A double-cell hybrid membrane coated biomimetic nanoparticle and a preparation method and application thereof

Abstract

The application belongs to the technical field of nanomedicine, and discloses a kind of double cell hybrid membrane coated biomimetic nanoparticles and its preparation method and application.Double cell hybrid membrane coated biomimetic nanoparticles of the application include drug nanoparticles and the biomimetic outer membrane coated the drug nanoparticles, the biomimetic outer membrane is hybrid cell membrane derived from tumor cell membrane and osteoclast membrane, the drug nanoparticles include mutually synergistic semiconductor polymer, Fe-based fenton catalyst and IDO1 inhibitor, the application integrates double-targeted delivery, magnetic resonance positioning, PTT, CDT, ferroptosis and synergistic immunotherapy, forms closed-loop diagnosis and treatment strategy, constructs a kind of biomimetic nanoprobe with magnetic resonance imaging ability and multi-mechanism synergistic therapy function, can realize the integration of positioning, treatment and efficacy monitoring of bone metastasis tumor, solves multiple bottlenecks in current bone metastasis treatment, provides a new mechanism-oriented treatment scheme for persistent control of tumor.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine technology, and more specifically, to a multifunctional bicellular hybrid membrane-coated biomimetic nanoparticle for the diagnosis and treatment of bone metastases and its preparation method. Background Technology

[0002] Bone metastases commonly occur in the late stages of breast, prostate, and lung cancer, often accompanied by complications such as pathological fractures, hypercalcemia, bone marrow failure, and spinal cord compression, severely impacting the quality of life for cancer patients. Biologically, cancer cells and the bone marrow microenvironment coordinate to create a tumor microenvironment conducive to metastasis. In bone metastases, cytokines and growth factors produced by cancer cells directly promote the proliferation and activity of osteoclasts, leading to bone resorption; conversely, osteoclasts release growth factors to promote the proliferation and survival of cancer cells, thus creating a vicious cycle between bone resorption and tumor growth. Accurate diagnosis and effective treatment of bone metastases are crucial for reducing patient mortality and improving survival rates.

[0003] Current treatments for bone metastases primarily rely on a model where imaging diagnosis and local or systemic therapy are independent. Imaging technologies such as magnetic resonance imaging (MRI) and computed tomography (CT) are used only for disease detection, while treatment methods (radiotherapy, chemotherapy, or surgery) typically cannot be monitored in real time. There is a lack of integrated diagnostic and therapeutic strategies that can simultaneously achieve precise imaging and treatment feedback. Furthermore, existing nanomedicines or targeted strategies often focus on a single cell type (such as tumor cells), making it difficult to simultaneously regulate the crucial bicellular axis of tumor cells and osteoclasts, resulting in insufficient synergistic control over bone destruction and tumor growth.

[0004] Chemodynamic therapy (CDT) relies on the generation of reactive oxygen species such as ·OH by Fenton or Fenton-like reactions. However, the concentration of H2O2 in the tumor microenvironment is limited and the level of glutathione (GSH) is high, which easily scavenges reactive oxygen species (ROS), thus significantly reducing the therapeutic efficiency of CDT.

[0005] Conventional photothermal therapy (PTT) mainly kills tumor cells directly through high temperature, but the single thermal effect can easily lead to tumor recurrence and may damage surrounding normal tissues. At the same time, it lacks the synergistic amplification effect with other treatment mechanisms, such as CDT or ferroptosis.

[0006] Bone metastases are often accompanied by a pronounced immunosuppressive microenvironment, in which the IDO1-mediated Trp / Kyn metabolic pathway can suppress T cell function and promote immune escape. Most current nanotherapy strategies rely primarily on directly killing tumor cells, lacking effective regulation of immunosuppressive metabolic pathways. Although some therapies can induce immunogenic cell death (ICD) in tumor cells, releasing warning signals such as adenosine triphosphate (ATP), calreticulin (CRT), and high-mobility group box 1 (HMGB1), these signals often fail to sustainably amplify an effective anti-tumor immune response in an immunosuppressive environment, making it difficult to maintain therapeutic efficacy long-term.

[0007] In addition, traditional nanomedicines often rely on the EPR effect to achieve passive enrichment, but in the complex bone metastasis microenvironment, their targeting and biodistribution are still limited, which can easily lead to low drug utilization and non-specific distribution. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide a dual-cell hybrid membrane-coated biomimetic nanoparticle for the diagnosis and treatment of bone metastases and its preparation method. This invention integrates dual-target delivery, magnetic resonance localization, PTT, CDT, ferroptosis, and synergistic immunotherapy to form a closed-loop treatment strategy. This approach solves several bottlenecks in the current treatment of bone metastases and provides a novel mechanism-guided treatment option for the long-term control of tumors.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0010] In a first aspect, the present invention provides a bicellular hybrid membrane-coated biomimetic nanoparticle, comprising a drug nanoparticle and a biomimetic outer membrane coating the drug nanoparticle, wherein the biomimetic outer membrane is a hybrid cell membrane derived from tumor cell membrane and osteoclast membrane, and the drug nanoparticle comprises semiconductor polymers (SPs), Fe-based Fenton catalysts, and IDO1 inhibitors.

[0011] Specifically, the semiconductor polymer is preferably a DA-type narrow bandgap conjugated semiconductor polymer with excellent photothermal effect, and can be selected from at least one of DPP-type semiconductor polymers, BBT-type semiconductor polymers, and BDT-type semiconductor polymers, with its structure comprising at least one of DPP units, BBT units, and BDT units. The excellent photothermal effect refers to a photothermal conversion efficiency of the semiconductor polymer reaching 70% or higher.

[0012] Specifically, the Fe-based Fenton catalyst is preferably an iron oxide nanomaterial with superparamagnetic properties, such as Fe3O4 or γ-Fe2O3.

[0013] Specifically, the IDO1 inhibitor is an immunomodulatory agent capable of regulating the Trp / Kyn metabolic pathway, selected from at least one of NLG919, 1-MT, and Epacadostat.

[0014] Specifically, the tumor cell membrane can be selected from the cell membrane of any tumor that can induce bone metastases, such as breast cancer, prostate cancer, lung cancer, nasopharyngeal carcinoma, liver cancer, leukemia, pancreatic cancer, osteosarcoma, skin cancer, cervical cancer, ovarian cancer, kidney cancer, and esophageal cancer.

[0015] Specifically, the drug nanoparticles are formed by polymer self-assembly and nano-coprecipitation, the Fe-based Fenton catalyst is modified with oleic acid, and the drug nanoparticles also contain amphiphilic block copolymers, preferably PEG-b-PPG-b-PEG.

[0016] Specifically, the particle size of the biomimetic nanoparticles coated with the dual-cell hybrid membrane of the present invention is 50-200 nm, and more preferably 50-150 nm.

[0017] In one specific embodiment, the semiconductor polymer, Fe-based Fenton catalyst, and IDO1 inhibitor are BBT-DPP copolymer, oleic acid-modified Fe3O4 particles, and NLG919, respectively. The tumor cell membrane is a 4T1 cell membrane. Through the hybrid cell membrane derived from 4T1 breast cancer cells and osteoclasts, with SPs, Fe3O4, and NLG919 as the core, homologous enrichment of the dual cell axis is achieved. Specifically, Fe3O4 is used as a T2WI contrast agent for lesion localization and monitoring; under 1064 nm laser irradiation, SPs mediate near-infrared II (NIR-II) photothermal therapy, amplifying Fe3O4-driven CDT and promoting ferroptosis through exogenous heat, synergistically inducing tumor cell apoptosis and ICD; simultaneously, NLG919, by inhibiting the IDO1-Trp / Kyn axis and reversing immunosuppression, synergistically enhances anti-tumor immune activation with ICD-induced immune induction.

[0018] Secondly, the present invention also provides a method for preparing the above-mentioned bicellular hybrid membrane-coated biomimetic nanoparticles, comprising:

[0019] Step 1: Extract tumor cell membranes and osteoclast membranes;

[0020] Step 2: Prepare the drug nanoparticles;

[0021] Step 3: Mix tumor cell membranes, osteoblast membranes and drug nanoparticles, then fuse them with ultrasound, and finally prepare the bicellular hybrid membrane-coated biomimetic nanoparticles by membrane extrusion.

[0022] Specifically, in step 2, the drug nanoparticles are prepared by polymer self-assembly and nano-coprecipitation. The surface of the Fe-based Fenton catalyst is modified with oleic acid. Specifically, the semiconductor polymer, the Fe-based Fenton catalyst and the IDO1 inhibitor are dissolved in an organic solvent to obtain a mixed solution. Then, the mixed solution is added to deionized water containing an amphiphilic block copolymer to obtain the drug nanoparticles.

[0023] Specifically, the mass ratio of the semiconductor polymer, Fe-based Fenton catalyst, IDO1 inhibitor, and amphiphilic block copolymer is 1:(0.5-2):(1-3):(80-150).

[0024] In one specific embodiment, the semiconductor polymer, Fe-based Fenton catalyst, IDO1 inhibitor, and amphiphilic block copolymer are BBT-DPP copolymer, oleic acid-modified Fe3O4 particles, NLG919, and PEG-b-PPG-b-PEG, respectively, with a mixing mass ratio of 1:1:2:100.

[0025] Specifically, the organic solvent is tetrahydrofuran.

[0026] Specifically, in step 3, tumor cell membranes, osteoclast membranes and drug nanoparticles are mixed, ultrasonically treated, and then the mixture is extruded through a polycarbonate porous membrane filter using an extruder. After ultrafiltration purification through an ultrafiltration tube, the biomimetic nanoparticles coated with the dual-cell hybrid membrane are obtained.

[0027] Specifically, the mixed mass ratio of tumor cell membrane and osteoclast membrane is (0.5-2):1, and the mixed mass ratio of the total mass of tumor cell membrane and osteoclast membrane to the mixed mass of drug nanoparticles is (1-3):1.

[0028] In one specific implementation, the mass ratio of the tumor cell membrane and the osteoclast membrane is 1:1, and the mass ratio of the total mass of the tumor cell membrane and the osteoclast membrane to the drug nanoparticles is 2:1.

[0029] Thirdly, the present invention also provides the application of the above-mentioned bicellular hybrid membrane-coated biomimetic nanoparticles in the preparation of diagnostic and / or therapeutic drugs for bone metastases.

[0030] Specifically, in practical applications, the bicellular hybrid membrane-coated biomimetic nanoparticles of the present invention are used in conjunction with laser irradiation for the diagnosis and treatment of bone metastases. The laser source is selected from near-infrared light sources, preferably with a wavelength of 700–1100 nm.

[0031] The beneficial effects of the technical solution of this invention are as follows:

[0032] The present invention utilizes a bicellular hybrid membrane to coat biomimetic nanoparticles. By constructing a hybrid biomimetic membrane using tumor cell membranes and osteoclast membranes, it achieves dual targeted enrichment of tumor cells and the bone microenvironment. Furthermore, it leverages the near-infrared photothermal effect of semiconductor polymers to promote a Fenton-like reaction in Fe-based nanomaterials, thereby enhancing reactive oxygen species generation and inducing lipid peroxidation and ferroptosis. Simultaneously, by loading an IDO1 inhibitor to regulate the Trp / Kyn metabolic pathway, it alleviates the tumor immunosuppressive microenvironment and enhances the anti-tumor immune response. This constructs a biomimetic nanoprobe that combines magnetic resonance imaging capabilities with multi-mechanism synergistic therapeutic functions, achieving integrated tumor localization, treatment, and efficacy monitoring. This addresses several bottlenecks in current bone metastasis treatment and provides a novel mechanism-guided treatment approach for sustained tumor control. Attached Figure Description

[0033] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 This is a schematic diagram of the synthesis route for biomimetic nanoparticles coated with a dual-cell hybrid membrane according to the present invention.

[0035] Figure 2 The nuclear magnetic resonance spectrum of SPs prepared in Example 1 ( 1 (H NMR) image.

[0036] Figure 3 The UV-Vis absorption spectrum characterization results of the SPs prepared in Example 1 are shown in the figure.

[0037] Figure 4 The graph shows the cell membrane protein assay results of NPs prepared in Example 3, CC@NPs prepared in Comparative Example 1, OS@NPs prepared in Comparative Example 2, and OS@CC@NPs prepared in Example 1. Figure 4 Figure a shows the results of BCA protein quantification. Figure 4 b is a protein band diagram from SDS-PAGE electrophoresis analysis.

[0038] Figure 5 The basic physical test results of the NPs prepared in Example 3, CC@NPs prepared in Comparative Example 1, OS@NPs prepared in Comparative Example 2, and OS@CC@NPs prepared in Example 1 are shown in the figure. Figure 5 a is a graph showing the TEM test results; Figure 5 b is the DLS detection result image; Figure 5 c shows the results of the colloidal stability test; Figure 5 d is the graph showing the zeta potential detection results; Figure 5 e is the graph showing the results of the ultraviolet absorption curve test.

[0039] Figure 6 The image shows the photothermal properties of the OS@CC@NPs prepared in Example 1. Figure 6 a, Figure 6 b、 Figure 6 c shows the temperature changes of the OS@CC@NPs solution under different nanomaterial concentrations, laser irradiation times, and laser power densities, respectively. Figure 6 d shows the temperature change of the OS@CC@NPs solution during the laser start-up and shut-down cycle; Figure 6 e shows the photothermal conversion efficiency of OS@CC@NPs.

[0040] Figure 7 The graph shows the in vivo photothermal conversion capability test results of OS@CC@NPs prepared in Example 1. Figure 7 Image a shows the infrared thermal imaging result. Figure 7 b is a graph showing the results of the quantitative analysis.

[0041] Figure 8 The in vitro magnetic resonance imaging performance test results of NPs prepared in Example 3, CC@NPs prepared in Comparative Example 1, OS@NPs prepared in Comparative Example 2, and OS@CC@NPs prepared in Example 1 are shown in the figure. Figure 8 a shows the T2-weighted MRI imaging results in PBS solution; Figure 8 Figure b shows the results of calculating the relaxation rate of the biomimetic nanocomposite material based on the concentration of Fe3O4 nanoparticles.

[0042] Figure 9 The in vivo magnetic resonance imaging performance test results of NPs prepared in Example 3, CC@NPs prepared in Comparative Example 1, OS@NPs prepared in Comparative Example 2, and OS@CC@NPs prepared in Example 1 are shown in the figure. Figure 9 Figure a shows the results of dynamic observation of T2-weighted MRI in the tibial lesion area of ​​mice; Figure 9 Figure b shows the results of quantitative analysis of the T2WI signal.

[0043] Figure 10 The graph shows the results of Fenton-like reactivity tests for different nanoparticles. Figure 10 a, Figure 10 b、 Figure 10 c. Figure 10 Figure d shows the Fenton-like reactivity test results of NPs prepared in Preparation Example 3, CC@NPs prepared in Comparative Example 1, OS@NPs prepared in Comparative Example 2, and OS@CC@NPs prepared in Example 1, respectively.

[0044] Figure 11 The results of the intracellular ROS generation ability test of NPs prepared for Example 3, CC@NPs prepared for Comparative Example 1, OS@NPs prepared for Comparative Example 2, and OS@CC@NPs prepared for Example 1 are shown in the figure. Figure 11 Figure a shows the results observed using a confocal microscope. Figure 11 Figure b shows the results of quantitative analysis of fluorescence intensity in 4T1 cells; Figure 11 Figure c shows the results of detecting intracellular ROS levels in osteoclasts using flow cytometry.

[0045] Figure 12 The results show the changes in intracellular MDA content in the tumor ferroptosis effect assessment test of NPs prepared in Preparation Example 3, CC@NPs prepared in Comparative Example 1, OS@NPs prepared in Comparative Example 2, and OS@CC@NPs prepared in Example 1. Figure 12 a shows the MDA content in 4T1 breast cancer cells with and without laser irradiation; Figure 12 b shows the MDA content in osteoclasts during and without laser irradiation.

[0046] Figure 13 The results show the changes in intracellular GSH content in the tumor ferroptosis effect assessment test of NPs prepared in Preparation Example 3, CC@NPs prepared in Comparative Example 1, OS@NPs prepared in Comparative Example 2, and OS@CC@NPs prepared in Example 1. Figure 13 a shows the GSH content in 4T1 breast cancer cells before and after laser irradiation. Figure 13 b shows the GSH content in osteoclasts during and without laser irradiation.

[0047] Figure 14 The image shows the Western blot analysis results of ferroptosis-related proteins in each group of cells in the tumor ferroptosis effect evaluation test of OS@CC@NPs prepared in Example 1.

[0048] Figure 15 The results of in vitro cell-killing ability tests of NPs prepared in Preparation Example 3, CC@NPs prepared in Comparative Example 1, OS@NPs prepared in Comparative Example 2, and OS@CC@NPs prepared in Example 1 under oxidative stress conditions are shown. Figure 15 a shows the killing effect on 4T1 breast cancer cells; Figure 15 b shows the killing effect on osteoclasts.

[0049] Figure 16 The graph shows the in vivo antitumor activity test results of NPs prepared in Preparation Example 3, CC@NPs prepared in Comparative Example 1, OS@NPs prepared in Comparative Example 2, and OS@CC@NPs prepared in Example 1.

[0050] Figure 17 The effects of different treatments on ICD-related markers (CRT, HMGB1, ATP) of 4T1 cells are shown for NPs prepared in Preparation Example 3, CC@NPs prepared in Comparative Example 1, OS@NPs prepared in Comparative Example 2, and OS@CC@NPs prepared in Example 1. Figure 17 a shows the impact on CRT; Figure 17 b shows the effect on HMGB1; Figure 17 c shows the effect on ATP.

[0051] Figure 18 Flow cytometry results of NPs prepared in Example 3, CC@NPs prepared in Comparative Example 1, OS@NPs prepared in Comparative Example 2, and OS@CC@NPs prepared in Example 1. Figure 18 a, Figure 18 b shows the flow cytometry analysis results and quantitative statistics of BMDCs and 4T1 cells after co-incubation with maturation markers such as CD80 / CD86 in different treatment groups.

[0052] Figure 19 The figure shows the IDO pathway activity evaluation test results of the NPs prepared in Example 3, CC@NPs prepared in Comparative Example 1, OS@NPs prepared in Comparative Example 2, and OS@CC@NPs prepared in Example 1. Detailed Implementation

[0053] To better explain the present invention, detailed descriptions of its embodiments are provided, and the main content of the invention is further clarified in conjunction with specific examples. However, the content of the present invention is not limited to the following embodiments. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0054] Cell membrane-coated biomimetic nanomaterials can be used for drug delivery, imaging, and therapy in the biomedical field. The surface of cell membrane-coated nanomaterials can not only reduce RES clearance and increase drug circulation time, but also specifically bind to cancer cells through a homologous targeting mechanism.

[0055] like Figure 1As shown, this invention provides a bicellular hybrid membrane-coated biomimetic nanoparticle that, with SPs, Fe-based Fenton-like catalysts, and an IDO1 inhibitor as its core components, mimics hybrid cell membranes derived from tumor cells and osteoclasts, achieving homologous enrichment of dual cell axes. Specifically, the superparamagnetic Fe-based Fenton-like catalyst serves as a T2WI contrast agent for lesion localization and monitoring; under laser irradiation, SPs mediate near-infrared II (NIR-II) photothermal therapy, amplifying the Fe-based Fenton-like catalyst-driven CDT and promoting ferroptosis through exogenous heat, synergistically inducing tumor cell apoptosis and ICD; simultaneously, the IDO1 inhibitor reverses immunosuppression by inhibiting the IDO1-Trp / Kyn axis, synergistically enhancing anti-tumor immune activation with ICD-induced immune induction. This platform integrates dual-target delivery, magnetic resonance localization, PTT, CDT, ferroptosis, and synergistic immunotherapy, forming a closed-loop treatment strategy that addresses multiple bottlenecks in current bone metastasis treatment, providing a novel mechanism-guided treatment approach for durable tumor control.

[0056] The present invention will be further described in detail below through examples and related performance tests.

[0057] Preparation Example 1: Induction of osteoclasts

[0058] Five-week-old BALB / c mice were sacrificed by cervical dislocation, and the intact femur and tibia were isolated. The bones were soaked in 75% ethanol for 15 minutes and washed with sterile PBS. The bone marrow was rinsed with DMEM medium. The bone marrow suspension was filtered through a 70µm cell sieve. The filtrate was centrifuged at 1400 rpm for 15 minutes, the supernatant was discarded, and erythrocyte lysis buffer was added to lyse the erythrocytes. The treated liquid was incubated in DMEM complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in a cell culture incubator for 12 hours. The culture medium was then resuspended and cultured in DMEM medium containing macrophage colony-stimulating factor (M-CSF, 50 ng / mL) and nuclear factor-κB ligand receptor activator (RANKL, 50 ng / mL). The medium was changed every 24 hours until osteoclasts were induced. Successful osteoclast synthesis was verified by microscopic morphological observation and TRAP staining.

[0059] Preparation Example 2: SPs Synthesis

[0060] SPs were prepared via Stille coupling polymerization. Benzobisthiadiazole (BBT, 25 mg, 0.023 mmol), 2,5-bis(2-ethylhexyl)-3,6-bis[5-(trimethylstanyl)thiophen-2-yl]-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione (DPP derivative, 21.3 mg, 0.025 mmol), Pd2(dba)3 (1.0 mg, 0.001 mmol), and tri(o-tolyl)phosphine (2.0 mg, 0.006 mmol) were added to a 50 mL Schlenk reaction flask and dissolved in chlorobenzene (4 mL). After three cycles of freezing-vacuuming-thawing to degas the reaction system, it was placed in a 100 °C oil bath for 2 h under nitrogen protection to carry out Stille polymerization. After the reaction was complete, the resulting reaction solution was slowly added dropwise to methanol to precipitate the polymer. The precipitate was then collected by centrifugation and washed three times with methanol to remove unreacted monomers and small molecule impurities. Finally, the product was dried overnight under vacuum to obtain the SPs polymer, specifically a BBT-DPP copolymer.

[0061] Through nuclear magnetic resonance spectroscopy ( 1 The molecular structure of the prepared SPs was characterized by ¹H NMR detection, and the test results are as follows: Figure 2 As shown. SPs 1 The 1H NMR spectrum showed aromatic hydrogen signals in the 6.8–8.3 ppm region, corresponding to protons on the benzobisthiadiazole (BBT) and thiophene units. A signal at approximately 3.9 ppm was attributed to the N-CH protons attached to the nitrogen atom of the DPP unit, while the strong signal in the 0.8–1.8 ppm region originated from aliphatic hydrogens on the 2-ethylhexyl side chain. Furthermore, the characteristic peak of the trimethylstannyl group (approximately 0.3 ppm) in the Stille coupling monomer was not observed in the spectrum, indicating that the tin-containing monomers were fully consumed during the reaction, suggesting that the polymerization reaction proceeded smoothly and the target polymers SPs were successfully obtained.

[0062] The UV-Vis absorption spectroscopy characterization results of SPs show that the material exhibits significant characteristic absorption peaks in the wavelength range of 800–1100 nm, and its absorption intensity shows a good linear positive correlation with the material concentration. Figure 3a). To establish a quantitative detection method for SPs concentration, the ultraviolet absorbance at a characteristic wavelength of 672 nm was selected for linear fitting analysis, and a concentration scale with high correlation was successfully constructed, providing a reliable experimental basis for the accurate quantitative detection of SPs concentration in subsequent experiments. Figure 3 b).

[0063] Preparation Example 3: Preparation of Drug Nanoparticles (NPs)

[0064] Preparation via nanoprecipitation: SPs (0.1 mg) obtained in Preparation Example 2, oleic acid-modified Fe3O4 nanoparticles (0.1 mg), and NLG919 (0.2 mg) were dissolved in 1.0 mL of tetrahydrofuran, followed by the addition of 10.0 mL of deionized water containing 10.0 mg of PEG-b-PPG-b-PEG. After sonication for 35 minutes, tetrahydrofuran was removed by evaporation, and the mixture was ultrafiltered through a 50 kDa ultrafiltration tube to finally obtain drug nanoparticles (NPs).

[0065] Example 1: Synthesis of OS@CC@NPs:

[0066] 4T1 breast cancer cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0067] Cell membranes of osteoclasts and 4T1 breast cancer cells were obtained using ultrasound and centrifugation.

[0068] Cell membranes of 4T1 breast cancer cells and osteoclasts were mixed at a 1:1 mass ratio. After membrane extraction, the membrane protein content was determined using the BCA method. Approximately 200 μg of total membrane protein (100 μg of each cell membrane) was mixed with 100 μg of nanoparticles (NPs) (total cell membrane to NPs mass ratio 2:1) and sonicated for 25 minutes. The mixture was then extruded 25 times through polycarbonate porous membrane filters (400 nm and 200 nm), and purified by ultrafiltration through a 50 kDa ultrafiltration tube to obtain a solution of bicellular hybrid membrane-coated biomimetic nanoparticles (OS@CC@NPs).

[0069] Comparative Example 1: Synthesis of CC@NPs

[0070] 4T1 breast cancer cells were extracted via membrane extraction, and the membrane protein content was determined by the BCA method. Approximately 200 μg of total membrane protein was mixed with 100 μg of nanoparticles (NPs) (mass ratio 2:1) and sonicated for 25 minutes. Subsequently, the mixture was extruded 25 times through a polycarbonate porous membrane filter (400 nm and 200 nm), and purified by ultrafiltration through a 50 kDa ultrafiltration tube to obtain a solution of tumor cell membrane-coated biomimetic nanoparticles (CC@NPs).

[0071] Comparative Example 2: Synthesis of OS@NPs

[0072] Osteoclasts were extracted via membrane extraction, and the membrane protein content was determined by the BCA method. Approximately 200 μg of total membrane protein was mixed with 100 μg of nanoparticles (NPs) at a mass ratio of 2:1, and the mixture was sonicated for 25 minutes. Subsequently, the mixture was extruded 25 times through a polycarbonate porous membrane filter (400 nm and 200 nm), and purified by ultrafiltration through a 50 kDa ultrafiltration tube to obtain a solution of osteoclast membrane-coated biomimetic nanoparticles (OS@NPs).

[0073] The technical solution of the present invention will be further explained through the following performance tests, and the technical effect of the technical solution of the present invention will be verified.

[0074] 1. Measurement of cell membrane proteins

[0075] Due to the presence of cell membrane proteins, measuring the relative content of these proteins can be used to evaluate the construction effect of cell membrane camouflage on the surface of nanomaterials. Cell membrane protein content was measured in NPs prepared in Example 3, CC@NPs prepared in Comparative Example 1, OS@NPs prepared in Comparative Example 2, and OS@CC@NPs prepared in Example 1. The results are as follows: Figure 4 As shown.

[0076] according to Figure 4 The BCA protein quantification results showed that the protein concentration levels of the CC@NPs, OS@NPs, and OS@CC@NPs groups were roughly similar, but significantly higher than those of the NPs group, indicating that the cell membrane successfully disguised itself as a nanomaterial. Further verification of the membrane protein composition was performed using SDS-PAGE analysis. Figure 4 b. It can be clearly observed that there are obvious membrane protein bands in each biomimetic nanocomposite sample, while the control group NPs group does not show the corresponding characteristic bands.

[0077] 2. Basic physical property testing

[0078] 2.1 Transmission Electron Microscopy (TEM) Testing

[0079] The results of transmission electron microscopy (TEM) observations are as follows: Figure 5 As shown in a, the NPs are slightly irregular spherical and significantly smaller in volume compared to CC@NPs, OS@NPs, and OS@CC@NPs. Furthermore, a membrane structure of approximately 11–14 nm thickness can be observed in the cell membrane-coated group.

[0080] 2.2 Dynamic Light Scattering (DLS) Detection

[0081] Figure 5The dynamic light scattering detection results of b showed that the average hydrated particle sizes of NPs, CC@NPs, OS@NPs and OS@CC@NPs were 58.6 nm, 107.86 nm, 104.5 nm and 113.35 nm, respectively.

[0082] 2.3 Colloidal stability test

[0083] like Figure 5 As shown in c, during the 14-day monitoring period, the average hydrated particle size of each group of nanomaterials NPs, CC@NPs, OS@NPs and OS@CC@NPs in PBS solution showed only slight fluctuations, confirming their good colloidal stability and suggesting that they are not prone to degradation during drug delivery.

[0084] 2.4 Zeta potential detection results

[0085] like Figure 5 The zeta potential detection results showed that the average potential of NPs was -12.3 mV, CC@NPs was -28.40 mV, OS@NPs was -23.90 mV, and OS@CC@NPs was -25.16 mV. Compared with CC@NPs, OS@NPs, and OS@CC@NPs, NPs had a significantly higher surface charge.

[0086] 2.5 Ultraviolet Absorption Curve Test

[0087] like Figure 5 As shown in Figure e, due to the presence of SPs, NPs, CC@NPs, OS@NPs and OS@CC@NPs all exhibit similar ultraviolet absorption curves, indicating that the above materials have significant NIR-II photothermal potential.

[0088] 3. Photothermal property testing

[0089] To verify the NIR-II photothermal properties of OS@CC@NPs nanomaterials, a 1064 nm laser was used to monitor the temperature change of OS@CC@NPs in real time. The test results are as follows: Figure 6 As shown.

[0090] The results showed that the temperature increased significantly with increasing nanomaterial concentration, prolonged laser irradiation time, or increased laser power density. Figure 6 When the OS@CC@NPs solution concentration was 100 μg / mL, the temperature stabilized after irradiation with a 1064nm laser at 0.75 W / cm² for 5 minutes. OS@CC@NPs consistently exhibited a stable temperature trend throughout five laser on / off cycles, confirming its reliable photothermal stability. Figure 6d). Quantitative measurements showed that the photothermal conversion efficiency (PCE) of OS@CC@NPs reached 43.13% ( Figure 6 e).

[0091] The above results indicate that OS@CC@NPs possess significant and stable photothermal heating characteristics, and have great application potential in the field of tumor hyperthermia.

[0092] 4. In vivo photothermal conversion capacity test

[0093] To verify the photothermal conversion capability of OS@CC@NPs in vivo, the tumor sites of 4T1 tumor-bearing mice were irradiated with laser 12 hours after tail vein injection of the material. Infrared thermal imaging results are shown below. Figure 7 As shown in Figure a, the tumor area temperature in the OS@CC@NPs group increased rapidly over time during irradiation, while the PBS group only showed a slight increase. Further quantitative analysis results are as follows... Figure 7 As shown in b, the OS@CC@NPs group reached approximately 48 °C within minutes and then stabilized; in contrast, the temperature of the PBS group remained at only around 37 °C.

[0094] The above results demonstrate that, under in vivo enrichment conditions, OS@CC@NPs can produce a significant photothermal effect under laser irradiation, providing a reliable temperature basis for subsequent photothermal synergistic therapy.

[0095] 5. In vitro magnetic resonance imaging performance measurement and testing

[0096] T2-weighted MRI imaging results of NPs, CC@NPs, OS@NPs, and OS@CC@NPs in PBS solution showed that image brightness decreased significantly with increasing iron ion concentration. Figure 8 a).

[0097] The relaxation rates of biomimetic nanocomposites were calculated based on the Fe3O4 nanoparticle concentration. The results showed that the transverse relaxation rates of NPs, CC@NPs, OS@NPs, and OS@CC@NPs were 141.6 mM⁻¹s⁻¹, 144.6 mM⁻¹s⁻¹, 156.3 mM⁻¹s⁻¹, and 170.0 mM⁻¹s⁻¹, respectively. Figure 8 b).

[0098] The results show that the composite nanomaterial OS@CC@NPs has excellent lateral relaxation rate and can be used as a negative contrast agent for T2-weighted imaging (T2WI). By shortening the lateral relaxation time, it can attenuate (darken) the signal in the nanomaterial-rich region, which is consistent with the T2WI imaging results.

[0099] 6. In vivo magnetic resonance imaging performance measurement and testing

[0100] Conventional T2WI imaging was used to detect the T2 signal attenuation in the lesion area before and after drug administration, reflecting the distribution and accumulation of nanoparticles in vivo. First, a mouse tibial bone metastasis model was established. Mouse 4T1 cells were digested and resuspended in sterile PBS. Under anesthesia and aseptic conditions, 10 μL of cell suspension (i.e., 1 × 10^5 cells) was slowly injected into the tibial medullary cavity of BALB / c mice. Four days after modeling, after different treatment groups, T2-weighted MRI was performed on the tibial lesion area of ​​the mice for dynamic observation. The results are as follows: Figure 9 As shown. Signal changes were observed in the tumor region at different time points after tail vein administration in each group. Figure 9 (a) Among them, the OS@CC@NPs group showed a more obvious signal change trend. Quantitative analysis of the T2WI signals revealed that the signal intensity of each group first decreased and then increased over time, with the most significant signal decrease occurring at 12 hours after intravenous administration. The OS@CC@NPs group showed more significant signal changes at multiple time points. Figure 9 b).

[0101] The above results indicate that there are certain differences in the enrichment and retention behavior of different materials in the bone metastasis area. The peak of drug enrichment occurred at 12 hours after drug administration. Among them, the OS@CC@NPs group showed more outstanding imaging effect, which can accurately detect small lesions, demonstrating the advantages of dual-cell targeting.

[0102] 7. Chemical performance testing

[0103] 7.1 Fenton-like Reactivity Test

[0104] Using tetramethylbenzidine (TMB) as a probe, the nanocomposite material was reacted with hydroxyl radicals (·OH) to generate blue oxidized TMB, which showed a characteristic absorption peak at 652 nm. The test results are as follows: Figure 10 As shown.

[0105] Compared with the TMB+H2O2 control group, the OS@CC@NPs+H2O2 group showed a significant characteristic absorption peak at 652 nm, indicating that the material has typical Fenton-like reactivity. Irradiation with a 1064 nm laser further enhanced the intensity of this absorption peak, suggesting that the photothermal effect can amplify its H2O2 activation ability, providing support for its application in chemokinetic therapy (CDT). Figure 10 ad).

[0106] 7.2 Test of the ability to induce intracellular reactive oxygen species (ROS) generation

[0107] To evaluate the ability of different nanosystems to induce reactive oxygen species (ROS) generation within cells, ROS levels in 4T1 breast cancer cells and osteoclasts were measured in a simulated bone metastasis microenvironment. Confocal microscopy results are shown below. Figure 11 As shown in Figure a, for 4T1 breast cancer cells, without laser irradiation, only weak fluorescence signals were observed in the cells of each treatment group. After laser irradiation, the fluorescence intensity of different material treatment groups increased to varying degrees, with the CC@NPs and OS@CC@NPs groups showing the most significant fluorescence signals, indicating a strong ROS generation ability. Figure 11 As shown in b, further quantitative analysis of the fluorescence intensity of 4T1 cells reveals the same trend. Meanwhile, as... Figure 11 As shown in c, the intracellular ROS level of osteoclasts was detected by flow cytometry, and the fluorescence signals of the OS@NPs and OS@CC@NPs groups were the most significant.

[0108] 8. In vitro tumor ferroptosis effect evaluation test

[0109] 8.1 Detection of intracellular MDA content

[0110] The changes in 4T1 breast cancer cells and osteoclasts were detected using MDA as an indicator in the presence of H2O2 (100 μM). Figure 12 As shown in Figure a, without laser irradiation, the MDA levels in all groups of 4T1 breast cancer cells were at a low level. After laser irradiation, the MDA content in all material treatment groups increased significantly, with the CC@NPs and OS@CC@NPs groups showing the most significant increase. Figure 12 As shown in b, a similar trend was observed in osteoclasts, with a significant increase in MDA levels after laser irradiation, but the magnitude was lower than that in 4T1 breast cancer cells, and the OS@NPs and OS@CC@NPs groups showed higher MDA contents. These results indicate that, in the presence of H2O2, laser triggering can significantly enhance the ability of different nanosystems to induce lipid peroxidation.

[0111] 8.2 Detection of intracellular GSH content

[0112] like Figure 13 As shown, since lipid peroxidation directly leads to a large depletion of intracellular GSH, the GSH content level in 4T1 breast cancer cells and osteoclasts is in stark contrast to the MDA content level.

[0113] 8.3 Western blot analysis

[0114] Western blot analysis was performed on ferroptosis-related proteins in cells from the PBS, PBS+laser, OS@CC@NPs, and OS@CC@NPs+laser groups. The results are as follows: Figure 14 As shown.

[0115] The results showed that, compared with the PBS and PBS+laser groups, OS@CC@NPs treatment upregulated ACSL4 expression while downregulated GPX4 and SLC7A11 expression. These changes were further aggravated after combined laser irradiation. These results indicate that OS@CC@NPs can significantly enhance lipid peroxidation-related pathways and weaken cellular antioxidant defenses under laser irradiation, confirming at the protein level that it can induce changes in ferroptosis-related molecular characteristics in cells.

[0116] 9. Evaluation of in vitro tumor and osteoclast killing effects

[0117] To further evaluate the cytotoxic ability of different nanomaterials under oxidative stress, a bone metastasis microenvironment was constructed using H2O2 (final concentration 50 μM), and the survival of 4T1 breast cancer cells and osteoclasts under different treatment conditions was compared and analyzed. (For example...) Figure 15 As shown in ab), without laser irradiation, each material treatment group only caused a limited decrease in cell activity compared to the PBS control group, and the overall activity remained at a high level, suggesting that the single administration had a relatively mild effect on cells under these conditions.

[0118] In contrast, after laser irradiation, cell activity decreased to varying degrees in all treatment groups, with significant differences observed between the different materials. The OS@CC@NPs group exhibited the most significant inhibitory effect in both cell types, with cell viability significantly lower than the NPs, CC@NPs, and OS@NPs groups, demonstrating stronger cytotoxicity. The PBS group showed no significant change before and after laser treatment, indicating that laser treatment alone did not cause significant cytotoxicity.

[0119] The above results indicate that laser triggering can significantly amplify the cell-killing effect of the nanosystem in the presence of H2O2, and that OS@CC@NPs coated with hybrid membranes exhibit the best therapeutic response in both 4T1 cells and osteoclasts.

[0120] 10. In vivo anti-tumor capacity detection

[0121] After establishing a mouse model of tibial bone metastases, the tumor in the leg was allowed to reach 100 mm. 3Mice were divided into two groups: those receiving no light treatment (PBS, NPs, CC@NPs, OS@NPs, and OS@CC@NPs) and those receiving laser treatment (PBS, NPs, CC@NPs, OS@NPs, and OS@CC@NPs). Mice were injected with either PBS or a corresponding nanomaterial solution (semiconductor polymer SPs concentration of 250 μg / mL, 200 μL). Twelve hours after administration, mice requiring laser treatment underwent local laser irradiation of the tumor-bearing tibia (laser parameters: 1.0 W / cm²). 2 (10 minutes). On day 16 post-treatment, all mice were euthanized, and H&E staining was performed on tumor tissue sections from 10 groups of 4T1 tumor-bearing mice with different treatments to evaluate the efficacy of nanoparticles in treating bone metastases under laser irradiation. The tumor tissues in the groups not treated with laser irradiation showed a relatively dense overall structure, with tightly packed tumor cells and largely intact nuclei, exhibiting only mild structural disorder. In contrast, under laser irradiation, the PBS, NPs, CC@NPs, and OS@NPs groups still showed a large number of densely distributed tumor cells, while the OS@CC@NPs+laser group showed significant tumor tissue structure destruction, loose cell arrangement, large-area cell reduction, and tissue vacuolar changes in local areas, exhibiting typical necrosis-like morphological characteristics. This indicates that under laser excitation, OS@CC@NPs can significantly aggravate tumor tissue damage in vivo, demonstrating a stronger anti-tumor effect.

[0122] 11. Detection of the ability of ICD to induce immunogenic cell death in tumor cells

[0123] 11.1 Tests on changes in ICD-related biomarkers under different treatment conditions

[0124] To assess changes in immunogenic cell death (ICD) markers in 4T1 cells under different treatment conditions, CRT exposure, HMGB1 release, and intracellular ATP levels were systematically measured. The results are as follows: Figure 17 As shown in the figure. Immunofluorescence imaging revealed that the CRT signal was generally weak in all groups of cells without laser irradiation; after the introduction of laser, the green fluorescence on the membrane surface of different material treatment groups was enhanced, with the OS@CC@NPs treatment group showing the most significant enhancement, indicating that the CRT had undergone significant eversion. Figure 17 a).

[0125] Further quantitative analysis of HMGB1 revealed that, under laser conditions, the HMGB1 level in the OS@CC@NPs group was significantly higher than that in the control group and other material groups, indicating that this treatment promoted the release of HMGB1 from the intracellular to the extracellular space. Figure 17 b). Meanwhile, ATP assay results showed that the intracellular ATP content in the OS@CC@NPs group decreased significantly after laser irradiation, with a greater decrease than in the other groups. Figure 17c) indicates that there is a significant leakage of intracellular ATP.

[0126] The above results indicate that under light conditions, OS@CC@NPs can simultaneously induce CRT eversion, HMGB1 release, and ATP reduction. These synergistic changes in typical DAMP signals suggest that this treatment effectively triggered ICD-related molecular events in 4T1 cells, providing a cellular basis for subsequent immune activation effects.

[0127] 11.2 Flow cytometry detection

[0128] To evaluate the effects of different treatments on dendritic cell maturation, flow cytometry was used to detect the proportion of CD80⁺CD86⁺ double-positive cells in BMDCs. The results are as follows: Figure 18 As shown. Without laser irradiation, the DC maturation rates in the PBS, NPs, CC@NPs, OS@NPs, and OS@CC@NPs treatment groups were 14.6%, 14.9%, 24.4%, 17.9%, and 19.5%, respectively, all at relatively low levels, with only the CC@NPs group showing a slight increase. Figure 18 a).

[0129] After laser irradiation, the maturity rate of DCs in all groups increased to varying degrees, with the PBS, NPs, CC@NPs, OS@NPs, and OS@CC@NPs groups reaching 14.7%, 25.8%, 38.6%, 27.8%, and 35.8%, respectively. It can be seen that the maturity rate of DCs in the OS@CC@NPs+laser group and the CC@NPs+laser group was significantly higher than that in the other treatment groups. Figure 18 b).

[0130] Quantitative analysis results further showed that OS@CC@NPs + laser treatment could increase the maturity rate of DCs to about 36%, which was significantly better than the PBS and NPs-related control groups, indicating that this treatment was more conducive to promoting the transformation of DCs to the mature phenotype in vitro.

[0131] 11.3 IDO pathway activity evaluation test

[0132] IDO pathway activity was evaluated by detecting the Kyn / Trp ratio in the culture supernatant of each group. Results are as follows: Figure 19 As shown, compared with the PBS group, the Kyn / Trp ratio of each material treatment group was reduced, with the CC@NPs and OS@CC@NPs groups showing a more significant decrease. After laser irradiation, the Kyn / Trp ratio of the CC@NPs and OS@CC@NPs groups decreased further, showing a significant difference compared with the control group, and there was no significant difference between the two groups.

[0133] The results suggest that CC@NPs and OS@CC@NPs have a stronger regulatory effect on the IDO pathway under illumination.

[0134] In summary, the test results show that the bicellular hybrid membrane-coated biomimetic nanoparticles OS@CC@NPs of this invention have a comprehensive relative advantage over single-cell membrane-coated drug nanoparticles CC@NPs and OS@NPs in various in vitro performance tests, while demonstrating an absolute advantage in in vivo imaging and in vivo anti-tumor tests. This invention constructs a hybrid biomimetic membrane by combining tumor cell membranes and osteoclast membranes to achieve dual-targeted enrichment of tumor cells and the bone microenvironment. It also utilizes the near-infrared photothermal effect of semiconductor polymers to promote the Fenton-like reaction of Fe-based nanomaterials, thereby enhancing reactive oxygen species generation and inducing lipid peroxidation and ferroptosis. Simultaneously, by loading an IDO1 inhibitor to regulate the Trp / Kyn metabolic pathway, it alleviates the tumor immunosuppressive microenvironment and enhances the anti-tumor immune response, thus constructing a biomimetic nanoprobe that combines magnetic resonance imaging capabilities with multi-mechanism synergistic therapeutic functions. This integrates tumor localization, treatment, and efficacy monitoring, solving several bottlenecks in current bone metastasis treatment and providing a novel mechanism-guided treatment approach for the long-term control of tumors.

Claims

1. A bicellular hybrid membrane-coated biomimetic nanoparticle, characterized in that, The invention includes drug nanoparticles and a biomimetic outer membrane coating the drug nanoparticles. The biomimetic outer membrane is a hybrid cell membrane derived from tumor cell membrane and osteoclast membrane. The drug nanoparticles contain a semiconductor polymer, a Fe-based Fenton catalyst, and an IDO1 inhibitor. The semiconductor polymer is a DA-type narrow bandgap conjugated semiconductor polymer.

2. The biomimetic nanoparticles coated with a dual-cell hybrid membrane according to claim 1, characterized in that, The semiconductor polymer is constructed by including at least one of DPP units, BBT units, and BDT units.

3. The biomimetic nanoparticles coated with a bicellular hybrid membrane according to claim 1, characterized in that, The Fe-based Fenton catalyst is selected from at least one of Fe3O4 and γ-Fe2O3.

4. The biomimetic nanoparticles coated with a bicellular hybrid membrane according to claim 1, characterized in that, The IDO1 inhibitor is selected from at least one of NLG919, 1-MT, and Epacadostat.

5. The biomimetic nanoparticles coated with a dual-cell hybrid membrane according to claim 1, characterized in that, The drug nanoparticles are formed by polymer self-assembly and nano-coprecipitation. The Fe-based Fenton catalyst is modified with oleic acid. The drug nanoparticles also contain amphiphilic block copolymers.

6. The bicellular hybrid membrane-coated biomimetic nanoparticles according to claim 5, characterized in that, The semiconductor polymer is a BBT-DPP copolymer, the Fe-based Fenton catalyst is Fe3O4, the IDO1 inhibitor is NLG919, and the amphiphilic block copolymer is PEG-b-PPG-b-PEG.

7. The method for preparing biomimetic nanoparticles coated with a bicellular hybrid membrane according to claim 1, characterized in that, include: Step 1: Extract tumor cell membranes and osteoclast membranes; Step 2: Prepare the drug nanoparticles; Step 3: Mix tumor cell membranes, osteoblast membranes and drug nanoparticles, then fuse them with ultrasound, and finally prepare the bicellular hybrid membrane-coated biomimetic nanoparticles by membrane extrusion.

8. The method for preparing biomimetic nanoparticles coated with a bicellular hybrid membrane according to claim 7, characterized in that, In step 2, the drug nanoparticles are prepared by polymer self-assembly and nano-coprecipitation. The Fe-based Fenton catalyst is modified with oleic acid. Specifically, the semiconductor polymer, the Fe-based Fenton catalyst, and the IDO1 inhibitor are dissolved in an organic solvent to obtain a mixed solution. Then, the mixed solution is added to deionized water containing an amphiphilic block copolymer to obtain the drug nanoparticles. The mass ratio of the semiconductor polymer, the Fe-based Fenton catalyst, the IDO1 inhibitor, and the amphiphilic block copolymer is 1:(0.5-2):(1-3):(80-150).

9. The method for preparing biomimetic nanoparticles coated with a bicellular hybrid membrane according to claim 7, characterized in that, In step 3, tumor cell membranes, osteoclast membranes, and drug nanoparticles are mixed, ultrasonically treated, and then the mixture is extruded through a polycarbonate porous membrane filter using an extruder. After ultrafiltration purification through an ultrafiltration tube, the biomimetic nanoparticles coated with the dual-cell hybrid membrane are obtained. The mixing mass ratio of tumor cell membranes and osteoclast membranes is (0.5-2):1, and the mixing mass ratio of the total mass of tumor cell membranes and osteoclast membranes to the mixing mass of drug nanoparticles is (1-3):

1.

10. The use of the bicellular hybrid membrane-coated biomimetic nanoparticles according to any one of claims 1-6 or the bicellular hybrid membrane-coated biomimetic nanoparticles prepared by the preparation method according to any one of claims 7-9 in the preparation of diagnostic and / or therapeutic drugs for bone metastases.

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