Cu-Lis self-assembled nano-drug as well as preparation method and application thereof

Abstract

The invention discloses a Cu-Lis self-assembled nano-drug as well as a preparation method and application thereof, and belongs to the technical field of nano-medicines. The nano-drug is formed by self-assembly of Cu < 2 + > and liensinine (Liensinine, Lis) under mediation of polyvinylpyrrolidone (PVP) at room temperature, the particle size is 11.14 nm, the surface potential is-8.04 mV, and the nano-drug has good physiological stability and biological safety. According to the invention, an LCD-pyroptosis cycle injury amplification effect is utilized to cooperate with a tumor killing and immune reprogramming mechanism of copper death for the first time, osteosarcoma cells are induced to generate strong pyroptosis and immunogenic cell death, and an immunosuppression microenvironment is reconstructed. The preparation method of the Cu-Lis self-assembled nano-drug is mild in condition, raw materials are easy to obtain, large-scale production can be achieved, and the Cu-Lis self-assembled nano-drug has remarkable application prospects in the field of osteosarcoma immunotherapy.

Description

A Cu-Lis self-assembled nanomedicine, its preparation method and application Technical Field

[0001] This invention relates to the field of nanomedicine technology, and in particular to a Cu-Lis self-assembled nanomedicine, its preparation method, and its application in osteosarcoma immunotherapy. Background Technology

[0002] Osteosarcoma is a refractory malignant tumor that commonly affects adolescents and is often accompanied by other complications (such as fractures or hypercalcemia), making treatment difficult and causing great suffering to patients. Despite the use of various treatment methods in recent years (including surgery and adjuvant chemotherapy), it remains difficult to effectively inhibit the proliferation and invasion of osteosarcoma. Therefore, finding effective drugs and strategies is an urgent problem to be solved. Recent research shows that pyroptosis-based immune cell death (ICD) has shown great potential in reshaping the immune microenvironment, redesigning the entire immune microenvironment into a highly unified system of immune function.

[0003] Lysosome-dependent cell death (LDC) is a type of cell death triggered by increased lysosomal membrane permeability. ROS and pro-inflammatory factors produced by pyroptosis can induce lysosomal membrane instability, thereby enhancing lysosomal membrane permeability and creating a positive feedback loop of ROS-lysosomal membrane permeability leading to cell damage. Simultaneously, as a crucial organelle regulating autophagy, lysosomal dysfunction inhibits autophagic flux, resulting in the effective accumulation of damaged mitochondria within tumor cells, further promoting ROS increases. During pyroptosis, impaired lysosomal membrane integrity and function trigger a strong tumor cell pyroptosis effect, thereby reshaping the tumor immune metabolic microenvironment. In this process, damaged mitochondria within tumor cells can help improve the poor efficacy of copper death alone in tumor therapy. Therefore, a combined therapy based on the LCD-pyroptosis cycle damage amplification effect and copper death for osteosarcoma immunotherapy shows great promise. Summary of the Invention

[0004] The purpose of this invention is to provide a Cu-Lis self-assembled nanomedicine that is simple to prepare, operates under mild conditions, and can be scaled up. Through a synergistic copper death strategy involving LCD-pyroptosis cycle damage amplification, Lis disrupts lysosomal membrane integrity, blocks autophagic flux, leading to the accumulation of damaged mitochondria and amplifying cell death signals; Cu... 2+ In an acidic environment, under the influence of H2O2, a Fenton-like reaction occurs to generate ·OH, which reduces GSH within the tumor, further increasing lysosomal membrane permeability and allowing it to enter mitochondria to trigger copper death. These two factors synergistically activate the caspase-3 / GSDME-N axis, inducing intense pyroptosis and immunogenic cell death in osteosarcoma cells, reconstructing the immunosuppressive microenvironment, and achieving effective treatment of osteosarcoma.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a Cu-Lis self-assembled nanomedicine, which is composed of Cu 2+ Cu is formed by the self-assembly of lotusine (Lis) at room temperature mediated by polyvinylpyrrolidone (PVP). 2+ The volume ratio of the feed to Lis is 2:1, and the concentration of polyvinylpyrrolidone is 1%.

[0007] Furthermore, the Cu-Lis self-assembled nanomedicine has a particle size of 11.14 nm, a PDI of < 0.25, a surface potential of -8.04 mV, and a particle size change of < 10% over 7 days in a physiological environment at pH 7.4.

[0008] Secondly, the present invention provides a method for preparing the above-mentioned Cu-Lis self-assembled nanomedicine, comprising the following steps:

[0009] (1) Prepare a stock solution of CuSO4·5H2O and lotus seed alkaloid with a concentration of 10 mg / mL;

[0010] (2) Cu 2+ The stock solution was added to a 1% polyvinylpyrrolidone aqueous solution and sonicated for 10 min.

[0011] (3) Add Lis stock solution to the system in step (2) and then sonicate for 10 min;

[0012] (4) Place the mixture from step (3) in a vortex mixer and incubate at 1000 rpm for 3 h;

[0013] (5) After collecting the reaction mixture from step (4), perform three ultrafiltration cycles using a 50 kDa ultrafiltration membrane;

[0014] (6) The supernatant from the final concentration in step (5) is freeze-dried to obtain Cu-Lis nanoparticles.

[0015] Thirdly, the present invention provides the application of the above-mentioned Cu-Lis self-assembled nanomedicines in the preparation of immunotherapy drugs for malignant tumors.

[0016] Furthermore, the malignant tumor is osteosarcoma.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] The Cu-Lis self-assembled nanomedicine provided by this invention is composed of Cu 2+The lysosomal cell was self-assembled with lotusine at room temperature under polyvinylpyrrolidone-mediated hydration, resulting in a particle size of 11.14 nm and a surface potential of -8.04 mV, exhibiting good physiological stability and biosafety. This invention is the first to utilize the LCD-pyroptosis cycle damage amplification effect to synergize with the tumor-killing and immune reprogramming mechanism of copper death: Lis disrupts lysosomal membrane integrity, blocking autophagic flux, leading to the accumulation of damaged mitochondria and amplifying cell death signals; Cu... 2+ In an acidic environment, Cu-Lis undergoes a Fenton-like reaction with H2O2 to generate ·OH, reducing glutathione (GSH) levels within the tumor. This further increases lysosomal membrane permeability and allows cytokines to enter the mitochondria, triggering copper death. These two factors synergistically activate the caspase-3 / GSDME-N axis, inducing intense pyroptosis and immunogenic cell death in osteosarcoma cells, thus reconstructing the immunosuppressive microenvironment. The Cu-Lis self-assembled nanomedicine preparation method provided by this invention is mild, uses readily available raw materials, and can be scaled up for production, showing significant application potential in the field of osteosarcoma immunotherapy. Attached Figure Description

[0019] Figure 1 is a schematic diagram of Cu-Lis synthesis provided in an embodiment of the present invention.

[0020] Figure 2 is a particle size diagram of Cu-Lis provided in an embodiment of the present invention.

[0021] Figure 3 is a potential diagram of Cu-Lis provided in an embodiment of the present invention.

[0022] Figure 4 is a TEM image of Cu-Lis provided in an embodiment of the present invention.

[0023] Figure 5 is a UV spectrum of Cu-Lis provided in an embodiment of the present invention.

[0024] Figure 6 is an XPS characterization diagram of Cu-Lis provided in an embodiment of the present invention.

[0025] Figure 7 shows the in vitro release characterization of Cu-Lis nanoparticles provided in the embodiments of the present invention.

[0026] Figure 8 shows the characterization of extracellular reactive oxygen species generation detection provided in the embodiments of the present invention.

[0027] Figure 9 shows the results of live / dead cell staining analysis provided in the embodiments of the present invention.

[0028] Figure 10 shows a schematic diagram of the synthesis of Cu-Lis nanomedicines and their anti-tumor immune mechanism. Detailed Implementation

[0029] The specific embodiments of the present invention will be described in detail below with reference to examples.

[0030] Example 1: Preparation of Cu-Lis self-assembled nanomedicines

[0031] As shown in Figure 1, the method for preparing Cu-Lis self-assembled nanomedicines includes the following steps: First, a CuSO4·5H2O and Lis stock solution with a concentration of 10 mg / mL is prepared. 20 μL of Cu... 2+ The stock solution was added to a 1% PVP aqueous solution and sonicated for 10 min. 10 μL of Lis stock solution was added to the above system, and sonication was performed again for 10 min. The mixture was then incubated in a vortex mixer at 1000 rpm for 3 h. After collecting the reaction mixture, it was subjected to three ultrafiltration cycles using a 50 kDa ultrafiltration membrane. The final concentrated supernatant was freeze-dried to obtain Cu-Lis self-assembled nanoparticles.

[0032] Example 2 Characterization of Cu-Lis nanoparticles

[0033] The size and zeta potential of Cu-Lis nanoparticles were measured by dynamic light scattering (DLS) and a zetasizer (NanoZS, Malvern Instruments). The size of Cu-Lis nanoparticles in PBS buffer over 7 days was also determined by DLS. Data are presented as mean ± standard deviation (SD) of three independent measurements. The morphology of Cu-Lis nanoparticles was observed by transmission electron microscopy (TEM) (JEOL JEM-2100).

[0034] Dynamic light scattering (DLS) was used to analyze the particle size and surface charge of Cu-Lis. The average particle size of Cu-Lis was 11.14 nm, and the polymer dispersion index (PDI) was 0.21, indicating uniform size, as shown in Figure 2. The zeta potential was -8.04 mV, as shown in Figure 3, which is conducive to aggregation and retention in tumor tissue. Transmission electron microscopy (TEM) was used to observe its morphology, as shown in Figure 4, and the results were consistent with the DLS data. To verify Cu... 2+ Cu-Lis successfully self-assembled with Lis. UV-Vis absorption spectra showed that the absorption peaks of Cu-Lis and Lis were close in position, as shown in Figure 5, indicating the formation of the complex. X-ray photoelectron spectroscopy (XPS) characterization, as shown in Figure 6, revealed characteristic peaks for Cu, C, N, and O throughout the spectrum. The peak separation results clearly indicated the valence states of each element, confirming the successful preparation of Cu-Lis.

[0035] Example 3: Simulated in vitro release of Cu-Lis nanoparticles

[0036] Hemolysis experiments were conducted to demonstrate the good stability of Cu-Lis in physiological environments. Fresh mouse whole blood was collected, and erythrocytes were collected by centrifugation. The erythrocytes were washed three times with 1% sterile NaCl to prepare a 10% (v / v) erythrocyte suspension. 100 µL of this suspension was mixed with 900 µL of Cu-Lis solutions of different concentrations (final Lis concentration 0-60 µg / mL), a negative control (1% NaCl), and a positive control (ultrapure water), respectively. The mixture was incubated at 37°C for 6 h, and the supernatant was collected by centrifugation. The absorbance was measured at 570 nm, and the hemolysis rate was calculated. Hemolysis rate (%) = (A... 样品 -A 阴性 ) / (A 阳性 -A 阴性 The result is shown in Figure 7. When the final concentration of Lis is 60 µg / mL, the hemolysis rate is still less than 5%, which proves that Cu-Lis has excellent blood compatibility and biosafety.

[0037] Cu-Li nanoparticles can release Cu under simulated tumor conditions. 2+ The release behavior of Cu-Lis was determined by dialysis at 37°C. One mL of Cu-Lis nanoparticle solution was placed in a dialysis bag with a molecular weight cutoff of 1000 Da and immersed in 9 mL of PBS solution. The sample was then placed in a 37°C water bath with continuous shaking. An equal volume of fresh PBS solution (pH 5.5 or 7.4) was added periodically. Cu... 2+ The concentrations of Lis and LiS were determined by inductively coupled plasma atomic emission spectrometry (ICP-OES) and high-performance liquid chromatography (HPLC), respectively. The release at different pH values ​​was measured; the cumulative release rate reached 92.45% over 24 h at pH 5.5; the release rate decreased significantly at pH 7.4, indicating tumor microenvironment-triggered release behavior.

[0038] Example 4 Detection of Extracellular Reactive Oxygen Species Generation

[0039] The Lis disrupts the integrity of the lysosomal membrane, causing damaged mitochondria to accumulate and amplify cell death signals step by step. 2+ Upon release, it can generate ·OH within tumor cells via a Fenton-like reaction, consuming intracellular GSH and further increasing lysosomal membrane permeability, leading to mitochondrial cell death. Both synergistically induce tumor cell death. The level of ROS generation was investigated using DCFH-DA as a probe.

[0040] K7M2 cells (1×10) 5Cells were seeded in 12-well plates and cultured at 37°C for 24 h. The original culture medium was then discarded and replaced with 1 mL of fresh medium containing a specific treatment agent, and the cells were co-incubated at 37°C for another 24 h. After 4 h of incubation, the cells were stained with medium containing 10 μM DCFH-DA and incubated in the dark for 20 min. Unbound probes were then washed with PBS. Finally, the intensity of green fluorescence was detected using a microplate reader (Mshot, MF52-N) and flow cytometry to verify the intensity of intracellular reactive oxygen species (ROS) fluorescence. The excitation wavelength λ for detecting intracellular ROS fluorescence intensity using flow cytometry was [not specified]. ex = 488nm, emission wavelength λ em = 528 nm.

[0041] The results are shown in Figure 8. The fluorescence of the Cu-Lis group increased by 5.54 times compared with the control, which was significantly higher than that of Cu alone. 2+ Or, in the Lis group alone (p < 0.0001), ROS formation with Cu 2+ Release and GSH depletion are synergistically correlated. Corresponding green fluorescence results show that the control and Cu... 2+ The fluorescence of the three groups (Li, Cu, and Lis) was weak, while the Cu-Lis group showed strong green fluorescence across the entire field of view, consistent with the flow cytometry data, indicating that it can significantly induce an increase in ROS within cells.

[0042] Example 5: Live / Dead Cell Staining Analysis

[0043] K7M2 cells (1×10) 5 (Each sample) was resuspended in 1 mL of culture medium and inoculated into a 35 mm glass petri dish. After 24 h of incubation, the original culture medium was discarded and replaced with 1 mL of medium containing different treatment agents (Cu). 2+ Lis, Cu-Lis nanomedicines, Cu in solution 2 + The cells were cultured in fresh medium (with Lis concentrations of 60 μg / mL and 30 μg / mL, respectively) at 37°C for 24 h. They were then cultured for another 4 h. Finally, the cells were stained using Calcein-AM / PI double staining and observed using a confocal laser scanning microscope.

[0044] Cu-Lis nanomedicine, in the culture and treatment of K7M2 osteosarcoma cells with antitumor activity, includes the following steps:

[0045] (1) K7M2 mouse osteosarcoma cells (accession number: GDC0675) from the China Center for Type Culture Collection were seeded in DMEM high glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin and cultured at 37°C and 5% CO2.

[0046] (2) After the cells reach the logarithmic growth phase, use 1×10 5 Cells / well were seeded into 12-well plates and cultured for 24 h;

[0047] (3) Replace with serum-free DMEM medium containing Cu-Lis nanomedicine, wherein Cu 2+ The final concentration was 60 μg / mL, and the final concentration of Lis was 30 μg / mL. The culture was continued for 24 h.

[0048] (4) Collect cells to detect ROS levels, cell death rate, CleavedCaspase-3 / GSDME-N expression and immunogenic cell death markers.

[0049] In the K7M2 osteosarcoma cell model, 30 μg / mL Lis and 60 μg / mL Cu were used. 2+ After treatment with Cu for 24 h, the number of dead cells was detected by Calcein-AM / PI double staining method, and the number was significantly higher than that of Cu alone. 2+ Alternatively, the Lis treatment group (p < 0.0001) showed experimental results as shown in Figure 9. This indicates that the Cu-Lis group exhibited the least green fluorescence, indicating the fewest live cells, and the most red fluorescence, indicating the most dead cells. The statistical mortality rate was significantly higher than the other groups, confirming its superior killing effect.

[0050] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A Cu-Lis self-assembled nanomedicine, characterized in that, The Cu-Lis self-assembled nanomedicine is composed of Cu 2+ Cu is formed by the self-assembly of lotusine (Lis) at room temperature mediated by polyvinylpyrrolidone (PVP). 2+ The volume ratio of the feed to Lis was 2:1, and the concentration of polyvinylpyrrolidone was 1%.

2. The Cu-Lis self-assembled nanomedicine as described in claim 1, characterized in that, The Cu-Lis self-assembled nanomedicine has a particle size of 11.14 nm, a PDI of < 0.25, a surface potential of -8.04 mV, and a particle size change of <10% over 7 days in a physiological environment at pH 7.

4.

3. The method for preparing Cu-Lis self-assembled nanomedicine as described in claim 1, characterized in that, The steps include: (1) preparing a CuSO4·5H2O and lotus seed alkaloid stock solution with a concentration of 10 mg / mL; (2) adding Cu 2+ Add the stock solution to a 1% polyvinylpyrrolidone aqueous solution and sonicate for 10 min; (3) Add the Lis stock solution to the system in step (2) and sonicate for another 10 min; (4) Place the mixture in step (3) in a vortex mixer and incubate at 1000 rpm for 3 h; (5) After collecting the reaction mixture in step (4), perform three ultrafiltration cycles using a 50 kDa ultrafiltration membrane; (6) Freeze-dry the supernatant that was finally concentrated in step (5) to obtain Cu-Lis nanoparticle powder.

4. The application of the Cu-Lis self-assembled nanomedicine as described in claim 1 in the preparation of immunotherapy drugs for malignant tumors.

5. The application as described in claim 1, characterized in that, The malignant tumor is osteosarcoma.

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