Bionic chelerythrine-loaded semiconductor polymer dot as well as preparation method and application thereof

Abstract

The invention discloses a bionic chelerythrine-loaded semiconductor polymer dot. The bionic chelerythrine-loaded semiconductor polymer dot comprises a functional polymer, a luminous polymer, chelerythrine, a 4T1 cell membrane and bacterial outer membrane vesicles. The invention also discloses a preparation method of the bionic chelerythrine-loaded semiconductor polymer dot and application of the bionic chelerythrine-loaded semiconductor polymer dot in preparation of antitumor drugs and as an imaging agent. The bionic chelerythrine-loaded semiconductor polymer dot disclosed by the invention has excellent optical performance, high biocompatibility and a remarkable anti-tumor treatment effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a bionic chelidonine-loaded semiconductor polymer dot and a preparation method and application thereof. BACKGROUND

[0002] Breast cancer is the most common and deadliest malignancy among women worldwide, requiring more precise, effective, and minimally invasive treatment strategies. Although traditional treatments such as surgery, chemotherapy, and radiotherapy have made significant progress in improving patient survival rates, these methods still have many limitations, including incomplete tumor resection, high recurrence rate, and severe side effects. Therefore, there is an urgent need for innovative treatment strategies to improve efficacy and reduce toxicity.

[0003] Photothermal therapy (PTT) can effectively kill tumor cells, but has limited tissue penetration and can cause thermal damage. Photodynamic therapy (PDT) is relatively safe, but often has suboptimal efficacy. Immuno-drug combination (IDC) therapy enhances immune responses through targeted drug delivery, but is limited by bioavailability and toxicity. Semiconducting conjugated polymer dots (Pdots) in the near-infrared-II (NIR-II, 1000-1700 nm) window have excellent optical properties, making them an ideal choice for drug delivery and PTT / PDT, and their modifiable surface can integrate phototherapy diagnosis and IDC effects, which is expected to overcome the limitations of traditional treatments.

[0004] Chelidonine (CTe) is a natural benzophenanthridine alkaloid with significant anti-cancer properties. Studies have shown that CTe can inhibit tumor growth by inducing mitochondrial dysfunction, triggering apoptosis pathways, and producing reactive oxygen species (ROS). In addition, CTe has high biocompatibility and low systemic toxicity, making it a safer alternative to many traditional chemotherapy drugs. However, the poor water solubility and low bioavailability of CTe limit its clinical application, which affects its distribution and tumor targeting ability.

[0005] IDC therapy induces a strong immune response against the tumor microenvironment by delivering anti-cancer drugs and stimulating the immune system. This approach not only amplifies the direct cytotoxic effects of drugs, but also activates key immune cells such as cytotoxic T lymphocytes (CTLs) and CD4+ helper T cells, which are essential for long-term tumor suppression and immune memory. However, the limitations of IDC therapy include low drug bioavailability and potential systemic toxicity.

[0006] Photothermal therapy (PTT) utilizes light-activated materials to generate local heat under near-infrared (NIR) light, which efficiently kills tumor cells. However, PTT is limited by its tissue penetration depth and the risk of thermal damage to normal tissues.

[0007] Photodynamic therapy (PDT) is a safer alternative that induces tumor cell apoptosis or necrosis by generating ROS, but it is limited by oxygen dependence and insufficient delivery of photosensitizers.

[0008] Bacterial outer membrane vesicles (OMVs) are natural secretory immunostimulants that can activate dendritic cells and T lymphocytes, enhancing the anti-tumor immune response. SUMMARY

[0009] To solve the above technical problems, the present application aims to provide a drug that can effectively treat tumors.

[0010] To achieve the above application purpose, the present application provides a biomimetic chelidonine-loaded semiconductor polymer dot, which comprises a functional polymer, a luminescent polymer, chelidonine, 4T1 cell membrane and bacterial outer membrane vesicles.

[0011] According to a preferred embodiment, the functional polymer is polystyrene-polyethylene glycol carboxylic acid.

[0012] According to a preferred embodiment, the luminescent polymer is PBTQ4F.

[0013] According to a preferred embodiment, the bacterial outer membrane vesicles are outer membrane vesicles from Escherichia coli.

[0014] In another aspect, the present application also provides a preparation method of the biomimetic chelidonine-loaded semiconductor polymer dot, which comprises the following steps: integrating a functional polymer, chelidonine and a luminescent polymer, and then ultrasonically coating 4T1 cell membrane and bacterial outer membrane vesicles on the surface to form the biomimetic chelidonine-loaded semiconductor polymer dot.

[0015] In another aspect, the present application also provides the use of the biomimetic chelidonine-loaded semiconductor polymer dot in the preparation of an anti-tumor drug.

[0016] According to a preferred embodiment, the tumor is breast cancer or cervical cancer.

[0017] In another aspect, the present application also provides the use of the biomimetic chelidonine-loaded semiconductor polymer dot as an imaging agent.

[0018] In the present application, a novel therapeutic carrier OCPdots@CTe is designed and synthesized, which contains chelerythrine (CTe) and is coated with 4T1 cell membrane and bacterial outer membrane vesicles (OMVs) on the surface. The present application integrates PTT, PDT and IDC therapies, and further enhances the therapeutic effect through the immunomodulatory effect of OMVs. By wrapping CTe in functionalized nanocarriers and modifying it with tumor cell membranes and OMVs, efficient drug delivery, enhanced immune activation and precise tumor targeting are achieved. The results show that OCPdots@CTe exhibits excellent optical performance, high biocompatibility and significant therapeutic effect in vitro and in vivo experiments. The combination of PTT, PDT and IDC improves the therapeutic effect and reduces side effects, solves the limitations of traditional methods, and provides a promising method for personalized, precise and minimally invasive cancer treatment. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The therapeutic mechanism of OCPdots@CTe is shown.

[0020] Figure 2 The synthesis and characterization of OCPdots@CTe are shown. (a) Chemical structure and preparation process of OCPdots@CTe. (b) UV-visible (UV-vis) absorption spectra of Pdots, CTe and Pdots@CTe. (c) UV-visible absorption spectra of Pdots, OPdots@CTe and OCPdots@CTe. (d) Emission spectra of Pdots, CTe and Pdots@CTe. (e) Representative dynamic light scattering (DLS) results of Pdots and Pdots@CTe. (f) Representative dynamic light scattering (DLS) results of OPdots@CTe and OCPdots@CTe. (g) Zeta potential results of Pdots, Pdots@CTe, OPdots@CTe, OCPdots@CTe, E. coli outer membrane vesicles (OMVs) and 4T1 cancer cell membrane (CCM). (h) Transmission electron microscopy (TEM) imaging of Pdots, Pdots@CTe, OPdots@CTe and OCPdots@CTe.

[0021] Figure 3Optical properties of OCPdots@CTe are shown. (a) Photothermal heating curves of OCPdots@CTe solutions with different concentrations (0, 12.5, 25, 50, 100 pg / mL) under 808 nm laser (power density 0.5 W / cm2) irradiation for 6 min. (b) Photothermal heating curves of OCPdots@CTe dispersion with a concentration of 25 pg / mL under different power densities (0.1, 0.25, 0.5, and 0.75 W / cm2) of 808 nm laser irradiation. (c) Photothermal properties of OCPdots@CTe and pure water (dWater) under 808 nm laser irradiation, including the heating process and the cooling process. (d) The relationship between the PA intensity of OCPdots@CTe at 808 nm and the concentration. The inset is the PA image of OCPdots@CTe with different concentrations (pg / mL). (e) Fluorescence intensity of OCPdots@CTe. (f) SDS-PAGE protein analysis of protein markers, Pdots, Pdots@CTe, cancer cell membrane (CCM), CPdots@CTe, OMVs, OPdots@CTe, and OCPdots@CTe.

[0022] Figure 4 Biocompatibility and cytotoxicity of OCPdots@CTe are shown. (a) Confocal fluorescence images of 4T1 cancer cells after co-incubation with PBS, Pdots, Pdots@CTe, OPdots@CTe, and OCPdots@CTe (all at a concentration of 25 pg / mL) for 12 h. Yellow fluorescence represents nanoparticles, the nucleus (blue) was stained with 4’,6-diamidino-2-phenylindole (DAPI), and the cell membrane was stained with Dil. Scale bar = 20 pm. (b) Flow cytometry analysis of 4T1 cancer cells after co-incubation with PBS and nanoparticles (at a concentration of 25 pg / mL) for 12 h. (c) Cell viability of 293T normal cells after treatment with different concentrations of nanoparticles for 24 h was detected by CCK-8 method. Error bars represent standard deviation (n = 3). (d) Cell viability assessment of 4T1 cells after co-incubation with nanoparticles at a concentration of 25 pg / mL for 12 h: with or without irradiation (808 nm NIR-II laser irradiation at a power density of 0.5 W / cm2for 10 min). Error bars represent standard deviation (n = 3). (e) Cell viability assessment of Hela and 4T1 cells after co-incubation with nanoparticles at a concentration of 25 pg / mL for 12 h: with irradiation (808 nm NIR-II laser irradiation at a power density of 0.5 W / cm2for 10 min) (+). Error bars represent standard deviation (n = 3).

[0023] Figure 5The effects of OCPdots@CTe on PTT, PDT are shown. (a) Live / dead cell fluorescence images of Hela cells after 12 h co-incubation with nanoparticles, followed by 808 nm laser irradiation at a power density of 0.5 W / cm2for 10 min. Scale bar = 200 μm. (b) 4T1 cancer cells after 12 h co-incubation with nanoparticles, followed by 808 nm laser irradiation at a power density of 0.5 W / cm2for 10 min, then stained with dichlorodihydrofluorescein-acetylacetic acid (DCFH-DA) and analyzed by flow cytometry. (c) 4T1 cancer cells after 12 h co-incubation with nanoparticles, followed by 808 nm laser irradiation at a power density of 0.5 W / cm2for 10 min, then stained with annexin V-fluorescein isothiocyanate / propidium iodide (ANNEXIN V-FITC / PI) and analyzed by flow cytometry.

[0024] Figure 6 The results of in vitro NIR-II photoacoustic imaging and fluorescence imaging are shown. (a) In vivo fluorescence (FL) imaging of nude mice at different time intervals after tail vein injection of nanoparticles (dose of 2.0 mg / kg). (b) Fluorescence signal intensity in the tumor region at different time points after injection of the drug. (c) Fluorescence intensity of each organ dissected from the mouse after 7 days of tail vein injection of nanoparticles. (d) Photoacoustic intensity in the tumor region at different time points after injection of the drug. (e) In vivo photoacoustic imaging of nude mice at different time intervals after tail vein injection of nanoparticles (dose of 2.0 mg / kg).

[0025] Figure 7 The in vivo therapeutic effect of OCPdots@CTe in the NIR-II window is shown. (a) Representative photographs of tumor-bearing mice in different experimental groups. (b) Representative photographs of tumor tissues ex vivo in different experimental groups. (c) Changes in the body weight of mice within 14 days after different treatments. Each data point represents the mean ± standard deviation of n = 5 animals. (d) Relative tumor volume change curve. Each data point represents the mean ± standard deviation of n = 5 animals. (e) H&E staining and Tunel analysis results of tumor tissues collected from mice in different experimental groups at the end of treatment. Scale bar = 200 μm.

[0026] Figure 8The application of OCPdots@CTe in PTT / PDT is shown. (a) The frequency and duration of the use of OCPdots@CTe for NIR-II phototherapy diagnosis and chemo-immunotherapy. (b) Changes in the body weight of mice within 14 days after different treatments. Each data point represents the mean ± standard deviation of n = 5 animals. (c) Relative tumor volume change curve of primary tumors. Each data point represents the mean ± standard deviation of n = 5 animals. (d) Relative tumor volume change curve of distal tumors. Each data point represents the mean ± standard deviation of n = 5 animals. (e) Flow cytometry analysis of infiltrated CD8+and CD4+cells in primary tumor tissues. (f) Weight of primary tumor tissues at the end of the treatment cycle, ± standard deviation (n = 5 animals). (g) Flow cytometry analysis of infiltrated CD8+and CD4+cells in distal tumor tissues. (h) Weight of distal tumor tissues at the end of the treatment cycle, ± standard deviation (n = 5 animals).

[0027] Figure 9 The determination of nanoparticle size at different time points is shown.

[0028] Figure 10 The absorption spectrum of DBPF with a concentration of 20 mM under 808 nm laser (power density 0.5 W / cm²) irradiation is shown.

[0029] Figure 11 The absorption spectrum of the mixed system of 2 mL OCPdots@CTe with a concentration of 25 μg / mL and DBPF under 808 nm laser (power density 0.5 W / cm²) irradiation is shown.

[0030] Figure 12 The temperature change of OCPdots@CTe dispersion with a concentration of 25 μg / mL after five "on / off" cycles under 808 nm laser (power density 0.5 W / cm²) irradiation is shown.

[0031] Figure 13 The plot of linear time data obtained from the cooling stage versus -lnθ is shown.

[0032] Figure 14 The ultraviolet-visible absorption spectra of CNPdots, CNPdots@CTe, OCNPdots@CTe, and OCCNPdots@CTe are shown.

[0033] Figure 15 The fluorescence spectra of CNPdots, CNPdots@CTe, OCNPdots@CTe, and OCCNPdots@CTe are shown.

[0034] Figure 16 Near-infrared fluorescence images of different organs dissected from mice 7 days after tail vein injection of Pdots@CTe, OPdots@CTe, and OCPdots@CTe (at a dose of 2.0 mg / kg) are shown.

[0035] Figure 17 A schematic diagram of a multispectral photoacoustic imaging system is shown.

[0036] Figure 18 The image shows the temperature rise at the tumor site in tumor-bearing mice (top image) and the corresponding infrared thermograph (bottom image) (808 nm laser dose: 2.0 W / cm², irradiation time 2.5 minutes).

[0037] Figure 19 The results of hematoxylin and eosin (H&E) staining of major organs after photothermal therapy are shown. The H&E staining of the liver, kidney, heart, spleen, and lungs in the PBS control group, PBS+ group, Pdots group, Pdots+ group, OCPdots@CTe group, and OCPdots@CTe+ group are presented after 14 days of near-infrared light irradiation ("+" indicates the use of an 808 nm laser with a power density of 0.3 W / cm²).

[0038] Figure 20 Representative digital images of tumor tissues from different experimental groups in vitro are shown (left: in situ tumor; right: distal tumor). Detailed Implementation

[0039] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0040] 1. Experimental Materials and Methods 1.1 Materials The semiconductor polymer poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinyl)-1,4-phenylene] (CN-PPV; average molecular weight: 25,000; polydispersity index: 2.1) was purchased from American DyeSource, Quebec, Canada.

[0041] The semiconducting polymer poly[4-(4,8-bis(5-(2-ethylhexyl)thiophen-2-yl)benzo[1,2-b:4,5-b']dithiophen-2-yl)-2-(2-ethylhexyl)-6,7-bis(3-((2-ethylhexyl)oxy)-4,5-difluorophenyl)-2H-[1,2,3]triazolo[4,5-g]quinoxaline] (PBTQ4F; average molecular weight: 30.2 kDa; polydispersity index: 1.38) was synthesized according to published literature (Liu, Y. et al., Advanced Functional Materials, 2020. 59(47): p. 21049-21057.). Fluorination Enhances NIR-II Fluorescence of Polymer Dots for Quantitative Brain Tumor Imaging. 2020.59(47): p. 21049-21057.).

[0042] The functional carboxyl-functionalized polymer polystyrene-grafted ethylene glycol (PS-PEG-COOH) was purchased from Polymer Source Inc., Quebec, Canada. Phosphate-buffered saline (PBS), Dulbecco's modified Eagle's medium (DMEM), penicillin / streptomycin, fetal bovine serum (FBS), and trypsin-EDTA were purchased from Thermo Fisher Scientific.

[0043] All materials were used as received without further purification unless otherwise stated.

[0044] Tetrahydrofuran (THF) was used for the preparation of polymer dots (Pdots).

[0045] Ultrapure water (resistivity of 18.25 MΩ·cm at 25 °C) was used throughout the study. −2 All other chemicals were used as received.

[0046] 1.2 Instruments and equipment Transmission electron microscope (TEM) images were taken by a JEM1200EX transmission electron microscope from JEOL. Particle size and zeta potential were measured on a Malvern Nano-ZS particle size analyzer from Malvern Instruments, UK. Fluorescence spectra were measured by a Fluorolog-3 fluorescence spectrometer from HORIBA Scientific, USA. Absorption spectra were obtained on a Shimadzu UV-1800 ultraviolet-visible spectrophotometer from Cole-Parmer, USA. Photoacoustic (PA) signal measurements in vitro and in vivo were performed using a multispectral photoacoustic imaging system. In vivo confocal fluorescence imaging was performed on a Carl Zeiss LSM710 confocal microscope from Carl Zeiss, Germany. In vitro fluorescence imaging was completed on a Raptor Photonics Ninox640 SUNIR CCD.

[0047] 1.3 Cell culture Human cervical cancer HeLa cells, human embryonic kidney 293T cells and 4T1 breast cancer cells were all placed in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin, and cultured in a humidified environment at 37°C, 5% CO2.

[0048] For in vitro experiments, cells were first seeded in T25 culture flasks for 24 hours, then trypsinized and seeded into 96-well plates or 48-well plates at a density of 5000-20000 cells per well. After 24 hours of seeding, nanoparticles were added, and samples were incubated for 12 hours before processing.

[0049] For in vivo experiments, cells were cultured in T75 culture flasks, and when the confluence reached 80-90%, they were digested for subsequent experiments.

[0050] If cell membranes need to be extracted, cells are cultured in T175 culture flasks, and when the confluence reaches 80-90%, they are digested to obtain cells for experiments.

[0051] 1.4 Preparation of Pdots, Pdots@CTe, CNPdots and CNPdots@CTe Polymer dots Pdots were prepared using the reprecipitation method. 0.1 mg of PBTQ4F and 0.1 mg of PS-PEG-COOH were dissolved in 1 mL of tetrahydrofuran (THF). Subsequently, the mixed solution was rapidly injected into 10 mL of solution (9 mL of Milli-Q water and 1 mL of 0.025% Triton-X100) under vigorous sonication. THF was removed by rotary evaporation and the solution was concentrated. Finally, a small amount of aggregated Pdots were removed by filtration through a 0.22 μm filter membrane.

[0052] Polymer dots CNPdots were also prepared using the reprecipitation method. 0.1 mg of CN-PPV, 0.1 mg of PBTQ4F and 0.2 mg of PS-PEG-COOH were dissolved in 2 mL of THF. Next, the mixed solution was rapidly injected into 10 mL of solution (9 mL of Milli-Q water and 1 mL of 0.025% Triton-X100) under vigorous sonication. THF was removed by rotary evaporation and the solution was concentrated. Afterwards, a small amount of aggregated Pdots were removed by filtration through a 0.22 μm filter membrane. Finally, the concentration of the solution was determined using a UV-Vis spectrophotometer.

[0053] Pdots@CTe were prepared by the reprecipitation method. PBTQ4F (0.1 mg), chelidonine (CTe, 0.03 mg) and PS-PEG-COOH (0.1 mg) were dissolved in 1 mL of THF solution. Then, the mixture was rapidly injected into 10 mL of solution (9 mL of Milli-Q water + 1 mL of 0.025% Trixon-X100) under strong sonication. Tetrahydrofuran was removed and the solution was concentrated by rotary evaporation. A small amount of aggregated Pdots were removed by filtration through a 0.22 μm membrane filter.

[0054] CNPdots@CTe were also prepared by the reprecipitation method. CN-PPV (0.1 mg), chelidonine (CTe, 0.03 mg), PBTQ4F (0.1 mg) and PS-PEG-COOH (0.2 mg) were dissolved in 2 mL of THF solution. Then, the mixture was rapidly injected into 10 mL of solution (9 mL of Milli-Q water + 1 mL of 0.025% Trixon-X100) under strong sonication. Tetrahydrofuran was removed and the solution was concentrated by rotary evaporation. A small amount of aggregated Pdots were removed by filtration through a 0.22 μm membrane filter. The concentration of the solution was then determined using a UV-Vis spectrophotometer.

[0055] 1.5 Preparation of cancer cell membranes To obtain 4T1 cancer cell membranes, the membrane protein and cytoplasmic protein extraction kit from Beyotime (China) was used.

[0056] Dissolve and mix the membrane protein extraction reagent A and reagent B at room temperature, then immediately place them on ice for standby. Prepare the appropriate amount of reagent A and reagent B according to the actual demand, and add PMSF (phenylmethylsulfonyl fluoride) a few minutes before use to make the final concentration 1 mM. Culture about 200 million to 500 million cells, wash once with phosphate buffer solution (PBS), then use a cell scraper to scrape the cells from the culture container, and use a pipette to collect the cells. Collect the cells by centrifugation, discard the supernatant, and set the cell pellet aside for later use.

[0057] Add 1 mL of membrane protein extraction reagent A with PMSF to 200 million to 500 million cells, gently and thoroughly resuspend the cells, and incubate on ice for 10 to 15 minutes. Freeze-thaw the cells twice between liquid nitrogen and room temperature. Then, take a small sample and observe it under a microscope to confirm that more than 70% of the cells have been lysed. To remove the nuclei and unbroken cells, centrifuge at 4°C and 700g for 10 minutes, and carefully transfer the supernatant to a new centrifuge tube. Centrifuge again at 4°C and 14000g for 30 minutes to precipitate the membrane fragments. Add 200 µL of membrane protein extraction reagent B to the precipitate, vortex at high speed for 5 seconds to resuspend the precipitate, and incubate on ice for 5 to 10 minutes. Repeat the vortexing and ice incubation steps 1 to 2 times to ensure complete extraction of the membrane proteins. Finally, centrifuge at 4°C and 14000g for 5 minutes to collect the supernatant as the final cancer cell membrane solution. Store it at -70°C for later use.

[0058] 1.6 Preparation of OMVs OMVs from the msbB (pBAD) strain (an E. coli mutant that lacks msbB and produces OMVs in large quantities) were prepared as follows. A colony was picked from LB agar and then placed in 20 mL of LB at 37 °C, 220 rpm, overnight. Then, it was diluted 1 : 100 into fresh LB (BD, 244620) and grown to an optical density (OD600) of between 0.5 and 0.55 (i.e., exponential phase). Expression of the ClyA-flag protein was induced by the addition of 0.1% arabinose (diluted 1 :200 in LB broth). After overnight incubation at 37 °C with shaking (220 rpm), the bacteria were centrifuged at 8000 g for 10 min. The supernatant was passed through a 0.45 μιη polyvinylidene fluoride filter (Millipore, R8SA47939, USA) and concentrated to 2 mL using a 100 kDa ultrafiltration membrane (Millipore, R3EA06699, USA). The concentrate was filtered through a 0.22 μιη filter to reduce contamination and then stored at -20 °C until use. Total protein concentration was quantified using a Nanodrop® spectrophotometer (Thermo Scientific, USA) and the bis-quinolinic acid protein assay.

[0059] 1.7 Preparation of OPdots@CTe, OCPdots@CTe, OCNPdots@CTe and OCCNPdots@CTe To coat the cell membrane onto the Pdots surface, 1 mL of 4T1 cancer cell membrane solution (1.0 mg / mL) was mixed with 1 mL of Pdots solution (1.0 mg / mL) and the mixture was sonicated for 10 min. Excess membrane fragments were removed by centrifugation and the resulting CPdots were stored at 4 °C for later use.

[0060] Preparation of OPdots@CTe: To coat the OMV onto the Pdots surface, 1 mL of OMV solution (1.0 mg / mL) was mixed with 1 mL of Pdots@CTe solution (1.0 mg / mL) and the mixture was sonicated for 10 min. Excess membrane fragments were removed by centrifugation and a 0.45 μιη polyvinylidene fluoride filter (Millipore, R8SA47939, USA) and the resulting OPdots@CTe were stored at 4 °C for later use.

[0061] Preparation of OCPdots@CTe: To coat OMV and cell membrane on the surface of Pdots, 4T1 cancer cell membrane solution 0.5 mL (1.0 mg / mL), OMV solution 0.5 mL (1.0 mg / mL) and Pdots@CTe solution 1 mL (1.0 mg / mL) were mixed, followed by 10 minutes of ultrasonic treatment. Excess membrane fragments were removed by centrifugation and 0.45 pm polyvinylidene fluoride filter (Millipore, R8SA47939, USA), and the resulting OCPdots@CTe was stored at 4 °C for subsequent use.

[0062] Preparation of OCNPdots@CTe: To coat OMV on the surface of Pdots, OMV solution 1 mL (1.0 mg / mL) and CNPdots@CTe solution 1 mL (1.0 mg / mL) were mixed, followed by 10 minutes of ultrasonic treatment. Excess membrane fragments were removed by centrifugation and 0.45 pm polyvinylidene fluoride filter (Millipore, R8SA47939, USA), and the resulting OCNPdots@CTe was stored at 4 °C for subsequent use.

[0063] Preparation of OCCNPdots@CTe: To coat OMV and cell membrane on the surface of Pdots, 4T1 cancer cell membrane solution 0.5 mL (1.0 mg / mL), OMV solution 0.5 mL (1.0 mg / mL) and CNPdots@CTe solution 1 mL (1.0 mg / mL) were mixed, followed by 10 minutes of ultrasonic treatment. Excess membrane fragments were removed by centrifugation and 0.45 pm polyvinylidene fluoride filter (Millipore, R8SA47939, USA), and the resulting OCCNPdots@CTe was stored at 4 °C for subsequent use.

[0064] 1.8 Agarose gel electrophoresis Agarose powder was dissolved in 1 x tris(hydroxymethyl)aminomethane / boric acid / ethylenediaminetetraacetic acid (TBE) buffer to prepare 2% agarose gel. 300 pL of cancer cell membrane, Pdots and CPdots were mixed with 10 pL of 4 x Laemmli sample buffer, followed by heating at 95 °C for 5 minutes and cooling in ice bath. Then, 20.0 pL of prepared sample was loaded into each well of 10.0% SDS-PAGE gel, and electrophoresis was performed at 180 V for 20 minutes. The resulting gel was then stained with Coomassie brilliant blue for 10 minutes (speed 90 rpm), and then left overnight to visualize protein bands.

[0065] 1.9 In vitro cytotoxicity assay 4T1 breast cancer cells and 293T cells cultured in 96-well plates were co-incubated with different concentrations of Pdots, CPdots for 24 h. After that, the cells were incubated in DMEM medium containing 10.0% CCK-8 reagent for 1 h. To calculate the cell viability, the absorbance of the cell culture solution was measured using a microplate reader.

[0066] 1.10 In vitro cell uptake experiment 4T1 breast cancer cells, 293T cells, GL261 cells and HeLa cells cultured in confocal cell culture dishes (4 x 10 4 cells / dish) were co-incubated with 25 μg / mL Pdots, CPdots in DMEM medium for 12 h. After treatment, the cells were washed and fixed, and then stained with DAPI.

[0067] 1.11 Experimental animals and tumor models DMEM medium (100 μL) containing 1 x 10 6 4T1 cells was subcutaneously injected into the right side of the back of 6-week-old female Balb / C mice to construct a xenograft tumor model. After 7 days of tumor growth, 4T1 tumor-bearing mice were used for fluorescence imaging, photoacoustic imaging, and cancer treatment experiments.

[0068] 1.12 In vivo cancer treatment 4T1 tumor-bearing mice were randomly divided into three groups, and were injected with PBS, Pdots, and CPdots through the tail vein, with an injection dose of 2.0 mg / kg, and the number of animals in each group was 5. Twelve hours after injection, the tumor site of the mice was irradiated with a laser with a wavelength of 980 nm for 5 minutes, and the laser power density was 0.5 W / cm². At the same time, the infrared thermal imager was used to record the temperature change of the tumor site every 30 seconds.

[0069] To monitor the treatment effect and biological safety, the tumor volume and body weight of the mice were measured every other day for 14 days. The tumor volume calculation formula is as follows: V = (length) x (width)² / 2. The relative tumor volume calculation method is V / V0 (V0 is the initial tumor volume).

[0070] 1.13 Histopathological evaluation For histological analysis, the collected mouse organs were fixed with 4% neutral buffered paraformaldehyde, and then embedded with paraffin. Hematoxylin-eosin (H&E) staining was performed on tissue sections of the main organs of the mice. Finally, optical microscopy was used to image and observe the histopathological sections.

[0071] 2. Results and discussion 2.1 Design, synthesis, and characterization of OCPdots@CTe High-concentration nanomaterials Pdots@CTe were prepared by mixing functional polymers (e.g., polystyrene-polyethylene glycol carboxylic acid (PS-PEG-COOH)), chelerythrine (CTe), and a second near-infrared (NIR-II) light-emitting polymer (PBTQ4F) using a recrystallization method. The chemical structures of these polymers are shown in Figure 2 Figure 2 , a).

[0072] 4T1 cell membranes were extracted from 4T1 cancer cells, and outer membrane vesicles (OMVs) were extracted from Escherichia coli (E. coli). Both membrane proteins were sonicated for 10 min and then coated on the surface of Pdots@CTe to produce OCPdots@CTe E. coli Figure 2 , a).

[0073] To confirm that CTe was successfully incorporated into Pdots, the absorption and emission spectra of CTe, Pdots, and Pdots@CTe were analyzed Figure 2 , b-d). Pdots@CTe exhibited characteristic peaks of both CTe and Pdots. In addition, the incorporation of CTe resulted in an increase in particle size and zeta potential Figure 2 , e, g). The successful coating of both membrane proteins on the surface of Pdots@CTe was confirmed by dynamic light scattering (DLS) measurements and transmission electron microscopy (TEM) imaging Figure 2 , h). The protein coating did not affect the absorption of Pdots at 950 nm Figure 2 , c). The coating also increased the particle size of the material Figure 2 , f). Figure 9 It was shown that the particle size of OCPdots@CTe remained stable over time. The zeta potential of OCPdots@CTe changed due to the potential difference between the membrane proteins and Pdots@CTe Figure 2 , g).

[0074] 2.2 Optical properties of OCPdots@CTe Under 808 nm laser irradiation, the photoacoustic (PA) signal of OCPdots@CTe linearly increased with increasing concentration (6.25-100 μg / mL) Figure 3 , d). Similarly, the fluorescence (FL) signal of OCPdots@CTe gradually increased with increasing concentration (3.125-100 μg / mL) Figure 3 ​​, e). In addition, all formulations (Pdots, Pdots@CTe, OPdots@CTe and OCPdots@CTe) emitted bright fluorescence in the NIR-II region Figure 3 , e). Subsequent SDS-PAGE protein analysis further confirmed that almost all cell membrane proteins were retained on the nanoparticle surface in large quantities Figure 3 , f). These results collectively indicate that the biomembrane modification and drug doping were successfully achieved without compromising the optical performance of OCPdots@CTe. In addition, as shown in Figure 10 and Figure 11 , the generation of reactive oxygen species (ROS) signals was detected under laser irradiation.

[0075] 2.3 In vitro uptake and therapeutic effect of OCPdots@CTe Given the significant and broad absorption properties of OCPdots@CTe in the near-infrared-II (NIR-II) window, they can serve as a contrast agent for NIR-II photoacoustic imaging (PAI) and a photothermal agent for NIR-II photothermal therapy (PTT). To evaluate their photothermal performance, the temperature change of OCPdots@CTe solution under 808 nm laser irradiation was studied in vitro. The temperature change at different concentrations (12.5, 25, 50 and 100 μg / mL) is shown in Figure 3 a. Even at a relatively low concentration, the solution temperature rises rapidly, and at a concentration of 100 μg / mL, the temperature rises to about 60°C after 5 minutes of laser irradiation Figure 3 , c). The photothermal performance under different laser power densities (0.1, 0.25, 0.5 and 0.75 W / cm²) was also evaluated, and OCPdots@CTe exhibited a strong laser power-dependent photothermal effect under continuous irradiation Figure 3 , b). To further evaluate the photothermal stability of OCPdots@CTe, a cyclic temperature change test was performed. Figure 12 The results in show that there is no significant change in the maximum temperature after five laser on / off cycles, which proves that OCPdots@CTe has excellent optical stability.

[0076] To determine the photothermal conversion efficiency, OCPdots@CTe solution was irradiated with 808 nm laser for 6 minutes until the stable temperature was reached. Then the laser was turned off and the solution was allowed to cool to room temperature, with pure water as a negative control.

[0077] Based on Figure 3 c and the following method, the photothermal conversion efficiency was calculated.

[0078] To measure the photothermal conversion efficiency, polymer dots (Pdots) with an optical density (OD) of 1 were added to a quartz cuvette. The Pdots were then exposed to 980 nm laser irradiation at a power density of 0.5 W / cm2until thermal equilibrium was reached, and then cooled to room temperature. The photothermal conversion efficiency can be calculated using the following equation. For the energy balance in the system, the total energy balance can be described as follows: (S1) where, is the product of the mass and the specific heat capacity of the system components, i is the total temperature of the system, T is time. t Q j j The energy term contains the laser-induced energy source term Q l (from the Pdots), Q 0 (from the solvent and cuvette), and the energy output term Q ext .

[0079] Ql is the photothermal energy input from the Pdots, which is expressed as: (S2) where, I is the incident laser power, A λ is the optical density at the laser wavelength (980 nm), η is the photothermal conversion efficiency. Q 0 represents the heat dissipated after the solvent and container absorb light energy, which can be measured separately by a container containing only a Pdot-free aqueous solution.

[0080] Q ext is the external heat flux in the system, which is approximately proportional to the linear thermal driving force with the proportionality constant being the heat transfer coefficient h : (S3) where, h is the heat transfer coefficient, A is the surface area of the cuvette. It can be determined by measuring the temperature drop rate after removing the light source hA .

[0081] In the absence of laser excitation (Ql= 0), equation (3) is substituted into equation (1) to obtain: Q l + 0 = 0, Q (S4)​​​ After sorting and integration, an expression for t can be obtained: (S5) where, m is the mass of water, C p is the specific heat capacity of water. To obtain the value of hA , a dimensionless driving force temperature θ is introduced, which is based on the system maximum temperature T max and the sample system time constant τ s is calibrated: (S6) (S7) where, τ s is the slope of the linear cooling phase time data: (S8) Therefore, the expression for the photothermal conversion efficiency η of Pdots is: (S9) The calculated photothermal conversion efficiency of OCPdots@CTe is about 52%.

[0082] This efficiency is significantly higher than that of other nanoparticles, as shown in Table 1.

[0083] Table 1. Photothermal conversion efficiency (PCE) of NIR-II nanoparticles

[0084] Figure 13 A plot of the linear time data obtained from the cooling phase versus -lnθ is shown.

[0085] Therefore, OCPdots@CTe has a high extinction coefficient, excellent photothermal stability, high NIR-II photothermal conversion efficiency, and excellent biocompatibility, making it an excellent candidate material for bimodal PA / FL imaging-guided PTT.

[0086] To evaluate the excellent biocompatibility, cellular uptake, and enhanced therapeutic effect of OCPdots@CTe, confocal imaging technology was first used to study the cellular uptake of biomimetic OCPdots@CTe (Fig. 6a). Figure 4, a). Due to the lack of NIR-II confocal imaging system, NIR-II luminescent polymers (m-PBTQ4F) and orange luminescent polymers (CN-PPV) were introduced to prepare nanoparticles with fluorescent properties in the visible light region. The absorption and excitation spectra of these nanoparticles are shown in Figure 14 and Figure 15 . After staining the cell membrane and nucleus, it was found that OCPdots@CTe was mainly taken up by 4T1 cells, which was further confirmed by flow cytometry analysis ( Figure 4 , b). In addition, the biocompatibility and cytotoxicity of OCPdots@CTe were evaluated using a 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid phenyl)-2H-tetrazole monosodium salt (WST-8) cell counting kit (CCK-8). The results showed that CTe had significant cytotoxicity to normal 293T cells, but when OCPdots@CTe was wrapped by membrane proteins, the toxicity was greatly reduced. After co-incubation with 293T cells for 24 hours, OCPdots@CTe did not show significant toxicity even at a concentration as high as 100 μg / mL ( Figure 4 , c).

[0087] To further evaluate the effect of PTT on living cells, CCK-8 kit and fluorescence imaging technology were used to perform photothermal ablation experiments on 4T1 cells and HeLa cancer cells. Different concentrations of nanoparticles were co-incubated with cancer cells under 808 nm laser irradiation. The results of CCK-8 experiment showed that as the amount of nanoparticles taken up by cells increased, the cell viability decreased significantly ( Figure 4 , d). Due to the specific targeting effect of 4T1 membrane proteins, HeLa cells took up less OCPdots@CTe than OPdots@CTe ( Figure 4 , e).

[0088] In addition, fluorescence microscopy imaging of calcein / propidium iodide (PI) co-stained HeLa cells showed that OCPdots@CTe exhibited high-efficiency photothermal ablation efficiency at very low laser power density ( Figure 5 , a), which indicates that they have great potential as efficient photothermal agents for enhanced PTT. Due to the drug mechanism of CTe and the photodynamic therapy (PDT) effect of OCPdots@CTe, in vitro treatment also induced apoptosis. As shown in the figure, different biofunctionalized nanoparticles produced different degrees of ROS signals at the same concentration, and the addition of CTe with strong absorption characteristics at 488 nm significantly enhanced the ROS signal ( Figure 5, b). Notably, OCPdots@CTe produced the best ROS signal. In addition, flow cytometry analysis of the treated cells stained with annexin V-FITC / PI found that up to 46% of the cells entered the apoptosis stage, and more than 70% of the cancer cells died ( Figure 5 , c).

[0089] 2.4 Dual-mode photoacoustic and fluorescence imaging of OCPdots@CTe in the NIR-II window To visually present the increased accumulation of OCPdots@CTe at the tumor site and the targeted homing ability, in vivo NIR-II photoacoustic (PA) imaging and fluorescence (FL) imaging studies were carried out. A 4T1 tumor-bearing nude mouse model was constructed, and the nanoparticles were injected into the mice via the tail vein (dose of 2.0 mg / kg). In the in vivo NIR-II fluorescence imaging experiment, within 1 hour after injection, a detectable fluorescence signal was observed at the tumor site, and the signal intensity gradually increased, reaching a peak at 24 hours ( Figure 6 , a, b). Notably, at each time point, OCPdots@CTe exhibited a stronger fluorescence signal than Pdots@CTe and OPdots@CTe ( Figure 6 , b). Even 7 days after injection, the strongest signal was detected at the tumor site of the mice injected with OCPdots@CTe ( Figure 6 , c; Figure 16 ).

[0090] Subsequently, the PA signal intensity at the tumor site was monitored at different time points using a NIR-II photoacoustic imaging system. Figure 6 The in vivo photoacoustic images of the tumor site and their corresponding PA signal intensities after injection of the nanoparticles and 808 nm laser irradiation are shown ( Figure 6 , d, e). The enhancement efficiency and effect of the PA signal were similar to those of the fluorescence imaging, indicating that due to the enhanced permeability and retention (EPR) effect and the targeted homing ability, the accumulation of the nanoparticles at the tumor site reached a peak at 24 hours. Since Pdots@CTe, OPdots@CTe, and OCPdots@CTe exhibited different absorption and fluorescence intensities at the same concentration, OCPdots@CTe exhibited a shorter tumor accumulation time and enhanced NIR-II PAI and FL abilities compared to Pdots@CTe and OPdots@CTe.

[0091] 2.5 In vivo therapeutic effect of OCPdots@CTe in the NIR-II window To further investigate the PTT and PDT effects of OCPdots@CTe in 4T1 tumor-bearing nude mice, the same mouse model as described in Section 2.4 was adopted, and all operations followed the relevant guidelines of the University of Macau Animal Care and Use Committee. Nude mice with tumor volume of about 100 mm3were randomly divided into six groups (n = 5 for each group), and each group was intravenously injected with Pdots and OCPdots@CTe at a dose of 2.0 mg / kg or an equivalent amount of phosphate buffer solution (PBS). Subsequently, the anti-tumor effects of different treatment methods (with or without laser irradiation) were observed, and the body weight and tumor volume of the mice were recorded every 2 days. After 14 days, the mice were sacrificed and tissue samples were collected.

[0092] The relative tumor volume curve shows that the “OCPdots@CTe + laser” group exhibits a better tumor inhibition effect ( Figure 7 , d). In addition, the size and weight of the tumor tissue in vivo are statistically consistent with the in vitro observation results ( Figure 7 , a, b). After treatment, the tumor tissue was collected, and hematoxylin-eosin (H&E) staining and fluorescence microscopy analysis found that there was a large amount of necrotic tissue in the “OCPdots@CTe + laser” group ( Figure 7 , e). However, no significant fluctuations in body weight were observed in all experimental groups ( Figure 7 , c). Nevertheless, to avoid thermal damage caused by high temperature, a lower power laser was used for treatment, which limited the local temperature rise to below 42°C ( Figure 17 , Figure 18 ). Therefore, complete tumor inhibition cannot be achieved solely by PTT and drug effects.

[0093] Recent studies have shown that the combination of PTT / PDT with immunotherapy is an effective synergistic strategy for treating solid tumors. Based on this, the potential of combining Pdots with PTT, PDT, CTe, and OMVs to enhance the immune response was explored ( Figure 8 ). A bilateral orthotopic breast cancer tumor-bearing mouse model was constructed, and the mice were divided into six groups (n = 5 for each group). When the primary tumor volume reached about 100 mm3and the distal tumor volume reached about 50 mm3, the mice in each experimental group were intravenously injected with Pdots, OCPdots@CTe, and PBS. After 24 hours, one group of mice received 808 nm laser irradiation, and the other group did not. On the 6th day of the treatment period, a second 808 nm laser irradiation was performed, and the body weight and tumor volume of the mice were recorded every 2 days ( Figure 8 , a). All tumor-bearing mice were sacrificed on the 14th day to collect tissue samples.

[0094] The relative volume change curves of primary tumors showed that the “OCPdots@CTe + laser” group almost achieved complete tumor ablation ( Figure 8 , c). The relative volume change curves of distal tumors indicated that only the “OCPdots@CTe + laser” group showed a significant tumor growth inhibition effect ( Figure 8 , d). The size and weight of the isolated tumor tissues were consistent with the in vivo measurements on day 14 ( Figure 8 , f, h; Figure 20 ).

[0095] Notably, OCPdots@CTe after phototherapy and biomimetic modification had high targeting and immune effects, and proved to be the most effective in inhibiting the growth of primary tumors, which highlighted the potential of Pdots-based PTT / PDT combined immunotherapy. Flow cytometry analysis of the isolated tumor tissues found that the proportions of CD8⁺, CD4⁺, and cytotoxic T lymphocytes (CTLs) in the primary and distal tumors of the “OCPdots@CTe + laser” group were significantly up-regulated (n = 3), which indicated that these cells were more activated and participated in the anti-tumor immunotherapy in the experimental group ( Figure 8 , e, g). There was no significant difference in the body weight of tumor-bearing mice in each group ( Figure 8 , b). In addition, H&E staining of important organs in vitro confirmed that Pdots-based PTT / PDT combined immunotherapy did not produce toxic side effects ( Figure 19 ). These results collectively indicated that OCPdots@CTe successfully achieved biomimetic modification and drug doping, and achieved excellent therapeutic effects without compromising its optical properties.

[0096] Figure 1 A schematic diagram showing the therapeutic mechanism of OCPdots@CTe is shown. The diagram demonstrates the process of preparing OCPdots@CTe by wrapping Pdots@CTe with 4T1 cell membranes and bacterial outer membrane vesicles (OMVs). After intravenous injection, OCPdots@CTe can actively target and accumulate in tumor tissues. Under laser irradiation, the photothermal effect of OCPdots@CTe is triggered, leading to the release of CTe and OMVs fragments. In combination with near-infrared (NIR) stimulation, this process promotes the maturation of dendritic cells (DCs) and induces tumor-specific T cell-mediated immunotherapy. In addition, CTe enhances reactive oxygen species (ROS) signaling, amplifying the efficacy of photothermal therapy (PTT) and further enhancing the immunotherapy response. In addition to therapeutic applications, OCPdots@CTe can also serve as an imaging agent to achieve fluorescence and photoacoustic imaging for precise positioning and tracking of tumors.

[0097] 3. Conclusion In the present application, a high-density nanocomposite OCPdots@CTe is successfully designed, synthesized and characterized. By recrystallization method, functionalized polystyrene-polyethylene glycol carboxylic acid (PS-PEG-COOH), Chelerythrine (CTe) and near-infrared two-zone (NIR-II) luminescent polymer (PBTQ4F) are integrated to prepare Pdots@CTe. Subsequently, membrane proteins extracted from 4T1 cancer cells and Escherichia coli outer membrane vesicles (OMVs) are ultrasonically coated on the surface of Pdots@CTe, thereby forming OCPdots@CTe.

[0098] Absorption and emission spectra confirm that CTe has been successfully incorporated into Pdots. Drug loading is verified by the increase in the size and zeta potential after encapsulation. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) further verify the stability of OCPdots@CTe and the effect of membrane protein coating on its particle size. The nanocomposite exhibits excellent photothermal performance under various laser power densities and maintains stable photothermal effect during multiple on-off cycles. Notably, its photothermal conversion efficiency (PCE) reaches about 52%, which is significantly better than previously reported materials, highlighting its potential in dual-modality photoacoustic (PA) and fluorescence (FL) imaging guided photothermal therapy (PTT).

[0099] Cell uptake and therapeutic efficacy evaluation results show that OCPdots@CTe can be efficiently taken up by 4T1 cells, and even at a concentration as high as 100 μg / mL, it only exhibits very low cytotoxicity to normal 293T cells. Photothermal ablation experiments further confirm that OCPdots@CTe can efficiently kill 4T1 and HeLa cancer cells at low laser power density. In addition, OCPdots@CTe also exhibits excellent photodynamic therapy (PDT) performance, significantly enhances the generation of reactive oxygen species (ROS), and induces a large number of apoptosis in cancer cells.

[0100] In vivo experiments show that OCPdots@CTe has excellent NIR-II PA and FL imaging capability in 4T1 tumor models. Within 1 hour after injection, fluorescence signals can be detected at the tumor site, and reach a peak at 24 hours. Compared with Pdots@CTe and OPdots@CTe, OCPdots@CTe shows significantly higher fluorescence and photoacoustic signals, which indicates that it has high efficient accumulation and targeted homing ability at the tumor site.

[0101] In the evaluation of the therapeutic effect in vivo, the "OCPdots@CTe + laser" group achieved significant tumor inhibition effect within 14 days, almost completely eradicated the primary tumor, and significantly inhibited the growth of the distal tumor. Flow cytometry analysis further confirmed that in the "OCPdots@CTe + laser" group, CD8⁺, CD4⁺ and cytotoxic T lymphocytes (CTLs) in the primary tumor and distal tumor were significantly up-regulated, indicating that the body's anti-tumor immune response was activated. Importantly, H&E staining of major organs showed that Pdot-based PTT / PDT combined immunotherapy did not cause observable toxic side effects.

[0102] In summary, the present application successfully developed OCPdots@CTe with biomimetic properties and drug loading, which retains excellent optical properties and exhibits excellent therapeutic effect in vitro and in vivo. These results highlight the broad clinical application prospects of OCPdots@CTe in the field of cancer treatment, marking an important step forward in cancer treatment research, and indicating that more precise and personalized treatment methods are on the horizon.

Claims

1. A biomimetic semiconductor polymer dot loaded with celandine, characterized in that... These include functional polymers, luminescent polymers, celandine, 4T1 cell membranes, and bacterial outer membrane vesicles.

2. The biomimetic supported celandine semiconductor polymer dot according to claim 1, characterized in that, The functional polymer is polystyrene-polyethylene glycol carboxylic acid.

3. The biomimetic supported celandine semiconductor polymer dot according to claim 1, characterized in that, The luminescent polymer is PBTQ4F.

4. The biomimetic supported celandine semiconductor polymer dot according to claim 1, characterized in that, The bacterial outer membrane vesicles are outer membrane vesicles derived from Escherichia coli.

5. The method for preparing biomimetic supported chelidonine semiconductor polymer dots as described in any one of claims 1 to 4, comprising the following steps: The functional polymer, celandine, and luminescent polymer were integrated, and then 4T1 cell membranes and bacterial outer membrane vesicles were ultrasonically coated on their surface to form the biomimetic celandine-loaded semiconductor polymer dot.

6. The use of the biomimetic supported celandine semiconductor polymer dots as described in any one of claims 1 to 4 in the preparation of antitumor drugs.

7. The application according to claim 6, characterized in that, The tumor is either breast cancer or cervical cancer.

8. The application of the biomimetic loaded celandine semiconductor polymer dots as described in any one of claims 1 to 4 as an imaging agent.

Images