Aptamer-modified nerve cell membrane hybrid liposome, preparation method thereof and application of aptamer-modified nerve cell membrane hybrid liposome in domoic acid detoxification

Abstract

The invention relates to the technical field of marine organisms, in particular to aptamer-modified nerve cell membrane hybrid lipidosome, a preparation method thereof and application of the aptamer-modified nerve cell membrane hybrid lipidosome in domoic acid detoxification. According to the invention, a cell membrane of a human hippocampal neuron HPPNCs cell line is hybridized with liposome, and then the surface of the cell membrane is modified with an aptamer which is specifically combined with domoic acid. High performance liquid chromatography detection and cellular level experiments both prove that after incubation treatment of the aptamer-modified nerve cell membrane hybrid liposome, the concentration of domoic acid in a system is obviously reduced, apoptosis and oxidative stress damage caused by domoic acid to nerve cells can be obviously improved, and the aptamer-modified nerve cell membrane hybrid liposome has detoxification capacity on domoic acid. The invention develops a new detoxification idea for the field of prevention and treatment of domoic acidosis, and has application potential.

Description

Technical Field

[0001] This invention relates to the field of marine biotechnology, specifically to an aptamer-modified hybrid liposome of the neural cell membrane, its preparation method, and its application in the detoxification of domoic acid. Background Technology

[0002] Toxins produced by marine algae and microorganisms can accumulate in marine shellfish, fish, and other organisms, and are transmitted through the food chain, causing significant negative impacts on aquaculture and posing a threat to human health. Domoic acid (DA) is a neurotoxin mainly produced by certain rhomboid algae and diatoms within rhomboid algae. It can directly bind to glutamate receptors, promoting the release of endogenous glutamate, thereby mediating damage to nerve tissue. Ingestion of DA in humans can cause poisoning symptoms such as abdominal pain, diarrhea, and vomiting. Severe cases may result in memory loss, confusion, and even coma and death.

[0003] Currently, research on dopamine (DA) focuses on its detection methods and neurotoxicity mechanisms, but there are still no effective antagonistic drugs to prevent or treat DA neurotoxicity. Clinically, acute neurological symptoms caused by DA are mostly treated symptomatically with anticonvulsants. Therefore, exploring DA detoxification methods will help in taking effective preventive and control measures, and improving food safety and biosafety levels.

[0004] In recent years, studies have utilized the interaction between specific receptors / ion channels on cell membranes and toxins to design various biomimetic nanoparticles and apply them to the neutralization and antagonism of various marine neurotoxins such as tetrodotoxin. However, obtaining large quantities of cell membranes is challenging. Liposomes have a similar structure to cell membranes and exhibit a strong affinity for them. Fusing liposomes with cell membranes creates cell membrane chimeric liposomes. Cell membrane chimeric liposomes can also function by binding toxins to membrane receptors, but with less cell membrane required. Furthermore, cell membrane chimeric liposomes have longer circulation times and better bioavailability than simple cell membrane nanoparticles, and the incorporated artificial lipid portion is easily functionally modified, thus exhibiting significant advantages.

[0005] Nucleic acid aptamers can bind to a variety of targets with high specificity and high affinity, enabling them to specifically recognize toxins. Their toxin-binding ability makes them potential applications in toxin detoxification and poisoning prevention. However, free nucleic acid aptamers are easily degraded by nucleases and rapidly filtered by the kidneys, thus limiting their therapeutic applications. Currently, there are no reports of using nucleic acid aptamers for domoic acid detoxification. Summary of the Invention

[0006] The purpose of this invention is to construct a novel doxorubicin neurotoxin antagonistic system, Apt-HPPNCs@lip, which couples the DA nucleic acid aptamer to a hybrid liposome on the hippocampal neuronal cell membrane. This system simultaneously exerts a dual-modal detoxification effect by binding the toxin to specific receptors on the neuronal cell membrane and the aptamer, providing a new approach for the effective prevention and treatment of DA poisoning.

[0007] To achieve the above objectives, the present invention employs the following... Figure 1 The technical solution shown:

[0008] 1. Preparation of hybrid liposomes of neural cell membranes (HPPNCs@lip): After obtaining the cell membrane of the human hippocampal neuronal HPPNCs cell line using the pure water hypotonic method, HPPNCs@lip was prepared by thin film hydration and co-extrusion methods;

[0009] 2. Conjugation of nucleic acid aptamers with HPPNCs@lip: First, the selected DA nucleic acid aptamer sequence (Apt) was amplified by PCR, with cholesterol linked to its 3′ end. Then, the 3′ end of the nucleic acid aptamer was conjugated to HPPNCs@lip via hydrophobic interactions to construct a nucleic acid aptamer-modified neural cell membrane hybrid liposome (Apt-HPPNCs@lip). The success of the nucleic acid aptamer conjugation was detected by urea-polyacrylamide gel electrophoresis. Fluorescent labeling was added to the 5′ end of the aptamer, and the fluorescence values ​​before and after ultrafiltration were measured. The ratio of these values ​​was used to calculate the nucleic acid aptamer conjugation efficiency.

[0010] 3. Safety and stability assays of Apt-HPPNCs@lip: cytotoxicity of Apt-HPPNCs@lip to mouse fibroblast L929 and microglia BV2; stability assay of Apt-HPPNCs@lip in phosphate-balanced saline containing 10% fetal bovine serum;

[0011] 4. Determination of the toxin neutralization efficacy of Apt-HPPNCs@lip: The toxin adsorption and neutralization efficiency of Apt-HPPNCs@lip was determined by ultra-high performance liquid chromatography-tandem mass spectrometry; the protective effect of Apt-HPPNCs@lip against DA neurotoxicity was determined in a microglia BV2 exposure model.

[0012] Based on the above technical solution, in a first aspect, the present invention provides an aptamer-modified hybrid liposome of neural cell membrane, which is prepared by hybridizing the cell membrane of human hippocampal neuronal HPPNCs cell line with liposomes to prepare a hybrid liposome of neural cell membrane (HPPNCs@lip), and then modifying its surface with a nucleic acid aptamer (M70-b) that specifically binds to doucolic acid (DA); the nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID NO.1.

[0013] Furthermore, the 3′ end of the nucleic acid aptamer is linked to cholesterol, and the 3′ end of the nucleic acid aptamer is coupled to HPPNCs@lip through hydrophobic interaction to construct an aptamer-modified neural cell membrane hybrid liposome (Apt-HPPNCs@lip).

[0014] A second aspect of the present invention provides a method for preparing aptamer-modified neural cell membrane hybrid liposomes as described above, comprising the following steps:

[0015] (A) Preparation of neural cell membrane hybrid liposomes (HPPNCs@lip): After obtaining the cell membrane of the human hippocampal neuronal HPPNCs cell line using the pure water hypotonic method, HPPNCs@lip was prepared by thin film hydration and co-extrusion methods;

[0016] (B) Coupling of nucleic acid aptamers with HPPNCs@lip: First, the DA nucleic acid aptamer sequence (Apt) was amplified by PCR, with cholesterol attached to its 3′ end; then, the 3′ end of the nucleic acid aptamer was coupled to HPPNCs@lip through hydrophobic interactions to construct a nucleic acid aptamer-modified neural cell membrane hybrid liposome (Apt-HPPNCs@lip).

[0017] Further, step (A) involves first lysing human hippocampal neurons in pure water at 4°C using hypotonic methods for 30 min, centrifuging at 2000×g for 20 min, collecting the supernatant, centrifuging at 4000×g for 45 min, dissolving the precipitate with PBS to obtain an HPPNCs cell membrane dispersion; dissolving lecithin, DSPE-PEG2000, and cholesterol together in chloroform, and rotary evaporating at 37°C to obtain a liposome membrane; adding HPPNCs cell membrane dispersion at a mass concentration ratio of 1:1 to the liposome membrane, and hydrating at 37°C for 1 h to obtain a homogeneous suspension; and sequentially pressing the suspension through polyester membranes of 400 nm, 200 nm, and 100 nm to finally obtain HPPNCs@lip.

[0018] Furthermore, the mass percentages of lecithin, DSPE-PEG2000, and cholesterol are 90%:5%:5%.

[0019] Further, during the coupling in step (B), 2 μM of nucleic acid aptamer M70-b was mixed with 1 mg / mL HPPNCs@lip and incubated at 37 °C for 12 h. The mixture was then ultrafiltered at 12,000 × g for 30 min using a 50 kD ultrafiltration tube. The liquid from the upper tube was collected to obtain aptamer-modified neural cell membrane hybrid liposomes.

[0020] A third aspect of the present invention provides the use of the aptamer-modified neural cell membrane hybrid liposomes as described above in the preparation of a medicament for preventing and treating domoic acid poisoning.

[0021] In a fourth aspect, the present invention provides the application of aptamer-modified neural cell membrane hybrid liposomes as described above in the preparation of products for the recognition, trapping, adsorption, and detoxification of doxorubicin.

[0022] In a fifth aspect, the present invention provides the use of the aptamer-modified neural cell membrane hybrid liposomes as described above in the preparation of a drug that protects against domoic acid-induced neural cell death.

[0023] In a sixth aspect, the present invention provides the use of aptamer-modified neural cell membrane hybrid liposomes as described above in the preparation of a drug to protect against oxidative stress damage to neural cells induced by domoic acid.

[0024] The advantages of this invention are:

[0025] 1. This invention provides a hybrid liposome of the neural cell membrane modified with a high-affinity DA nucleic acid aptamer. It involves hybridizing the cell membrane of a human hippocampal neuronal (HPPNC) cell line with a liposome, and then modifying the surface of the liposome with an aptamer that specifically binds to domoic acid. This increases the stability of the nucleic acid aptamer and allows for the synergistic effect of natural toxin binding receptors on the neural cell membrane and the DA aptamer in recognizing and trapping toxins, potentially significantly enhancing the detoxification effect.

[0026] 2. High-performance liquid chromatography (HPLC) and cell-level experiments both demonstrated that the concentration of domoic acid in the system after incubation with the aptamer-modified hybrid liposomes of the present invention was significantly reduced, which can significantly improve the apoptosis and oxidative stress damage of domoic acid to nerve cells and has the ability to detoxify domoic acid.

[0027] 3. This invention expands new detoxification strategies for the prevention and treatment of domoic acid poisoning and has application potential. Attached Figure Description

[0028] Figure 1 A schematic diagram of nucleic acid aptamers coupled with hybrid liposomes of neural cell membranes.

[0029] Figure 2 Fusion verification of hybrid liposomes HPPNCs@lip for neural cell membranes. (A) Physical mixing of liposomes and neural cell membranes with fluorescence colocalization images of hybrid liposomes for neural cell membranes; (B) Fluorescence spectra of liposomes labeled with the fluorescent dyes Rhodamine B and anthocyanin Cy5 after hybridization with neural cell membranes; (C) Comparison of Fourier transform infrared spectra of neural cell membranes, blank liposomes, and HPPNCs@lip; (D) Comparison of protein spectra of HPPNCs@lip and cell membranes; (E) Expression level of GLUK1 protein in HPPNCs@lip.

[0030] Figure 3 Nucleic acid aptamer ligation verification. (A) is the mass spectrum of DA nucleic acid aptamer M70-b; (B) is the urea polyacrylamide gel electrophoresis image, lane 1 is M70-b, and lane 2 is Apt-HPPNCs@lip.

[0031] Figure 4 Characterization of nucleic acid aptamer-coupled neural cell membrane hybrid liposomes Apt-HPPNCs@lip. (A) Potential magnitudes of blank liposomes (lip), neural cell membrane hybrid liposomes (HPPNCs@lip), and nucleic acid aptamer-coupled neural cell membrane hybrid liposomes (Apt-HPPNCs@lip) measured by dynamic light scattering (DLS); (B) Particle sizes of lip, HPPNCs@lip, and Apt-HPPNCs@lip; (C) Morphological characteristics of Apt-HPPNCs@lip observed by transmission electron microscopy (TEM).

[0032] Figure 5 Safety assay of nucleic acid aptamer-coupled neural cell membrane hybrid liposomes Apt-HPPNCs@lip. (A) Cell viability after co-incubation of L929 cells with different concentrations of Apt-HPPNCs@lip; (B) Cell viability after co-incubation of BV2 cells with different concentrations of Apt-HPPNCs@lip.

[0033] Figure 6 Stability assay of nucleic acid aptamer-coupled neural cell membrane hybrid liposomes Apt-HPPNCs@lip. (A) Changes in particle size and zeta potential of Apt-HPPNCs@lip in PBS over 7 days were measured using DLS; (B) Degradation of Apt-HPPNCs@lip aptamers after treatment with 10% fetal bovine serum.

[0034] Figure 7 Detoxification efficiency of nucleic acid aptamer-coupled neural cell membrane hybrid liposomes Apt-HPPNCs@lip. (A) High-performance liquid chromatography-mass spectrometry (HPLC-MS) multiple reaction detection chromatograms of residual DA after co-incubation of 5 mg / mL HPPNCs@lip and Apt-HPPNCs@lip with 0.1 μM DA; (B) Quantitative analysis of characteristic peak area (n=3, P < 0.01; P < 0.001.

[0035] Figure 8Changes in mitochondrial membrane potential in microglia after detoxification of nucleic acid aptamer-coupled neural cell membrane hybrid liposomes Apt-HPPNCs@lip. (A) Detection of mitochondrial membrane potential in BV2 microglia after co-incubation of 5 mg / mL HPPNCs@lip and Apt-HPPNCs@lip with 0.1 μM DA (scale bar 100 μm); (B) Average quantitative fluorescence analysis (n=3, P < 0.0001.

[0036] Figure 9 Changes in antioxidant enzymes in microglia after detoxification of aptamer-coupled neural cell membrane hybrid liposomes Apt-HPPNCs@lip. (A) Changes in the expression of heme oxygenase-1 (HO-1) mRNA in BV2 microglia after co-incubation of 5 mg / mL HPPNCs@lip and Apt-HPPNCs@lip with 0.1 μM DA; (B) Changes in the activity of superoxide dismutase (SOD) in BV2 microglia after co-incubation of 5 mg / mL HPPNCs@lip and Apt-HPPNCs@lip with 0.1 μM DA. Detailed Implementation

[0037] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments.

[0038] Example 1: Preparation and characterization of hybrid liposomes HPPNCs@lip for neural cell membranes

[0039] like Figure 1 As shown, human hippocampal neuronal cells (HPPNCs) were extracted and then used to prepare hybrid liposomes using a membrane hydration and co-extrusion method. Specifically, human hippocampal neuronal cells were lysed in pure water at 4°C under hypotonic conditions for 30 min, centrifuged at 2000×g for 20 min, and the supernatant was collected and centrifuged at 4000×g for 45 min. The precipitate was dissolved in PBS. Lecithin, DSPE-PEG2000, and cholesterol (90%, 5%, and 5% by mass, respectively) were dissolved in chloroform and evaporated at 37°C in a rotary evaporator to obtain liposome membranes. HPPNCs cell membrane dispersion (1:1 mass ratio) was added to the liposome membranes, and the mixture was hydrated at 37°C for 1 h until a homogeneous suspension was obtained. The suspension was then sequentially extruded through polyester membranes of 400 nm, 200 nm, and 100 nm to obtain HPPNCs@lip.

[0040] like Figure 2 As shown in Figure A, the nerve cell membrane is stained with blue fluorescent dye, and the blank liposomes are stained with red fluorescent dye. Fluorescence colocalization shows that the blue and red fluorescence overlap. Figure 2As shown in B, the position of the fluorescence spectrum peak changes after liposomes simultaneously labeled with the fluorescent dyes Rhodamine B and anthocyanin Cy5 are fused with the nerve cell membrane. Figure 2 As shown in C, the peak positions of the Fourier transform infrared spectra of the nerve cell membrane and HPPNCs@lip are basically coincident. Figure 2 As shown in D, the protein maps of HPPNCs@lip and nerve cell membranes are basically consistent, and Figure 2 E indicates that HPPNCs@lip retains the GLUK1 protein, the receptor for DA toxin action on the nerve cell membrane; in summary, hybrid liposomes of the nerve cell membrane were successfully prepared.

[0041] Example 2: Preparation of Apt-HPPNCs@lip

[0042] First, the DA aptamer M70-b, which is reported to have high affinity in the literature, was selected. Then, the above sequence was amplified by PCR, and cholesterol was added to the 3′ end of the aptamer, and different fluorescent groups were added to the 5′ end, specifically:

[0043] M70-b: 5′-AGGGGGGAGGGTGGTATGTGTCGGTGGGAGGGGTGC-3′ (SEQ ID NO.1)

[0044] The correctness of the synthesized aptamer was verified by mass spectrometry. The molar mass of M70-b was 11141.21 μg / μmol, the molar mass after 3-terminal modification with cholesterol was 11767.21 μg / μmol, and the molar mass after 3-terminal modification with cholesterol and 5-terminal modification with FAM fluorescent group was 12304.81 μg / μmol.

[0045] Figure 3 As shown in Figure A, no multiplets appeared on the mass spectrum, indicating that the sequence does not contain degenerate bases; and the molecular weight error is ≤0.05%, indicating that the aptamer sequence is correct.

[0046] 100 μM nucleic acid aptamer M70-b (2 μL) was mixed with 3 mg / mL HPPNCs@lip (100 μL) and incubated overnight at 37 °C. The mixture was then ultrafiltered at 12,000 × g for 30 min using a 100 kD ultrafiltration tube. The liquid in the upper tube was collected to obtain nucleic acid aptamer-coupled neural cell membrane hybrid liposomes. Figure 3 As shown in Figure B, the aptamer coupled with the neural cell membrane hybrid liposome had an increased molecular weight, and its band was positioned above the uncoupled aptamer during gel electrophoresis, indicating that aptamer M70-b was successfully coupled to the HPPNCs@lip surface. The total fluorescence value of the liquid before ultrafiltration and the fluorescence value of the liquid after ultrafiltration were measured using a fluorescence microplate reader, and the ratio was calculated, yielding an aptamer coupling rate of approximately 65% ​​at this feed ratio.

[0047] Example 3: Characterization and detection of Apt-HPPNCs@lip

[0048] The particle size and zeta potential of lip, HPPNCs@lip, and Apt-HPPNCs@lip were detected by dynamic light scattering, such as... Figure 4 As shown in A and B, the diameter of Apt-HPPNCs@lip is approximately 110 nm, and the zeta potential on its surface is approximately -35 mV. Figure 4 C shows that transmission electron microscopy reveals that Apt-HPPNCs@lip has a distinct lipid bilayer structure.

[0049] Example 4: Safety and stability testing of Apt-HPPNCs@lip

[0050] L929 and BV2 cells were incubated with different concentrations of Apt-HPPNCs@lip for 24 h, and cell viability was detected by CCK-8 assay. Figure 5 A and Figure 5 B shows that Apt-HPPNCs@lip has no cytotoxicity against L929 cells and BV2 cells, indicating that it has good safety at the cellular level.

[0051] Apt-HPPNCs@lip were dissolved in PBS and incubated at 37°C for 7 days. Particle size and potential were continuously monitored on days 1, 3, 5, and 7 using DLS. Figure 6 As shown in Figure A, the particle size and potential of Apt-HPPNCs@lip did not change significantly within 7 days. Apt-HPPNCs@lip was dissolved in a complete culture medium solution containing 10% fetal bovine serum and incubated at 37 °C for 48 h. Ultrafiltration was performed at different time points (0 h, 6 h, 12 h, 24 h, 48 h) using 3 kD ultrafiltration tubes at 12,000 × g for 30 min. The total fluorescence value before ultrafiltration and the fluorescence value of the tube after ultrafiltration were measured, and the ratio was calculated to determine the aptamer degradation rate. Figure 6 B showed that the degradation rate of the aptamer coupled to HPPNCs@lip was only 6% within 48 h. In summary, Apt-HPPNCs@lip exhibits good stability in both PBS and complete culture medium.

[0052] Example 5: Determination of the toxin neutralizing efficacy of Apt-HPPNCs@lip

[0053] 5 mg / mL HPPNCs@lip and Apt-HPPNCs@lip were incubated with 0.1 μM DA at 37 °C for 30 min. The bound Apt-HPPNCs-lip was removed by centrifugation at 12,000 × g for 30 min in a 30 kD ultrafiltration tube. The liquid in the tube was collected and the content of the remaining DA in the solution was determined by LC-MS / MS.

[0054] The LC-MS / MS detection conditions are as follows:

[0055] A 5TC-C18 column (4.6 mm × 12.5 mm, 5 μm) was used as the enrichment column, and a Zorbax Eclipse, XDB-C18 column (3 mm × 100 mm, 1.8 μm) was used as the analytical column. The column temperature was 25℃, the flow rate was 0.4 mL / min, the injection volume was 2 μL, and gradient elution was used.

[0056] Mass spectrometry conditions: AJS-ESI sources in both positive and negative modes were used to determine DA. The parameters used were as follows: AJS-ESI positive ion mode: capillary voltage: 4000 kV; sheath gas flow rate: 11 L / min; sheath gas temperature: 350 °C; nebulizer pressure: 40 psi; drying gas temperature: 300 °C; drying gas flow rate: 7 L / min. Multiple reaction monitoring (MRM) mode: DA precursor ion m / z: 312.1, fragmentation voltage: 130 V; daughter ion m / z: 266.2, 248.2, and 193.2, with 266.2 being the quantitative ion, and collision energies of 16 eV, 18 eV, and 20 eV, respectively; negative ion mode: DA precursor ion m / z: 310.1, fragmentation voltage: 95 V; daughter ion m / z: 266 and 222, with collision energies of 13 eV and 22 eV, respectively.

[0057] like Figure 7 As shown in Figure A, the three characteristic fragment peaks 193, 248, and 266 generated by the secondary mass spectrometry analysis all showed good responses, proving that the solution is a DA solution. Figure 7 B shows that HPPNCs@lip and Apt-HPPNCs@lip can effectively adsorb DA. At a concentration of 5 mg / mL, the adsorption efficiencies of HPPNCs@lip and Apt-HPPNCs@lip for DA are 53% and 80%, respectively. This is equivalent to 5 mg of Apt-HPPNCs@lip being able to adsorb approximately 25 ng of DA.

[0058] 0.1 μM DA and 5 mg / mL HPPNCs@lip and Apt-HPPNCs@lip were incubated in serum-free medium at 37°C for 2 h before ultrafiltration to remove bound nanoparticles. BV2 cells were seeded at a density of 5000 cells / well in 96-well plates, treated with ultrafiltration post-treatment solution for 48 h, and then incubated with the staining working solution according to the JC-1 fluorescent probe instructions in the dark. After washing with buffer to remove unbound probes, the cells were observed under a fluorescence microscope. Changes in the ratio of the fluorescence intensity of the JC-1 monomer to polymer were used to determine changes in mitochondrial membrane potential. Figure 8 A. Compared with the DA-only group, the 5 mg / mL Apt-HPPNCs-lip treatment group showed a significantly lower ratio of JC-1 monomer to polymer fluorescence intensity, effectively resisting DA-induced changes in mitochondrial membrane potential.

[0059] 0.1 μM DA and 5 mg / mL HPPNCs@lip and Apt-HPPNCs@lip were incubated in serum-free medium at 37°C for 2 h before ultrafiltration to remove the bound nanoparticles. BV2 cells were then incubated at 10... 5 Seeds were planted at a density of 1000 RNA molecules per well in 24-well plates. After ultrafiltration and liquid conditioning for 48 h, total RNA was extracted using the Molpure Cell RNA Kit. RNA concentration and purity were determined using a NanoDrop spectrophotometer, and then reverse transcription was performed using the Hifair AdvanceFast 1 reverse transcription kit. st Strand cDNA synthesis SuperMix for qPCR was performed for cDNA synthesis. The total reaction volume was 20 μL, containing 10 μL HieffUNICON Universal Blue qPCR SYBR Green Master Mix (2×), 0.4 μL forward primer (10 μM), 0.4 μL reverse primer (10 μM), 2 μL cDNA template, and 6.2 μL ddH2O. Pre-denaturation was performed at 95℃ for 2 min, followed by 40 cycles, each consisting of 95℃ denaturation for 10 s and 60℃ annealing / extension for 30 s. Melting curve analysis was then performed. Relative quantification was performed using the 2^(-ΔΔCt) method, with β-actin as an internal reference gene. Figure 9 A shows that, with DA processing group HO -1 Compared to significantly increased gene expression, the Apt-HPPNCs@lip treatment group effectively suppressed the effect of DA on its expression.

[0060] The primer sequences are as follows:

[0061]

[0062] BV2 cells were at 10 6 Cells were seeded at a density of [number] cells / well in 6-well plates. After ultrafiltration and liquid treatment for 48 h, the cell pellet was collected, lysed, and protein concentration was measured. SOD activity in each group was measured according to the instructions of the SOD kit (WST-1 method). Figure 9 As shown in Figure B, the SOD activity in the DA-treated group was significantly lower than that in the untreated group, while treatment with 5 mg / mL HPPNCs@lip and Apt-HPPNCs@lip significantly resisted the effect of DA on SOD activity. Figure 9 This indicates that Apt-HPPNCs@lip can significantly improve oxidative damage to nerve cells caused by DA and has a good detoxification effect.

[0063] In summary, Apt-HPPNCs-lip has a certain adsorption and trapping ability for DA, which can significantly reduce its neurotoxicity.

[0064] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. An aptamer-modified neural cell membrane hybrid liposome, characterized in that, The method involves hybridizing the cell membrane of human hippocampal neuronal HPPNCs cell line with liposomes to prepare neuronal cell membrane hybrid liposomes HPPNCs@lip, and then modifying the surface of these liposomes with a nucleic acid aptamer that specifically binds to domoic acid; the nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID NO.

1.

2. The aptamer-modified neural cell membrane hybrid liposome according to claim 1, characterized in that, The nucleic acid aptamer has cholesterol attached to its 3′ end, and the 3′ end of the nucleic acid aptamer is coupled to HPPNCs@lip through hydrophobic interaction to construct an aptamer-modified neural cell membrane hybrid liposome Apt-HPPNCs@lip.

3. A method for preparing aptamer-modified neural cell membrane hybrid liposomes as described in claim 1 or 2, characterized in that, Includes the following steps: (A) Preparation of hybrid liposomes HPPNCs@lip: After obtaining the cell membrane of the human hippocampal neuronal HPPNCs cell line using the pure water hypotonic method, HPPNCs@lip was prepared by thin film hydration and co-extrusion methods; (B) Coupling of nucleic acid aptamers with HPPNCs@lip: First, the domoic acid aptamer sequence was amplified by PCR, with cholesterol attached to its 3′ end; then, the 3′ end of the nucleic acid aptamer was coupled to HPPNCs@lip through hydrophobic interaction to construct a nucleic acid aptamer-modified neural cell membrane hybrid liposome Apt-HPPNCs@lip.

4. The preparation method according to claim 3, characterized in that, Step (A) involves first lysing human hippocampal neurons in pure water at 4°C using hypotonic methods for 30 min, centrifuging at 2000×g for 20 min, collecting the supernatant, centrifuging at 4000×g for 45 min, dissolving the precipitate in PBS to obtain an HPPNCs cell membrane dispersion; dissolving lecithin, DSPE-PEG2000, and cholesterol together in chloroform, and rotary evaporating at 37°C to obtain a liposome membrane; adding HPPNCs cell membrane dispersion at a mass concentration ratio of 1:1 to the liposome membrane, and hydrating at 37°C for 1 h until a homogeneous suspension is obtained; and then sequentially pressing the suspension through polyester membranes of 400 nm, 200 nm, and 100 nm to finally obtain HPPNCs@lip.

5. The preparation method according to claim 4, characterized in that, The mass percentages of lecithin, DSPE-PEG2000, and cholesterol are 90%:5%:5%.

6. The preparation method according to claim 3, characterized in that, In step (B), during coupling, 2 μM of nucleic acid aptamer M70-b and 1 mg / mL of HPPNCs@lip were mixed and incubated at 37 °C for 12 h. The mixture was then ultrafiltered at 12,000 × g for 30 min using a 50 kD ultrafiltration tube. The liquid from the upper tube was collected to obtain aptamer-modified neural cell membrane hybrid liposomes.

7. The use of an aptamer-modified neural cell membrane hybrid liposome as described in claim 1 or 2 in the preparation of a drug for preventing and treating domoic acid poisoning.

8. The application of an aptamer-modified neural cell membrane hybrid liposome as described in claim 1 or 2 in the preparation of a product for the recognition, trapping, adsorption, and detoxification of doxorubicin.

9. The use of an aptamer-modified neural cell membrane hybrid liposome as described in claim 1 or 2 in the preparation of a drug that protects against domoic acid-induced neural cell death.

10. The use of an aptamer-modified neural cell membrane hybrid liposome as described in claim 1 or 2 in the preparation of a drug to protect against oxidative stress damage to neural cells induced by domoic acid.

Images