AS1411 aptamer functionalized liposome as well as preparation method and application thereof

Abstract

The invention discloses an AS1411 aptamer functionalized liposome. The AS1411 aptamer functionalized liposome comprises a lipid, indocyanine green, chelerythrine and an AS1411 aptamer. The invention also discloses a preparation method and an application of the AS1411 aptamer functionalized liposome. The AS1411 aptamer functionalized liposome loaded with ICG and chelerythrine provided by the invention has excellent fluorescence imaging and chemical-photo-thermal treatment effects, is expected to become an ideal diagnosis and treatment nano system, and has the potential of being clinically applied to cancer treatment in the future.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to an AS1411 aptamer functionalized liposome, a preparation method thereof, and an application thereof. Background Art

[0002] Breast cancer is a common malignancy 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 have problems such as poor tissue selectivity, high toxicity, and drug resistance. To meet the growing demand for precise and personalized treatment, integrated diagnosis and treatment technologies have been developed. However, limited tumor accumulation limits the effectiveness of diagnostic and therapeutic agents. Therefore, various active targeting ligands or recognition motifs, such as peptides, aptamers, and membrane proteins, have been developed.

[0003] DNA aptamers have been extensively studied as a novel targeting agent. Aptamers are single-stranded oligonucleotides that bind to specific cellular molecules with extremely high affinity. Aptamers exhibit excellent stability and safety, and have potential clinical applications. 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 for real-time cancer diagnosis and treatment has developed rapidly. Among various imaging techniques, fluorescence imaging in the near-infrared region II (NIR-II, 1000-1700 nm) 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 preparation with excellent anti-tumor effect and fluorescence imaging effect in response to the above technical problems to be solved.

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

[0007] Another object of the present invention is to provide the use of the preparation.

[0008] In order to achieve the above object of the invention, the present invention provides an AS1411 aptamer-functionalized liposome, which comprises lipids, indocyanine green, chelerythrine and AS1411 aptamer.

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

[0010] On the other hand, the present invention also provides a method for preparing the AS1411 aptamer-functionalized liposomes, which comprises the following steps: Step 1. Dissolve 5 mg of DOPC and 1 mg of DSPE-PEG-MAL in 1 mL of chloroform, and dissolve 300 μg of chelerythrine in 1 mL of methanol. After mixing, remove the organic solvent by rotary evaporation to form a lipid film. Add 1 mL of a 300 μg / mL aqueous solution of ICG to dissolve the lipid film. Extrude the solution 20 times through polycarbonate membranes with 200 nm and 100 nm pore sizes to prepare ICG- and chelerythrine-loaded liposomes (I / C@Lipo). Step 2. The aptamer HS-AS1411 was covalently linked to the surface of I / C@Lipo at a mass ratio of 10:1 by stirring overnight. The liposomes were then ultrafiltered and resuspended in distilled water to remove small molecular weight residues, yielding AS1411 aptamer-functionalized liposomes loaded with ICG and chelerythrine.

[0011] Preferably, the sequence of the aptamer HS-AS1411 is shown as SEQ ID NO. 1.

[0012] On the other hand, the present invention also provides the use of the AS1411 aptamer functionalized liposome in the preparation of anti-tumor drugs.

[0013] Preferably, the tumor is breast cancer.

[0014] Preferably, the drug is a chemo-photothermal therapy drug.

[0015] On the other hand, the present invention also provides the use of the AS1411 aptamer-functionalized liposome as an imaging agent.

[0016] Preferably, the imaging agent is a fluorescent imaging agent.

[0017] Preferably, the imaging agent is a near-infrared second region fluorescent imaging agent.

[0018] Chelerythrine is a potent apoptosis inducer, and indocyanine green (ICG) can generate significant heat under laser irradiation, effectively enabling photothermal therapy (PTT) and enabling diagnosis through fluorescence imaging. Liposomes are one of the most successful drug delivery systems, exhibiting excellent biocompatibility and biodegradability.

[0019] The AS1411 aptamer-functionalized liposomes I / C@Lipo-A loaded with ICG and chelerythrine provided by this invention exhibit remarkable imaging and therapeutic potential. Modification with AS1411 significantly enhances the chemotherapeutic efficacy of chelerythrine and the photothermal therapeutic effect of ICG. Furthermore, ICG can be used as a near-infrared II (NIR-II) fluorescence imaging contrast agent for breast cancer monitoring. Overall, I / C@Lipo-A demonstrates high efficacy in both fluorescence imaging and chemo-photothermal therapy, demonstrating its potential as an ideal diagnostic and therapeutic nanosystem with potential for future clinical application in cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Shown are the preparation and characterization of I / C@Lipo-A. (A) Schematic diagram of the preparation process of I / C@Lipo-A. (B) Particle size distribution of I / C@Lipo-A measured by dynamic light scattering (DLS). Inset: Transmission electron microscopy (TEM) image of I / C@Lipo-A. Scale bar: 200 nm. (C) UV-visible 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. (H) Temperature changes of I / C@Lipo-A solution under near-infrared laser irradiation of different powers.

[0021] Figure 2 Figure 3 shows the in vitro chemo-photothermal combined treatment of I / C@Lipo-A. (A) Confocal fluorescence imaging of 4T1 cells after incubation with different formulations. Scale bar: 20 μm. (B) Flow cytometric analysis of cellular uptake of different formulations. (C) Flow cytometric analysis of reactive oxygen species (ROS) production in 4T1 cells after incubation with different formulations. (D) Flow cytometric 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 apoptotic 4T1 cells. (G) Cell viability after treatment with different formulations (n = 3).

[0022] Figure 3Figure 2 shows in vivo fluorescence imaging of I / C@Lipo-A. (A) In vivo fluorescence imaging of tumor-bearing mice following injection using near-infrared 1 (NIR-I). (B) In vivo fluorescence imaging of tumor-bearing mice following injection using near-infrared 2 (NIR-II). White circles indicate the tumor area. (C) Ex vivo fluorescence imaging of tumor tissue using near-infrared 1 (NIR-I). (D) Ex vivo fluorescence imaging of tumor tissue using near-infrared 2 (NIR-II).

[0023] Figure 4 The in vivo antitumor effects of I / C@Lipo-A are shown. (A) Infrared thermal imaging of injected mice during near-infrared laser (808 nm, 1 W / cm²) irradiation. (B) Photos of tumors excised from 4T1 tumor-bearing mice after treatment with different formulations. (C) Corresponding tumor weights.

[0024] Figure 5 Immunohistochemical analysis of I / C@Lipo-A and its anti-metastatic effects are shown. (A) H&E staining 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) Photos 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.

[0025] Figure 6 The mechanism of I / C@Lipo-A as an outstanding theranostic nanosystem is shown. (A) Schematic diagram of the preparation process of I / C@Lipo-A. (B) Schematic diagram of I / C@Lipo-A for near-infrared zone II imaging-guided chemo-photothermal therapy of breast cancer.

[0026] Figure 7 Shown are the particle size distributions of I / C@Lipo and I / C@Lipo-A measured by dynamic light scattering (DLS).

[0027] Figure 8 The particle size distribution of I / C@Lipo-A in water, phosphate buffered saline (PBS), and 10% fetal bovine serum (FBS) measured by dynamic light scattering (DLS) is shown.

[0028] 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).

[0029] Figure 10Cell viability after incubation with different preparations is shown (n=3). (A) Cell viability of HT22 cells. (B) Cell viability of Shsy5y cells.

[0030] Figure 11 Live / dead staining of 4T1 cells after incubation with different formulations is shown. Scale bar: 100 μm.

[0031] Figure 12 Shown are ex vivo fluorescence imaging of major organs in the near-infrared region 1 (NIR-I) 12 hours after injection.

[0032] Figure 13 Shown is ex vivo fluorescence imaging of major organs in the near-infrared II region (NIR-II) 12 hours after injection.

[0033] Figure 14 Figure 2 shows the changes in tumor volume in 4T1 tumor-bearing mice treated with different formulations. Data are presented as mean ± standard deviation (SD).

[0034] Figure 15 Figure 2 shows the changes in body weight of 4T1 tumor-bearing mice treated with different formulations. Data are expressed as mean ± standard deviation (SD).

[0035] Figure 16 Representative H&E-stained images of major organs are shown. Scale bar: 100 μm. DETAILED DESCRIPTION

[0036] The present invention will be further described below with reference to specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0037] 1. Materials and Methods 1.1 Materials 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) was purchased from Avanti (Alabama, USA), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]-maleimide (DSPE-PEG2000-MAL) was purchased from Xi'an Ruixi Biotechnology Co., Ltd.

[0038] Indocyanine green (ICG) and chelerythrine were purchased from Sigma-Aldrich (USA).

[0039] Fetal bovine serum (FBS), high-glucose DMEM medium, trypsin-EDTA, and penicillin / streptomycin (pen / strep) were purchased from Gibco Life Technologies (USA).

[0040] 4,6-diamidino-2-phenylindole (DAPI), cell counting kit-8 (CCK-8) kit, Calcein-AM / PI staining kit, annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit, and reactive oxygen species (ROS) detection kit were purchased from Beyotime Biotechnology Co., Ltd. (China).

[0041] 1.2 Cell culture 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 atmosphere with 5% CO2.

[0042] 1.3 Preparation and characterization of I / C@Lipo-A The preparation process was carried out at room temperature.

[0043] First, I / C@Lipo was prepared using a thin film hydration method. 5 mg of DOPC and 1 mg of DSPE-PEG2000-MAL were dissolved in 1 mL of chloroform, and 300 μg of chelerythrine was dissolved in 1 mL of methanol. After mixing, the organic solvent was removed by rotary evaporation to form a lipid film. 1 mL of an aqueous solution of ICG (300 μg / mL) was added to dissolve the lipid film. The solution was extruded 20 times through polycarbonate membranes with 200 nm and 100 nm pore sizes to prepare I / C@Lipo liposomes loaded with both ICG and chelerythrine.

[0044] Then, the aptamer HS-AS1411 (nucleotide sequence: HS-C6-5 ’ -TTGGTGGTGGTGGTTGTGGTGGTGGTGG-3', purchased from Shanghai Biotech, was covalently attached to the surface of I / C@Lipo 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 ICG- and chelerythrine-loaded AS1411 aptamer-functionalized liposomes, I / C@Lipo-A.

[0045] The prepared I / C@Lipo-A was stored at 4°C until use.

[0046] In addition, the preparation methods of I@Lipo and C@Lipo are basically the same as those of I / C@Lipo, with the only difference being that I@Lipo is a liposome containing ICG but not chelerythrine, while C@Lipo is a liposome containing chelerythrine but not ICG.

[0047] 1.4 Photothermal Effect of I / C@Lipo-A Free ICG and aqueous solutions of I / C@Lipo-A at different concentrations were irradiated with laser light (808 nm, varying powers). The temperature was monitored every 10 seconds using an infrared thermal imager (Fluke) for 300 seconds.

[0048] 1.5 In vitro cellular uptake The cellular uptake of I / C@Lipo and I / C@Lipo-A in 4T1 cells (mouse breast cancer cells) was evaluated by confocal laser scanning microscopy (CLSM) and flow cytometry.

[0049] For CLSM studies, 4T1 cells were seeded onto confocal microscopy dishes and cultured for 24 hours. Subsequently, the cells were incubated with I / C@Lipo and I / C@Lipo-A (ICG concentration, 20 μg / mL) for 4 hours. After incubation, the cells were washed with phosphate-buffered saline (PBS), fixed with paraformaldehyde, and stained with DAPI for 15 minutes. The samples were observed using a CLSM (Zeiss, LSM700).

[0050] For flow cytometry studies, 4T1 cells were seeded into 12-well plates and cultured for 24 hours. Subsequently, the cells were incubated with I / C@Lipo and I / C@Lipo-A (ICG concentration, 20 μg / mL) for 4 hours. After incubation, the cells were washed, collected, and resuspended in PBS. Samples were analyzed by flow cytometry using the APC-700 channel.

[0051] 1.6 Cell viability 4T1 cell viability was assessed by cell counting kit-8 (CCK-8) assay and Calcein-AM / PI staining assay.

[0052] For the CCK-8 assay, 4T1 cells were seeded in 96-well plates and cultured for 24 hours. Subsequently, the cells were incubated with C@Lipo, I@Lipo, I / C@Lipo-A, and I / C@Lipo-A + Laser for 12 hours. Following incubation, the cells were incubated with CCK-8 reagent for 30 minutes according to the manufacturer's instructions, and cell viability was analyzed.

[0053] For Calcein-AM / PI staining, 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 minutes), the cells were stained using the Calcein-AM / PI kit according to the manufacturer's instructions and visualized using an EVOS (M7000).

[0054] 1.7 Apoptosis Apoptosis in 4T1 cells after various treatments was assessed using the annexin V / PI assay. 4T1 cells were seeded in 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 minutes), the cells were washed, collected, and resuspended in PBS. The cells were then stained with annexin V and PI for 15 minutes. The samples were analyzed by flow cytometry using the FITC and PE channels.

[0055] 1.8 Animals and Tumor Models Female Balb / c mice (weight 20 ± 2 g, six weeks old) were purchased from the University of Macau (Macao SAR, China). All animal experiments were performed in accordance with the regulations for animal research care of the University of Macau.

[0056] 4T1 cells (1 × 10 6 Tumor-bearing mouse models were established using 100 cells / mouse. When the solid tumor volume (tumor length × tumor width² / 2) reached approximately 100 mm³, mice were used for subsequent experiments.

[0057] 1.9 In vivo fluorescence imaging of I / C@Lipo-A To investigate the in vivo biodistribution of I / C@Lipo-A, I / C@Lipo and I / C@Lipo-A (ICG concentration, 0.5 mg / kg) were injected into tumor-bearing mice via the tail vein. 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. Concurrently, mice were sacrificed 12 hours after injection, and ex vivo NIR-I and NIR-II fluorescence images of major organs (heart, liver, spleen, lung, kidney, and tumor) were collected.

[0058] 1.10 Antitumor effect of I / C@Lipo-A in vivo The in vivo therapeutic efficacy of I / C@Lipo-A was evaluated in 4T1 tumor-bearing mice when tumors reached approximately 100 mm³. Mice were randomly divided into six groups and intravenously injected with saline, saline + laser, C@Lipo, I@Lipo + laser, I / C@Lipo + laser, or I / C@Lipo-A + laser (ICG concentration of 1 mg / kg and chelerythrine concentration of 1 mg / kg) on days 0 and 2. Twelve hours after injection, each mouse was irradiated with 808 nm laser (1 W / cm², 5 minutes). Simultaneously, body temperature was monitored and images were taken using an infrared thermal imager for the following treatments: 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 using H&E staining and immunofluorescence staining for Ki67 and Bcl-2.

[0059] 1.11 Anti-pulmonary metastasis effect of I / C@Lipo-A After treatment, lungs were collected and fixed in paraformaldehyde for 24 hours, followed by photography. Furthermore, H&E staining was performed to assess lung metastatic nodules and further analyze metastatic status.

[0060] 1.12 In vivo safety analysis On days 0 and 2, healthy mice were intravenously injected with normal saline, I@Lipo, C@Lipo, I / C@Lipo, and I / C@Lipo-A (ICG at 1 mg / kg and chelerythrine at 1 mg / kg), respectively. The mice were observed for their health. Finally, the mice were sacrificed, and major organs (heart, liver, spleen, lungs, and kidneys) were harvested for H&E staining to analyze the biocompatibility of the different formulations.

[0061] 1.13 Statistical Analysis All experiments were repeated at least three times, and data are presented as mean ± standard deviation (SD) unless otherwise stated. Data were compared by Student's t-test, and differences were considered statistically significant at *p < 0.05, **p < 0.01, and ***p < 0.001.

[0062] 2. Results 2.1 Preparation and characterization of I / C@Lipo-A like Figure 1As shown in Figure 1, I / C@Lipo-A was prepared. First, indocyanine green (ICG) and chelerythrine-loaded liposomes (I / C@Lipo) were prepared by thin-film hydration. This was followed by continuous physical extrusion. Next, the aptamer HS-AS1411 was covalently attached to the I / C@Lipo surface via Michael addition to form I / C@Lipo-A.

[0063] 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) showed that its morphology was uniform spherical ( Figure 1 , B).

[0064] Next, the particle size stability of I / C@Lipo-A was studied. Figure 8 As shown, the particle size of I / C@Lipo-A in water, phosphate buffered saline (PBS), and 10% fetal bovine serum (FBS) remained stable over 5 days, indicating its excellent stability.

[0065] In addition, the photophysical properties of I / C@Lipo-A were studied. The absorption spectrum of I / C@Lipo-A is similar to that of free ICG ( Figure 1 , C). Compared with ICG, the fluorescence emission peak of I / C@Lipo-A red-shifted from 780 nm to 792 nm ( Figure 1 , D). Therefore, it is assumed that the fluorescence intensity of I / C@Lipo-A in NIR-II may be higher than that of ICG. As expected, the fluorescence intensity of I / C@Lipo-A at 1000 nm is 4.28 times higher than that of ICG ( Figure 1 , E), which supports the application of I / C@Lipo-A in NIR-II fluorescence imaging.

[0066] Next, the photothermal properties of I / C@Lipo-A were examined. The temperature of I / C@Lipo-A increased rapidly and was significantly higher than that of ICG ( Figure 1 , F). In addition, the temperature of I / C@Lipo-A increases with the increase of laser concentration and power ( Figure 1 , G, H), which is beneficial for the subsequent photothermal therapy.

[0067] 2.2 In vitro chemo-photothermal therapy 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, the red fluorescence signal of ICG was observed to be stronger in the I / C@Lipo-A treated cells ( Figure 2 , A).

[0068] In addition, quantitative analysis by flow cytometry showed that the fluorescence intensity of cells treated with I / C@Lipo-A was higher ( Figure 2 , B, E), demonstrating the active targeting functionalization of AS1411-modified I / C@Lipo-A.

[0069] Next, the chemotherapeutic and photothermal effects of I / C@Lipo-A on 4T1 cells were investigated. Chelerythrine has been reported to mediate cell apoptosis by generating reactive oxygen species (ROS). Therefore, ROS production was detected using the 2′, 7′-dichlorofluorescin diacetate (DCFH-DA) indicator. As shown in the figure, each chelerythrine-based formulation enhanced the fluorescence intensity of ROS, demonstrating the chemotherapeutic mechanism of I / C@Lipo-A ( Figure 2 , C).

[0070] Then, Calcein-AM / PI double staining was used to distinguish live cells from dead cells. Calcein-AM has high cell membrane permeability and is esterified in live cells and emits green fluorescence. In contrast, PI cannot pass through the membrane of live cells, but can penetrate the membrane of dead cells to reach the cell nucleus and emit red fluorescence. Figure 11 As shown, I / C@Lipo-A combined with laser irradiation significantly reduced the viability of 4T1 cells, as manifested by a significant increase in the red fluorescence of dead cells.

[0071] To further verify the cell killing effect, Annexin V-FITC / PI assay was performed to evaluate the apoptosis of 4T1 cells. As shown in the figure, compared with C@Lipo or I / C@Lipo-A incubation alone, I / C@Lipo-A combined with laser irradiation had the strongest ability to induce apoptosis ( Figure 2 , D, F).

[0072] In addition, the therapeutic effect of I / C@Lipo-A on 4T1 cells was evaluated by CCK-8 assay. The survival rate of cells incubated with I / C@Lipo-A combined with laser irradiation was significantly reduced ( Figure 2, G), demonstrating the potent 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 deleterious effects in 4T1 breast cancer cells.

[0073] 2.3 Active targeted fluorescence imaging of I / C@Lipo-A in vivo The in vivo distribution of I / C@Lipo-A directly impacts tumor imaging and therapeutic outcomes. I / C@Lipo-A modified with AS1411 aptamers accumulates more efficiently at tumor sites, thereby enhancing imaging and therapy.

[0074] 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 and reached a maximum value at 12 hours after injection ( Figure 3 , A, B).

[0075] In addition, compared with I / C@Lipo-treated mice, I / C@Lipo-A-treated mice showed brighter fluorescence signals in the tumors, indicating greater uptake of the nanomedicine, which facilitated chemotherapy and photothermal therapy. In addition, fluorescence images of tumor tissues isolated from I / C@Lipo-A-treated mice showed stronger signals ( Figure 3 , C, D), further demonstrating the excellent 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 has the potential to be used for imaging deeper biological structures beyond subcutaneous tumors.

[0076] 2.4 Antitumor effect of I / C@Lipo-A in vivo As mentioned above, I / C@Lipo-A can serve as a delivery system for a photothermal agent and chelerythrine 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 minutes). As shown in the figure, I@Lipo and I / C@Lipo exhibited similar temperature changes under laser irradiation ( Figure 4 , A). However, the body temperature of mice treated with I / C@Lipo-A increased more rapidly, indicating that more nanomedicine accumulated at the tumor site and generated more heat under laser irradiation. Previous studies have shown that high temperatures above 43°C can induce tumor cell death. Therefore, I / C@Lipo-A is expected to exhibit excellent tumor-destroying activity under laser irradiation.

[0077] 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, the tumor volume of mice treated with laser alone increased rapidly due to the continued temperature increase. As expected, the tumor growth of mice treated with I / C@Lipo-A combined with laser treatment was most significantly inhibited, which was attributed to its excellent targeting properties and chemo-photothermal 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 recovered completely and their tumors were ablated ( Figure 4 , B). In addition, the weight of the isolated tumor was also consistent with the observation results ( Figure 4 , C). During the treatment period, the mice did not lose significant weight, indicating the safety of the nanomedicine ( Figure 15 ).

[0078] 2.5 Immunohistochemical analysis and anti-metastatic effect of I / C@Lipo-A 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, tumors were analyzed by immunohistochemistry. As shown in the figure, H&E staining images show that after treatment with I / C@Lipo-A combined with laser irradiation, tumor cells showed significant rarefaction and necrosis ( Figure 5 , A), which further proves its excellent therapeutic effect.

[0079] Next, Ki-67 staining was performed to detect cell proliferation in the tumor after treatment. A higher Ki-67 expression rate indicates a stronger proliferation activity of the tumor cells, which is directly related to the patient's prognosis. As shown in the figure, laser-irradiated I / C@Lipo-A can inhibit the expression of Ki-67 ( Figure 5 , B), which is confirmed by the decrease of green fluorescence.

[0080] In addition, Bcl-2 is a proto-oncogene associated with programmed cell necrosis and apoptosis. Studies have shown that overexpression of Bcl-2 protein increases the invasion and metastasis of breast cancer, so it is crucial to evaluate its efficacy in breast cancer recovery. As expected, after treatment with I / C@Lipo-A combined with laser irradiation, the green fluorescence intensity of Bcl-2 was significantly reduced ( Figure 5 , C), which showed that the expression of Bcl-2 was reduced.

[0081] Therefore, based on the above studies, it is expected that lung metastasis will also be inhibited. As shown in the figure, the 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 treatment group. This suggests 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.

[0082] This metastasis inhibition was further confirmed by H&E staining analysis. In the lungs of mice treated with I / C@Lipo-A combined with laser, the size of metastatic lesions was significantly reduced ( Figure 5 , E). Overall, the study showed that I / C@Lipo-A combined with laser irradiation had the best effect in inhibiting tumor metastasis to the lungs.

[0083] 3. Discussion In this study, a theranostic nanosystem was developed that enables near-infrared (NIR-I) and near-infrared (NIR-II) imaging-guided chemo-photothermal therapy. The prepared I / C@Lipo-A was able to target tumors and provide near-infrared imaging-guided photothermal therapy. Furthermore, chelerythrine inhibited tumor growth by generating reactive oxygen species (ROS). Due to the modification with the AS1411 aptamer, more I / C@Lipo-A accumulated at the tumor site, resulting in enhanced therapeutic efficacy after 808 nm laser irradiation, as confirmed in both in vitro and in vivo experiments. Notably, lung metastasis was also suppressed. Furthermore, I / C@Lipo-A has the potential to be used as a NIR-II fluorescence imaging agent, potentially for deep tissue imaging. Therefore, NIR-II fluorescence imaging holds significant promise for advancing clinical diagnostic and therapeutic strategies.

Claims

1. An AS1411 aptamer functionalized liposome, characterized in that Includes lipids, indocyanine green, chelerythrine and AS1411 aptamer.

2. The AS1411 aptamer-functionalized liposome according to claim 1, wherein The AS1411 aptamer functionalized liposomes are obtained by first preparing liposomes loaded with ICG and chelerythrine, and then modifying them with the AS1411 aptamer.

3. The method for preparing the AS1411 aptamer-functionalized liposome according to claim 1 or 2, characterized in that The following steps are involved: Step 1. Dissolve 5 mg of DOPC and 1 mg of DSPE-PEG-MAL in 1 mL of chloroform, and dissolve 300 μg of chelerythrine in 1 mL of methanol. After mixing, remove the organic solvent by rotary evaporation to form a lipid film. Add 1 mL of a 300 μg / mL aqueous solution of ICG to dissolve the lipid film. Extrude the solution 20 times through polycarbonate membranes with 200 nm and 100 nm pore sizes to prepare ICG- and chelerythrine-loaded liposomes (I / C@Lipo). Step 2. The aptamer HS-AS1411 was covalently linked to the surface of I / C@Lipo at a mass ratio of 10:1 by stirring overnight. The liposomes were then ultrafiltered and resuspended in distilled water to remove small molecular weight residues, yielding AS1411 aptamer-functionalized liposomes loaded with ICG and chelerythrine.

4. The method according to claim 3, characterized in that The sequence of the aptamer HS-AS1411 is shown in SEQ ID NO.

1.

5. Use of the AS1411 aptamer-functionalized liposomes according to claim 1 or 2 in the preparation of anti-tumor drugs.

6. The use according to claim 5, characterized in that The tumor is breast cancer.

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

8. Use of the AS1411 aptamer-functionalized liposomes according to claim 1 or 2 as an imaging agent.

9. The use according to claim 8, characterized in that The imaging agent is a fluorescent imaging agent.

10. The use according to claim 8, characterized in that The imaging agent is a near-infrared second-region fluorescent imaging agent.

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