u003csub\u003emax,mix\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;ΔT\u003csub\u003emax,H2O\u003c/sub\u003e)/I(1\u0026thinsp;\u0026minus;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;A808\u003c/sup\u003e) (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eθ defined the dimensionless driving force temperature, m was 2\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e kg, Cp was 4.2\u0026times;10\u003csup\u003e3\u003c/sup\u003e J/(kg\u0026middot;℃), ζ is the slope of T and -Lnθ, ΔTmax,mix is the maximum temperature change of PDA\u0026amp;DOX@LAC, ΔTmax,H\u003csub\u003e2\u003c/sub\u003eO is the maximum temperature change of water. I is the laser power. Where A808 is the absorbance of PDA\u0026amp;DOX@LAC at the wavelength of 808 nm in aqueous solution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003e4T1 cells (murine breast cancer cells), GES-1 cells (human gastric mucosa epithelial cells), and LAC (Lactobacillus) were procured from SUNNCELL Biological Technology Co., Ltd. The cells were cultured in RPMI Medium Modified (RPMI-1640) supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin. Cell cultures were maintained at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere and were approved for use by the Northern Jiangsu People\u0026rsquo;s Hospital.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eThe evaluation of O\u003csub\u003e2\u003c/sub\u003e generation ability from the PDA\u0026amp;DOX@LAC\u003c/h2\u003e \u003cp\u003eThe oxygen generation capability of PDA\u0026amp;DOX@LAC nanoplatform was assessed by dissolved oxygen meter (JPBJ-608, INESL, Shanghai, China).\u003csup\u003e22\u003c/sup\u003e Initially, 10 mL of PDA\u0026amp;DOX@LAC dispersion solution (200 \u0026micro;g/mL) was added to a 20 mL beaker. Subsequently, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (30 \u0026micro;M) was introduced into the PDA\u0026amp;DOX@LAC dispersion solution. The dissolved oxygen meter probe was then inserted into the dispersion of PDA\u0026amp;DOX@LAC and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e mix dispersion solution to monitor real-time changes in oxygen concentration. PBS solution, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution, and PDA\u0026amp;DOX@LAC dispersion served as control experiments.\u003c/p\u003e \u003cp\u003e[Ru(dpp)\u003csub\u003e3\u003c/sub\u003e]Cl\u003csub\u003e2\u003c/sub\u003e(RDPP) serves as a fluorescent probe for dissolved oxygen detection, where its fluorescence signal is quenched in the presence of oxygen.\u003csup\u003e23\u003c/sup\u003e Thus, 2 mg of PDA\u0026amp;DOX@LAC nanoplatform was added to 20 mL PBS solution containing 30 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and 10 \u0026micro;M RDPP. Fluorescence measurements were taken at predetermined time points by fluorescence spectrometer to detect oxygen concentration in the dispersion of PDA\u0026amp;DOX@LAC. Additionally, 10 \u0026micro;M RDPP fluorescent dye was separately added to 20 mL PBS solution, PBS solution containing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (30 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), and PDA\u0026amp;DOX@LAC dispersion (200 \u0026micro;g/mL) as control groups to analyze oxygen concentration in the above solutions.\u003c/p\u003e \u003cp\u003e4T1 cells were co-incubated with PDA\u0026amp;DOX@LAC, and the oxygen generation capability within the cells was assessed by observing the intensity of RDPP fluorescence signals using fluorescence microscopy. The specific steps were as follows: Initially, 4T1 cells (1\u0026times;10\u003csup\u003e7\u003c/sup\u003e) were seeded into cell culture dishes and co-incubated with 10 \u0026micro;M RDPP for 4 hours. Then, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (30 \u0026micro;M), PDA\u0026amp;DOX@LAC (200 \u0026micro;g/mL), and DA\u0026amp;DOX@LAC (200 \u0026micro;g/mL)\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (30 \u0026micro;M) were separately added to the aforementioned culture, followed by 4 h incubation period and multiple washes with PBS. Finally, fluorescence imaging of 4T1 cells in each treatment group was observed using fluorescence microscopy.\u003csup\u003e24\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eThe detection of extracellular and intracellular ROS\u003c/h2\u003e \u003cp\u003e1,3-diphenylisobenzofuran (DPBF) serves as a chemical probe for monitoring extracellular reactive oxygen species (ROS), exhibiting decreased absorbance when exposed to ROS environments.\u003csup\u003e25\u0026ndash;26\u003c/sup\u003e Initially, PDA\u0026amp;DOX@LAC (200 \u0026micro;g/ml) was added to a mixture of DPBF (1 mM) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (30 \u0026micro;M), followed by irradiation under 808 nm NIR for 10 minutes. UV-vis spectroscopy was then utilized to detect the UV-visible absorption peaks of DPBF at predetermined time points. DPBF in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution (30 \u0026micro;M), dispersed solution of PDA\u0026amp;DOX@LAC (200 \u0026micro;g/ml)\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (30 \u0026micro;M), PDA\u0026amp;DOX@LAC (200 \u0026micro;g/ml)\u0026thinsp;+\u0026thinsp;NIR, and UV-visible absorption peaks in PDA\u0026amp;DOX@LAC served as control groups.\u003c/p\u003e \u003cp\u003eAdditionally, the intracellular levels of ROS were assessed using the cell-permeable probe 2',7'-dichlorofluorescein diacetate (DCFH-DA).\u003csup\u003e27\u0026ndash;28\u003c/sup\u003e DCFH-DA does not exhibit any fluorescence signal under normal conditions; however, in the presence of abundant ROS within cells, DCFH-DA is rapidly oxidized to yield green fluorescence of 2,7-dichlorofluorescein (DCF). To further validate whether co-incubation of PDA\u0026amp;DOX@LAC with 4T1 cells could induce ROS generation under 808 nm NIR irradiation, 4T1 cells (1\u0026times;10\u003csup\u003e7\u003c/sup\u003e) were seeded into 6-well plates and subjected to different treatments (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, PDA\u0026amp;DOX@LAC\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, PDA\u0026amp;DOX@LAC\u0026thinsp;+\u0026thinsp;NIR, and PDA\u0026amp;DOX@LAC\u0026thinsp;+\u0026thinsp;NIR\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e). After 6 h, the cells were exposed under 2.0 W/cm\u003csup\u003e2\u003c/sup\u003e NIR irradiation for 10 minutes, followed by removal of the culture medium and replacement with fresh medium containing DCFH-DA. After 30 min, the medium was discarded, and the cells were washed three times with PBS before fluorescence signals within 4T1 cells were observed using fluorescence microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eThe temperature regulates cell membrane permeability\u003c/h2\u003e \u003cp\u003eDifferent concentrations of PDA\u0026amp;DOX@LAC (100 \u0026micro;g/ml and 200 \u0026micro;g/ml) were co-incubated with 4T1 cells and placed in a cell culture incubator (37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e). After 6 h, they were subjected to NIR irradiation at different power densities (1.5 and 2.0 W/cm\u003csup\u003e2\u003c/sup\u003e) for 10 minutes, followed by 3 h incubation period. Subsequently, the culture medium was discarded, and the cells were washed three times with PBS. Then, 1 mL of 4% paraformaldehyde was added to each cell culture dish for fixation for 15 minutes. After discarding the fixative, 1 mL of DAPI staining solution was added to each dish and gently shaken on a horizontal shaker. After 15 min, the DAPI staining solution was removed, and the cells were washed three times with PBS. A small amount of PBS was then added, and the fluorescence intensity of 4T1 cells and GES-1 cells was observed using confocal fluorescence microscopy.\u003csup\u003e29\u0026ndash;30\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003ecytotoxicity\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe cytotoxicity of PDA\u0026amp;DOX@LAC combined with NIR was evaluated using the CCK-8 assay. 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e 4T1 cells and GES-1 cells were seeded into 96-well plates and incubated for 12 hours. Subsequently, different concentrations of PDA\u0026amp;DOX@LAC, DOX, PDA, and PDA\u0026amp;DOX nanoparticles were added to each well and co-incubated with the cells for 6 h, followed by irradiation under 808 nm NIR (2.0 W/cm\u003csup\u003e2\u003c/sup\u003e, 10 min). Untreated cells were used as a control group. After 24 h, the cells were washed several times with PBS, and then 10 \u0026micro;l of CCK-8 reagent was added to each well and further incubated for 3 h. The absorbance values (OD) of each well in the 96-well plate were measured using a microplate reader at an excitation wavelength of 450 nm.\u003csup\u003e31\u0026ndash;32\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnimal welfare\u003c/h2\u003e \u003cp\u003e In this study, the animal experiment was conducted in accordance with the guidelines outlined in the Animal Management Rules of the ARRIVE guidelines. Healthy female C57BL/6 mice, aged 6 to 8 weeks, were procured from the Model Animal Genetics Research Center of Yangzhou University (Yangzhou, People\u0026rsquo;s Republic of China). Every effort was made to minimize animal suffering and reduce the number of animals used in the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eThe construction and treatment of 4T1 mouse subcutaneous xenografts model\u003c/h2\u003e \u003cp\u003eMale Balb/c mice were subcutaneously injected with 0.2 mL of 4T1 cells (5\u0026times;10\u003csup\u003e7\u003c/sup\u003e) in the groin area to establish the 4T1 subcutaneous tumor model.\u003csup\u003e31\u0026ndash;32\u003c/sup\u003e When the tumor volume reached approximately 100 mm\u003csup\u003e3\u003c/sup\u003e, the 4T1 tumor-bearing mice were randomly divided into 6 groups (n\u0026thinsp;=\u0026thinsp;6/group): PBS, PBS\u0026thinsp;+\u0026thinsp;NIR, DOX (4 mg/kg), PDA (4 mg/kg)\u0026thinsp;+\u0026thinsp;NIR, PDA\u0026amp;DOX (4 mg/kg)\u0026thinsp;+\u0026thinsp;NIR, and PDA\u0026amp;DOX@LAC\u0026thinsp;+\u0026thinsp;NIR groups. The aforementioned drugs were administered intravenously via the tail vein to the tumor-bearing mice. After 24 h post-injection, NIR irradiation at a power density of 2.0 W/cm\u003csup\u003e2\u003c/sup\u003e was applied for 10 minutes. Injections were administered every 3 days, with real-time monitoring of each mouse's body weight and tumor volume. Tumor volume was calculated using the formula: 0.5 \u0026times; width\u003csup\u003e2\u003c/sup\u003e \u0026times; length. Subsequently, tumor tissues and major organs (heart, liver, spleen, lungs, and kidneys) were fixed in 4% formalin buffer and embedded in paraffin. Specimens underwent hematoxylin-eosin (H\u0026amp;E) staining and Terminal Deoxynucleotidyl Transferase Dutp Nick End Labeling (TUNEL) staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHistological analysis\u003c/h2\u003e \u003cp\u003eAfter 15 d, all mice were euthanized using cervical dislocation, and the major organs (heart, liver, spleen, lungs, and kidneys) along with tumor tissues of the 4T1 tumor-bearing mice were collected for histological analysis. The collected tissues were fixed in 4% formalin, followed by embedding in paraffin. Tissue sections were prepared and stained with H\u0026amp;E staining. Tumor tissues were also subjected to TUNEL staining. The prepared tissue sections were placed on a slide scanner to capture tissue images, which were then evaluated by experienced pathologists.\u003csup\u003e33\u0026ndash;34\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eThe detection of mouse blood samples\u003c/h2\u003e \u003cp\u003eBefore treatment, blood tests were conducted on 4T1 tumor-bearing mice in the PDA\u0026amp;DOX@LAC\u0026thinsp;+\u0026thinsp;NIR group. Subsequently, PDA\u0026amp;DOX (4 mg/kg) was injected intravenously via the tail vein into the 4T1 tumor-bearing mice. Blood samples were collected from the mice on days 1, 7, and 14 of treatment to evaluate hematological and biochemical parameters, assessing the biocompatibility of PDA\u0026amp;DOX@LAC.\u003csup\u003e35\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTumor dendritic cells and infiltrating T cells analysis\u003c/h2\u003e \u003cp\u003eAfter treatment, tumor tissues were collected from each mouse in every treatment group. These tissues were minced and digested using Dulbecco's Modified Eagle's Medium (DMEM) supplemented with hyaluronidase (100 \u0026micro;g/mL), DNase I (100 \u0026micro;g/mL), 10% fetal bovine serum (FBS), and collagenase type IV (1 mg/mL) under continuous agitation (200 rpm) in an incubator at 37\u0026deg;C. Following digestion, the cells were filtered through a nylon mesh (500 mesh), then centrifuged at 500 g for 5 minutes. The resulting cell pellet was subjected to red blood cell lysis and purified using a 40% Percoll (GE) solution. Dendritic cells isolated from the extracted cell population were stained with anti-mouse CD11c, CD80, and CD86 antibodies for flow cytometric analysis. Additionally, cytotoxic T lymphocytes present in the single-cell suspension obtained from 4T1 xenograft tumors were stained with anti-mouse CD3, CD4, and CD8a antibodies for subsequent flow cytometric analysis.\u003csup\u003e36\u0026ndash;37\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of tumor dendritic cells and infiltrating T cells\u003c/h2\u003e \u003cp\u003eAfter treatment, tumor tissues were collected from each treatment group of each mouse, and continuously shaken in an oven at 37℃ (200 rpm, 40 minutes). Then, they were chopped and digested with Dulbecco modified Dulbecco medium (DMEM) containing hyaluronidase (100 \u0026micro;g/mL), deoxyribonuclease I(100\u0026micro;g/mL), 10% fetal bovine serum (FBS) and collagenase IV (1 mg/mL). Subsequently, the digested cells were filtered with a nylon mesh (500 mesh), collected by centrifugation at 500 g for 5 minutes, and then purified by using erythrocyte lysis and 40% Percoll (GE) solution. The extracted dendritic cells were collected and stained with anti-mouse CD11c, anti-mouse CD80 and anti-mouse CD86 for flow cytometry analysis. In addition, cytotoxic T lymphocytes in single cell suspension were collected from 4T1 xenograft tumors and stained with anti-mouse CD3, anti-mouse CD4 and anti-mouse CD8a for flow cytometry analysis.\u003csup\u003e36\u0026ndash;37\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe experimental data were analyzed by SPSS (17.0) software and OriginPro (2017). All data were presented by mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Student\u0026rsquo;s t-test was used to analyze Two-group comparison and the data from more than three groups were compared and analyzed by one-way analysis of variance (ANOVA). The p value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant. \u0026lsquo;*** \u0026rsquo; means p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u0026lsquo;**\u0026rsquo; means p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and \u0026lsquo;*\u0026rsquo; means p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cp\u003eThe preparation and characterization of PDA\u0026amp;DOX@LAC nanosystem\u003c/p\u003e\n\u003cp\u003eSince the pioneering work of W. Busch and W. Coley utilizing bacteria in tumor therapy, bacterial-mediated tumor treatment has been extensively explored and utilized.\u003csup\u003e38\u0026ndash;39\u003c/sup\u003e With advancements in biotechnology, materials science, and immunology, bacteria have been genetically engineered to serve as specific immunotherapeutic agents, capable of ameliorating the tumor immunosuppressive microenvironment and enhancing anti-tumor immune responses.\u003csup\u003e40\u0026ndash;41\u003c/sup\u003e However, the safety concerns associated with genetically engineered bacteria, including the risk of gene transfer and mutation, have hindered their widespread application as therapeutic vectors.\u003csup\u003e42\u003c/sup\u003e In recent years, inspired by advances in materials science, researchers have employed methods such as covalent coupling, supramolecular interactions, and physical encapsulation to modify bacteria, effectively enhancing the accumulation and retention of therapeutic drugs at tumor sites and imparting additional biological functions.\u003csup\u003e43\u003c/sup\u003e Motivated by these strategies, this study utilized the oxidative polymerization property of dopamine in alkaline environments to coat chemotherapeutic drug DOX onto the surface of LAC bacteria. TEM images demonstrated the successful modification of LAC surface with dopamine and DOX at concentrations of 1 mg/mL, resulting in the formation of uniform nanoparticles with a core-shell structure, termed PDA\u0026amp;DOX@LAC nanosystem (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). Dynamic light scattering results indicated an increase in average particle size from 1803\u0026thinsp;\u0026plusmn;\u0026thinsp;158 nm to 2586\u0026thinsp;\u0026plusmn;\u0026thinsp;312 nm upon PDA loading (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb). Additionally, electrochemical potential observations revealed a shift in potential from \u0026minus;\u0026thinsp;3.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69 mV (LAC) to -10.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26 mV (PDA@LAC) after PDA coating, and further elevation to -5.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93 mV upon successful loading of DOX onto the surface of PDA\u0026amp;DOX@LAC nanosystem (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec). To further confirm the successful loading of DOX into the PDA\u0026amp;DOX@LAC nanocarrier, UV/Vis spectroscopic results shown a significant increase in absorbance in the suspension of PDA\u0026amp;DOX@LAC nanosystem, with the absorption peak position matching that of DOX (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed). Furthermore, fourier transform infrared (FTIR) spectroscopy results revealed similar vibrational characteristic peaks at 3327 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 2977 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 1590 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for DOX, PDA, and PDA\u0026amp;DOX@LAC nanosystem, corresponding to -OH, C-H, and C\u0026thinsp;=\u0026thinsp;O stretching vibrations, respectively. Moreover, DOX and PDA\u0026amp;DOX@LAC nanosystem exhibited similar absorption peaks between 900\u0026ndash;1500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, confirming the successful preparation of the PDA\u0026amp;DOX@LAC nanosystem (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ee). Subsequently, the biocompatibility of the PDA\u0026amp;DOX@LAC nanosystem during the preparation process was further validated. Colony counting showed that the survival rate of LAC after encapsulation remained around 94.5% (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ef and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eg). Finally, the impact of the PDA shell on bacterial growth was evaluated through optical density (OD) measurements, revealing that the PDA coating effectively delayed LAC growth without affecting its viability (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eh). In conclusion, we successfully prepared a core-shell structured PDA\u0026amp;DOX@LAC nanosystem.\u003c/p\u003e\n\u003cp\u003eIn order to evaluate the environmentally responsive release performance of the PDA\u0026amp;DOX@LAC nanosystem, we conducted X-ray photoelectron spectroscopy (XPS) analysis. The results revealed that the PDA\u0026amp;DOX@LAC nanosystem was composed of Na, Mn, O, N, and C elements, with manganese ions primarily existing in the forms of Mn\u003csup\u003e3+\u003c/sup\u003e and Mn\u003csup\u003e4+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee). Subsequently, the PDA\u0026amp;DOX@LAC nanosystem was immersed in five different simulated solutions: PBS, pH\u0026thinsp;=\u0026thinsp;5.5, 30 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 10 mM GSH, and a simulated tumor microenvironment (TME) (pH\u0026thinsp;=\u0026thinsp;5.5, 30 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 10 mM GSH). As depicted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea, only approximately 15% of DOX was released in the normal physiological environment (PBS), mainly due to the flow of the solution causing DOX release from the surface of the PDA\u0026amp;DOX@LAC nanosystem. However, in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution, around 22.3% of DOX was released from the surface of the PDA\u0026amp;DOX@LAC nanosystem. This release was primarily attributed to the accumulation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e on the surface of the PDA\u0026amp;DOX@LAC nanosystem, where the manganese ions present in the nanosystem underwent Fenton reaction with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, weakening the interaction between DOX and the PDA\u0026amp;DOX@LAC nanosystem and resulting in DOX release. When the PDA\u0026amp;DOX@LAC nanosystem was immersed in the GSH solution, nearly 60% of DOX was released. This significant release was attributed to the redox reaction between GSH and the PDA\u0026amp;DOX@LAC nanosystem. Although the elemental composition of PDA\u0026amp;DOX@LAC remained unchanged, a large amount of Mn\u003csup\u003e3+\u003c/sup\u003e and Mn\u003csup\u003e4+\u003c/sup\u003e was reduced to Mn\u003csup\u003e2+\u003c/sup\u003e, causing structural changes in the PDA\u0026amp;DOX@LAC nanosystem and subsequently leading to DOX releasing (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ef). However, when the PDA\u0026amp;DOX@LAC nanosystem was immersed in an acidic environment, the release rate approached 100% after 24 hours. Fourier-transform infrared spectroscopy (FTIR) spectra results shown that while the absorption peak positions of PDA\u0026amp;DOX@LAC and PDA\u0026amp;DOX@LAC\u0026thinsp;+\u0026thinsp;TME were the same, the absorption peak of DOX between 900\u0026ndash;1500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in PDA\u0026amp;DOX@LAC\u0026thinsp;+\u0026thinsp;TME was weakened (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb). This weakening was mainly attributed to the protonation of DOX in the acidic environment, leading to its release from the surface of the PDA\u0026amp;DOX@LAC nanosystem.\u003csup\u003e44\u0026ndash;45\u003c/sup\u003e When the PDA\u0026amp;DOX@LAC nanosystem was immersed in an \u003cem\u003ein vitro\u003c/em\u003e simulated TME, nearly 100% of DOX was released within 18 hours. The rapid release of DOX was attributed to the combined effects of multiple factors. Thus, the prepared PDA\u0026amp;DOX@LAC nanosystem exhibited environmentally responsive release performance.\u003c/p\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n\u003ch2\u003eEnzyme-like property of PDA\u0026amp;DOX@LAC nanosystem\u003c/h2\u003e\n\u003cp\u003eDue to the inclusion of Mn ions in the PDA\u0026amp;DOX@LAC nanosystem, which possess catalase-like activity, they can react with the high concentration of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in tumor tissues, generating oxygen and hydroxyl radicals. This process effectively alleviates the hypoxic microenvironment of tumor tissues and the hydroxyl radicals produced further damage tumor cells.\u003csup\u003e46\u0026ndash;47\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eIn order to investigate the oxygen-generating capacity of the PDA\u0026amp;DOX@LAC nanosystem in response to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, the nanosystem was immersed in five different simulated solutions: PBS, pH\u0026thinsp;=\u0026thinsp;5.5, 30 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 10 mM GSH, and a simulated tumor microenvironment (TME) (pH\u0026thinsp;=\u0026thinsp;5.5, 30 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 10 mM GSH). The oxygen content in each solution was monitored in real-time using a dissolved oxygen meter. Over time, as the PDA\u0026amp;DOX@LAC nanosystem was immersed in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution and TME, the oxygen content gradually increased. After 26 min, the oxygen content in these two solutions reached 9.87 mg/mL and 9.93 mg/mL, respectively, while the oxygen content in the other solutions remained relatively unchanged (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). Photographs were taken of each reaction system, revealing a greater number of bubbles when the PDA\u0026amp;DOX@LAC nanosystem was immersed in the solution containing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). RDPP, serving as a typical oxygen fluorescence probe, is oxidized in the presence of oxygen, leading to quenching of its fluorescence signal. As shown in Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec \u0026minus;\u0026thinsp;3f, when RDPP was immersed in a mixture of PDA\u0026amp;DOX@LAC and 30 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution, the fluorescence signal of RDPP gradually weakened. However, in the solution containing RDPP, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and PDA\u0026amp;DOX@LAC, there was no significant change in the RDPP fluorescence signal. This result indicates that the dispersion of PDA\u0026amp;DOX@LAC in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution generated oxygen, causing the oxidation of RDPP and subsequent reduction in its fluorescence signal. In an \u003cem\u003ein vitro\u003c/em\u003e simulated high-concentration H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e environment, PDA\u0026amp;DOX@LAC generated a significant amount of oxygen, prompting further evaluation of intracellular oxygen production. Subsequently, PDA\u0026amp;DOX@LAC was co-incubated with 4T1 cells to assess intratumoral oxygen generation. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eg, when 4T1 cells were co-incubated with PDA\u0026amp;DOX@LAC, the RDPP fluorescence signal exhibited noticeable quenching under fluorescence microscopy. Conversely, when 4T1 cells were co-incubated with PDA\u0026amp;DOX@LAC and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, the red fluorescence signal was barely detected. In comparison, strong red fluorescence signals were observed when 4T1 cells were solely incubated with RDPP or co-incubated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. These results suggest that PDA\u0026amp;DOX@LAC possesses catalase-like properties, capable of generating oxygen through its reaction with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n\u003ch2\u003ePhotothermal Properties of PDA\u0026amp;DOX@LAC nanosystem\u003c/h2\u003e\n\u003cp\u003eThe bacterial suspension of the PDA\u0026amp;DOX@LAC nanosystem exhibits absorbance in the near-infrared (NIR) region, and dopamine nanoparticles demonstrate excellent photothermal conversion properties. This prompted us to further evaluate the photothermal conversion performance of PDA\u0026amp;DOX@LAC. Therefore, bacterial suspensions of different concentrations of PDA\u0026amp;DOX@LAC nanosystem (100 \u0026micro;g/mL and 200 \u0026micro;g/mL) were subjected to irradiation under different power densities of NIR light (1.5 W/cm2, 1.75 W/cm\u003csup\u003e2\u003c/sup\u003e, and 2.0 W/cm\u003csup\u003e2\u003c/sup\u003e) for 10 minutes. As shown in Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb, the photothermal conversion performance of the PDA\u0026amp;DOX@LAC nanosystem is positively correlated with time, concentration, and power density. When the concentration of PDA\u0026amp;DOX@LAC nanosystem was 200 \u0026micro;g/mL and irradiated with 2.0 W/cm\u003csup\u003e2\u003c/sup\u003e NIR light for 10 min, the temperature of the PDA\u0026amp;DOX@LAC suspension could increase to 53.2 ℃, with minimal change compared to the temperature of PDA nanoparticles (55.9 ℃) under the same conditions. Furthermore, the photothermal conversion efficiency of the PDA\u0026amp;DOX@LAC nanosystem remained at 35.6% (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed). Thus, PDA\u0026amp;DOX@LAC exhibits superior photothermal conversion performance suitable for photothermal therapy of tumors. To further validate the photothermal stability of the PDA\u0026amp;DOX@LAC nanosystem for repeated irradiation, the 200 \u0026micro;g/mL PDA\u0026amp;DOX@LAC suspension was subjected to 2.0 W/cm\u003csup\u003e2\u003c/sup\u003e NIR irradiation for 10 min, followed by NIR excitation shutdown for 10 min. This cycle was repeated four times, and temperature changes were recorded using an infrared thermal imaging instrument (recorded every 2 min). As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ee, after four cycles of irradiation, the temperature of the PDA\u0026amp;DOX@LAC suspension remained at 54.1\u0026deg;C, with a slight increase in temperature observed after each cycle, attributed to water evaporation caused by temperature rise. Therefore, the PDA\u0026amp;DOX@LAC nanoplatform prepared in this study demonstrates superior photothermal conversion performance and can withstand multiple repeated irradiation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n\u003ch2\u003eGeneration of ROS inside and outside PDA-DOX @ LAC cells stimulated by near infrared spectroscopy\u003c/h2\u003e\n\u003cp\u003eThe enzyme-like property and photothermal response property of PDA\u0026amp;DOX@LAC nanosystem prompted us to further explore its ability to generate ROS under the stimulation of near-infrared light for photodynamic therapy of tumors. As a typical extracellular active oxygen probe, DPBF can be oxidized to an oxidation product (O-dibenzoylbenzene) in the presence of ROS, resulting in the decrease of its UV absorption values. To explore whether PDA\u0026amp;DOX@LAC could produce ROS under NIR irradiation, DPBF was respectively immersed in solutions of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, PDA\u0026amp;DOX@LAC\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, PDA\u0026amp;DOX@LAC\u0026thinsp;+\u0026thinsp;NIR, and PDA\u0026amp;DOX@LAC\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea-\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ed). Compared with the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group in the control group, the UV absorption values of DPBF in other groups decreased to some extent over time. It was mainly attributed to the Fenton reaction between PDA\u0026amp;DOX@LAC and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, which generated hydroxyl radicals and caused the decrease of absorption value of DPBF. PDA\u0026amp;DOX@LAC produced a large amount of heat after NIR irradiation, which was then transferred to the oxygen in the surrounding tissue. The molecular oxygen transited to the ground state oxygen, causing the decrease of UV absorption values DPBF. However, after H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was added into PDA\u0026amp;DOX@LAC solution and placed in the NIR for 10 minutes, the UV absorption values of DPBF exhibited a more significant decrease, indicating that Fenton reaction occurred after PDA\u0026amp;DOX@LAC was dispersed in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution, resulting in the generation of hydroxyl free radicals and a large amount of oxygen. After NIR irradiation, more ROS generated.\u003c/p\u003e\n\u003cp\u003ePDA\u0026amp;DOX@LAC produced large amounts of ROS after extracellular stimulation with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and NIR, which prompted us to further evaluate the production of ROS in cells. DCFH-DA, as a cell permeability dye, has no fluorescence itself. Once it enters cells, it is easily hydrolyzed by cell esterase into DCFH, which is then rapidly oxidized by ROS to DCF with a strong green fluorescence product. Therefore, DCFH-DA was co-incubated with 4T1 cells to test whether PDA\u0026amp;DOX@LAC could produce ROS in 4T1 cells. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ee, 4T1 cells were randomly divided into 6 groups as control groups, PDA\u0026amp;DOX@LAC\u0026thinsp;+\u0026thinsp;NIR, NIR Only, PDA\u0026amp;DOX@LAC\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and PDA\u0026amp;DOX@LAC\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NIR. It is found by fluorescence microscope that the green fluorescence signal could be clearly observed when the 4T1 cells were incubated with PDA\u0026amp;DOX@LAC and irradiated with NIR for 10 minutes. In addition, after adding 30 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e into the 4T1 cell culture medium and placing it under NIR irradiation for 10 min, the green fluorescence signal was the strongest compared with the control group. The main reason is that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e reacts with PDA\u0026amp;DOX@LAC to produce more oxygen and a large amount of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e is produced after NIR irradiation, which leads to the enhancement of green fluorescence signal. However, in other treatment groups like the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group and the PDA\u0026amp;DOX@LAC group, the green fluorescence signal could hardly be detected, mainly due to the small production of ROS in 4T1 cells, which is consistent with that result of \u003cem\u003ein vitro\u003c/em\u003e DPBF detection of ROS. Therefore, PDA\u0026amp;DOX@LAC could produce a large amount of ROS under the excitation of NIR when there was sufficient oxygen around the tissue. Thus PDA\u0026amp;DOX@LAC can be used as an ideal material for PDT treatment.\u003c/p\u003e\n\u003cp\u003eThe killing effect of PDA\u0026amp;DOX@LAC on cells\u003c/p\u003e\n\u003cp\u003ePDA\u0026amp;DOX@LAC has superior environmental response performance and the research shows that with the increase of temperature, it can effectively enhance the permeability of cell membrane, and thus enhance the uptake of drugs by cells and enhance the killing performance of cells.\u003csup\u003e29\u0026ndash;30\u003c/sup\u003e Therefore, we incubated 200 \u0026micro;g/mL PDA\u0026amp;DOX@LAC with 4T1 cells and GES-1 cells for 12 h respectively, and then treated with NIR(1.5, 1.75 and 2.0 W/cm\u003csup\u003e2\u003c/sup\u003e) with different powers. The results of confocal microscopy shown that strong fluorescence signals could be detected in 4T1 cells and the fluorescence signal of DOX was also becoming stronger with the gradual increase of NIR power. It was mainly because the simulated tumor microenvironment constructed by 4T1 cells triggers the release of DOX from the surface of PDA\u0026amp;DOX@LAC. Subsequently, NIR causes the temperature to rise, which increases the permeability of 4T1 cells and promotes the uptake of DOX by 4T1 cells. (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea). However, the fluorescence signal of DOX in GES-1 cells did not become stronger with the increase in NIR power (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb). It is mainly due to the fact that the normal tissue microenvironment of GES-1 cells can't release DOX from the surface of PDA\u0026amp;DOX@LAC, which leads to that GES-1 cells can't absorb DOX and its fluorescence signal doesn't get stronger with the increase of NIR power.\u003c/p\u003e\n\u003cp\u003eThanks to the effective environmental responsiveness of drug release exhibited by PDA\u0026amp;DOX@LAC, coupled with the efficient drug absorption capability of tumor cells, we have delved deeper into its cytotoxic effects on tumor cells. Specifically, the viability of both GES-1 and 4T1 cells was assessed through CCK-8 experiments to gauge the anti-tumor efficacy of the chemo-photothermal synergistic therapy mediated by PDA\u0026amp;DOX@LAC. As depicted in Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ec and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed, the escalation in PDA\u0026amp;DOX@LAC concentration corresponded to a gradual decline in the cell viability of both GES-1 and 4T1 cells. Upon exposure to NIR (10 min, 2.0 W/cm\u003csup\u003e2\u003c/sup\u003e) under identical concentrations and conditions, the viability of 4T1 cells notably plummeted compared to that of GES-1 cells. Specifically, at a concentration of 200 \u0026micro;g/mL of PDA\u0026amp;DOX@LAC, the cell viability of GES-1 cells remained approximately at 50%, whereas that of 4T1 cells dipped below 20%. These findings underscore the potent cytotoxicity of the chemo-photothermal synergistic therapy mediated by PDA\u0026amp;DOX@LAC against tumor cells, primarily attributed to the effective tumor microenvironment-driven DOX release and subsequent enhancement of 4T1 cell permeability facilitated by photothermal therapy, thereby augmenting DOX absorption. In addition, when DOX and PDA\u0026amp;DOX@LAC were in the same concentration, the killing effect of DOX on GES-1 cells was significantly higher than that of PDA\u0026amp;DOX@LAC. Furthermore, when DOX and PDA\u0026amp;DOX@LAC were administered at equivalent concentrations, the cytotoxic efficacy of DOX against GES-1 cells significantly surpassed that of PDA\u0026amp;DOX@LAC. Hence, PDA\u0026amp;DOX@LAC achieves a multifaceted approach encompassing chemotherapy, photothermal therapy, and photodynamic therapy, bolstered by NIR irradiation, effectively eradicating tumor cells while mitigating DOX-induced damage to normal cells.\u003c/p\u003e\n\u003cp\u003eAntitumor Effect of PDA\u0026amp;DOX@LAC \u003cem\u003ein Vivo\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBased on the excellent photothermal conversion performance of the PDA\u0026amp;DOX@LAC nanosystem \u003cem\u003ein vitro\u003c/em\u003e, we further evaluated its aggregation performance in tumor tissues of 4T1 tumor-bearing mice to examine its anti-tumor effect \u003cem\u003ein vivo\u003c/em\u003e. To begin with PBS, PDA nanoparticles, PDA\u0026amp;DOX, and PDA\u0026amp;DOX@LAC were injected into mice with 4T1 xenografts through their tail vein. After 24 h, the 4T1 tumor-bearing mice in each group were irradiated with NIR (2.0W/cm\u003csup\u003e2\u003c/sup\u003e) for 10 min, and the changes in tumor tissue temperature in each group were recorded by infrared thermal imaging (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea and Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eb). When 4T1 tumor-bearing mice were treated with PDA-DOX@LAC, the temperature of tumor tissue increased to 54.3 ℃. After PDA and PDA-DOX treatment, the temperature of tumor tissue only increased to 46.5℃ and 46.1 ℃. To sum up, the PDA\u0026amp;DOX@LAC nanosystem increased the targeted delivery of PDA\u0026amp;DOX@LAC to tumor tissues with the help of the anaerobic feature of LAC.\u003c/p\u003e\n\u003cp\u003eThen we further evaluated PDA\u0026amp;DOX@LAC nanosystem anti-tumor performance. When the 4T1 xenografts grew to ~\u0026thinsp;100 mm\u003csup\u003e3\u003c/sup\u003e, 4T1 tumor-bearing mice were randomly divided into six groups (n\u0026thinsp;=\u0026thinsp;6/group) and treated with PBS, PBS\u0026thinsp;+\u0026thinsp;NIR, PDA\u0026thinsp;+\u0026thinsp;NIR, DOX, PDA\u0026amp;DOX\u0026thinsp;+\u0026thinsp;NIR and PDA\u0026amp;DOX@LAC\u0026thinsp;+\u0026thinsp;NIR respectively. The changes in tumor tissues and body weight of these mice were recorded every day. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ec shown that synergistic treatment of PDA\u0026amp;DOX@LAC\u0026thinsp;+\u0026thinsp;NIR effectively inhibits the growth of xenografts in mice. After 15 d of treatment, the average size of tumor tissues was only 135mm\u003csup\u003e3\u003c/sup\u003e in the PDA\u0026amp;DOX@LAC\u0026thinsp;+\u0026thinsp;NIR group, compared to 405.6 mm\u003csup\u003e3\u003c/sup\u003e, 442.6 mm\u003csup\u003e3\u003c/sup\u003e, and 387.6 mm\u003csup\u003e3\u003c/sup\u003e for the PDA\u0026thinsp;+\u0026thinsp;NIR, DOX, and PDA\u0026amp;DOX\u0026thinsp;+\u0026thinsp;NIR groups, respectively. In addition, NIR irradiation alone did not significantly inhibit the growth of xenografts in mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ed and Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ee). It was found that the body weight of the mice decreased rapidly after treatment with DOX, and they even became depressed (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ef). In groups of PDA\u0026thinsp;+\u0026thinsp;NIR, PDA\u0026amp;DOX\u0026thinsp;+\u0026thinsp;NIR, and PDA\u0026amp;DOX@LAC\u0026thinsp;+\u0026thinsp;NIR, only a slight change in the body weight of tumor-bearing mice can be observed and the life cycle of the mice was longer than that in the DOX treatment group. In particular, after 40 days of PDA\u0026amp;DOX@LAC\u0026thinsp;+\u0026thinsp;NIR synergistic therapy, the survival rate of mice in this group remained around 30%, while mice in all the other treatment groups had already died (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eg). In a word, PDA\u0026amp;DOX@LAC\u0026thinsp;+\u0026thinsp;NIR synergistic treatment of 4T1 tumor-bearing mice could not only effectively inhibit the growth of xenografts in mice, but also greatly reduce the toxic and side effects of DOX and extend their life.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n\u003ch2\u003eAnalysis of Antitumor Mechanism of PDA\u0026amp;DOX@LAC in Vivo\u003c/h2\u003e\n\u003cp\u003eGiven that PDA\u0026amp;DOX@LAC\u0026thinsp;+\u0026thinsp;NIR synergistic therapy effectively inhibits the growth of mouse xenografts, we further analyzed its anti-tumor mechanism. After 15 d of treatment, the tumor tissues of mice in each treatment group were collected for TUNEL and H\u0026amp;E staining, and the necrosis and apoptosis of tumor cells in the tumor tissues were analyzed. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ea, no obvious necrosis and apoptosis of tumor cells were observed in either the PBS or PBS\u0026thinsp;+\u0026thinsp;NIR treatment groups when compared with the PDA\u0026thinsp;+\u0026thinsp;NIR, DOX, PDA\u0026amp;DOX\u0026thinsp;+\u0026thinsp;NIR, and PDA\u0026amp;DOX@LAC\u0026thinsp;+\u0026thinsp;NIR treatment groups. H\u0026amp;E staining results indicated that chemo-photothermal synergistic therapy of PDA\u0026amp;DOX@LAC was highly destructive to tumor cells, which is featured by vacuolation, karyopycnosis, and karyolysis. In addition, TUNEL staining showed that a green fluorescence signal was only detected on the surface of tumor tissue in PDA\u0026thinsp;+\u0026thinsp;NIR and PDA\u0026amp;DOX\u0026thinsp;+\u0026thinsp;NIR treatment groups. while the green fluorescence signal of the tumor tissue was weaker after DOX treatment. However, after chemo-photothermal synergistic therapy of PDA\u0026amp;DOX@LAC\u0026thinsp;+\u0026thinsp;NIR group, a large number of green fluorescence signals could be found. To conclude, the PDA\u0026amp;DOX@LAC nanosystem could effectively transport chemotherapeutic drugs and photothermal carriers to the deep part of tumor tissue to release DOX and kill tumor cells in the deep part of tumor tissue under LAC's ability to easily survive in anoxic and hypoxic environments. And then, apoptotic tumor cells release tumor-associated antigens, thereby promoting the immune response of the body. Therefore, tumor cells from each treatment group were collected to analyze the immune cells in tumor tissues, which helps to explore the immune regulation in the nanosystem PDA\u0026amp;DOX@LAC chemo-photothermal synergistic therapy. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eb, the proportion of dendritic cells (PDA\u0026amp;DOX@LAC) in the PDA\u0026amp;DOX@LAC treatment group exposed to NIR was 39.7%, which was higher than that in other treatment groups (PBS, PBS\u0026thinsp;+\u0026thinsp;NIR, PDA\u0026thinsp;+\u0026thinsp;NIR, DOX, and PDA\u0026amp;DOX\u0026thinsp;+\u0026thinsp;NIR). That results from the efficacy of PDA\u0026amp;DOX@LAC in killing tumor cells and promoting the release of tumor-related antigens after PDA\u0026amp;DOX@LAC chemo-photothermal synergistic therapy, thereby increasing dendritic cells\u0026rsquo; uptake of it, eventually leading to the maturation of dendritic cells. However, cytotoxic T lymphocytes (CD8\u003csup\u003e+\u003c/sup\u003e T cells) playing an important role in the anti-tumor immune response by being activated by tumor-derived antigens and then directly killing tumor cells. Therefore, the expression of CD8\u003csup\u003e+\u003c/sup\u003e T cells in tumor tissue was analyzed. The percentages of CD8\u003csup\u003e+\u003c/sup\u003e T cells in the PBS, PBS\u0026thinsp;+\u0026thinsp;NIR, PDA\u0026thinsp;+\u0026thinsp;NIR, DOX, and PDA\u0026amp;DOX\u0026thinsp;+\u0026thinsp;NIR treatment groups were 8.38%, 8.4%, 9.91%, 10.4%, and 14.6%, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ec). However, chemo-photothermal synergistic therapy of PDA\u0026amp;DOX@LAC resulted in the highest percentage of CD8\u003csup\u003e+\u003c/sup\u003e T cells among all groups, which was 18.4%. These results indicated that the PDA\u0026amp;DOX@LAC nanosystem, as a potential in situ vaccine, could significantly activate the immune system, promote DC cell maturation and CD8\u003csup\u003e+\u003c/sup\u003e T cell activation, and then kill tumor cells and inhibit the growth of tumors.\u003c/p\u003e\n\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n\u003ch2\u003ePDA\u0026amp;DOX@LAC Biocompatibility\u003c/h2\u003e\n\u003cp\u003eChemo-photothermal synergistic therapy of the PDA\u0026amp;DOX@LAC nanosystem exhibits superior anti-tumor effect, so it is used for the treatment of mouse xenografts and future clinical application, which requires low toxicity or even non-toxicity \u003cem\u003ein vivo\u003c/em\u003e. So the biocompatibility should be analyzed through animal experiments. The biocompatibility of PDA\u0026amp;DOX@LAC \u003cem\u003ein vivo\u003c/em\u003e was analyzed by H\u0026amp;E staining during the treatment of tumor-bearing mice. After the treatment, the H\u0026amp;E staining was performed on the main organs (heart, liver, spleen, lung, and kidney) of mice in each treatment group to compare with the control group. It was found that when the mice were injected with PDA\u0026amp;DOX@LAC nanosystem for 15 days, no significant inflammation or tissue damage could be found in the pathological sections. Moreover, the PDA\u0026amp;DOX@LAC nanosystem could effectively alleviate the toxic and side effects of DOX in the tumor treatment process, and especially reduce the damage of DOX to heart tissues (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e). Then, blood samples of the PDA\u0026amp;DOX@LAC\u0026thinsp;+\u0026thinsp;NIR treatment group were collected for routine blood tests and serum biochemical indicators tests. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e, blood was drawn from 4T1 tumor-bearing mice on days 0, 1, 7, and 14 of PDA\u0026amp;DOX@LAC\u0026thinsp;+\u0026thinsp;NIR treatment, respectively. The results shown that there were no obvious changes in routine and biochemical blood test results during the PDA\u0026amp;DOX@LAC\u0026thinsp;+\u0026thinsp;NIR treatment. The above results indicated that PDA\u0026amp;DOX@LAC exhibits excellent biocompatibility as a drug carrier during tumor treatment.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, the investigations detailed above have led to the development of a live bacterial drug carrier endowed with enzyme-like attributes, tailored for synergistic therapy targeting tumors. The PDA\u0026amp;DOX@LAC nanosystem employed exhibits notable advantages: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) mitigating the toxicities and adverse effects of DOX on normal tissues \u003cem\u003ein vivo\u003c/em\u003e during tumor intervention; (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) targeted delivery to tumor sites and effective tumor cell eradication facilitated by LAC's adaptability within anoxic environments; (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) harnessing the catalase property of the PDA\u0026amp;DOX@LAC nanosystem to ameliorate tumor tissue hypoxia, thereby augmenting oxygen availability for photodynamic therapy; (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) enabling the synergistic photothermal therapy of PTT and PDT via NIR laser activation in a single procedure; (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) fostering the generation of tumor-associated antigens and eliciting a robust anti-tumor immune response through the chemo-photothermal synergistic therapy mediated by the PDA\u0026amp;DOX@LAC nanosystem. Consequently, outcomes derived from both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e treatments underscore the innovative and efficacious role of the formulated PDA\u0026amp;DOX@LAC nanosystem in advancing the landscape of tumor synergistic therapy platforms, thus furnishing a theoretical foundation and design paradigm for future iterations of tumor treatment modalities.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures were approved by the Institutional Animal Care and Use Committee of Northern Jiangsu People\u0026rsquo;s Hospital (no. UJS-IACUC-AP-20190314002).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach coauthor has read the manuscript and approves its submission. This work is being submitted exclusively to your journal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets supporting the results of this article are included within the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by Natural Science Foundation of Jiangsu Province (SBK2020022937).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLu Liu are responsible for the experimental operations, data collection and analysis. Xuefen Zhao are responsible for the experimental design and writing of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe acknowledged are included within the article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYanji Chu, Xiao-Qi Xu, Yapei Wang. Ultradeep Photothermal Therapy Strategies. Ultradeep Photothermal Therapy Strategies. \u003cem\u003eJ. Phys. Chem. Lett., \u003c/em\u003e2022. 13: 9564-9572.\u003c/li\u003e\n\u003cli\u003eZhenglin He, Yihan Wang, Liang Han, et al. The mechanism and application of traditional Chinese medicine extracts in the treatment of lung cancer and other lung-related diseases. \u003cem\u003eFront Pharmacol.,\u003c/em\u003e 2023. 14: 1330518.\u003c/li\u003e\n\u003cli\u003eXingshu Li, Jonathan F Lovell, Juyoung Yoon, et al. 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Eng.,\u003c/em\u003e 2021. 7: 1686-1692.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"dopamine, combination therapy, environmental response, precise release","lastPublishedDoi":"10.21203/rs.3.rs-4251041/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4251041/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe development of intelligent, environmentally responsive, and biocompatible photothermal system holds significant importance for the photothermal combined therapy of tumors. In this study, inspired by lactobacillus, we prepared a biomimetic nanoplatform, PDA\u0026amp;DOX@LAC, for tumor photothermal-chemotherapy by integrating the chemotherapeutic drug doxorubicin (DOX) with dopamine through oxidative polymerization to form polydopamine on the surface of LAC. The PDA\u0026amp;DOX@LAC nanoplatform not only achieves precise and controlled release of DOX based on the slightly acidic microenvironment of tumor tissues, but also exhibits enzyme-like properties to alleviate tumor hypoxia. Under near-infrared light irradiation, it effectively induces photothermal ablation of tumor cells, enhances cellular uptake of DOX with increasing temperature, and thus efficiently inhibits tumor cell growth. Moreover, \u003cem\u003ein vivo\u003c/em\u003e experiments further confirmed that photothermal therapy combined with PDA\u0026amp;DOX@LAC induces tumor cells apoptosis, releases tumor-associated antigens, and is engulfed by dendritic cells, activating cytotoxic T lymphocytes, thereby effectively suppressing tumor growth and prolonging the survival period of 4T1 tumor-bearing mice. Therefore, the PDA\u0026amp;DOX@LAC nanoplatform holds immense potential in precise tumor targeting and photothermal combined therapy, providing valuable insights and theoretical foundations for the development of novel tumor treatment strategies based on endogenous substances within the body.\u003c/p\u003e","manuscriptTitle":"Preparation of Environmentally Responsive PDA\u0026amp;DOX@LAC Live Drug Carrier for synergistic tumor therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-07 15:22:38","doi":"10.21203/rs.3.rs-4251041/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-29T06:52:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-21T12:07:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-14T06:02:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-06T20:37:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"d1199cfc-1746-4bf6-be26-56545c326aa8","date":"2024-05-06T11:23:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"bdb19dff-3292-49ce-a985-aee235c8c3e2","date":"2024-05-06T11:23:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"dd06d7d1-847e-4d3c-a02e-f438a4e288af","date":"2024-05-06T11:00:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-06T10:52:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-01T09:25:37+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-04-30T13:50:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-30T13:48:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-04-11T08:07:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d9be2bff-7f31-48cb-969c-dde9fc1734f5","owner":[],"postedDate":"May 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":31587581,"name":"Biological sciences/Cancer"},{"id":31587582,"name":"Biological sciences/Drug discovery"},{"id":31587583,"name":"Biological sciences/Immunology"},{"id":31587584,"name":"Physical sciences/Materials science"}],"tags":[],"updatedAt":"2024-07-13T13:07:14+00:00","versionOfRecord":{"articleIdentity":"rs-4251041","link":"https://doi.org/10.1038/s41598-024-66966-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-07-10 13:07:14","publishedOnDateReadable":"July 10th, 2024"},"versionCreatedAt":"2024-05-07 15:22:38","video":"","vorDoi":"10.1038/s41598-024-66966-2","vorDoiUrl":"https://doi.org/10.1038/s41598-024-66966-2","workflowStages":[]},"version":"v1","identity":"rs-4251041","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4251041","identity":"rs-4251041","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}