Plant exosome-liposome composite targeted nanoparticle drug delivery system, preparation method and application thereof

Abstract

This invention discloses a plant exosome-liposome composite targeted nanoparticle drug delivery system, its preparation method, and its applications. The nanoparticle drug delivery system comprises plant exosomes and liposomes, as well as a targeting polypeptide linked to the surface of the liposomes and an antioxidant drug encapsulated within the liposomes. The plant exosome-liposome composite targeted nanoparticle drug delivery system prepared by this invention can penetrate the blood-brain barrier, possesses the ability to actively target nerve cells, exhibits good drug carrier stability, and is not easily degraded by macrophages or the reticuloendothelial system. Combining the dual therapeutic advantages of the active substances carried by plant exosomes and the antioxidant drugs, it achieves both the regulation of autophagy in nerve cells and the inhibition of neuroinflammation, realizing multi-target therapy and providing a new approach and drug for the efficient treatment of Parkinson's disease.

Description

Technical Field

[0001] This invention pertains to novel bio-nanomaterials, specifically relating to a plant exosome-liposome composite targeted nanoparticle drug delivery system, its preparation method, and its applications. Background Technology

[0002] Parkinson's disease (PD), a common neurodegenerative disease, is increasingly prevalent, becoming one of the most serious threats to human health and life. Clinically and preclinically, PD is primarily treated by compensating for the loss of dopaminergic neurons in the substantia nigra or inhibiting abnormal aggregation of α-synuclein, but the efficacy remains unsatisfactory. In recent years, the decline in autophagy function in nerve cells and the resulting cascade of neuroinflammation have been proven to be key factors in the development and progression of PD. Currently, strategies such as using small molecule inducers or stimulating gating proteins to activate autophagy, or activating anti-inflammatory signaling pathways, have made some progress. However, these single treatment strategies often overlook the complex interaction and crosstalk between autophagy function and neuroinflammation in nerve cells, leading to limited efficacy in treating PD.

[0003] Exosomes are extracellular vesicles with a diameter between 30 and 300 nm, secreted by most cells, and possessing a lipid bilayer. They share the same topological structure as their originating cells, enabling them to carry harmless chemicals into the biological environment and play a role in regulating gene transcription and translation, influencing cell proliferation and apoptosis, and modulating cell differentiation and metabolism. They are playing an important role in anti-tumor and anti-inflammatory fields and are attracting increasing attention from researchers. Plant exosomes, derived from plant cells, are rich in proteins, RNA, and other pharmacologically active molecules, offering advantages such as low production cost, low immunogenicity, and easy availability. They can regulate complex intracellular pathways involved in various diseases across species. Furthermore, due to their unique endogenous function, plant exosomes easily penetrate natural biological barriers such as the blood-brain barrier, blood-testis barrier, and placental barrier, and are considered potential next-generation natural nanocarriers. However, plant exosomes lack natural targeting capabilities; after intravenous administration, most plant exosomes are consumed by the liver, resulting in low exosome drug delivery efficiency, which is a bottleneck problem that urgently needs to be solved in researching their use as a drug delivery system.

[0004] Liposomes are composed of a lipid bilayer membrane. In the aqueous phase, liposomes usually spontaneously aggregate and close vesicles in a tail-to-tail configuration with hydrophobic tails and hydrophilic heads side-by-side. Their particle size ranges from 10 to 200 nm. Liposomes have shown significant advantages as drug carriers in the diagnosis and treatment of diseases: (1) They have a wide drug loading range. Water-soluble drugs are encapsulated in the internal aqueous phase, lipid-soluble drugs are encapsulated in the lipid bilayer membrane, and amphiphilic drugs can be inserted at the interface between the aqueous phase and the membrane. The same liposome can also encapsulate both hydrophilic and hydrophobic drugs. (2) They improve drug stability, prolong the half-life of drugs in vivo, and achieve controlled release of drugs at the lesion site. (3) Their particle size is adjustable, they have good biocompatibility, and they are low in cost. (4) Various active targeting ligands can be modified on the surface of liposomes. By utilizing the specific recognition and binding of the modifiers to cell receptors, active targeting of liposomes can be achieved. Currently, several liposome preparations have been applied in clinical practice, including doxorubicin liposomes, vincristine liposomes, and irinotecan liposomes. However, liposomes are mechanically unstable and prone to drug leakage. Furthermore, as exogenous substances, liposomes are easily phagocytosed and cleared by macrophages or the reticuloendothelial system in the body, severely impacting their therapeutic efficacy.

[0005] The delivery of multiple drugs using exosome-liposome composite nanoparticles is gradually becoming a hot topic, but current research reports mainly focus on the combination of animal-derived exosomes and liposomes. There are almost no reports on the preparation of composite nanoparticles using plant-derived exosomes and liposomes. Summary of the Invention

[0006] Purpose of the invention: To address the shortcomings and defects of existing technologies, this invention provides a plant exosome-liposome composite targeted nanoparticle drug delivery system. The composite nanoparticles of this invention retain the active substances carried by the plant exosomes themselves, while encapsulating antioxidant drugs, thereby achieving the effects of both regulating the autophagy function of nerve cells and inhibiting neuroinflammation. It has active targeting, can penetrate the blood-brain barrier, has high biocompatibility, good drug carrier stability, and is not easily degraded by macrophages or the reticuloendothelial system, thus achieving the goal of highly effective treatment of PD.

[0007] The system provided by this invention effectively solves the problems in treatment strategies: it ignores the complex relationship of interaction and crosstalk between autophagy function and neuroinflammation in nerve cells, and current single treatment strategies cannot simultaneously activate the autophagy function of nerve cells and inhibit the inflammatory response; at the same time, it solves the problems in formulation strategies: plant exosomes have poor targeting, liposomes have poor stability, are not easy to penetrate the blood-brain barrier, and are easily cleared.

[0008] This invention also provides a method for preparing and applying a plant exosome-liposome composite targeted nanoparticle drug delivery system.

[0009] Technical solution: In order to achieve the above solution, the present invention provides a plant exosome-liposome composite targeted nanoparticle drug delivery system, which includes plant exosomes and liposomes fused together, as well as a targeted polypeptide connected to the surface of the liposomes and an antioxidant drug encapsulated in the liposomes.

[0010] The plant exosomes are obtained by differential centrifugation; the plant is derived from any one of ginger, garlic, blueberry, lemon, grape, broccoli, dandelion, spinach, dogwood, and aloe vera.

[0011] The plant exosomes contain one or more of the following: small molecules, miRNA, lipids, and proteins.

[0012] The liposomes include lecithin, cholesterol, and phosphatidylethanolamine-polyethylene glycol with functional terminals.

[0013] Furthermore, the mass ratio of the lecithin, cholesterol, and phosphatidylethanolamine-polyethylene glycol with functional terminals is 50-150:20-40:5.

[0014] The phospholipids include any one or more of lecithin, phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid; the functional terminals in the phosphatidylethanolamine-polyethylene glycol with functional terminals include any one or more of maleimide terminals, succinimide terminals, amino terminals, carboxyl terminals, or thiol terminals.

[0015] Preferably, the phospholipid is soybean lecithin; the phosphatidylethanolamine-polyethylene glycol with a functional terminus is phosphatidylethanolamine-polyethylene glycol 2000-maleimide (DSPE-PEG2000-Mal).

[0016] The targeting peptide is covalently linked to the liposome surface via amino acid residues; the targeting peptide is any one or more of CIKRG, NGR, CPPs, NYZL1, PCM, and PTP.

[0017] The antioxidant drug is any one or more of quercetin, vitamin C, vitamin E, beta-carotene, astaxanthin, proanthocyanidins, resveratrol, and catechins.

[0018] Furthermore, the antioxidant drugs are coated with hydrophobic drugs in the hydrophobic layer of the liposomes and hydrophilic drugs in the hydrophilic layer of the liposomes through hydrophobic interactions. The coating method is thin-film dispersion.

[0019] The composite targeted drug delivery system is in the form of particles with a particle size of 100–200 nm.

[0020] The preparation method of the plant exosome-liposome composite targeted nanoparticle drug delivery system of the present invention includes the following steps:

[0021] (1) Prepare liposomes coated with antioxidant drugs by dissolving antioxidant drugs and liposomes in an organic solvent;

[0022] (2) The targeting peptide is covalently linked to the surface of the liposomes coated with antioxidant drugs obtained in step (1) by amino acid residues to prepare targeted liposomes coated with antioxidant drugs.

[0023] (3) After washing and crushing the plant, the buffer solution was centrifuged multiple times to obtain exosome precipitate, and then purified by sucrose gradient centrifugation to obtain plant exosomes.

[0024] (4) After mixing and incubating the plant exosomes obtained in step (3) with the targeted liposomes coated with antioxidant drugs obtained in step (2), a plant exosome-liposome composite targeted nanoparticle drug delivery system is obtained.

[0025] As a preferred embodiment, the specific steps for preparing the liposomes coated with antioxidant drugs in step (1) are as follows: the antioxidant drugs and liposomes are mixed at a mass ratio of 1:2 to 1:10. Within this mass ratio range, the antioxidant drugs and liposomes are dissolved in an organic solvent, stirred for 5-10 minutes, vacuumed by rotary evacuation to remove the organic solvent, 10-20 mL of PBS is added, and stirred at 40-60°C for 30-60 minutes.

[0026] The organic solvents include acetonitrile, methanol, dichloromethane, chloroform, etc.

[0027] As a preferred option, the specific steps for preparing the targeted liposomes coated with antioxidant drugs in step (2) are as follows: add the targeted peptide to the liposome solution coated with antioxidant drugs at a mass ratio of 1:50 to 1:100 of the targeted peptide to the liposomes, react for 12-24 hours under inert gas and 20-40℃, and dialyze 5-10 times using an ultrafiltration tube with a pore size of 3-10KD for 30-60 minutes each time to remove the uncoated antioxidant drugs and residual targeted peptides in the solution, thereby obtaining the targeted liposomes coated with antioxidant drugs.

[0028] As a preferred option, the specific steps for preparing the plant exosome storage solution in step (3) are as follows: After slicing the plant, stir and homogenize it in PBS buffer, centrifuge at 3000-5000g for 10-20min, repeat twice to remove large fibers, then take the supernatant and continue to centrifuge at 8000-12000g for 30-50min to remove small fibers, then centrifuge at 150000-200000g for 120min to obtain plant exosome precipitate, finally suspend the plant exosome precipitate in PBS buffer and purify the plant exosome precipitate by centrifuging at 150000-200000g for 120min under a sucrose gradient of 8%, 30%, 45%, and 60%, and take the liquid with 8%-30% sucrose to obtain the plant exosome storage solution.

[0029] In step (4), plant exosomes and targeted liposomes coated with antioxidant drugs are fused using any one or more of the following methods: membrane coating, co-extrusion technology, and ultrasonic fusion technology.

[0030] As a preferred option, the specific steps for preparing plant exosome-liposome composite nanoparticles in step (4) are as follows: plant exosome storage solution and targeted liposomes coated with antioxidant drugs are mixed at a particle ratio of 1:1 to 1:6, sonicated for 30-60 seconds, incubated at 0-4℃ for 6-24 hours, and then extruded 5-10 times to obtain the plant exosome-liposome composite targeted nanoparticle drug delivery system.

[0031] The application of the plant exosome-liposome composite targeted nanoparticle drug delivery system described in this invention in the preparation of drugs that activate the autophagy function of nerve cells and inhibit neuroinflammation.

[0032] The application of the plant exosome-liposome composite targeted nanoparticle drug delivery system described in this invention in the preparation of drugs for treating neurodegenerative diseases.

[0033] Furthermore, the neurodegenerative disease is Parkinson's disease (PD).

[0034] This invention innovatively combines plant exosomes with liposomes coated with antioxidant drugs to prepare a composite targeted nanoparticle drug delivery system that can penetrate the blood-brain barrier and actively target nerve cells. The drug carrier has good stability and is not easily degraded by macrophages or the reticuloendothelial system. More importantly, this composite targeted nanoparticle drug delivery system combines the dual therapeutic advantages of the active substances carried by plant exosomes and antioxidant drugs, achieving both the regulation of autophagy function of nerve cells and the inhibition of neuroinflammation. It has a bidirectional regulatory function and produces a significant synergistic effect, achieving the goal of multi-target therapy. This provides a new approach for the efficient treatment of neurodegenerative diseases such as Parkinson's disease.

[0035] The composite targeted nanoparticle drug delivery system prepared in this invention achieves effective treatment of neuropathic disorder (PD) by simultaneously activating neuronal autophagy and inhibiting neuroinflammation. This invention utilizes 6-shogaol (a calcium channel activator) to open TRPV1 channels (calcium channels), and ginger exosomes are rich in 6-shogaol, which activates neuronal autophagy by promoting calcium ion influx. Simultaneously, this system is based on membrane fusion technology, fusing quercetin-coated targeted liposomes with ginger exosomes to deliver quercetin and inhibit neuroinflammation. The membrane structure of ginger exosomes enhances the stability of the liposomes, facilitating penetration of the blood-brain barrier and reducing their clearance. The targeted liposomes improve the targeting specificity of ginger exosomes.

[0036] This invention presents a novel plant exosome-liposome composite targeted nanoparticle drug delivery system that, for the first time, simultaneously activates autophagy in neural cells and inhibits inflammatory responses. This invention is the first to combine plant exosomes and liposomes, leveraging their combined advantages to overcome their respective shortcomings, improving the targeting and stability of the drug carrier, facilitating its penetration of the blood-brain barrier, and reducing its susceptibility to clearance. Existing technologies cannot simultaneously activate autophagy in neural cells and inhibit inflammatory responses, and drug carriers often suffer from the aforementioned problems. This invention achieves highly efficient drug delivery, ultimately improving the treatment efficacy of Parkinson's disease (PD).

[0037] The plant exosome-liposome composite targeted nanoparticle drug delivery system prepared in this invention addresses the lack of natural targeting ability and low drug delivery efficiency of plant exosomes by adding targeting peptides. Mouse brain targeting experiments have demonstrated that the targeting effect of this invention is better than that of ginger exosomes alone. Furthermore, the plant exosome membrane structure overcomes the mechanical instability and susceptibility to clearance of liposomes.

[0038] The plant exosomes in this invention have good stability due to their own phospholipid bilayer membrane structure. By fusing with the liposome membrane, their stability can be improved. Furthermore, plant exosomes have the advantages of naturally penetrating the blood-brain barrier and being difficult to clear. By introducing targeting peptides on the surface of the liposomes, the entire carrier system has good targeting. In addition, plant exosomes do not carry zoonotic diseases or human pathogens, are easy to obtain, inexpensive, and easy to prepare.

[0039] Beneficial results: Compared with the prior art, the present invention has the following advantages:

[0040] (1) This invention provides a plant exosome-liposome composite targeted nanoparticle drug delivery system, which utilizes the active substances carried by the plant exosomes to interact with the TRPV1 channel protein on the surface of nerve cells, resulting in an increase in intracellular calcium ion concentration, thereby activating and causing cell autophagy. At the same time, the antioxidant drugs are encapsulated by liposomes to scavenge reactive oxygen species and inhibit neuroinflammation, ultimately achieving the goal of synergistic treatment of PD.

[0041] (2) This invention combines plant exosomes with liposomes with surface-modified targeting peptides to form a plant exosome-liposome composite targeted nanoparticle drug delivery system. The composite targeted nanoparticles combine the advantages of plant exosomes and liposomes, overcome their respective disadvantages, actively target and deliver drugs, and can escape phagocytosis by immune cells and reticuloendothelial tissue. At the same time, the composite targeted nanoparticles can penetrate the blood-brain barrier, have high biocompatibility, good drug carrier stability, and improve drug delivery efficiency. The preparation method of the composite targeted nanoparticles is simple and controllable, and can realize mass production, which promotes the promotion and use of the product and has broad clinical application prospects. Attached Figure Description

[0042] Figure 1 Mass spectrometry (A) and high-performance liquid chromatography (B) images of the targeted peptide prepared in Example 1;

[0043] Figure 2 Transmission electron microscopy (TEM) morphology images of the quercetin-coated targeted liposomes prepared in Example 1;

[0044] Figure 3 The nanoparticle tracking analysis (NTA) image shows the particle size characterization of the quercetin-coated targeted liposomes prepared in Example 1.

[0045] Figure 4 Zeta potential was investigated for the quercetin-coated targeting liposomes prepared in Example 1.

[0046] Figure 5 The images show fluorescence images of the quercetin-coated targeting liposomes prepared in Example 1, where A is a Dil-labeled liposome, B is a FITC-labeled targeting peptide, and C is a superimposed image of A and B.

[0047] Figure 6 Fluorescence spectrum image of the quercetin-coated targeting liposomes prepared in Example 1;

[0048] Figure 7 TEM morphology images of ginger exosomes prepared in Example 2;

[0049] Figure 8 Image showing the NTA particle size characterization of ginger exosomes prepared in Example 2;

[0050] Figure 9 TEM morphology characterization image of the ginger exosome-liposome composite targeted nanoparticles prepared in Example 3;

[0051] Figure 10 The image shows the NTA particle size characterization of the ginger exosome-liposome composite targeted nanoparticles prepared in Example 3.

[0052] Figure 11 Zeta potential of ginger exosome-liposome composite targeted nanoparticles prepared in Example 3;

[0053] Figure 12 The fluorescence spectrum of the ginger exosome-liposome composite targeted nanoparticles prepared in Example 3 is shown below.

[0054] Figure 13 Fluorescent images of ginger exosome-liposome composite targeted nanoparticles with different proportions prepared in Example 3;

[0055] Figure 14 In Example A, the stability evaluation of the ginger exosome-liposome composite targeted nanoparticles prepared in Example 3 is presented; in Example B, the stability evaluation of the targeted liposomes coated with quercetin is presented.

[0056] Figure 15 A shows the therapeutic effect of different nanoparticles prepared in Example 3 on SH-SY5Y nerve cells; B shows the therapeutic effect of different concentrations of ginger exosome-liposome composite targeted nanoparticles prepared in Example 3 on SH-SY5Y nerve cells.

[0057] Figure 16 The effects of different nanoparticles prepared in Example 3 on the SH-SY5Y activation of autophagy in nerve cells were investigated.

[0058] Figure 17 The different nanoparticles prepared in Example 3 were used to investigate their targeting effects on the mouse brain. The bar chart from left to right shows free Cy5, ginger exosomes, quercetin-coated targeting liposomes, and ginger exosome-liposome composite targeting nanoparticles.

[0059] Figure 18 The therapeutic effects of different nanoparticles prepared in Example 3 on mice were evaluated. Detailed Implementation

[0060] To make the present invention easier to understand, the present invention will be further described below with reference to specific embodiments. These embodiments are not intended to limit the present invention in any way. They are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Any modifications or changes to the present invention that are easily implemented by those skilled in the art without departing from the technical solution of the present invention will fall within the scope of the claims of the present invention.

[0061] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Experimental methods not specifically described in the examples are generally performed under standard conditions or as recommended by the manufacturer.

[0062] Soybean lecithin, cholesterol, phosphatidylethanolamine-polyethylene glycol 2000-maleimide (DSPE-PEG2000-Mal), quercetin, and other raw materials or reagents were purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd. or other commercially available manufacturers.

[0063] The targeted peptides are either synthesized by biotechnology companies or obtained directly from commercial products.

[0064] Example 1

[0065] The specific steps for preparing quercetin-coated targeted liposomes are as follows:

[0066] (1) Weigh 10 mg of soybean lecithin, 3 mg of cholesterol, and 0.5 mg of phosphatidylethanolamine-polyethylene glycol 2000-maleimide (DSPE-PEG2000-Mal). Weigh 2.3 mg of the antioxidant drug quercetin. Dissolve all materials and drugs together in 5 mL of dichloromethane solution, stir for 10 min, remove dichloromethane by rotary vacuum, add 10 mL of PBS (pH 7.4), and stir at 60 °C for 30 min to obtain a liposome solution coated with antioxidant drugs.

[0067] (2) A peptide targeting nerve cells was synthesized using solid-phase synthesis technology. The peptide has the sequence CIKRG and has a thiol terminus, which can covalently react with the maleimide group exposed on the surface of the DSPE-PEG2000-Mal in the liposomes coated with antioxidant drugs. Specifically, 0.3 mg of the targeting peptide was dissolved in 0.2 mL of PBS (pH 7.4) under a nitrogen atmosphere and added to the liposome solution prepared in step (1). The reaction was carried out at 25 °C for 24 h. The solution was dialyzed five times using a 3 KD pore size ultrafiltration tube for 30 min each time to remove uncoated quercetin and residual targeting peptides, thus obtaining a quercetin-coated targeting liposome solution.

[0068] The targeted peptides prepared in this example were subjected to structural identification and purity analysis using mass spectrometry and high-performance liquid chromatography, such as... Figure 1 As shown, the mass spectra of the obtained targeting peptides are: C 23 H 45 N9O6S[M+H] + :575.37, with a purity of over 95%, it can be used for subsequent experiments.

[0069] The morphology of the quercetin-coated targeted liposomes prepared in this example was characterized using TEM, such as... Figure 2 As shown, Figure 2The quercetin-coated targeted liposomes formed nanoparticles that were uniform in size and dispersed individually. The particle size of the quercetin-coated targeted liposomes prepared in this example was characterized using NTA. Figure 3 As shown, by Figure 3 As can be seen, the average particle size of the quercetin-coated targeted liposomes prepared in this embodiment is 158 nm. The zeta potential of the quercetin-coated targeted liposomes prepared in this embodiment was investigated, as shown below. Figure 4 As shown, group a consists of liposomes, group b consists of liposomes coated with quercetin (i.e., liposomes of antioxidant drugs), group c consists of targeting peptides, and group d consists of targeting liposomes coated with quercetin (i.e., targeting liposomes coated with quercetin). Figure 4 It can be seen that the liposome potentials before and after coating with quercetin are both negative and change little. The potential of the targeting peptide is 14 mV. This is attributed to the presence of positively charged amino acid residues in the targeting peptide. After the targeting peptide is covalently linked to the liposome surface coated with quercetin, the potential decreases to -22 mV.

[0070] The drug encapsulation efficiency of the quercetin-coated targeted liposomes prepared in this embodiment was calculated, and the encapsulation efficiency of quercetin was found to be 45%.

[0071] The quercetin-coated targeted liposomes prepared in this example were observed using an inverted fluorescence microscope. Figure 5 As shown, Figure 5 In the diagram, A represents a Dil-labeled liposome. Figure 5 B is a FITC-labeled targeting peptide. Figure 5 C is Figure 5 China A and Figure 5 The result shows the overlay of the image with B in the middle. Figure 5 The red fluorescence of Dil in A and Figure 5 After superposition of the green fluorescence of FITC in B Figure 5 The presence of orange fluorescence in C indicates that the targeting peptide was successfully covalently attached to the surface of the quercetin-coated liposomes. The grafting rate of the targeting peptide on the quercetin-coated liposomes prepared in this example was investigated using fluorescence spectroscopy. The fluorescence intensity of the FITC-labeled quercetin-coated liposomes was measured at an excitation wavelength of 490 nm and an emission wavelength of 520 nm. Figure 6 As shown, the results indicate that the grafting rate of the targeted peptide was 70.6%. This example demonstrates the successful preparation of targeted liposomes coated with quercetin.

[0072] Example 2

[0073] The specific steps for preparing ginger exosomes are as follows:

[0074] Weigh 500g of fresh ginger, wash it with deionized water, peel it, and cut it into uniform slices. Add 100mL of water. PBS (pH 7.4) was homogenized in a high-speed homogenizer for 1 min. The resulting ginger juice was centrifuged at 3000g for 20 min at 4℃, and this process was repeated twice to remove large fibers. The supernatant was then collected and centrifuged at 10000g for 40 min to remove small fibers. The supernatant was then collected and centrifuged at 150000g for 120 min to obtain ginger exosome precipitate. Finally, the ginger exosome precipitate was resuspended in 2 mL of PBS (pH 7.4) buffer. 8 mL of sucrose solutions with mass fractions of 8%, 30%, 45%, and 60% were added to centrifuge tubes, respectively. The plant exosome precipitate suspension was then added and centrifuged at 150000g for 120 min for purification. The liquid between 8% and 30% sucrose was collected to obtain the ginger exosome solution (i.e., when different concentrations of sucrose (8%, 30%, 45%, and 60%) are added to the same centrifuge tube, stratification occurs during centrifugation. After ultra-high-speed centrifugation, the plant exosomes are located in the layer between the 8% and 30% sucrose concentrations). The morphology of the ginger exosomes prepared in this example was characterized using TEM, such as... Figure 7 As shown, Figure 7 The ginger exosomes prepared in this example formed nanoparticles that were uniform in size and dispersed individually. The particle size of the ginger exosomes was characterized using NTA. Figure 8 As shown, by Figure 8 It can be seen that the average particle size of the ginger exosomes prepared in this embodiment is 126 nm.

[0075] Example 3

[0076] The specific steps for preparing ginger exosome-liposome composite targeted nanoparticles are as follows:

[0077] Take 1 mL of the ginger exosome solution prepared in Example 2 and 880 μL of the quercetin-coated targeted liposomes prepared in Example 1. Calculate the exosome to liposome ratio to be 1:1 according to NTA. Sonicate for 60 s, incubate at 4 °C for 24 h, and then extrude 10 times using a 0.2 μm pore size needle extruder to prepare ginger exosome-liposome composite targeted nanoparticles, which is the plant exosome-liposome composite targeted nanoparticle drug delivery system of this invention.

[0078] The morphology of the ginger exosome-liposome composite targeted nanoparticles prepared in this example was characterized using TEM, such as... Figure 9 As shown, the ginger exosome-liposome composite targeted nanoparticles formed regular morphology and were individually dispersed. The particle size of the ginger exosome-liposome composite targeted nanoparticles prepared in this example was characterized using NTA, as shown... Figure 10As shown, the average particle size of the ginger exosome-liposome composite targeting nanoparticles prepared in this embodiment is 161 nm. The zeta potential of the ginger exosome-liposome composite targeting nanoparticles prepared in this embodiment was investigated, as shown below. Figure 11 As shown, group a consists of quercetin-coated targeted liposomes, group b consists of ginger exosomes, and group c consists of ginger exosome-liposome composite targeted nanoparticles. Figure 11 It can be seen that ginger exosomes have a negative potential due to the presence of negatively charged groups. The potential change between ginger exosomes and liposomes coated with quercetin-targeting liposomes is small.

[0079] Example 4

[0080] 4.4 mL and 8.8 mL of the quercetin-coated targeted liposomes obtained in Example 1 were taken, along with 1 mL of ginger exosomes stained at a Dil to Did molar ratio (1:1). The ratios of liposomes to exosomes were calculated according to NTA as 5:1 and 10:1, respectively. After sonication for 60 s and incubation at 4°C for 24 h, the composite targeted nanoparticles were obtained by extrusion 10 times using a 0.2 μm pore size extruder. Fluorescence detection of the ginger exosome-liposome composite targeted nanoparticles prepared in this example was performed using a fluorescence spectrometer, with an excitation wavelength of 525 nm and an emission wavelength of 550-800 nm. Figure 12 As shown, with the increase of liposome ratio, the degree of complexation increases, the distance between the donor Dil and the receptor Did dye increases, and the fluorescence intensity of Did decreases between 650-700 nm, indicating that ginger exosomes and quercetin-coated targeted liposomes are successfully complexed.

[0081] Ginger exosomes were labeled with the dye Dio, and quercetin-coated targeting liposomes were labeled with the dye Dil. The exosome-to-liposome particle ratios were calculated to be 1:1, 1:3, 1:6, 3:1, and 6:1 according to NTA. After sonication for 60 s and incubation at 4°C for 24 h, composite targeting nanoparticles were prepared by extrusion 10 times using a 0.2 μm pore size extruder. The ginger exosome-liposome composite targeting nanoparticles prepared in this example were observed using a fluorescence microscope. Figure 13 As shown, the composite efficiency is 82% when the particle number ratio is 1:1, which is the best result.

[0082] Example 5

[0083] The ginger exosome-liposome composite targeting nanoparticles prepared in Example 3 and the quercetin-coated targeting liposomes prepared in Example 1 were incubated in PBS buffer at 37°C, pH 7.4 for 5 days, during which time the particle size was measured daily. The results are as follows: Figure 14As shown in Figure A, the particle size of the ginger exosome-liposome composite targeting nanoparticles remained essentially unchanged, indicating structural stability. This is attributed to the stable phospholipid bilayer membrane structure of ginger exosomes. The particle size of the quercetin-coated targeting liposomes gradually decreased over 5 days, indicating structural degradation. Figure 14 B.

[0084] Example 6

[0085] 10 5 / mL of human neuroblastoma cells SH-SY5Y were seeded in 96-well plates at a volume of 200 μL per well. Six wells were set up as a blank control group, and the remaining wells were filled with 100 μM H2O2. The cells were cultured at 37°C and 5% CO2 for 24 h to induce oxidative stress. The drug-treated groups included: quercetin-coated targeted liposomes (Example 1), ginger exosomes (Example 2), and ginger exosome-liposome composite targeted nanoparticles (Example 3). The number of particles in each of the quercetin-coated targeted liposomes, ginger exosomes, and ginger exosome-liposome composite targeted nanoparticles was 5*10. 6 / mL. Six wells were used in each group, and cells were cultured at 37℃ and 5% CO2 for 24 h. Cell viability in each group was assessed using the MTT assay. Experimental results are shown below. Figure 15 As shown in A, compared with the H2O2 group, the other three groups could restore the cell viability of human neuroblastoma cells SH-SY5Y, among which the cell viability of ginger exosome-liposome composite targeted nanoparticles was 86%.

[0086] Following the experimental procedures described above, different numbers of ginger exosome-liposome composite targeted nanoparticles were added to the drug-treated groups, including: 0.1*10 6 0.5*10 6 1.0*10 6 5.0*10 6 10*10 6 50*10 6 / mL. Experimental results are as follows: Figure 15 As shown in B, when the number of particles is 10*10 6 At a concentration of 92% per mL, the cell viability was 92%, resulting in the best effect.

[0087] Example 7

[0088] 10 4 / mL SH-SY5Y cells were seeded in 12-well plates, with 6 wells set up as a blank control group, and cultured at 37℃ and 5% CO2 for 24 h. The drug-treated groups included: quercetin-coated targeted liposomes (Example 1), ginger exosomes (Example 2), and ginger exosome-liposome composite targeted nanoparticles (Example 3), wherein the number of particles in each of the quercetin-coated targeted liposomes, ginger exosomes, and ginger exosome-liposome composite targeted nanoparticles was 1*10-1. 7 / mL. Six wells were used in each group, and cells were cultured at 37℃ and 5% CO2 for 24 h. Cells were collected, washed three times with PBS, and centrifuged at 1200g for 10 minutes to collect the cell pellet. Western blotting was used to detect the expression of autophagy-related proteins LC3-I and LC3-II. Experimental results are as follows: Figure 16 As shown, ginger exosome-liposome composite targeted nanoparticles significantly upregulated LC3-II / LC3-I expression, indicating that they effectively activated the autophagy function of SH-SY5Y cells.

[0089] Through Examples 6 and 7, the ginger exosome-liposome composite targeted nanoparticles prepared in this invention utilize the active substances carried by plant exosomes to interact with the TRPV1 channel protein on the surface of nerve cells, leading to an increase in intracellular calcium ion concentration, thereby activating and inducing autophagy. At the same time, the encapsulated antioxidants play a role in scavenging reactive oxygen species to inhibit neuroinflammation, exhibiting a significant synergistic effect of bidirectional regulation, and ultimately achieving the goal of synergistic treatment of PD.

[0090] Example 8

[0091] Male black mice aged 6-8 weeks were used to establish a PD mouse model via routine intraperitoneal injection of MPTP. The PD model mice were divided into four groups. Different samples labeled with 200 μL of Cy5 (free Cy5, ginger exosomes (Example 2), quercetin-coated targeted liposomes (Example 1), and ginger exosome-liposome composite targeted nanoparticles (Example 3)) were injected via tail vein (200 μL per mouse, concentration 1 x 10⁻⁶). 9 Small animal brain imaging was performed at 1, 5, 9, and 13 hours (particles / mL). The experimental results are as follows: Figure 17As shown, free Cy5 and ginger exosomes exhibit poor brain targeting ability, while targeted liposomes demonstrate better brain targeting effects compared to free Cy5. Furthermore, the targeting effect of ginger exosome-liposome composite targeted nanoparticles is further improved compared to targeted liposomes, indicating that the composite drug delivery system maintains its targeting function and achieves better results. Therefore, the composite targeted nanoparticles prepared in this invention combine the advantages of both plant exosomes and liposomes, overcoming their respective shortcomings, actively targeting and delivering drugs. They can escape phagocytosis by immune cells and reticuloendothelial tissue, and simultaneously penetrate the blood-brain barrier, exhibiting high biocompatibility, good drug carrier stability, and improved drug delivery efficiency.

[0092] Example 9

[0093] PD model mice were divided into four groups: PBS, ginger exosomes (Example 2), quercetin-coated targeted liposomes (Example 1), and ginger exosome-liposome composite targeted nanoparticles (Example 3). The mice were administered via tail vein injection (200 μL per mouse, concentration 1 x 10⁻⁶). 9 (particles / mL), with a sham surgery as a blank control. Mice were tested in a 120cm diameter water maze. The water temperature was maintained at 20±2℃. There were four equidistant points on the pool wall, dividing the maze into four quadrants. The platform was fixed 1cm underwater and was invisible. Each mouse was trained twice a day in the dark for 5 days, with a 20-minute interval between each session. Mice from different groups were placed in the same starting position in different quadrants for training. If a mouse successfully found the platform within 60 seconds, it was allowed to stay on the platform for 10 seconds; if it did not find the platform, it was guided to stay on the platform for 30 seconds. Twenty-four hours after the location training ended and the platform was removed, the mice's spatial orientation ability was tested. Mice from different groups were placed in the same starting position in the quadrant opposite to the platform, and each trained mouse was allowed to swim freely for 60 seconds. The trajectory of each mouse was recorded and analyzed using the ANY maze video analysis system, recording parameters such as escape time, time spent in the area, average speed, and number of times the mouse crossed the platform. The results are as follows: Figure 18 As shown, compared with PBS, ginger exosomes, quercetin-coated targeted liposomes, and ginger exosome-liposome composite targeted nanoparticles can all effectively improve the memory and learning abilities of mice. Moreover, the ginger exosome-liposome composite targeted nanoparticles have the best therapeutic effect, further demonstrating that the composite targeted system prepared in this invention can both regulate the autophagy function of nerve cells and inhibit neuroinflammation, exhibiting bidirectional regulatory function and producing a significant synergistic effect, achieving the goal of multi-target therapy. This provides a new approach for the efficient treatment of neurodegenerative diseases such as Parkinson's disease.

[0094] Example 10

[0095] Example 10 uses the preparation method of Example 1, except that: the functional terminus of the phosphatidylethanolamine-polyethylene glycol with a functional terminus is succinimide (phosphatidylethanolamine-polyethylene glycol 2000-succinimide), the targeting peptide is NGR, the antioxidant drug is vitamin C, and the mass ratio of lecithin, cholesterol and phosphatidylethanolamine-polyethylene glycol with a functional terminus is 50:20:5.

[0096] Example 11

[0097] Example 11 uses the preparation method of Example 1, except that: the functional terminus of the phosphatidylethanolamine-polyethylene glycol with a functional terminus is amino (phosphatidylethanolamine-polyethylene glycol 2000-aminoDSPE-PEG2000-NH2), the targeting polypeptide is CPPs, the antioxidant drug is β-carotene, and the mass ratio of lecithin, cholesterol and phosphatidylethanolamine-polyethylene glycol with a functional terminus is 150:40:5.

[0098] Example 12

[0099] Example 12 uses the preparation method of Example 1, except that: the functional terminal of the phosphatidylethanolamine-polyethylene glycol with a functional terminal is a carboxyl group (phosphatidylethanolamine-polyethylene glycol 2000-carboxylDSPE-PEG2000-COOH), the targeting peptide is NYZL1, and the antioxidant is resveratrol.

[0100] Example 13

[0101] Example 13 uses the preparation method of Example 2, except that: plant exosomes are obtained by extracting plants by differential centrifugation; the plant is garlic.

[0102] Example 14

[0103] Example 14 uses the preparation method of Example 2, except that: plant exosomes are obtained by extracting plants by differential centrifugation; the plant is lemon.

[0104] Example 15

[0105] Example 15 uses the preparation method of Example 2, except that: the targeted liposomes coated with antioxidant drugs prepared in Example 10 and the plant exosomes prepared in Example 14 are prepared by membrane coating with an exosome-liposome composite nanoparticle with a ratio of 1:1 based on NTA calculation.

Claims

1. The application of a plant exosome-liposome composite targeted nanoparticle drug delivery system in the preparation of drugs for treating neurodegenerative diseases, wherein the plant exosomes are ginger exosomes, and the preparation of the plant exosome-liposome composite targeted nanoparticle drug delivery system includes the following steps: The specific steps for preparing quercetin-coated targeted liposomes are as follows: (1) Weigh 10 mg of soybean lecithin, 3 mg of cholesterol and 0.5 mg of phosphatidylethanolamine-polyethylene glycol 2000-maleimide, and weigh 2.3 mg of quercetin. Dissolve all materials and drugs together in 5 mL of dichloromethane solution, stir for 10 min, rotate and vacuum to remove dichloromethane, add 10 mL of pH 7.4 PBS, stir at 60 °C for 30 min to obtain liposome solution coated with antioxidant drugs; (2) Synthesize a peptide targeting nerve cells with the sequence CIKRG. Dissolve 0.3 mg of the peptide in 0.2 mL of pH 7.4 PBS under a nitrogen atmosphere and add it to the liposome solution in step (1). React at 25 °C for 24 h. Dialyze the solution five times using a 3 KD pore size ultrafiltration tube for 30 min each time to remove uncoated quercetin and residual targeting peptides, and obtain a quercetin-coated targeting liposome solution. The specific steps for preparing ginger exosomes are as follows: Weigh 500g of fresh ginger, wash it with deionized water, peel it, and cut it into uniform slices. Add 100mL of pH7.4 PBS and stir in a high-speed homogenizer for 1min. Centrifuge the obtained ginger juice at 3000g for 20min at 4℃, repeating twice to remove large fibers. Then, take the supernatant and centrifuge at 10000g for 40min to remove small fibers. Take the supernatant and centrifuge at 150000g for 120min to obtain ginger exosome precipitate. Finally, suspend the ginger exosome precipitate in 2mL of pH7.4 PBS buffer. Add 8mL of sucrose solution with a mass fraction of 8%, 30%, 45%, and 60% to centrifuge tubes, respectively. Then, add the plant exosome precipitate suspension and centrifuge at 150000g for 120min for purification. Collect the liquid between 8% and 30% sucrose to obtain ginger exosome solution. The specific steps for preparing ginger exosome-liposome composite targeted nanoparticles are as follows: Take 1 mL of the prepared ginger exosome solution and 880 μL of the prepared quercetin-coated targeted liposomes, with a particle ratio of exosomes to liposomes of 1:

1. After sonication for 60 s and incubation at 4 °C for 24 h, the ginger exosome-liposome composite targeted nanoparticles, i.e., the plant exosome-liposome composite targeted nanoparticle drug delivery system, are prepared by extrusion 10 times using a 0.2 μm pore size needle extruder.

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