AS1411 aptamer functionalized liposome and preparation method and application thereof

Abstract

The application discloses an AS1411 aptamer functionalized liposome which comprises lipids, indocyanine green, chelidonium red base and AS1411 aptamer. The application also discloses a preparation method and application of the AS1411 aptamer functionalized liposome. The AS1411 aptamer functionalized liposome provided by the application has excellent fluorescence imaging and chemical-photothermal treatment effects, and is expected to become an ideal diagnosis and treatment nano system and has the potential for future clinical application in cancer treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an AS1411 aptamer-functionalized liposome, its preparation method, and its application. Background Technology

[0002] Breast cancer is a common malignant tumor with a poor prognosis and a high risk of recurrence and death. In clinical practice, surgery combined with chemotherapy or radiotherapy is used as a promising treatment strategy. However, these treatments suffer from poor tissue selectivity, high toxicity, and drug resistance. To meet the growing demand for precision and personalized medicine, therapeutic technologies have been developed. However, limited tumor accumulation restricts the effectiveness of therapeutic agents. Therefore, various actively targeted ligands or recognition motifs, such as peptides, aptamers, and membrane proteins, have been developed.

[0003] DNA aptamers have been extensively studied as novel targeting agents. Aptamers are single-stranded oligonucleotides that bind with extremely high affinity to specific cellular molecules. Aptamers exhibit good stability and safety, and possess potential clinical application value. Among them, AS1411 is the most advanced aptamer and the first to enter clinical trials.

[0004] In recent years, the integration of therapeutic drugs and imaging agents into nanomedicine has seen rapid development for the real-time diagnosis and treatment of cancer. Among various imaging techniques, near-infrared II (NIR-II, 1000-1700 nm) fluorescence imaging is an advanced optical imaging technique with high sensitivity, fast response, and excellent resolution. Summary of the Invention

[0005] One object of the present invention is to provide a formulation with excellent anti-tumor effect and fluorescence imaging effect in order to solve the above-mentioned technical problems.

[0006] Another object of the present invention is to provide a method for preparing the formulation.

[0007] Another object of the present invention is to provide the application of the said formulation.

[0008] To achieve the above-mentioned objectives, the present invention provides an AS1411 aptamer-functionalized liposome, comprising lipids, indocyanine green, celandine, and AS1411 aptamer.

[0009] In a preferred embodiment, the AS1411 aptamer-functionalized liposomes are obtained by first preparing liposomes loaded with ICG and chelidonine, and then modifying them with AS1411 aptamers.

[0010] On the other hand, the present invention also provides a method for preparing the AS1411 aptamer-functionalized liposomes, which includes the following steps:

[0011] Step 1. Dissolve 5 mg DOPC and 1 mg DSPE-PEG-MAL in 1 mL chloroform, and dissolve 300 μg chelidonine in 1 mL methanol; after mixing, remove the organic solvent by rotary evaporation to form a lipid membrane; add 1 mL of an aqueous solution of ICG with a concentration of 300 μg / mL to dissolve the lipid membrane in the solution; extrude the solution through polycarbonate membranes with pore sizes of 200 nm and 100 nm 20 times to prepare liposomes I / C@Lipo loaded with ICG and chelidonine;

[0012] Step 2. The aptamer HS-AS1411 was covalently attached to the I / C@Lipo surface at a mass ratio of 10:1 by stirring overnight. Then, it was removed by ultrafiltration centrifugation and resuspended in distilled water to remove small molecular weight residues, thus obtaining AS1411 aptamer-functionalized liposomes loaded with ICG and celandine.

[0013] Preferably, the sequence of the aptamer HS-AS1411 is as shown in SEQ ID NO. 1.

[0014] On the other hand, the present invention also provides the application of the AS1411 aptamer-functionalized liposomes in the preparation of antitumor drugs.

[0015] Preferably, the tumor is breast cancer.

[0016] Preferably, the drug is a chemophotothermal therapy drug.

[0017] On the other hand, the present invention also provides the application of the AS1411 aptamer-functionalized liposomes as imaging agents.

[0018] Preferably, the imaging agent is a fluorescent imaging agent.

[0019] Preferably, the imaging agent is a near-infrared II fluorescent imaging agent.

[0020] Cheerythrine is a potent apoptosis inducer, and indocyanine green (ICG) generates a large amount of heat under laser irradiation, effectively achieving photothermal therapy (PTT) and enabling diagnosis via fluorescence imaging. Liposomes are one of the most successful drug delivery systems, exhibiting excellent biocompatibility and biodegradability.

[0021] The AS1411 aptamer-functionalized liposome I / C@Lipo-A, loaded with ICG and chelidonine, provided by this invention, possesses excellent imaging and therapeutic potential. Modification with AS1411 significantly enhances the chemotherapeutic effect of chelidonine and the photothermal therapeutic effect of ICG. Furthermore, ICG can also serve as a near-infrared II (NIR-II) fluorescence imaging contrast agent for monitoring breast cancer. In summary, I / C@Lipo-A exhibits high efficiency in both fluorescence imaging and chemo-photothermal therapy, and holds promise as an ideal diagnostic and therapeutic nanosystem with potential for future clinical applications in cancer treatment. Attached Figure Description

[0022] Figure 1 The preparation and characterization of I / C@Lipo-A are shown. (A) Schematic diagram of the I / C@Lipo-A preparation process. (B) Particle size distribution of I / C@Lipo-A determined by dynamic light scattering (DLS). Inset: Transmission electron microscopy (TEM) image of I / C@Lipo-A. Scale bar: 200 nm. (C) UV-Vis absorption spectra of free ICG and I / C@Lipo-A. (D) Fluorescence spectra of free ICG and I / C@Lipo-A in the near-infrared region I (NIR-I). (E) Fluorescence spectra of free ICG and I / C@Lipo-A in the near-infrared region II (NIR-II). (F) Temperature changes of free ICG and I / C@Lipo-A solutions under near-infrared laser irradiation. (G) Temperature changes of I / C@Lipo-A solutions of different concentrations under near-infrared laser irradiation. Temperature changes of (H)I / C@Lipo-A solution under near-infrared laser irradiation of different powers.

[0023] Figure 2 This study demonstrates the in vitro chemophotothermal combined therapy of I / C@Lipo-A. (A) Confocal fluorescence imaging of 4T1 cells after incubation with different formulations. Scale bar: 20 μm. (B) Flow cytometry analysis of cellular uptake by different formulations. (C) Flow cytometry analysis of reactive oxygen species (ROS) generation in 4T1 cells after incubation with different formulations. (D) Flow cytometry assessment of apoptosis in 4T1 cells after incubation with different formulations. (E) Quantitative analysis of ICG fluorescence intensity after incubation with different formulations (n=3). (F) Percentage of apoptosis in 4T1 cells. (G) Cell viability after treatment with different formulations (n=3).

[0024] Figure 3The following images show the in vivo fluorescence imaging of I / C@Lipo-A. (A) In vivo fluorescence imaging of tumor-bearing mice in the near-infrared region I (NIR-I) after injection. (B) In vivo fluorescence imaging of tumor-bearing mice in the near-infrared region II (NIR-II) after injection. The tumor area is marked by a white circle. (C) Ex vivo fluorescence imaging of tumor tissue in the near-infrared region I. (D) Ex vivo fluorescence imaging of tumor tissue in the near-infrared region II.

[0025] Figure 4 The in vivo antitumor effects of I / C@Lipo-A are shown. (A) Infrared thermographic images of mice after injection during near-infrared laser (808 nm, 1 W / cm²) irradiation. (B) Photographs of tumors excised from 4T1 tumor-bearing mice after treatment with different formulations. (C) Weight of the tumor.

[0026] Figure 5 Immunohistochemical analysis of I / C@Lipo-A and its anti-metastatic effect are shown. (A) H&E staining images of tumor tissues after different treatments. Scale bar: 50 μm. (B) Immunofluorescence imaging of Ki67 in tumor tissues after different treatments. Scale bar: 50 μm. (C) Immunofluorescence imaging of Bcl-2 in tumor tissues after different treatments. Scale bar: 50 μm. (D) Photographs of lung tissues from 4T1 tumor-bearing mice after different treatments. (E) H&E staining of lung tissues from 4T1 tumor-bearing mice after different treatments. Scale bar: 500 μm.

[0027] Figure 6 The mechanism of I / C@Lipo-A as an outstanding diagnostic and therapeutic nanosystem is illustrated. (A) Schematic diagram of the I / C@Lipo-A preparation process. (B) Schematic diagram of I / C@Lipo-A used for near-infrared II imaging-guided chemophotothermal therapy of breast cancer.

[0028] Figure 7 The particle size distributions of I / C@Lipo and I / C@Lipo-A, as determined by dynamic light scattering (DLS), are shown.

[0029] Figure 8 The particle size distribution of I / C@Lipo-A in water, phosphate-buffered saline (PBS), and 10% fetal bovine serum (FBS) is shown by dynamic light scattering (DLS).

[0030] Figure 9 Fluorescence imaging of the solution is shown. (A) Fluorescence imaging of the solution in the near-infrared region I (NIR-I); (B) Fluorescence imaging of the solution in the near-infrared region II (NIR-II).

[0031] Figure 10Cell viability (n=3) after incubation with different formulations is shown. (A) Cell viability of HT22 cells. (B) Cell viability of Shsy5y cells.

[0032] Figure 11 Viable / dead staining images of 4T1 cells after incubation with different formulations are shown. Scale bar: 100 μm.

[0033] Figure 12 This study shows in vitro fluorescence imaging of major organs in the near-infrared region I (NIR-I) 12 hours after injection.

[0034] Figure 13 This study shows in vitro fluorescence imaging of major organs in the near-infrared II (NIR-II) region 12 hours after injection.

[0035] Figure 14 Tumor volume changes in 4T1 tumor-bearing mice treated with different formulations are shown. Data are presented as mean ± standard deviation (SD).

[0036] Figure 15 The changes in body weight of 4T1 tumor-bearing mice treated with different formulations are shown. Data are presented as mean ± standard deviation (SD).

[0037] Figure 16 Representative H&E staining images of major organs are shown. Scale bar: 100 μm. Detailed Implementation

[0038] 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.

[0039] 1. Materials and Methods

[0040] 1.1 Materials

[0041] 1,2-Dioleoyl-sn-glycerol-3-phosphocholine (DOPC) was purchased from Avanti, Inc. (Alabama, USA), and 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]-maleimide (DSPE-PEG2000-MAL) was purchased from Xi'an Ruixi Biotechnology Co., Ltd.

[0042] Indocyanine green (ICG) and celandine were purchased from Sigma-Aldrich (USA).

[0043] Fetal bovine serum (FBS), high-glucose DMEM medium, trypsin EDTA, and penicillin / streptomycin were purchased from Gibco Life Sciences, Inc. (USA).

[0044] 4,6-Diamidinyl-2-phenylindole (DAPI), Cell Count Kit-8 (CCK-8), Calcein-AM / PI staining kit, annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit, and reactive oxygen species (ROS) detection kit were all purchased from Beyotime Biotechnology Co., Ltd. (China).

[0045] 1.2 Cell Culture

[0046] Mouse breast cancer (4T1) cells were cultured in high-glucose DMEM medium. All media contained 10% fetal bovine serum (FBS) and 1% antibiotics (100 IU / mL penicillin and 100 μg / mL streptomycin). Cells were incubated at 37°C in a humidified environment with 5% CO2.

[0047] 1.3 Preparation and Characterization of I / C@Lipo-A

[0048] The preparation process is carried out at room temperature.

[0049] First, I / C@Lipo was prepared using a thin-film hydration method. 5 mg DOPC and 1 mg DSPE-PEG2000-MAL were dissolved in 1 mL chloroform, and 300 μg chelidonine was dissolved in 1 mL methanol. After mixing, the organic solvent was removed by rotary evaporation to form a lipid membrane. 1 mL of an aqueous solution of ICG (300 μg / mL) was added to dissolve the lipid membrane. The solution was then extruded 20 times through polycarbonate membranes with pore sizes of 200 nm and 100 nm to prepare liposomes I / C@Lipo simultaneously loaded with ICG and chelidonine.

[0050] Then, the aptamer HS-AS1411 (nucleotide sequence: HS-C6-5) was used. ’ -TTGGTGGTGGTGGTTGTGGTGGTGGTGG-3' (purchased from Shanghai Sangon Biotech) was covalently attached to the I / C@Lipo surface at a mass ratio of 10:1 by stirring overnight. Finally, the solution was centrifuged by ultrafiltration (3000 MWCO, Amicon, Millipore Corporation, Bedford, USA) and resuspended in distilled water to remove low molecular weight residues, yielding AS1411 aptamer-functionalized liposomes I / C@Lipo-A loaded with ICG and celandine.

[0051] The prepared I / C@Lipo-A was stored at 4°C for later use.

[0052] Furthermore, the preparation methods of I@Lipo and C@Lipo are basically the same as those of I / C@Lipo, the only difference being that I@Lipo is a liposome containing ICG but not celandine, while C@Lipo is a liposome containing celandine but not ICG.

[0053] 1.4 Photothermal effect of I / C@Lipo-A

[0054] Free ICG and aqueous solutions of I / C@Lipo-A at different concentrations were irradiated with lasers (808 nm, different powers). Temperature was monitored every 10 seconds using an infrared thermal imager (Fluke) for 300 seconds.

[0055] 1.5 In vitro cellular uptake

[0056] Cellular uptake of I / C@Lipo and I / C@Lipo-A in 4T1 cells (mouse breast cancer cells) was assessed using confocal laser scanning microscopy (CLSM) and flow cytometry.

[0057] For CLSM studies, 4T1 cells were seeded into confocal dishes and cultured for 24 hours. Subsequently, cells were incubated for 4 hours with I / C@Lipo and I / C@Lipo-A (ICG concentration, 20 μg / mL), respectively. After incubation, cells were washed with phosphate-buffered saline (PBS), fixed with paraformaldehyde, and stained with DAPI for 15 minutes. Samples were observed using a CLSM (Zeiss, LSM700).

[0058] For flow cytometry studies, 4T1 cells were seeded into 12-well plates and cultured for 24 hours. Subsequently, the cells were incubated for 4 hours each with I / C@Lipo and I / C@Lipo-A (ICG concentration, 20 μg / mL). After incubation, the cells were washed, collected, and resuspended in PBS. Samples were analyzed by flow cytometry using an APC-700 channel analyzer.

[0059] 1.6 Cell viability

[0060] 4T1 cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay and Calcein-AM / PI staining assay.

[0061] For the CCK-8 assay, 4T1 cells were seeded in 96-well plates and cultured for 24 hours. Subsequently, the cells were incubated for 12 hours each with C@Lipo, I@Lipo, I / C@Lipo-A, and I / C@Lipo-A + laser, respectively. After incubation, the cells were incubated with the CCK-8 reagent for 30 minutes according to the manufacturer's instructions, and cell viability was analyzed.

[0062] For Calcein-AM / PI staining assays, 4T1 cells were seeded into 96-well plates and cultured for 24 hours. After incubation with C@Lipo, I@Lipo, I / C@Lipo-A, and "I / C@Lipo-A + laser" (808 nm, 1 W / cm², 5 min), cells were stained with the Calcein-AM / PI kit according to the manufacturer's instructions and observed using EVOS (M7000).

[0063] 1.7 Apoptosis

[0064] Apoptosis in 4T1 cells after different treatments was assessed using an annexin V / PI assay. 4T1 cells were seeded into 12-well plates and cultured for 24 hours. After incubation for 24 hours with C@Lipo, I@Lipo, I / C@Lipo-A, and "I / C@Lipo-A + laser" (1 W / cm², 5 min), cells were washed, collected, and resuspended in PBS, followed by annexin V and PI staining for 15 min. Samples were analyzed by flow cytometry using FITC and PE channels.

[0065] 1.8 Animal and Tumor Models

[0066] Female Balb / c mice (weighing 20 ± 2 g, six weeks old) were purchased from the University of Macau (Macao Special Administrative Region, China). All animal experiments were conducted in accordance with the University of Macau's animal research care regulations.

[0067] 4T1 cells (1×10⁻⁶) were injected subcutaneously into the right hind leg of 4T1 tumor-bearing mice. 6 A tumor-bearing mouse model was established using cells per mouse. When the solid tumor volume (tumor length × tumor width² / 2) reached approximately 100 mm³, it was used for subsequent experiments.

[0068] 1.9 In vivo fluorescence imaging of I / C@Lipo-A

[0069] To investigate the biodistribution of I / C@Lipo-A in vivo, I / C@Lipo and I / C@Lipo-A (ICG concentration, 0.5 mg / kg) were injected into tumor-bearing mice via tail vein injection. NIR-I and NIR-II fluorescence images were acquired at selected time points (0, 6, 12, and 24 hours) using an AniView Kirin (Bltlux, China) imaging system. Simultaneously, mice were sacrificed 12 hours post-injection, and ex vivo NIR-I and NIR-II fluorescence imaging was performed on major organs (heart, liver, spleen, lung, kidney, and tumor).

[0070] 1.10 Antitumor effects of I / C@Lipo-A in vivo

[0071] The in vivo therapeutic effect of I / C@Lipo-A was evaluated in 4T1 tumor-bearing mice when the tumor size reached approximately 100 mm³. Mice were randomly divided into six groups and administered intravenous injections of saline, saline + laser, C@Lipo, I@Lipo + laser, I / C@Lipo + laser, and I / C@Lipo-A + laser (ICG concentration 1 mg / kg, chelidonine concentration 1 mg / kg) on ​​days 0 and 2, respectively. Twelve hours after injection, each mouse was irradiated with an 808 nm laser (1 W / cm², 5 min). Simultaneously, the body temperature of mice receiving the following treatments was monitored and imaged using infrared thermal imaging: saline + laser, I@Lipo + laser, I / C@Lipo + laser, and I / C@Lipo-A + laser. Tumor size and weight were measured and recorded. In vivo tumor cell apoptosis and necrosis were further assessed by H&E staining and Ki67 and Bcl-2 immunofluorescence staining.

[0072] 1.11 Anti-lung metastasis effect of I / C@Lipo-A

[0073] Following treatment, lung samples were collected and fixed in paraformaldehyde solution for 24 hours, followed by photography. Furthermore, lung metastatic nodules were assessed using H&E staining to further analyze the metastatic status.

[0074] 1.12 In vivo safety analysis

[0075] On days 0 and 2, healthy mice were intravenously injected with saline, I@Lipo, C@Lipo, I / C@Lipo, and I / C@Lipo-A (ICG concentration 1 mg / kg, chelidonine concentration 1 mg / kg), respectively. The health status of the mice was observed. Finally, the mice were euthanized, and major organs (heart, liver, spleen, lungs, and kidneys) were collected for H&E staining to analyze the biocompatibility of the different formulations.

[0076] 1.13 Statistical Analysis

[0077] All experiments were repeated at least three times. Unless otherwise stated, data are expressed as mean ± standard deviation (SD). Data were compared using Student's t-test, and differences were considered statistically significant at *p<0.05, **p<0.01, and ***p<0.001.

[0078] 2. Results

[0079] 2.1 Preparation and Characterization of I / C@Lipo-A

[0080] like Figure 1As shown in Figure A, I / C@Lipo-A was prepared. First, liposomes (I / C@Lipo) loaded with indocyanine green (ICG) and celandine were prepared using a thin-film hydration method, followed by continuous physical extrusion. Next, the aptamer HS-AS1411 was covalently attached to the surface of I / C@Lipo via Michael addition to form I / C@Lipo-A.

[0081] The dynamic particle size of the prepared I / C@Lipo-A was 132.9 ± 0.9 nm, slightly larger than that of I / C@Lipo ( Figure 1 B; Figure 7 Transmission electron microscopy (TEM) shows that its morphology is a uniform sphere. Figure 1 B).

[0082] Next, the particle size stability of I / C@Lipo-A was investigated. For example... Figure 8 As shown, the particle size of I / C@Lipo-A remained stable for 5 days in water, phosphate-buffered saline (PBS), and 10% fetal bovine serum (FBS), indicating its excellent stability.

[0083] Furthermore, the photophysical properties of I / C@Lipo-A were investigated. The absorption spectrum of I / C@Lipo-A is similar to that of free ICG ( Figure 1 Compared to ICG, the fluorescence emission peak of I / C@Lipo-A redshifted from 780 nm to 792 nm. Figure 1 Therefore, it was assumed that the fluorescence intensity of I / C@Lipo-A in NIR-II might be higher than that of ICG. As expected, the fluorescence intensity of I / C@Lipo-A at 1000 nm was 4.28 times higher than that of ICG (D). Figure 1 This supports the application of I / C@Lipo-A in NIR-II fluorescence imaging.

[0084] Next, the photothermal properties of I / C@Lipo-A were examined. The temperature of I / C@Lipo-A increased rapidly, and during 808 nm laser irradiation, the temperature was significantly higher than that of ICG (…). Figure 1 Furthermore, the temperature of I / C@Lipo-A increases with increasing laser concentration and power (F). Figure 1 (G, H), which is beneficial for subsequent photothermal therapy.

[0085] 2.2 In Vitro Chemotherapy-Photothermal Combined Therapy

[0086] First, the active targeting efficiency of I / C@Lipo-A on 4T1 cells was investigated using confocal laser scanning microscopy (CLSM) and flow cytometry. As shown in the figure, after incubation with I / C@Lipo and I / C@Lipo-A for 4 hours, respectively, a stronger red fluorescence signal of ICG was observed in cells treated with I / C@Lipo-A. Figure 2 A).

[0087] Furthermore, quantitative analysis by flow cytometry showed that cells treated with I / C@Lipo-A exhibited higher fluorescence intensity. Figure 2 (B, E), demonstrating the active targeting functionalization of AS1411-modified I / C@Lipo-A.

[0088] Next, the chemotherapeutic and photothermal effects of I / C@Lipo-A on 4T1 cells were investigated. Chelidonine has been reported to mediate apoptosis by generating reactive oxygen species (ROS). Therefore, ROS generation was detected using the 2′,7′-dichlorofluorescin diacetate (DCFH-DA) indicator. As shown in the figure, each chelidonine-based formulation enhanced the fluorescence intensity of ROS, demonstrating the chemotherapeutic mechanism of I / C@Lipo-A. Figure 2 C).

[0089] Then, Calcein-AM / PI double staining was used to distinguish between live and dead cells. Calcein-AM has high cell membrane permeability, esterifies in live cells, and emits green fluorescence. Conversely, PI cannot pass through the cell membrane of live cells but can penetrate the cell membrane of dead cells to reach the nucleus and emit red fluorescence. Figure 11 As shown, I / C@Lipo-A combined with laser irradiation significantly reduced the survival rate of 4T1 cells, as evidenced by a significant increase in red fluorescence of dead cells.

[0090] To further verify the cell-killing effect, the Annexin V-FITC / PI assay was performed to assess apoptosis in 4T1 cells. As shown in the figure, compared with C@Lipo or I / C@Lipo-A incubation alone, I / C@Lipo-A showed the strongest ability to induce apoptosis by combining with laser irradiation. Figure 2 (D, F).

[0091] Furthermore, the therapeutic effect of I / C@Lipo-A on 4T1 cells was evaluated using a CCK-8 assay. Cells incubated with laser irradiation after being combined with I / C@Lipo-A showed significantly reduced cell viability (…). Figure 2(G), demonstrating the strong cytotoxic effect of I / C@Lipo-A on 4T1 cells. Overall, these results strongly demonstrate the ability of I / C@Lipo-A to enhance the destructive effects of 4T1 breast cancer cells.

[0092] 2.3 Active-targeted fluorescence imaging of I / C@Lipo-A in vivo

[0093] The in vivo distribution of I / C@Lipo-A directly affects tumor imaging and treatment outcomes. I / C@Lipo-A modified with the AS1411 aptamer accumulates more effectively at the tumor site, thereby enhancing imaging and treatment.

[0094] To evaluate the active targeting effect, the biodistribution of I / C@Lipo and I / C@Lipo-A in subcutaneous 4T1 tumor-bearing mice was observed, including the NIR-I and NIR-II regions. As shown in the figure, the fluorescence signal in the tumor gradually increased over time, reaching a maximum at 12 hours post-injection. Figure 3 (A, B).

[0095] Furthermore, compared to mice treated with I / C@Lipo, mice treated with I / C@Lipo-A showed brighter fluorescence signals in tumors, indicating greater uptake of the nanomedicine, which is beneficial for chemotherapy and photothermal therapy. Additionally, fluorescence images of tumor tissue isolated from I / C@Lipo-A-treated mice showed a stronger signal (…). Figure 3 (C, D) further demonstrate the superior targeting performance of I / C@Lipo-A. Due to its superior tissue penetration and reduced background autofluorescence, NIR-II fluorescence imaging is a powerful tool in medical research. Therefore, I / C@Lipo-A holds promise for imaging deep biological structures beyond subcutaneous tumors.

[0096] 2.4 Antitumor effects of I / C@Lipo-A in vivo

[0097] As described above, I / C@Lipo-A can serve as a photothermal agent and a delivery system for chelidonine, and is expected to exhibit significant antitumor activity. Therefore, the photothermal capacity was first evaluated in tumor-bearing mice. Temperature changes at the tumor site were monitored under laser irradiation (808 nm, 1 W / cm², 5 min). As shown in the figure, the temperature changes of I@Lipo and I / C@Lipo under laser irradiation were similar ( Figure 4 (A). However, mice treated with I / C@Lipo-A showed a faster increase in body temperature, indicating that more nanomedicine accumulated at the tumor site and generated more heat under laser irradiation. Previous studies have shown that temperatures above 43°C can induce tumor cell death. Therefore, I / C@Lipo-A is expected to exhibit excellent tumor-destructive activity under laser irradiation.

[0098] Subsequently, the in vivo therapeutic effect of I / C@Lipo-A was evaluated. 4T1 tumor-bearing mice were randomly divided into six groups: saline, laser, C@Lipo, I@Lipo + laser, I / C@Lipo + laser, and I / C@Lipo-A + laser. Figure 14 As shown, in mice treated only with laser, tumor volume increased rapidly due to continuous heating. As expected, mice treated with I / C@Lipo-A combined with laser showed the most significant tumor growth inhibition, attributed to its excellent targeting properties and chemophotothermal therapy. After treatment, the mice were sacrificed and the tumors were harvested. Surprisingly, three mice treated with I / C@Lipo-A combined with laser irradiation fully recovered, and their tumors were ablated. Figure 4 B). Furthermore, the weight of the isolated tumor was consistent with the observed results ( Figure 4 (C). During treatment, mice did not experience significant weight loss, indicating the safety of the nanomedicine. Figure 15 ).

[0099] 2.5 Immunohistochemical analysis and anti-metastatic effect of I / C@Lipo-A

[0100] Studies have shown that I / C@Lipo-A combined with laser irradiation exhibits good tumor targeting and anti-tumor activity. To further investigate the therapeutic effect, immunohistochemical analysis was performed on the tumors. As shown in the figure, H&E staining images reveal significant thinning and necrosis of tumor cells after treatment with I / C@Lipo-A combined with laser irradiation. Figure 5 (A), which further proves its remarkable therapeutic effect.

[0101] Next, Ki-67 staining was performed to detect cell proliferation in the tumor after treatment. Higher Ki-67 expression levels indicate stronger tumor cell proliferative activity, which is directly related to patient prognosis. As shown in the figure, laser-irradiated I / C@Lipo-A can inhibit Ki-67 expression (…). Figure 5 (B), which can be confirmed by the reduction of green fluorescence.

[0102] Furthermore, Bcl-2 is a proto-oncogene associated with programmed cell death and apoptosis. Studies have shown that overexpression of Bcl-2 protein increases the invasiveness and metastasis of breast cancer, thus assessing its role in breast cancer recovery is crucial. As expected, after treatment with I / C@Lipo-A in combination with laser irradiation, the green fluorescence intensity of Bcl-2 was significantly reduced ( Figure 5 (C), indicating that Bcl-2 expression is reduced.

[0103] Therefore, based on the above research, it is expected that lung metastasis will also be suppressed. As shown in the figure, metastatic nodules in the lungs are marked with black arrows. Figure 5 (D). In the saline group, numerous metastatic nodules were clearly visible. However, no metastasis was observed in the I / C@Lipo-A combined with laser therapy group, indicating that I / C@Lipo-A combined with laser irradiation can significantly inhibit the metastasis of 4T1 breast cancer to the lungs and reduce the number and size of tumor nodules.

[0104] H&E staining analysis further confirmed this metastasis inhibition. In the lungs of mice treated with I / C@Lipo-A combined with laser, the size of metastatic lesions was significantly reduced. Figure 5 Overall, the study indicates that I / C@Lipo-A combined with laser irradiation is most effective in inhibiting tumor metastasis to the lungs.

[0105] 3. Discussion

[0106] In this invention, a therapeutic nanosystem was developed that enables chemophotothermal therapy guided by near-infrared I (NIR-I) and near-infrared II (NIR-II) imaging. The prepared I / C@Lipo-A can target tumors and guide photothermal therapy via NIR imaging. Simultaneously, chelidonine inhibits tumor growth by generating reactive oxygen species (ROS). Due to modification with the AS1411 aptamer, more I / C@Lipo-A accumulates at the tumor site, achieving better therapeutic effects when combined with 808 nm laser irradiation, as confirmed in both in vitro and in vivo experiments. Notably, lung metastasis was also inhibited. Furthermore, I / C@Lipo-A shows potential as an NIR-II fluorescent imaging agent, which could be used for imaging deep tissues in the future. Therefore, NIR-II fluorescent imaging holds significant promise for advancing clinical diagnostic and therapeutic strategies.

Claims

1. An AS1411 aptamer functionalized liposome, characterized in that lipids, indocyanine green, chelerythrine and AS1411 aptamer; The AS1411 aptamer functionalized liposome is prepared by the following steps: Step 1. 5 mg of DOPC and 1 mg of DSPE-PEG-MAL are dissolved in 1 mL of chloroform, and 300 μg of chelerythrine is dissolved in 1 mL of methanol; after mixing, the organic solvent is removed by rotary evaporation to form a lipid film; 1 mL of an aqueous solution of ICG with a concentration of 300 μg / mL is added to dissolve the lipid film in the solution; the solution is extruded 20 times through polycarbonate membranes with pore sizes of 200 nm and 100 nm to prepare I / C@Lipo loaded with ICG and chelerythrine; Step 2. The aptamer HS-AS1411 is covalently linked to the surface of I / C@Lipo by stirring overnight at a mass ratio of 10:1, and then removed by ultrafiltration centrifugation and resuspended in distilled water to remove small molecular weight residues to obtain AS1411 aptamer functionalized liposome loaded with ICG and chelerythrine.

2. The AS1411 aptamer functionalized liposome of claim 1, wherein, The sequence of the aptamer HS-AS1411 is shown in SEQ ID NO.

1.

3. The use of the AS1411 aptamer functionalized liposome of claim 1 in the preparation of an anti-tumor drug.

4. Use according to claim 3, characterized in that, The tumor is breast cancer.

5. Use according to claim 3, characterized in that, The drug is a chemo-photothermal therapy drug.

6. The use of the AS1411 aptamer functionalized liposome of claim 1 as an imaging agent.

7. Use according to claim 6, characterized in that, The imaging agent is a fluorescent imaging agent.

8. Use according to claim 6, characterized in that, The imaging agent is a near-infrared two-zone fluorescent imaging agent.

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