Grapefruit extracellular vesicle modified bionic phase change nano-particles as well as preparation method and application thereof

Abstract

The invention discloses a grapefruit extracellular vesicle modified bionic phase change nano-particle EV-NP, which is obtained by compounding a phase change nano-particle NP and a grapefruit extracellular vesicle GFEV, and relates to the technical field of nano-drug delivery systems and intracranial nerve disease treatment. The phase-change nano-particles provided by the invention can be coated by a membrane through the grapefruit-derived extracellular vesicles to prepare a bionic phase-change nano-drug carrier, and the bionic phase-change nano-drug carrier is used for in-vivo drug delivery and directly acts on nerve cells by carrying drugs or functional molecules, so that the drug delivery efficiency is improved. Therefore, a new treatment scheme is provided for effective treatment of intracranial nervous system diseases such as glioma.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine delivery systems and intracranial neurological disease treatment technology, particularly to a drug delivery system targeting malignant gliomas, and especially to biomimetic phase transition nanoparticles modified with grapefruit extracellular vesicles and their preparation method, thereby further applicable to the field of glioma treatment. Background Technology

[0002] Gliomas are among the most aggressive and deadliest tumors of the central nervous system, characterized by high invasiveness, heterogeneity, and recurrence. Besides surgery, chemotherapy is a standard clinical treatment for gliomas. However, the presence of the physiological blood-brain barrier (BBB) ​​and the blood-tumor barrier (BTB) often hinders the effective entry of chemotherapy drugs into glioma cells, thus limiting their therapeutic efficacy. To address this challenge, researchers have explored various nanoparticle drug delivery systems aimed at improving the efficacy of glioma treatment by bypassing the BBB / BTB.

[0003] Extracellular vesicles (EVs) are widely distributed in almost all body fluids. With the continuous advancement of nanotechnology, plant-secreted EVs have gradually become a research hotspot in recent years due to their inexpensive and abundant raw material sources and good regeneration capabilities. The biogenesis of EVs typically involves the vacuolar pathway, the multivesicular body pathway, and the extracellular positive organelle pathway. As natural nanoparticles, plant-derived extracellular vesicles (EVs) have been shown to efficiently cross the brain border (BBB / BTB), providing strong support for their use as intracranial drug carriers. The mechanism by which these EVs can cross the BBB / BTB is mainly attributed to the membrane proteins abundant on their surface, which promote uptake by endothelial cells. For example, recent studies have shown that EVs from ginseng and grapefruit can spontaneously promote the entry of chemotherapy drugs into the brain.

[0004] Cell membrane-coated biomimetic nanoparticles are a novel type of nanocarrier whose surface is mimicked by the biomembrane structure of specific cell membranes. Due to their cell membrane-like properties, these nanoparticles offer significant advantages and potential applications in drug delivery, cancer therapy, and immunomodulation. By inheriting the components and properties of cell membranes, these particles typically exhibit good biocompatibility, reducing immune responses and toxic side effects. Furthermore, because receptors and molecules on the cell membrane can recognize specific cells, they can achieve highly efficient targeted delivery in the bloodstream by binding to receptors on the surface of target cells, increasing drug concentration at the target site while minimizing impact on non-target tissues. In addition, the cell membrane provides a natural barrier, protecting drug-loaded molecules from premature degradation, thereby improving drug stability and bioavailability. Therefore, using plant-derived extracellular vesicles to modify the surface of nanoparticles may provide a more effective strategy for the treatment of intracranial neurological diseases such as gliomas. Summary of the Invention

[0005] The purpose of this invention is to provide a class of biomimetic phase transition nanoparticles modified with grapefruit extracellular vesicles. These nanoparticles can autonomously cross the BBB / BTB, overcoming the biological barrier to intracranial drug delivery. Furthermore, by carrying drugs or functional molecules, they can directly act on nerve cells, thus providing a new treatment option for the effective treatment of intracranial neurological diseases such as gliomas. Specifically, this invention adopts the following technical solution:

[0006] Firstly, this invention discloses a biomimetic phase change nanoparticle modified with grapefruit extracellular vesicles, named EV@NP. This nanoparticle is obtained by combining phase change nanoparticles NP carrying a sonosensitive agent with grapefruit extracellular vesicles GFEV. The NP is composed of disulfide-bridged polyphosphate ester SS-PPE, perfluoropentane PFP, and hematoporphyrin monomethyl ether HMME. HMME is the sonosensitive agent, and perfluoropentane is the phase change material. The main function of the sonosensitive agent is to generate reactive oxygen species to kill cells upon ultrasonic stimulation (i.e., sonodynamic therapy).

[0007] The EV@NP surface has a membrane structure, with grapefruit extracellular vesicles (GFEVs) coating the surface of phase change nanoparticles (NPs). The diameter of the EV@NP is 110-150 nm, preferably 110-120 nm.

[0008] Secondly, the present invention also provides a method for preparing the biomimetic phase change nanoparticles modified with grapefruit extracellular vesicles, including the preparation of NP, the preparation of GFEV, and the preparation of EV@NP.

[0009] In the above-described preparation method, preferably, the NP is prepared by simultaneously dissolving SS-PPE, PFP and HMME in anhydrous chloroform at a mass ratio of 8:2:1, then mixing them evenly with water, emulsifying them using an ultrasonic cell disruptor, and then rotary evaporating under reduced pressure to obtain the NP.

[0010] For the preparation method described above, preferably, the GFEV is prepared by juicing grapefruit pulp and then centrifuging it four times at 4 °C under the following conditions: 500 g, 0.16 h; 2000 g, 0.33 h; 5000 g, 0.5 h; 10000 g, 1 h. The supernatant is collected each time. Then, ultracentrifugation at 100000 g is performed for 2 h. After centrifugation, the precipitate is collected and density gradient centrifugation is performed using a sucrose gradient system. The sucrose concentrations from low to high are 8%, 30%, 45%, and 60%. The centrifugation temperature is 4 °C, the centrifugal force is 150000 g, and the centrifugation time is 2 h. Finally, the product located between the 30% and 45% concentration gradients is collected.

[0011] For the preparation method described above, preferably, the EV@NP is prepared by mixing NP and GFEV protein in water at a mass ratio of 1:1 (GFEV is calculated by protein mass), treating with an ultrasonic cleaner for 2 min, then performing ten consecutive extrusions through a liposome extruder (pore size of 400 nm, 400 nm is the pore size of the polycarbonate filter membrane), followed by centrifugation to collect the precipitate at a centrifugation force of 3000 g for 0.25 h, finally obtaining grapefruit extracellular vesicle modified biomimetic phase change nanoparticles EV@NP.

[0012] The method for preparing the grapefruit extracellular vesicle-modified biomimetic phase change nanoparticles EV@NP described in this invention involves using a lipid extruder to extrude grapefruit-derived extracellular vesicles through a polycarbonate membrane, then mixing them with the aforementioned phase change nanoparticles in water, and extruding again through a polycarbonate membrane to obtain a biomimetic phase change nanomedicine carrier. In this invention, the nanocore is prepared through monoemulsification at a suitable temperature, and grapefruit extracellular vesicles are encapsulated on the particle surface through extrusion using a 400 nm polycarbonate membrane. Experiments have shown that a pore size of 400 nm is necessary for the particle size suitable for this invention; pore sizes that are too large or too small are not conducive to forming the grapefruit extracellular vesicle-modified biomimetic phase change nanoparticles EV@NP required by this invention.

[0013] Thirdly, the grapefruit extracellular vesicle modified biomimetic phase change nanoparticles described in this invention can generate a large amount of ROS and kill glioma cells, while also regulating the ferroptosis effect of glioma cells, thus making them suitable for the treatment of nervous system diseases.

[0014] When the surface of the phase change nanoparticles (NP) in this invention is not modified with a cell membrane, they are easily cleared from the blood and have difficulty penetrating the blood-brain barrier to reach glioma lesions. However, EV@NP modified with grapefruit extracellular vesicles (GFEV) can better penetrate the blood-brain barrier to reach glioma lesions, overcoming the biological barrier of intracranial drug delivery. This allows them to be used to prepare biomimetic phase change nanomedicine carriers and to deliver drugs in vivo. By carrying drugs or functional molecules, they can directly act on nerve cells, thus providing a new treatment option for the effective treatment of intracranial nervous system diseases such as gliomas. Attached Figure Description

[0015] Figure 1 The particle size and morphology of the phase change nanoparticles (NP) carrying the acoustic sensor in Example 1 of the present invention are shown.

[0016] Figure 2 The particle size and morphology of grapefruit extracellular vesicle-modified biomimetic phase change nanoparticles (EV@NP) in Example 2 of the present invention are shown.

[0017] Figure 3 In Embodiment 3 of the present invention, the acoustic dynamics effect of EV@NP after FUS treatment is the amount of ROS generated.

[0018] Figure 4 In Example 4 of the present invention, the death of mouse GL261 glioma cells after treatment with NP(A) or EV@NP(B) in combination with FUS.

[0019] Figure 5 In Example 5 of the present invention, the ferroptosis effect in mouse GL261 glioma cells after treatment with NP(A) or EV@NP(B) in combination with FUS.

[0020] Figure 6 This is a comparison of the contents of NP and EV@NP in the lower ventricle of an in vitro blood-brain barrier model in Example 6 of the present invention. Detailed Implementation

[0021] The following embodiments are further illustrations of the present invention and serve as explanations of the technical content of the present invention. However, the essence of the present invention is not limited to the embodiments described below. Those skilled in the art can and should know that any simple changes or substitutions based on the spirit of the present invention should fall within the protection scope claimed by the present invention.

[0022] The abbreviations used in this embodiment have the following meanings:

[0023] PFP, perfluoropentane

[0024] HMME, hematoporphyrin monomethyl ether

[0025] SS-PPE, disulfide-bridged polyphosphate

[0026] ddH2O, double-distilled water

[0027] GFEV, grapefruit-derived extracellular vesicles

[0028] BCA, diquinoline carboxylic acid

[0029] DCFH-DA, 2',7'-Dichlorodihydrofluorescein diacetate

[0030] DCFH, 2',7'-dichlorodihydrofluorescein, was obtained by alkaline hydrolysis of DCFH-DA (10 mM sodium hydroxide) at 25 °C for 0.5 h.

[0031] DCF, 2',7'-dichlorofluorescein

[0032] FUS, Focused Ultrasound

[0033] DLS, Dynamic Light Scattering

[0034] PDI, Dispersion

[0035] ROS, reactive oxygen species

[0036] GSH, glutathione

[0037] RIPA, radioimmunoprecipitation assay

[0038] MDA, malondialdehyde

[0039] Raw material sources and processing methods in the examples:

[0040] PFP, Shanghai Aladdin Chemical Reagent Co., Ltd.

[0041] HMME, Shanghai Aladdin Chemical Reagent Co., Ltd.

[0042] DCFH-DA, Shanghai Aladdin Chemical Reagent Co., Ltd.

[0043] Grapefruit, Zhangzhou Jieyin E-commerce Co., Ltd.;

[0044] Reduced glutathione content assay kit, Beijing Solarbio Science & Technology Co., Ltd.;

[0045] CCK-8 reagent kit, Shanghai Beyotime Biotechnology Co., Ltd.;

[0046] RIPA lysis buffer, Shanghai Beyotime Biotechnology Co., Ltd.;

[0047] Lipid oxidation (MDA) detection kit, Shanghai Beyotime Biotechnology Co., Ltd.;

[0048] BCA Protein Concentration Assay Kit (Enhanced Version), Shanghai Beyotime Biotechnology Co., Ltd.

[0049] Anti-TSG101 antibody, Thermo Fisher Scientific (China) Co., Ltd.;

[0050] Anti-Alix antibody, Abogen (Shanghai) Trading Co., Ltd.;

[0051] GL261 mouse glioma cells, ATCC;

[0052] bEnd.3 mouse brain microvascular endothelial cells, ATCC;

[0053] Dulbecco's Modified Eagle Medium (DMEM) Complete Culture Medium, Thermo Fisher Scientific (China) Co., Ltd.

[0054] SS-PPE: Ethyl dichlorophosphate (16.3 g, 0.1 mol) and triethylamine (11.1 g, 0.11 mol) were dissolved in 100 mL of tetrahydrofuran. Then, 2-hydroxyethyl disulfide (15.4 g, 0.1 mol) was slowly added dropwise at -20 °C. The reaction was stirred at 25 °C for 48 h, followed by precipitation twice in excess diethyl ether at 0 °C. Finally, SS-PPE was obtained by vacuum drying.

[0055] Unless otherwise specified, all other reagents are analytical grade reagents that can be purchased from regular chemical reagent companies and should be used directly.

[0056] Example 1: Preparation of phase change nanoparticles carrying the acoustic sensitizer HMME

[0057] First, SS-PPE (80.0 mg), PFP (20.0 mg), and HMME (10.0 mg) were simultaneously dissolved in anhydrous chloroform (12.0 mL), and then mixed thoroughly with 60.0 mL ddH2O. Next, emulsification was performed using an ultrasonic cell disruptor at 65 W (5 min). Finally, chloroform was removed by rotary evaporation under reduced pressure, and the resulting phase change nanoparticles were named NP. Figure 1 As shown, the DLS results indicate that the hydrodynamic diameter of the NP is approximately 96.75 nm, with a uniform size distribution and a PDI of 0.189. Transmission electron microscopy observation shows that the NP exhibits a spherical morphology, and the particle size is consistent with the above DLS results.

[0058] Example 2: Preparation of biomimetic phase transition nanoparticles modified with grapefruit extracellular vesicles

[0059] Grapefruit pulp was juiced and then centrifuged four times at 4 °C under the following conditions: 500 g, 0.16 h; 2000 g, 0.33 h; 5000 g, 0.5 h; 10000 g, 1 h. The supernatant was collected after each centrifugation. Next, ultracentrifugation was performed at 100,000 g for 2 h. The precipitate was collected and subjected to density gradient centrifugation using a sucrose gradient system. The sucrose concentrations were increased from 8% to 60%, with a centrifugation force of 150,000 g and a centrifugation time of 2 h. Finally, the product located between the 30% and 45% concentration gradients was collected to obtain GFEV.

[0060] Quantitative analysis of proteins in GFEV was performed using the BCA method. Subsequently, NP and GFEV were mixed uniformly in ddH2O at a 1:1 mass ratio (GFEV was calculated based on protein mass), and treated with an ultrasonic cleaner for 2 min. Next, the mixture was extruded ten times consecutively using a liposome extruder (pore size 400 nm). The precipitate was then collected by centrifugation at a force of 3000 g for 0.25 h. The precipitate was then collected, finally yielding biomimetic phase transition nanoparticles EV@NP modified with grapefruit extracellular vesicles.

[0061] like Figure 2 As shown, DLS results indicate that the hydrodynamic diameter of EV@NP is approximately 112.6 nm, with a PDI of 0.173. Transmission electron microscopy reveals a membrane-like structure on its surface, and Western blot analysis confirms the presence of Alix and TSG101, characteristic proteins of GFEV, in EV@NP.

[0062] Example 3: Acoustodynamic effects of grapefruit extracellular vesicle-modified biomimetic phase change nanoparticles

[0063] DCFH and EV@NP were mixed thoroughly in ddH2O at 25 °C, and the solution was irradiated with FUS under ultrasonic power of 2.0 W / cm². 2 The transducer frequency was 1.0 MHz, and the ultrasonic duty cycle was 50%. After irradiation, the fluorescence emission spectrum of the DCF under 488 nm excitation was measured. Figure 3 As shown, after FUS treatment, EV@NP generates a large amount of ROS, and the amount of ROS generated is positively correlated with the FUS irradiation time.

[0064] Example 4: Killing effect of EV@NP on glioma cells

[0065] GL261 mouse glioma cells were seeded at a density of 5,000 cells / well in 96-well plates and incubated at 37 °C for 12 h. The culture medium was then replaced with fresh medium containing NP or EV@NP, and after 12 h of incubation, the cells were again replaced with fresh medium and irradiated with FUS at a sonication power of 2.0 W / cm². 2 The transducer frequency was 1.0 MHz, the ultrasonic duty cycle was 50%, the ultrasonication time was 10 min, and the incubation was continued for 36 h. Finally, the culture medium was replaced with CCK-8 working solution and incubated for 1 h. The absorbance was measured at 450 nm using a microplate reader. The results are as follows: Figure 4As shown, cell viability decreased after EV@NP combined with FUS treatment, and cell viability was negatively correlated with the concentration of EV@NP (the measured concentration of HMME). Compared with NP without grapefruit extracellular vesicle modification, EV@NP showed significantly increased cytotoxicity, mainly due to the relatively weak cellular uptake of NP.

[0066] Example 5: EV@NP combined with FUS regulates ferroptosis in glioma cells

[0067] GL261 mouse glioma cells were seeded at a density of 1,000,000 cells / well in 6-well plates and incubated at 37 °C for 12 h. The culture medium was then replaced with fresh medium containing NP or EV@NP, and after 12 h of incubation, the cells were again replaced with fresh medium and irradiated with FUS at a sonication power of 2.0 W / cm². 2 The transducer frequency was 1.0 MHz, the sonication duty cycle was 50%, the sonication time was 10 min, and the cells were incubated for another 12 h. Cells were collected after freeze-thaw lysis, and the cell lysate was centrifuged at 4 °C at 12000 g for 10 min. The supernatant was collected, and the protein and GSH content were quantitatively analyzed using a BCA assay kit and a reduced GSH assay kit.

[0068] GL261 mouse glioma cells were seeded at a density of 1,000,000 cells / well in 6-well plates and incubated at 37 °C for 12 h. The culture medium was then replaced with fresh medium containing NP or EV@NP, and after 12 h of incubation, the cells were again replaced with fresh medium and irradiated with FUS at a sonication power of 2.0 W / cm². 2 The transducer frequency was 1.0 MHz, the ultrasonic duty cycle was 50%, the ultrasonic time was 10 min, and the cells were incubated for 12 h. Subsequently, cells were lysed using RIPA lysis buffer, and the cell lysate was centrifuged at 4 °C at 10000 g for 10 min. The supernatant was collected, mixed with MDA detection working solution, heated to 100 °C for 0.25 h, cooled to 25 °C in a water bath, and finally centrifuged at 10000 g for 10 min. The supernatant was collected, and the absorbance was measured at 532 nm.

[0069] like Figure 5As shown, compared with the PBS control group, the levels of GSH and MDA in the EV@NP combined with FUS experimental group were reduced, demonstrating that EV@NP combined with FUS accelerates cell death by regulating the ferroptosis pathway in GL261 glioma cells. Similar to the trend in Example 4, due to the lack of surface modification of grapefruit extracellular vesicles, NP's regulatory effect on the ferroptosis pathway in GL261 cells was weak.

[0070] Example 6: EV@NP combined with FUS regulates ferroptosis in glioma cells

[0071] bEnd.3 cells were fed at a rate of 1×10 4 GL261 cells were seeded at a density of 1.0 × 10⁶ cells / well at the bottom of the upper chamber of the Transwell. After 5 days of culture, GL261 cells were seeded at a density of 1.0 × 10⁶ cells / well. 4 Cells were seeded at a density of 10 cells / well on the bottom side of the lower chamber, and after 24 hours of culture, PBS, NP, or EV@NP ([HMME] = 10 μg / mL) were added to the upper chamber. After 12 hours of incubation, the fluorescence intensity of HMME loaded on different nanoparticles in the lower chamber culture medium was measured using a fluorescence spectrophotometer. Figure 6 As shown, compared with the PBS control group, the content of NP in the lower chamber was increased, indicating that NP can penetrate the blood-brain barrier in small amounts. Furthermore, due to the unique interaction between GFEV and vascular endothelial cells, the content of EV@NP penetrating the bEnd.3 endothelial cell layer into the lower chamber was significantly increased, demonstrating the function of GFEV in NP surface modification.

[0072] It should be noted that the above-described technical content of this invention is merely an explanation and clarification to enable those skilled in the art to understand the technical essence of this invention, and therefore is not intended to limit the scope of protection of this invention. The scope of protection of this invention should be determined by the claims. Those skilled in the art should understand that any modifications, equivalent substitutions, and improvements made based on the essential spirit of this invention should be within the scope of protection of this invention.

Claims

1. A biomimetic phase change nanoparticle modified with grapefruit extracellular vesicles, named EV@NP, is obtained by combining phase change nanoparticles NP carrying a sonosensitive agent with grapefruit extracellular vesicles GFEV. The NP is obtained by combining disulfide-bridged polyphosphate ester SS-PPE, perfluoropentane PFP and hematoporphyrin monomethyl ether HMME. The surface of the EV@NP has a film-like structure, and GFEV is coated on the surface of the NP with a diameter of 110-150 nm.

2. The method for preparing biomimetic phase change nanoparticles according to claim 1 includes the preparation of NP, the preparation of GFEV, and the preparation of EV@NP.

3. The preparation method according to claim 2, characterized in that, The NP was prepared by simultaneously dissolving SS-PPE, PFP and HMME in anhydrous chloroform at a mass ratio of 8:2:1, then mixing it evenly with water, emulsifying it using an ultrasonic cell disruptor, and then rotary evaporating it under reduced pressure.

4. The preparation method according to claim 2, characterized in that, The GFEV was prepared by juicing grapefruit pulp and then centrifuging it four times at 4 °C under the following conditions: 500 g, 0.16 h; 2000 g, 0.33 h; 5000 g, 0.5 h; 10000 g, 1 h. The supernatant was collected each time. Then, ultracentrifugation was performed at 100000 g for 2 h. After centrifugation, the precipitate was collected and density gradient centrifugation was carried out using a sucrose gradient system. The sucrose concentrations from low to high were 8%, 30%, 45%, and 60%. The centrifugation temperature was 4 °C, the centrifugal force was 150000 g, and the centrifugation time was 2 h. Finally, the product located between the 30% and 45% concentration gradients was collected.

5. The preparation method according to claim 2, characterized in that, The EV@NP was prepared by mixing NP and GFEV in water at a mass ratio of 1:1, treating the mixture with an ultrasonic cleaner for 2 min, and then performing ten consecutive extrusions through a liposome extruder with a pore size of 400 nm. After centrifugation, the precipitate was collected at a centrifugation force of 3000 g for 0.25 h. The precipitate was then collected to obtain grapefruit extracellular vesicle modified biomimetic phase change nanoparticles EV@NP.

6. The application of grapefruit extracellular vesicle-modified biomimetic phase change nanoparticles as described in claim 1 or prepared by any one of claims 2-5 in the generation of ROS.

7. The use of grapefruit extracellular vesicle-modified biomimetic phase change nanoparticles as described in claim 1 or prepared by any one of claims 2-5 in the preparation of a drug for killing glioma cells.

8. The use of grapefruit extracellular vesicle-modified biomimetic phase change nanoparticles as described in claim 1 or prepared by any one of claims 2-5 in the preparation of a drug for regulating ferroptosis in glioma cells.

9. The use of grapefruit extracellular vesicle-modified biomimetic phase change nanoparticles as described in claim 1 or prepared by any one of claims 2-5 in the preparation of drugs for treating nervous system diseases.

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