An engineered biomimetic nanoparticle for inducing calcification of glioblastoma and preparation method and application thereof

Abstract

The application belongs to the technical field of medicine, and particularly relates to an engineered biomimetic nanoparticle for inducing glioblastoma calcification and a preparation method and application thereof. 2+ Pi homeostasis and force tumor calcification. Firstly, the pre-engineered macrophage membrane is used to display MLV-derived receptor binding domain RBD-XPR1, which is specifically combined with phosphate efflux protein XPR1, so that tumor targeting and local Pi efflux block are realized across the blood-brain barrier. At the same time, the nanoparticle is loaded with a high-load CaCO3 inner core, which can quickly release Ca 2+ in the intracellular acid environment, forming an acute cytoplasmic calcium shock; co-delivered curcumin further weakens the organelle buffering capacity, promotes endogenous Ca 2+ mobilization, thereby synergistically amplifying Ca 2+ overload. The strategy redirects the Ca 2+ signal to a lethal factor driving uncontrolled calcification, forcing tumor cells to enter an irreversible terminal calcification state, thereby inhibiting tumor progression and remodeling the tumor microenvironment.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, specifically relating to an engineered biomimetic nanoparticle that induces calcification of glioblastoma, its preparation method, and its application. Background Technology

[0002] Glioblastoma (GBM) is one of the most aggressive primary tumors of the central nervous system, characterized by high heterogeneity, strong invasiveness, and significant treatment resistance, with a median survival of less than 15 months. In addition to the blood-brain barrier (BBB) ​​restricting drug delivery, its refractory nature stems from the persistent neurobiological driving signals in the brain microenvironment, placing the tumor in a highly dynamic and facilitative niche.

[0003] In this niche, GBMs do not grow in isolation, but rather form a highly coupled interactive network with neurons, glial cells, and the vascular system. Neuronal electrical activity can continuously act on tumor cells through synaptic-like structures and paracrine signals, causing them to translate physiological neural activity into pro-tumor intracellular responses, thereby constructing a neuro-tumor interaction network that significantly enhances the tumor's signal amplification and adaptive evolutionary capabilities.

[0004] In this network, Ca 2+ Signals occupy a central and pivotal position. Neural activity can remodel tumor cell calcium. 2+ Homeostasis is achieved, thereby activating classic oncogenic pathways such as EGFR, PI3K-Akt, and MAPK, and driving cytoskeleton remodeling, metabolic reprogramming, and enhanced invasion and migration capabilities. Simultaneously, Ca... 2+ Signals can propagate among cell populations via gap junctions and tumor microtubule networks, achieving population-scale coordinated regulation and thus maintaining the stability of the overall malignant phenotype of the tumor. Therefore, Ca 2+ It is not only a classic second messenger, but also a key hub connecting neural input, intracellular signal amplification, and population-based coordinated regulation. It is worth further noting that in the GBM microenvironment, Ca... 2+ It possesses dual properties as both a signaling molecule and a calcification precursor. On one hand, it participates in driving tumor proliferation and invasion; on the other hand, when Ca... 2+ When inorganic phosphate (Pi) is abnormally enriched locally and reaches a supersaturated state, calcification can occur, forming deposits. Clinical and pathological studies have shown that brain tumor calcification is usually associated with local metabolic homeostasis imbalance and is more common in slow-growing, low-grade tumors, suggesting lower invasiveness and a better prognosis.

[0005] However, high-level GBM, despite its high dependence on Ca 2+While the tumor signals calcification, it rarely occurs. This is due to several factors: firstly, rapid tumor progression limits the time window for calcification formation; secondly, and more importantly, GBM maintains intracellular Pi homeostasis and prevents calcium loss by finely regulating phosphate (Pi) metabolic flux and enhancing its uptake and turnover capacity. 2+ - Calcification process triggered by aberrant Pi coupling. This metabolic system, while ensuring the needs of nucleic acid, phospholipid, and energy metabolism, also objectively inhibits the occurrence of terminal calcification. Summary of the Invention

[0006] This invention addresses the challenges encountered in the treatment of glioblastoma by constructing engineered biomimetic nanoparticles (pM-CaC) that induce calcification in glioblastoma, along with their preparation method and applications for rapidly breaking down cellular calcification. 2+ - This system promotes Pi metabolic homeostasis and induces tumor calcification. It utilizes pre-engineered macrophage membranes to display an MLV-derived receptor-binding domain (RBD-XPR1), specifically binding to the phosphate efflux protein XPR1, achieving tumor targeting across the blood-brain barrier and local Pi efflux blocking. Simultaneously, nanoparticles encapsulate a highly loaded CaCO3 core, enabling rapid release of CaCO3 in the acidic intracellular environment. 2+ , forming acute cytoplasmic Ca 2+ Impact; co-delivered curcumin (Cur) further weakens organelle buffering capacity and promotes endogenous calcium. 2+ Mobilization, thereby synergistically amplifying Ca 2+ Overload. Ultimately, this strategy redirects Ca2+, which was originally intended to serve tumor progression. 2+ Signal redirection acts as a lethal factor driving uncontrolled calcification, forcing tumor cells into an irreversible terminal calcification state, thereby inhibiting tumor progression and reshaping the tumor microenvironment, providing an intervention pathway for glioblastoma treatment that differs from traditional killing modes.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] An engineered biomimetic nanoparticle for inducing glioblastoma calcification has a core of calcium carbonate nanoparticles loaded with curcumin and an outer layer of macrophage membranes engineered to express XPR1 inhibitory binding domain fragments; the nanoparticles have a particle size of 110-120 nm.

[0009] The method for preparing the engineered biomimetic nanoparticles that induce glioblastoma calcification includes the following steps: (1) Construct a recombinant lentiviral expression vector lenti-CMV-SP-RBD-mFc-SS1-PGK-Puro that expresses the truncated fragment RBD (Δsp) of the XPR1 receptor binding domain; (2) The recombinant lentiviral expression vector and the helper packaging plasmid were co-transfected into HEK-293T human embryonic kidney cells for lentiviral packaging. The supernatant was collected and concentrated by centrifugation, filtration and ultrafiltration to obtain lentiviral concentrate. (3) The concentrated lentivirus solution was used to infect RAW264.7 macrophages, and an engineered cell line that stably expressed RBD was obtained after antibiotic screening; (4) The engineered cells were subjected to flow cytometry and RT-qPCR to verify the expression of XPR1-RBD; (5) Cell membrane components were extracted from engineered RAW264.7 macrophages by differential centrifugation, and engineered cell membrane M was obtained by lysis, sonication and fractional centrifugation purification. (6) DOPE-based pH-sensitive liposomes pH-V were prepared using a thin-film hydration-ultrasound method; (7) The liposomes are fused and reconstructed with engineered cell membranes to obtain a liposome-cell membrane hybrid membrane pM; (8) A calcium-based drug-loaded core, CaC, was prepared using a gas diffusion-induced method; (9) The hybrid membrane pM is coated on the surface of CaC and ultrasonically encapsulated to form core-membrane structured nanoparticles pM-CaC.

[0010] Preferably, in step (1), the CMV promoter drives the RBD (Δsp) expression cassette, fuses the mFc tag and SS1 linker sequence, and sets the PGK promoter to drive the puromycin resistance gene. The vector is transformed and amplified after the ligation reaction at 16-20 ℃ for 10-20 min to obtain the recombinant plasmid.

[0011] Preferably, in step (2), HEK-293T human embryonic kidney cells are seeded in 10 cm culture dishes and cultured at 37 ℃ and 5% CO2 until 70%-80% confluency before transfection. Each transfection system contains 850 μL of DMEM, 10 μg of recombinant lentiviral vector plasmid, 10 μL of lentiviral packaging helper plasmid mixture, and 60 μg of nucleic acid transfection reagent. After mixing, the mixture is allowed to stand at room temperature for 15-20 min before being added to the cell culture system. 10-12 h after transfection, 120 μL of 100-fold concentrated enhancement buffer is added, and the complete culture medium is replaced after 18-20 h. The supernatant is collected after 48 h, centrifuged at 4 ℃ and 3500 g for 5 min to remove cell debris, filtered through a 0.45 μm filter membrane, and then concentrated by ultrafiltration centrifugation at 4 ℃ and 3000 g for 10 min. The process is repeated 2-3 times to obtain the concentrated virus solution.

[0012] Preferably, in step (3), RAW264.7 macrophages are 1×10 5Cells / wells were seeded in 12-well plates and cultured to 20%-30% confluence. Lentiviral virus (800 μL total volume) was added at MOI = 200, and the cells were infected at 37 ℃ for 16 h. The medium was then replaced with 1 mL of complete medium for further culture. On day 5, puromycin was added for selection, with a lethal concentration of 1 μg / mL and a maintenance concentration of 0.5 μg / mL. The selection cycle was 2 rounds, 2 days per round, to obtain a stable RAW264.7 macrophage cell line expressing RBD.

[0013] Preferably, in step (4), the PE anti-mouse Fc antibody is used for flow cytometry detection, diluted with 1% BSA / PBS at a ratio of 1:250 (1 μL antibody + 249 μL diluent); 100 μL of cell suspension is mixed with 100 μL of antibody working solution, incubated at 4 ℃ in the dark for 30 min, washed with 500 μL of 1% BSA / PBS, centrifuged at 1380×g for 1 min, washed twice, and finally resuspended in 300 μL of 1% BSA / PBS and 100 μL is used for flow cytometry detection; in RT-qPCR, 1×10 6 RNA was extracted from each cell using 350 μL of lysis buffer LB. The reverse transcription system used 500 ng of RNA, with a total system volume of 10 μL. The reaction conditions were 37 ℃ for 2 min, 55 ℃ for 15 min, and 85 ℃ for 5 min.

[0014] Preferably, in step (5), after the cells are cultured to a confluence of >100%, they are collected, centrifuged at 250 g for 4 min, washed 3 times with PBS, and resuspended in 0.1×TM hypotonic lysis buffer (containing protease and phosphatase inhibitors), and lysed overnight at 4 ℃; the sonication conditions are 300 W for 45 min; then centrifuged at 3,200 g for 5 min, 20,000 g for 25 min, and 100,000 g for 60 min in sequence to obtain membrane component M, and the protein concentration is determined by BCA method.

[0015] Preferably, in step (6), lecithin, cholesterol and DOPE are dissolved in chloroform at a mass ratio of 10:5:2, and the mixture is rotary evaporated at 37°C and 0.08-0.1 MPa for 2-4 h and then vacuum dried. Subsequently, the mixture is hydrated at 40°C for 30-60 min to form a liposome suspension, and then sonicated at 200 W for 30 min (4°C ice bath) to obtain liposomes with uniform particle size.

[0016] Preferably, in step (7), liposomes and cell membranes are mixed at a lipid mass to membrane protein mass ratio of 1:2, and ultrasonically treated at 180 W for 5 min at 4°C (intermittent mode, working for 5 s and pausing for 5 s) to promote membrane fusion and reconstruction, thereby obtaining a hybrid membrane pM.

[0017] Preferably, in step (8), 150 mg of CaCl2·H2O, 10 mg of dopamine and 40 mg of curcumin are dissolved in 100 mL of anhydrous ethanol and reacted for 12-16 h under stirring at 1500 rpm. Then, 10 g of ammonium bicarbonate is added and reacted at 40 °C for 18 h. The container is loosely sealed to maintain a gas diffusion environment. After the reaction, the precipitate is collected by centrifugation at 12000 rpm for 10 min and washed twice each with anhydrous ethanol and deionized water.

[0018] Preferably, in step (9), CaC is dispersed by ultrasonication at 480 W for 15 min and then mixed with pM at a mass ratio of 1:1. The mixture is premixed under ice bath conditions for 2-3 min, then encapsulated by ultrasonication at 140 W for 5 min (intermittent mode, working for 5 s / pause for 5 s), and centrifuged at 5000 rpm for 5 min to remove free membrane components, thereby obtaining pM-CaC nanoparticles.

[0019] The present invention also provides the use of nanoparticles prepared by the above preparation method in the preparation of drugs for treating glioblastoma.

[0020] The room temperature mentioned in this invention refers to 25°C.

[0021] Compared with the prior art, the present invention has the following advantages: (1) This invention achieves the directional display of functional protein (XPR1-RBD) on the membrane surface through engineered cell membrane modification, organically combining the biomimetic cell membrane targeting ability with the molecular function of the receptor binding domain.

[0022] (2) The process for preparing nanoparticles in this invention is relatively simple, has high drug loading efficiency and strong repeatability, and has good feasibility for batch preparation. At the same time, the system has high stability under blood circulation and storage conditions and good overall biosafety.

[0023] (3) This invention achieves synergistic regulation of Ca 2+ It works with inorganic phosphate (Pi) metabolic homeostasis to achieve specific calcification induction within the tumor microenvironment, thus enabling functional intervention in glioblastoma. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the recombinant lentiviral expression vector structure.

[0025] Figure 2 Figure 1 shows the results of flow cytometry analysis of XPR1-RBD expression on the macrophage membrane surface obtained in Example 1; a) fluorescence intensity of cells in different treatment groups; b) median fluorescence value of cells in different treatment groups.

[0026] Figure 3 The figure shows the results of RT-qPCR investigation of the expression of XPR1-RBD on the surface of macrophage membranes obtained in Example 1.

[0027] Figure 4 Figure 1 shows the particle size characterization results of the pM-CaC nanoparticles obtained in Example 1; a) Particle size change of nanoparticles obtained by DLS measurement; b) TEM image of nanoparticles.

[0028] Figure 5 Figures showing the results of the retention of macrophage membrane proteins in nanoparticles of each group; a) SDS-PAGE results of the retention of all proteins; b) Western Blot results of the retention of key proteins; where 1 is the protein marker, 2 is Comparative Example 5, 3 is Example 1, and 4 is Comparative Example 4.

[0029] Figure 6 The image shows the elemental distribution of the pM-CaC nanoparticles obtained in Example 1; a) elemental scan image; b) elemental content determination results.

[0030] Figure 7 The figure shows the stability test results of the pM-CaC nanoparticles obtained in Example 1.

[0031] Figure 8 The figure shows the pH sensitivity test results of the pM-CaC nanoparticles obtained in Example 1.

[0032] Figure 9 Figure 1 shows the targeting results of the nanoparticles obtained in Example 1 and Comparative Example 2; a) Permeation efficiency of nanoparticles in an in vitro blood-brain barrier model; b) Permeation amount of nanoparticles with loaded probes in an in vitro blood-brain barrier model; c) Fluorescence signal intensity of nanoparticles at different time points in vivo; d) Fluorescence signal intensity of nanoparticles in isolated brain tissue after 24 h.

[0033] Figure 10 The graph shows the results of the functional evaluation of the nanoparticles inhibiting phosphate efflux obtained in the examples, comparative examples, and control group.

[0034] Figure 11 Figure 1 shows the functional evaluation results of nanoparticles rapidly inducing calcium overload in the examples, comparative examples, and control groups; a) Intracellular Ca2+ after treatment in different groups. 2+ Level; b) Intracellular Ca after treatment 2+ Imaging.

[0035] Figure 12 The figure shows the results of the functional investigation of pM-CaC nanoparticles obtained in Example 1 in triggering tumor calcification at the cellular level.

[0036] Figure 13The images show the results of the functional investigation of the nanoparticles obtained in Example 1 and the control group in triggering tumor calcification at the animal level; a) Micro-CT image; b) Silver nitrate staining image of tissue sections. Detailed Implementation

[0037] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, the embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0038] Example 1 An engineered biomimetic nanoparticle for inducing glioblastoma calcification comprises a core of calcium carbonate nanoparticles loaded with curcumin and an outer layer coated with a macrophage membrane engineered to express an XPR1 inhibitory binding domain fragment. The preparation method includes the following steps: (1) Construction of recombinant lentiviral vector The recombinant lentiviral expression vector lenti-CMV-SP-RBD-mFc-SS1-PGK-Puro, expressing the XPR1-RBD fragment, was constructed using molecular cloning methods. A nucleic acid sequence encoding a truncated fragment of the XPR1 receptor-binding domain (RBD, Δsp) was used as the target insert fragment and recombinated with a lentiviral backbone vector containing a CMV promoter-driven expression cassette to construct a fusion expression structure. The vector was ligated at 20 °C for 15 min, followed by transformation and amplification to obtain the recombinant plasmid.

[0039] The expression carrier consists of the following functional modules: A CMV promoter-driven SP-RBD(Δsp)-mFc fusion expression cassette is used to achieve high-level constitutive expression of exogenous proteins in mammalian cells; The SS1 linker sequence is used to provide structural flexibility for the fusion protein and to assist in transmembrane localization and secretion. mFc tag sequences are used for immunological detection, signal amplification, and subsequent purification of fusion proteins; A PGK promoter-driven puromycin resistance selection cassette is used for resistance selection and construction of stable mammalian cell lines.

[0040] The recombinant lentiviral expression vector structure obtained by construction is as follows: Figure 1 As shown.

[0041] The target inserted fragment sequence is shown in SEQ ID NO: 1.

[0042] (2) Lentiviral packaging and concentration Lentiviral cells were packaged and prepared using the HEK-293T human embryonic kidney cell transient transfection system.

[0043] One day before transfection, HEK-293T cells were seeded in 10 cm culture dishes at an appropriate ratio and cultured at 37 ℃ and 5% CO2 until the cell density reached 70%-80% for transfection.

[0044] The transfection system for each dish is as follows: 850 μL of DMEM; 10 μg of recombinant lentiviral expression vector plasmid; 10 μL of lentiviral packaging helper plasmid mixture; 60 μg of nucleic acid transfection reagent.

[0045] After mixing all components, allow them to stand at room temperature for 15 min, then add them evenly to the cell culture system. Ten h after transfection, add 120 μL of 100x concentration enhancement buffer to each dish to enhance transfection efficiency; 20 h after transfection, replace with fresh complete culture medium and continue culturing. Forty-eight h after transfection, collect the cell culture supernatant, centrifuge at 3500 g for 5 min at 4 °C to remove cell debris, and filter through a 0.45 μm filter to obtain clear viral supernatant. Transfer the filtered viral supernatant to a 100 kDa ultrafiltration centrifuge tube and concentrate by centrifugation at 3000 g for 10 min at 4 °C. Repeat the ultrafiltration process three times until the target volume of viral concentrate is obtained. The final lentivirus concentrate is aliquoted and stored in [location missing]. Keep at 80℃ for later use.

[0046] Lentiviral functional titer assay HEK-293T human embryonic kidney cells at 2 × 10 5 Cells / wells were seeded in 12-well plates and cultured at 37 °C until 20%-30% confluence was achieved before being used for infection experiments.

[0047] The viral fluid was thawed in an ice bath and then serially diluted using serum-free DMEM, as follows: Diluent 1: 12 μL virus solution + 108 μL DMEM; Diluent #2: 12 μL Diluent #1 + 108 μL DMEM; Diluent #3: 12 μL Diluent #2 + 108 μL DMEM; Diluent #4: 12 μL of diluent #3 + 108 μL of DMEM.

[0048] Add 100 μL of virus dilution to each well and incubate overnight at 37 °C.

[0049] Cells were cultured for another 5 days post-infection, and then collected for total RNA extraction and subsequent RT-qPCR analysis. Total RNA was purified using a column chromatography method, with approximately 1 × 10⁻⁶ cells purified. 6 After complete lysis with 350 μL of lysis buffer LB, cells were purified by column chromatography, sequentially passing through a DNA removal column, binding buffer BD, protein removal buffer, and wash buffer. Finally, RNA was eluted and collected. Store at 80℃ for later use.

[0050] 500 ng of RNA was used for reverse transcription, and the system is as follows: RNA 500 ng; All-in-One First-Strand Synthesis MasterMix 4 μL; dsDNase 1μL; RNase-free ddH2O make up to 10 μL.

[0051] The reverse transcription conditions were: 37 ℃ for 2 min → 55 ℃ for 15 min → 85 ℃ for 5 min.

[0052] The product was diluted with ultrapure water to a final volume of 50 μL. RT-qPCR was then performed using the SYBR Green assay.

[0053] PCR system (20 μL): Taq SYBR Green qPCR Premix 10 μL; upstream primer (10 μM) 0.2 μL; downstream primer (10 μM) 0.2 μL; template cDNA 2 μL; ultrapure water 7.6 μL.

[0054] PCR conditions: 95 ℃ for 30 s; followed by 40 cycles (95 ℃ for 10 s, 60 ℃ for 60 s); melting curve analysis (60 ℃-95 ℃).

[0055] The primer sequences are as follows: WPRE-F is shown in SEQ ID NO: 2; WPRE-R is shown in SEQ ID NO: 3; GAPDH-F is shown in SEQ ID NO: 4; GAPDH-R is shown in SEQ ID NO: 5.

[0056] The measured titer was 2.48 × 10⁻⁶. 9 TU / mL, lentivirus packaging successful.

[0057] (3) Establishment of stable RBD-RAW264.7 macrophage cell line RAW264.7 macrophages at a rate of 1 × 10 5 Cells / wells were seeded in 12-well plates and cultured overnight at 37 °C with 5% CO2 until 20%-30% confluence, placing them in the logarithmic growth phase. Lentiviral infection was performed the following day. The virus solution stored at -80 °C was slowly thawed in an ice bath and diluted with complete culture medium at MOI = 200 (total volume 800 μL). After discarding the original culture medium, the diluted virus solution (500 μL / well) was added to the cells, and the cells were cultured at 37 °C. Sixteen hours after infection, the medium was replaced with 1 mL of fresh complete culture medium. Antibiotic selection began on day 5 post-infection. Puromycin was added to a final concentration of 1 μg / mL for selection, maintaining a concentration of 0.5 μg / mL for two rounds (approximately 2 days per round). A stable positive cell population, i.e., the stable RBD-RAW264.7 macrophage line, was obtained after selection. After stabilizing the cells, they were maintained in a complete culture medium containing DMEM (10% FBS, 1% PS, 0.5 μg / mL puromycin).

[0058] (4) Flow cytometry and RT-qPCR were used to verify the expression of XPR1-RBD. The selected cells were collected and resuspended in 1% BSA / PBS for flow cytometry analysis. PE anti-mouse Fc secondary antibody was prepared at a ratio of 1:250 (1 μL antibody + 249 μL 1% BSA / PBS) and thoroughly mixed. 100 μL of the cell suspension was mixed with an equal volume of the secondary antibody working solution and incubated at 4 ℃ in the dark for 30 min. After incubation, 500 μL of 1% BSA / PBS was added for washing, followed by centrifugation at 1380 × g for 1 min. The supernatant was discarded, and the washing was repeated twice. Finally, the cells were resuspended in 300 μL of 1% BSA / PBS, and 100 μL was used for flow cytometry analysis.

[0059] Total RNA was extracted using a column chromatography method: 1 × 10⁻⁶ 6 Cells were lysed with 350 μL of lysis buffer, then purified by column chromatography, and the RNA solution was obtained after standard washing. Store at 80 ℃. Take 500 ng RNA for reverse transcription (same system and conditions as described above), total system 10 μL, reaction conditions are 37 ℃ for 2 min, 55 ℃ for 15 min, 85 ℃ for 5 min, and then perform RT-qPCR detection after obtaining cDNA.

[0060] The RT-qPCR system and conditions are as follows (same as the aforementioned standard system).

[0061] The RBD (Δsp) primer sequences are as follows: The forward primer sequence is shown in SEQ ID NO: 6; The reverse primer sequence is shown in SEQ ID NO: 7; The primers for the internal reference gene GAPDH are as follows: The forward primer sequence is shown in SEQ ID NO: 8; The reverse primer sequence is shown in SEQ ID NO: 9.

[0062] The relative expression levels of each gene were calculated using the ΔΔCt method, with GAPDH as an internal reference. That is: ΔΔCt = (average CT of the target gene in the test sample - average CT of the internal reference gene in the test sample) - (average CT of the target gene in the control sample - average CT of the internal reference gene in the control sample). The relative gene expression level F = 2. -△△Ct The efficiency of the target gene is 1-2. -△△Ct .

[0063] The verification results are as follows Figure 2 , Figure 3 As shown.

[0064] (5) Extraction of RBD-RAW264.7 macrophage membrane (M) After cells reached >100% confluence, they were centrifuged at 250 g for 4 min, and the cell pellet was collected. The pellet was washed three times with PBS to remove residual culture medium. The cell pellet was resuspended in 0.1×TM hypotonic lysis buffer (containing protease and phosphatase inhibitors) and lysed overnight at 4°C. The lysate was then sonicated at 300 W for 45 min (4°C) to further disrupt cell structure.

[0065] Subsequently, differential centrifugation was performed to purify the membrane components: centrifugation at 3,200 g for 5 min removed cell nuclei and unbroken cells; centrifugation at 20,000 g for 25 min removed organelle components; and centrifugation at 100,000 g for 60 min enriched cell membrane components. The resulting membrane precipitate was resuspended in PBS for BCA protein quantification and stored at -80 ℃ for later use.

[0066] (6) Preparation of pH-sensitive liposomes (pH-V) Lecithin, cholesterol, and DOPE were accurately weighed at a mass ratio of 10:5:2 and dissolved in chloroform, then transferred to a round-bottom flask to form a homogeneous organic phase system. Subsequently, the mixture was rotary evaporated at 37 °C and 0.1 MPa under reduced pressure for 4 hours to remove the organic solvent, allowing the lipids to spread uniformly on the flask wall to form a continuous, dense lipid film. Further vacuum drying was then performed to completely remove residual chloroform. Ultrapure water or isotonic solution was then added, and the mixture was hydrated at 40 °C for 40 minutes under gentle rotation to fully hydrate the lipid film, spontaneously forming a multilayered liposome structure. After hydration, the resulting suspension was sonicated in a 200W ice bath for 30 minutes to break up large aggregates and obtain a uniformly sized and stably dispersed liposome system.

[0067] (7) Preparation of hybrid membrane (pM) The pre-prepared DOPE-based liposomes were precisely mixed with the cell membrane derived from RBD-RAW264.7 macrophages at a lipid mass to membrane protein quantitative ratio of 1:2.

[0068] The hybrid system was subjected to ultrasonic treatment at 180 W for 5 min at 4 ℃ to form a structurally stable and compositionally homogeneous hybrid membrane system.

[0069] (8) Preparation of calcium-based drug delivery core (CaC) CaCl2·H2O (150 mg), dopamine (10 mg), and curcumin (40 mg) were dissolved in 100 mL of anhydrous ethanol and thoroughly mixed under magnetic stirring (1500 rpm) overnight to obtain a homogeneous precursor reaction system. Ammonium bicarbonate (10 g) was then added to the system, and a gas diffusion-induced reaction was carried out at 40 °C for 18 h, allowing NH3 / CO2 to gradually diffuse into the reaction system and drive in-situ deposition of CaCO3 to co-assemble with the drug to form a nanostructure. The reaction system was loosely sealed to maintain gas exchange equilibrium. After the reaction, the precipitate was collected by centrifugation at 12000 rpm for 10 min and then thoroughly washed with anhydrous ethanol and deionized water to remove unreacted small molecules and free drug. Finally, the obtained calcium-based nanoparticles were resuspended in ultrapure water or isotonic solution for later use; they were brownish-red in color.

[0070] (9) Preparation of pM-CaC nanoparticles The prepared calcium-based drug-loaded core (CaC) nanoparticles were ultrasonically dispersed at 480 W for 15 min to fully depolymerize and form a homogeneous dispersion. Subsequently, based on membrane protein quantification, CaC was mixed with a pre-prepared hybrid membrane (pM) at a mass ratio of 1:1 and premixed under ice bath conditions for 2-3 min. The mixture was further ultrasonically treated at 140 W in an ice bath for 5 min (intermittent mode, 5 s on, 5 s off) to promote the self-assembly, coating, and reconstruction of the hybrid membrane on the CaC surface. The mixture was then centrifuged at 5000 rpm for 5 min to remove free membrane components, forming stable core-membrane structured nanoparticles. The final pM-CaC nanoformulation was resuspended in ultrapure water or isotonic solution, appearing light brown.

[0071] pM-CaC refers to the nanoparticles obtained in Example 1, while the control group was treated with physiological saline.

[0072] Comparative Example 1 Following the same preparation process as in Example 1, steps (6) and (7) were omitted, and the encapsulation operation in step (9) was performed directly to obtain pH-insensitive nanoparticles (M-CaC) that were only encapsulated by engineered macrophage membranes.

[0073] Comparative Example 2 Following the same preparation process as in Example 1, steps (5) and (7) were omitted, and the encapsulation operation in step (9) was performed directly to obtain nanoparticles (p-CaC) with no targeting and Pi regulation function that are only encapsulated by liposomes.

[0074] Comparative Example 3 Following the same preparation process as in Example 1, but without adding curcumin in step (8), drug-free nanoparticles (pM-Ca) were obtained.

[0075] Comparative Example 4 Following the same preparation process as in Example 1, the hybrid membrane (pM) was directly obtained in step (7).

[0076] Comparative Example 5 Following the same preparation process as in Example 1, the drug-loaded core (CaC) was directly obtained in step (8).

[0077] The structural characteristics of pM-CaC nanoparticles (Example 1) are as follows: Figure 4 As shown, the hydrated particle size of the nanoparticles is approximately 110-120 nm (e.g., Figure 4 As shown in a), it is uniformly distributed and has a regular morphology. TEM observation shows that it is a uniform sphere with a complete structure (as shown in a diagram). Figure 4 (as shown in b).

[0078] SDS-PAGE results showed that the basic components and key proteins of the RBD-RAW264.7 macrophage membrane remained intact. Figure 5 EDS elemental mapping and energy dispersive spectroscopy results showed that Ca and P were uniformly distributed, confirming that pM-CaC nanoparticles have a core-membrane structure. Figure 6 ).

[0079] Experimental Example 1 The stability of pM-CaC nanoparticles was investigated. The results are as follows: Figure 7 As shown: The DLS particle size distribution of nanoparticles was measured for 7 consecutive days. The results showed that the particle size and polydispersity index of the nanoparticles did not change significantly over the 7 days, indicating that they have stability.

[0080] Experimental Example 2 The pH response characteristics of pM-CaC nanoparticles were investigated. The nanoparticles were incubated for a certain period in simulated physiological environments (pH = 7.4), tumor microenvironments (pH = 6.8), and intracellular lysosomal acidic environments (pH = 5.5), respectively. Particle size and morphology were analyzed using DLS and TEM. DLS results showed that at pH = 7.4, the nanoparticle size distribution remained stable without significant change; when the pH decreased to 6.8, the particle size increased to some extent and was accompanied by slight aggregation; further decreasing to pH = 5.5, the particle size distribution became multimodal, and the system exhibited significant disintegration. Figure 8 a). TEM also confirmed that the nanoparticles maintained an intact core-membrane structure at pH = 7.4; the outer hybrid membrane structure was partially disrupted at pH = 6.8; and the calcium-based core structure underwent significant disintegration at pH = 5.5, resulting in the overall disintegration of the nanoparticle structure. Figure 8 b).

[0081] The above results indicate that pM-CaC nanoparticles exhibit pH-dependent structural response characteristics, with the outer membrane breaking down and the core disintegrating sequentially in neutral to weakly acidic and strongly acidic environments, achieving a stepwise structural change response.

[0082] Experimental Example 3 Targeting ability analysis of pM-CaC nanoparticles. The cross-barrier transport ability of pM-CaC nanoparticles was evaluated using an in vitro blood-brain barrier (BBB) ​​model. bEnd.3 cells were used at a concentration of 1×10⁻⁶. 5 Cells / well were seeded at a density of 1 μm pore size and 0.33 cm² membrane area. 2Transwell cell culture inserts were cultured continuously at 37 °C and 5% CO2 for 26 days to form a monolayer BBB model with a complete and tightly connected structure. Fluorescently labeled pM-CaC nanoparticles (Example 1) and untargeted p-CaC nanoparticles (Comparative Example 2) were added to the donor side of the upper chamber of the Transwell cell culture insert to evaluate their cross-BBB transport efficiency. The results showed that the cross-barrier transport capacity and efficiency of pM-CaC nanoparticles were significantly higher than those of the p-CaC nanoparticle group. Figure 9 ab). Further evaluation of its in vivo distribution was conducted in an orthotopic GBM-bearing mouse model. Results showed that, 24 h after administration, the fluorescence signal intensity of pM-CaC nanoparticles in the brain tumor region was significantly higher than that of the p-CaC nanoparticle group, and this consistency was verified in semi-quantitative fluorescence analysis of isolated brain tissue. Figure 9 cd).

[0083] The above results indicate that the macrophage membrane coating strategy can significantly improve the ability of nanoparticles to cross the blood-brain barrier and enhance their targeted enrichment ability in brain tumor regions.

[0084] Test Example 4 The ability of pM-CaC nanoparticles to inhibit cellular phosphate efflux was investigated. GL261 glioma cells were subjected to a 3×10⁻⁶ thiocarcinoma incubation. 5 Cells were seeded at a density of 5 / well in 6-well plates and cultured at 37 °C and 5% CO2 for 24 h to ensure full adhesion. Then, pM-CaC nanoparticles (50 μg / mL), comparative nanoparticles, and physiological saline were added as controls for further treatment, followed by another 24 h of culture. After treatment, cells were collected, washed once with PBS, scraped, and centrifuged at 500 g for 5 min. The supernatant was discarded, and the cell pellet was collected. The cells were resuspended in pre-cooled PBS and sonicated on ice (300 W, 10 min), followed by centrifugation at 12,000 g and 4 °C for 10 min to separate the supernatant and cell pellet. The supernatant was used to detect extracellular free phosphate levels: an equal volume of protein precipitant was added to remove protein, and the mixture was centrifuged at 12,000 g for 10 min. The supernatant was collected, neutralized, and then phosphate was quantitatively detected using the malachite green method. The cell pellet assay was used to detect intracellular total phosphate levels: the pellet was dissolved in 0.2 M HCl for 10–15 min and then fully neutralized, followed by quantification using the malachite green colorimetric method. All results were normalized to the total protein content determined by the BCA method.

[0085] The results showed that ( Figure 10 Treatment with pM-CaC nanoparticles can significantly increase intracellular phosphate levels.

[0086] Experimental Example 5 The ability of pM-CaC nanoparticles to induce calcium overload was investigated. GL261 glioma cells were subjected to a calcium overload induction assay at 5 × 10⁻⁶ cells / day. 5 Cells / wells were seeded in 6-well plates and cultured at 37 °C and 5% CO2 for 24 h to ensure full adhesion before subsequent experiments. After the experiments, the 6-well plates were removed and gently rinsed once with physiological saline to remove residual free calcium ions and background from the culture medium. Then, 100 μL of lysis buffer was added per well, and the cells were repeatedly pipetted to ensure complete lysis. After lysis, the samples were centrifuged at 10,000-14,000 g for 3-5 min at 4 °C to separate the supernatant and precipitate. The supernatant was used as free calcium. 2+ The components were stored on ice for later use and analyzed using the OCPC colorimetric method. The precipitate fraction was used to detect bound and total Ca. 2+ Level: Add 0.2 M HCl (50-100 μL) to the precipitate, incubate in an ice bath for 10-15 min to allow the precipitate to fully dissolve, then neutralize. Repeat the Ca reaction using the OCPC method. 2+ Quantitative detection. All the above detection results were normalized in conjunction with the total cellular protein content determined by the BCA method.

[0087] The results showed that pM-CaC nanoparticles could significantly increase intracellular Ca2+. 2+ level( Figure 11 a). Further through Ca 2+ Fluorescent probe staining results showed that intracellular Ca2+ could be observed approximately 15 minutes after treatment. 2+ The signal significantly increased and remained at a high level for the next 12 hours. Figure 11 b). The above results suggest that pM-CaC nanoparticles can rapidly induce acute calcium overload in tumor cells, and this calcium homeostasis imbalance is persistent, indicating that cells have difficulty restoring normal calcium levels through endogenous buffering mechanisms. 2+ Steady state.

[0088] Experimental Example 6 The ability of pM-CaC nanoparticles to induce tumor calcification was investigated: GL261 glioma cells from different groups were prepared as slides and treated. After fixation with 4% paraformaldehyde for 15 min, the slides were washed with PBS. They were then incubated in 1-2% silver nitrate solution in the dark for 60 min, and developed with formaldehyde until a brownish-black deposit appeared. The reaction was immediately terminated by thorough rinsing with distilled water. The slides were then stained with nuclear red solution for 1-5 min, followed by mild differentiation with alcohol to lighten the background and highlight the cell nuclei as bright red. Finally, the slides were dehydrated with graded alcohols, cleared with xylene, mounted with neutral resin, and observed under a microscope. Brain tissue sections from GBM model mice after drug administration were treated using the same method.

[0089] The results showed that obvious black calcification deposits were observed in cells within 12 h in the pM-CaC nanoparticle treatment group, and the calcification signal was further enhanced and widely distributed after 24 h. Figure 12 The tissue section results also showed significant black calcification deposits in the tumor area. Figure 13 b). Micro-CT results further showed that, compared with the control group, the pM-CaC nanoparticle treatment group exhibited significant and specific high-density calcification signals in the tumor lesion area (b). Figure 13 a). The above results indicate that pM-CaC nanoparticles can effectively induce specific calcification deposition in tumor tissues both in vivo and in vitro.

Claims

1. An engineered biomimetic nanoparticle for inducing glioblastoma calcification, characterized in that, The core consists of calcium carbonate nanoparticles loaded with curcumin, and the outer layer is coated with macrophage membranes engineered to express XPR1 inhibitory binding domain fragments; the nanoparticles have a particle size of 110~120 nm.

2. The method for preparing engineered biomimetic nanoparticles that induce glioblastoma calcification according to claim 1, characterized in that, Includes the following steps: (1) Construct a recombinant lentiviral expression vector lenti-CMV-SP-RBD-mFc-SS1-PGK-Puro that expresses the truncated fragment RBD (Δsp) of the XPR1 receptor binding domain; (2) The recombinant lentiviral expression vector and the helper packaging plasmid were co-transfected into HEK-293T human embryonic kidney cells for lentiviral packaging. The supernatant was collected and concentrated by centrifugation, filtration and ultrafiltration to obtain lentiviral concentrate. (3) The concentrated lentivirus solution was used to infect RAW264.7 macrophages, and an engineered cell line that stably expressed RBD was obtained after antibiotic screening; (4) The engineered cells were subjected to flow cytometry and RT-qPCR to verify the expression of XPR1-RBD; (5) Cell membrane components were extracted from engineered RAW264.7 macrophages by differential centrifugation, and engineered cell membrane M was obtained by lysis, sonication and fractional centrifugation purification. (6) DOPE-based pH-sensitive liposomes pH-V were prepared using a thin-film hydration-ultrasound method; (7) The liposomes are fused and reconstructed with engineered cell membranes to obtain a liposome-cell membrane hybrid membrane pM; (8) A calcium-based drug-loaded core, CaC, was prepared using a gas diffusion-induced method; (9) The hybrid membrane pM is coated on the surface of CaC and ultrasonically encapsulated to form core-membrane structured nanoparticles pM-CaC.

3. The method for preparing engineered biomimetic nanoparticles that induce glioblastoma calcification according to claim 2, characterized in that, In step (1), the CMV promoter is used to drive the RBD (Δsp) expression cassette, which is fused with the mFc tag and SS1 linker sequence. The PGK promoter is set to drive the puromycin resistance gene. The vector is transformed and amplified after the ligation reaction at 16-20 ℃ for 10-20 min to obtain the recombinant plasmid.

4. The method for preparing engineered biomimetic nanoparticles that induce glioblastoma calcification according to claim 2, characterized in that, In step (2), HEK-293T human embryonic kidney cells were seeded in 10 cm culture dishes and cultured at 37 ℃ and 5% CO2 until 70%-80% confluency before transfection. Each transfection system contained 850 μL of DMEM, 10 μg of recombinant lentiviral vector plasmid, 10 μL of lentiviral packaging helper plasmid mixture, and 60 μg of nucleic acid transfection reagent. After mixing, the mixture was allowed to stand at room temperature for 15-20 min before being added to the cell culture system. 10-12 h after transfection, 120 μL of 100-fold concentrated enhancement buffer was added to each dish. The complete culture medium was replaced after 18-20 h. The supernatant was collected after 48 h, centrifuged at 3500 g at 4 ℃ for 5 min to remove cell debris, filtered through a 0.45 μm filter membrane, and then concentrated by ultrafiltration centrifugation at 3000 g at 4 ℃ for 10 min. The process was repeated 2-3 times to obtain the concentrated virus solution.

5. The method for preparing engineered biomimetic nanoparticles that induce glioblastoma calcification according to claim 2, characterized in that, In step (3), RAW264.7 macrophages were induced at a dose of 1×10⁻⁶. 5 Cells / wells were seeded in 12-well plates and cultured to 20%-30% confluence. Lentiviral virus was added at MOI = 200, with a total volume of 800 μL. After infection at 37 ℃ for 16 h, the medium was replaced with 1 mL of complete medium for continued culture. On day 5, puromycin was added for selection, with a lethal concentration of 1 μg / mL and a maintenance concentration of 0.5 μg / mL. The selection cycle was 2 rounds, 2 days per round, to obtain a stable RAW264.7 macrophage cell line expressing RBD.

6. The method for preparing engineered biomimetic nanoparticles that induce glioblastoma calcification according to claim 2, characterized in that, In step (4), PE anti-mouse Fc antibody was used for flow cytometry detection. It was diluted with 1% BSA / PBS at a ratio of 1:250, meaning 1 μL of antibody corresponded to 249 μL of diluent. 100 μL of cell suspension was mixed with 100 μL of antibody working solution and incubated at 4 °C in the dark for 30 min. After washing with 500 μL of 1% BSA / PBS, the mixture was centrifuged at 1380 × g for 1 min. This washing was repeated twice. Finally, the suspension was resuspended in 300 μL of 1% BSA / PBS, and 100 μL was used for flow cytometry detection. In RT-qPCR, 1 × 10⁻⁶ cells were used. 6 RNA was extracted from each cell using 350 μL of lysis buffer LB. The reverse transcription system used 500 ng of RNA, with a total system volume of 10 μL. The reaction conditions were 37 ℃ for 2 min, 55 ℃ for 15 min, and 85 ℃ for 5 min.

7. The method for preparing engineered biomimetic nanoparticles that induce glioblastoma calcification according to claim 2, characterized in that, In step (5), cells were cultured to a confluence of >100% and then collected. They were centrifuged at 250 g for 4 min, washed three times with PBS, and resuspended in 0.1×TM hypotonic lysis buffer containing protease and phosphatase inhibitors. The cells were lysed overnight at 4 °C. The sonication conditions were 300 W for 45 min. Subsequently, the cells were centrifuged at 3,200 g for 5 min, 20,000 g for 25 min, and 100,000 g for 60 min to obtain membrane component M. The protein concentration was determined by the BCA method.

8. The method for preparing engineered biomimetic nanoparticles that induce glioblastoma calcification according to claim 2, characterized in that, In step (6), lecithin, cholesterol and DOPE are dissolved in chloroform at a mass ratio of 10:5:2, and the mixture is rotary evaporated at 37 ℃ and 0.08-0.1 MPa for 2-4 h and then vacuum dried. Subsequently, the mixture is hydrated at 40 ℃ for 30-60 min to form a liposome suspension, and then sonicated at 200 W for 30 min in an ice bath at 4 ℃ to obtain liposomes with uniform particle size.

9. The method for preparing engineered biomimetic nanoparticles that induce glioblastoma calcification according to claim 2, characterized in that, In step (7), liposomes and cell membranes are mixed at a lipid mass to membrane protein mass ratio of 1:2 and treated with ultrasound at 180 W for 5 min at 4 ℃. The ultrasound is performed in an intermittent mode, working for 5 s and pausing for 5 s, to promote membrane fusion and reconstruction and obtain hybrid membrane pM. Preferably, in step (8), 150 mg of CaCl2·H2O, 10 mg of dopamine, and 40 mg of curcumin are dissolved in 100 mL of anhydrous ethanol and reacted at 1500 rpm for 12-16 h. Then, 10 g of ammonium bicarbonate is added and reacted at 40 °C for 18 h. The container is loosely sealed to maintain a gas diffusion environment. After the reaction, the precipitate is collected by centrifugation at 12000 rpm for 10 min and washed twice each with anhydrous ethanol and deionized water. Preferably, in step (9), CaC is dispersed by ultrasonication at 480 W for 15 min and then mixed with pM at a mass ratio of 1:

1. The mixture is premixed under ice bath conditions for 2-3 min and then encapsulated by ultrasonication at 140 W for 5 min. The ultrasonication is performed in intermittent mode, working for 5 s and pausing for 5 s. The free membrane components are removed by centrifugation at 5000 rpm for 5 min to obtain pM-CaC nanoparticles.

10. The use of engineered biomimetic nanoparticles for inducing glioblastoma calcification prepared by the method according to claim 1 or any one of claims 2-9 in the preparation of drugs for treating glioblastoma.

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