Omeprazole nano-liposome and preparation method thereof

Abstract

The invention discloses an omeprazole nano-liposome and a preparation method thereof. The omeprazole nano-liposome is prepared in a film hydration method active packaging mode, targeted peptide modification is carried out, the generated modified omeprazole nano-liposome can successfully penetrate through a blood brain barrier, and sepsis related encephalopathy is effectively treated.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to omeprazole nanoliposomes and their preparation methods. Background Technology

[0002] Existing technologies utilize thin-film hydration to prepare drug nanoliposomes. Thin-film hydration methods are divided into passive and active packaging methods. One existing technology uses a passive method to prepare omeprazole nanoliposomes, which involves placing omeprazole, lecithin, cholesterol, and DSPE into a flask for rotary evaporation, followed by hydration, sonication, filtration, centrifugation, and lyophilization. The stability of omeprazole nanoliposomes produced by passive packaging needs improvement, as they are prone to denaturation and discoloration.

[0003] No existing technologies have reported the preparation of omeprazole nanoliposomes using active packaging. Summary of the Invention

[0004] This invention provides an omeprazole nanoliposome, the omeprazole nanoliposome comprising a nanoliposome and an omeprazole encapsulated within the nanoliposome;

[0005] Preferably, the nanoliposomes include phospholipids, cholesterol, and DSPE-PEG2000-Mal.

[0006] Preferably, the phospholipid is lecithin.

[0007] Preferably, the lecithin is soybean lecithin.

[0008] The present invention also provides a modified omeprazole nanoliposome, wherein the modified omeprazole nanoliposome comprises the omeprazole nanoliposome described above, and a targeting peptide linked to the nanoliposome in the omeprazole nanoliposome;

[0009] The "targeting peptides" described herein include, but are not limited to, "tumor-targeting peptides," "cell-penetrating peptides," and "tumor cell-targeting membrane-penetrating peptides." Modified derivatives of these peptides can be produced using conventional methods in the existing technical field. These modification methods include modification of the amino or carboxyl terminus of the peptide, replacement of intermediate amino acid residues, and side chain modification. For example, methods for modifying the peptide chain terminus include N-terminal acetylation and C-terminal amidation, thereby protecting the terminal amino or carboxyl groups. Fatty acids of different lengths can also be attached to the peptide chain terminus. PEG molecules can be glycosylated to increase the relative molecular weight and steric hindrance of the peptide molecule, improve its stability to peptide hydrolases, and prolong its residence time in the in vivo circulation system. Simultaneously, the half-life of the peptide drug can be prolonged or improved by replacing individual easily enzymatically hydrolyzed amino acid residues or replacing L-type amino acids with D-type amino acids.

[0010] "Tumor-targeting peptides" refer to a class of short peptide sequences that are artificially mutated and optimized from the amino acid sequence of the S3 mimic peptide that binds to EGFR, resulting in a tumor-targeting peptide structure with better specific binding ability. These peptides can be used for the marker-based recognition and diagnosis of tumor cells, competitively bind to EGFR, or carry tumor therapeutic drug molecules to the surface of tumor cells to exert targeted therapeutic effects.

[0011] Tumor-targeting peptides, when conjugated with molecular imaging reagents (such as radioactive isotope labeling and fluorescent dye labeling), can be used for tumor diagnosis and analysis; or, when conjugated with tumor therapeutic drugs, they can be formulated into tumor cell-targeting therapeutic drugs for targeted treatment of malignant tumors, thereby reducing the impact of tumor therapeutic drugs on normal cells, reducing the occurrence of adverse drug reactions, and improving treatment efficacy; or, by directly utilizing the property that their own polypeptide structure can have a high affinity for the target site (EGFR), they can competitively bind to and mask the EGFR site, preferentially inhibiting the growth of tumor cells, thereby achieving the purpose of treating tumor diseases.

[0012] Cell penetrating peptides (CPPs), also known as protein transduction domains or membrane transduction peptides, are polypeptides composed of 30 or fewer amino acids capable of penetrating cell membranes. CPPs with cell penetrating peptide activity include, but are not limited to, sequences such as, but not limited to, human immunodeficiency virus transcription activator TAT, herpes simplex virus type I VP22 transcription factor, Drosophila homologous antennal protein (Antp) penetrantin penetrantin penetrants ... The heparin-binding domain is one of the following: antigen, HATF3, hCT, pVEC, Integrin, DPV6, S413PV, Poly-P, heparin-binding domain, etc., preferably TAT (with the amino acid sequence YGRKKRRQRRR) or EC-SOD carboxyl-terminal heparin-binding domain (with the amino acid sequence GPGLWERQAREHSERKKRRRESECKAA) or its variants, such as the cell-penetrating peptides contained in Chinese patents CN201210587097.1 and CN1049111; it can also be the heparin-binding domain of HBEGF derived from HBEGF.

[0013] "Tumor cell-targeting membrane-penetrating peptides" refer to a class of short peptide sequences containing CPP domains with cell-penetrating peptide activity and the aforementioned "tumor-targeting peptides".

[0014] Preferably, the targeting peptide is a TAT peptide.

[0015] Preferably, the nanoliposomes comprise phospholipids, cholesterol, and DSPE-PEG2000-TAT peptide, wherein the DSPE-PEG2000-TAT peptide is formed by a Michael addition reaction between the maleimide of DSPE-PEG2000-Mal and the thiol group of the N-terminal cysteine ​​residue of the TAT peptide.

[0016] Preferably, the phospholipid is lecithin.

[0017] Preferably, the lecithin is soybean lecithin.

[0018] Furthermore, the omeprazole nanoliposomes have an average particle size of 139.5 nm, a PDI of 0.208, and a Zeta potential of -11.4 ± 0.6 mV.

[0019] Furthermore, the modified omeprazole nanoliposomes have an average particle size of 166.7 nm, a PDI of 0.169, and a PDI of -17.9 ± 0.4 mV.

[0020] This invention provides a method for preparing the aforementioned omeprazole nanoliposomes, characterized in that the preparation method includes the following steps:

[0021] The organic solvent-dissolved DSPE-PEG2000-Mal, lecithin, and cholesterol were rotary evaporated to remove the organic solvent, resulting in a uniform film, which was then dried.

[0022] Omeprazole and an hydration solution were added to the container containing the film to hydrate it, resulting in a suspension.

[0023] The suspension was sonicated in an ice bath to obtain the omeprazole nanoliposomes;

[0024] Preferably, the mass ratio of DSPE-PEG2000-Mal, lecithin, cholesterol, and omeprazole is 2.5:4:2:2;

[0025] Preferably, the masses of DSPE-PEG2000-Mal, lecithin, cholesterol, and omeprazole are 12.5 mg, 20 mg, 10 mg, and 10 mg, respectively.

[0026] Preferably, the organic solvent is at least one selected from chloroform, anhydrous ethanol, methanol, and dimethyl sulfoxide.

[0027] Preferably, the chloroform is tritium-substituted chloroform-d;

[0028] Preferably, the temperature of the rotary evaporation is 37 degrees Celsius;

[0029] Preferably, the drying conditions are vacuum drying for 2 hours;

[0030] Preferably, the hydration solution is one or more of the following: deionized water, drip water, sterile sodium chloride solution, phosphate buffer, 10% glucose solution, and 10% sucrose solution;

[0031] Preferably, the hydration solution is a phosphate buffer solution;

[0032] Preferably, the amount of phosphate buffer added is 20 ml;

[0033] Preferably, the ice bath ultrasound conditions are: 80W power, 2s operation, 2s interval, 5min total.

[0034] Furthermore, the preparation method also includes a filtration step, preferably, sterilization by filtration through a syringe filter to remove unbound active ingredients from the mixture.

[0035] Furthermore, the preparation method also includes a centrifugation step. Preferably, centrifugation is performed in a centrifuge at 4500 rpm, 4°C, and 10 min to remove omeprazole that is not encapsulated, and the supernatant is taken as omeprazole nanoliposomes, i.e., OM@LD.

[0036] This invention provides a method for preparing the modified omeprazole nanoliposomes described above, the method comprising the following steps:

[0037] The DSPE-PEG2000-TAT peptide, lecithin, and cholesterol dissolved in organic solvent were rotary evaporated to remove the organic solvent, resulting in a uniform film, which was then dried.

[0038] Omeprazole was added to the container containing the film, and the hydration solution was hydrated to obtain a suspension.

[0039] The suspension was sonicated in an ice bath to obtain the modified omeprazole nanoliposomes;

[0040] Preferably, the mass ratio of DSPE-PEG2000-TAT peptide, lecithin, cholesterol, and omeprazole is 1:10:5:5;

[0041] Preferably, the masses of DSPE-PEG2000-TAT peptide, lecithin, cholesterol, and omeprazole are 2 mg, 20 mg, 10 mg, and 10 mg, respectively.

[0042] Preferably, the organic solvent is at least one selected from chloroform, anhydrous ethanol, methanol, and dimethyl sulfoxide.

[0043] Preferably, the chloroform is tritium-substituted chloroform-d;

[0044] Preferably, the organic solvent is tritium-chloroform-d;

[0045] Preferably, the temperature of the rotary evaporation is 37 degrees Celsius;

[0046] Preferably, the drying conditions are vacuum drying for 2 hours;

[0047] Preferably, the hydration solution is one or more of the following: deionized water, drip water, sterile sodium chloride solution, phosphate buffer, 10% glucose solution, and 10% sucrose solution;

[0048] Preferably, the hydration solution is a phosphate buffer solution;

[0049] Preferably, the amount of phosphate buffer added is 20 ml;

[0050] Preferably, the ice bath ultrasound conditions are: 80W power, 2s operation, 2s interval, 5min total;

[0051] Preferably, the preparation method of the DSPE-PEG2000-TAT peptide includes the following steps: reacting the TAT peptide with the sequence YGRKKRRQRRR with DSPE-PEG2000-Mal;

[0052] Preferably, the reaction conditions are: uniform stirring and standing at room temperature for 24 hours;

[0053] Preferably, the TAT peptide is dissolved in an organic solvent; the DSPE-PEG2000-Mal is dissolved in an organic solvent.

[0054] Preferably, 10 mg of TAT peptide is dissolved in 5 ml of methanol; 12.5 mg of DSPE-PEG2000-Mal is dissolved in 3 ml of tritium-chloroform-d.

[0055] Preferably, the preparation method further includes a filtration step; preferably, the mixture is sterilized by filtration through a syringe filter to remove unbound active ingredients.

[0056] Preferably, the preparation method further includes a centrifugation step; preferably, centrifugation is performed in a centrifuge at 4500 rpm, 4°C, and 10 min to remove omeprazole that is not encapsulated, and the supernatant is taken as the modified omeprazole nanoliposomes, namely OM@LDT.

[0057] The present invention also provides the use of the omeprazole nanoliposomes described above or the modified omeprazole nanoliposomes described above in the preparation of a medicament for treating sepsis-associated encephalopathy.

[0058] The present invention also provides the use of the omeprazole nanoliposomes described above or the modified omeprazole nanoliposomes described above in the preparation of drugs with antioxidant, anti-inflammatory and anti-apoptotic properties.

[0059] In addition to the active ingredient, omeprazole nanoliposomes or modified omeprazole nanoliposomes, this invention also includes pharmaceutically acceptable carriers. These pharmaceutically acceptable carriers include (but are not limited to) diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants.

[0060] The ingredients include diluents such as lactose, sodium chloride, glucose, urea, starch, and water; binders such as starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginate and alginates, xanthan gum, hydroxypropyl cellulose, and hydroxypropyl methylcellulose; surfactants such as polyethylene oxide sorbitan fatty acid esters, sodium lauryl sulfate, glyceryl monostearate, and hexadecyl alcohol; humectants such as glycerin and starch; adsorbents such as starch, lactose, bentonite, silica gel, kaolin, and soap clay; and lubricants such as zinc stearate and monostearate. The ingredients include: glyceryl fatty acids, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearate fumarate, polyoxyethylene monostearate, monolauric sucrose ester, sodium lauryl sulfate, magnesium lauryl sulfate, magnesium dodecyl sulfate, etc.; fillers such as mannitol (granular or powdered), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polysaccharides, coupled sugars, glucose, lactose, sucrose, dextrin, starch, sodium alginate, kelp polysaccharide powder, agar powder, calcium carbonate and sodium bicarbonate, etc.; disintegrants such as crosylvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, crosylcarboxymethyl cellulose sodium, soybean polysaccharides, etc.

[0061] The drug in this invention may also include additives such as stabilizers, bactericides, buffers, isotonic agents, chelating agents, pH control agents, and surfactants.

[0062] Stabilizers include human serum albumin, L-amino acids, sugars, and cellulose derivatives. L-amino acids may also include any one of glycine, cysteine, and glutamic acid. Sugars include monosaccharides such as glucose, mannose, galactose, and fructose; sugar alcohols such as mannitol, cellulose alcohol, and xylitol; disaccharides such as sucrose, maltose, and lactose; and polysaccharides such as dextran, hydroxypropyl starch, chondroitin sulfate, hyaluronic acid, and their derivatives. Cellulose derivatives include methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, and sodium hydroxymethylcellulose. Surfactants include ionic or nonionic surfactants such as alkyl polyethylene oxide esters, sorbitan monoacyl esters, and fatty acid glycerides. Additive buffers may include boric acid, phosphoric acid, acetic acid, citric acid, glutamic acid, and their corresponding salts (their alkali metal or alkaline rare earth metal salts, such as sodium, potassium, calcium, and magnesium salts). Isotonic agents include potassium chloride, sodium chloride, sugars, and glycerol. Chelating agents include sodium ethylenediaminetetraacetate and citric acid.

[0063] The medicaments of this invention can be administered orally, non-gastrointestinally, via inhalation spray, topically, rectally, nasally, buccally, vaginally, or through an implanted drug delivery device. Oral or injectable administration is preferred. The pharmaceutical compositions of this invention may contain any commonly used, non-toxic, pharmaceutically acceptable carriers, excipients, or excipients.

[0064] During treatment, the dosage of the drug of this invention can be adjusted according to the severity of symptoms, the frequency of recurrence, and the physiological response of the treatment regimen. Attached Figure Description

[0065] Figure 1 Showing a schematic diagram of the synthesis of OM@LD and OM@LDT;

[0066] Figure 2 The results of characterization of OM@LD and OM@LDT are shown in the following figures: (A): Schematic diagram of OM@LD and OM@LDT, with the TAT peptide sequence structure being (YGRKKRRQRRR); (B): TEM image of OM@LD and OM@LDT, with a scale bar of 200 nm; (C): Zeta potential results of L (blank liposome carrier), OM@LD and OM@LDT; (DF): Hydrodynamic diameter and polydispersity index (PDI) results of L (blank liposome carrier), OM@LD and OM@LDT; (GH): CCK-8 cytotoxicity curves of ordinary omeprazole, L (blank liposome carrier), OM@LD and OM@LDT, and different concentrations of... Line graph of CCK-8 cytotoxicity of OM@LDT; (IL): Schematic diagram of full-wavelength spectral scanning of OM@LD and OM@LDT; (I): Detection range of 230nm-980nm, interval of 25nm; (J): Detection range of 230nm-330nm, interval of 25nm; (K): Detection range of 230nm-330nm, interval of 3nm; (L): Detection range of 230nm-280nm, interval of 3nm; (M): Omeprazole dissolved in PBS, omeprazole dissolved in NaOH, L (blank liposome carrier), appearance changes of OM@LD and OM@LDT, and comparison of the appearance of synthesized OM@LD and OM@LDT;

[0067] Figure 3 The diagram shows the penetration test results of OM@LD and OM@LDT. Among them, (AB): the content distribution of OM@LD and OM@LDT in various ex vivo organs 6 hours, 12 hours and 24 hours after intraperitoneal injection, and the control group was injected with PBS for 6 hours; (C): the distribution of HT22 (mouse hippocampal neurons) of OM@LD and OM@LDT.

[0068] Figure 4 The diagram shows the protective effect of OM@LDT on LPS-induced HT22 cells. (A): OM@LDT antioxidant damage graph; (B): Statistical analysis of OM@LDT antioxidant damage graph (n=3, mean ± standard deviation, one-way ANOVA, **p<0.01, ***p<0.001); (C): OM@LDT anti-apoptotic effect graph; (D): Statistical analysis of OM@LDT anti-apoptotic effect graph. Annexin V is marked in green, representing early apoptosis. P I is marked in red, representing late apoptosis or death (n=3, mean ± standard deviation, one-way ANOVA, *p<0.05, ****p<0.0001); (EG): the levels of cytokines IL-1β(E), IL-6(F), and TNF-α(G) in HT22 cells 24 hours after LPS stimulation and LPS+OM@LDT stimulation (n=3, mean ± standard deviation, one-way ANOVA, *p<0.05, ****p<0.0001);

[0069] Figure 5 The images show the results of OM@LD and OM@LDT improving the survival rate of CLP-induced septic encephalopathy mice and the changes in H&E staining and Nissl staining histopathology. Among them, (A): CLP-induced SAE mouse model protocol, 0.5 ml (0.5 mg / ml) of OM@LD and OM@LDT were administered intraperitoneally 2 hours and 6 hours after CLP induction; (B): Survival rate of CLP-induced SAE mice treated with OM@LD and OM@LDT (n=10); (C): H&E images of the hippocampus, liver, kidney, and spleen of Sham, CLP, and CLP-induced SAE mice treated with OM@LD and OM@LDT (n=3); (D): Nissl images of the hippocampus of Sham, CLP, and CLP-induced SAE mice treated with OM@LD and OM@LDT (n=3).

[0070] Figure 6 The figure shows the results of OM@LD and OM@LDT improving motor function decline and cognitive impairment in CLP-induced SAE mice. (A): Behavioral experimental protocol in CLP-induced SAE mice, with the same administration regimen as above. Figure 4(A) Off-the-fist (OFT) was performed on the first postoperative day, Y-maze on the third postoperative day, and NORT on the seventh postoperative day (n=8, mean ± standard deviation, one-way ANOVA, *p<0.05, **p<0.01, ***p<0.001); (B) Representative records of mouse walking paths in OFT (green line). The starting and ending points of mouse movement are represented by blue and red dots, respectively; (C) Walking distance, average speed, and the proportion of time spent in the central region to the total time in OFT (n=8); (D) Mouse walking paths in Y-maze. Representative records (green line): The starting and ending points of mouse movement are represented by blue and red dots, respectively; (E): The time, number of new arm visits, and percentage of the total new arm visit distance in the Y-maze (n=8, mean ± standard deviation, one-way ANOVA, *p<0.05, **p<0.01, ***p<0.001); (F): Representative records of mouse walking paths in NORT (green line): The starting and ending points of mouse movement are represented by blue and red dots, respectively; (G): Cognitive index, walking distance, and average speed of mice in NORT. Detailed Implementation

[0071] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0072] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0073] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0074] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0075] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0076] Unless otherwise specified, the "%" mentioned in this invention refers to a percentage by mass.

[0077] Example: Preparation method of omeprazole nanoliposomes

[0078] I. Experimental Materials: Omeprazole (Shandong Keyuan Biochemical Co., Ltd.), Lecithin (Shanghai Maclean Biochemical Technology Co., Ltd.), Cholesterol (Shanghai Maclean Biochemical Technology Co., Ltd.)

[0079] Tritium-chloroform-d Chloroform-d Shanghai Maclean Biotechnology Co., Ltd. DSPE-PEG2000-Mal Shanghai Yuanye Biotechnology Co., Ltd.

[0080] TAT peptide (YGRKKRRQRRR), Hubei Qiangyao Biotechnology Co., Ltd., PBS buffer, Beijing Solarbio Co., Ltd.

[0081] FITC (fluorescein isothiocyanate) (P0639M) Shanghai Beyotime Biotechnology Co., Ltd.

[0082] II. Preparation Method

[0083] Omeprazole nanoliposomes were synthesized using the hydration thin film method.

[0084] (1) Dissolve 10 mg of TAT peptide (YGRKKRRQRRR) in 5 ml of methanol and define it as solution 1;

[0085] (2) Dissolve 12.5 mg of DSPE-PEG2000-Mal in 3 ml of tritium-chloroform-d and define it as solution 2;

[0086] (3) Slowly add solution 2 to solution 1, stir evenly at room temperature and let stand for 24 hours. Through Michael addition reaction, couple the thiol group at the N-terminus of TAT peptide with the maleimide group of DSPE-PEG2000-Mal to prepare the synthesized DSPE-PEG2000-TAT material for later use.

[0087] (4) Weigh 20 mg of lecithin, 10 mg of cholesterol, and 2 mg of DSPE-PEG2000-TAT into a flask and dissolve them in tritium chloroform-d.

[0088] (5) The organic solvent was removed by rotary evaporation at 37°C to obtain a uniform film, which was then vacuum dried for 2 hours.

[0089] (6) Add 10 mg of omeprazole to the flask and add 20 ml of PBS (phosphate buffer) to hydrate and obtain a nano suspension;

[0090] (7) Under ice bath conditions, ultrasonic dispersion was performed at a certain power of 80w for 5 minutes (working for 2 seconds, then pausing for 2 seconds) to obtain a uniform nanoliposome solution.

[0091] (8) The obtained nanoliposomes were sterilized by filtering through a syringe filter (Millipore, Billerica, MA, USA) to remove unbound active ingredients from the mixture.

[0092] (9) Centrifuge at 4500 rpm, 4℃, for 10 min to remove omeprazole that was not encapsulated. Take the supernatant as omeprazole nanoliposomes, i.e. OM@LDT.

[0093] (10) The synthesis method of OM@LD is basically the same as above, that is, the third step is cancelled, and the fourth step is to add DSPE-PEG2000-Mal. The remaining steps are the same, and OM@LD can be obtained.

[0094] III. Characterization of OM@LD and OM@LDT

[0095] (1) Take 10 μl of TEM OM@LD and OM@LDT and drop them onto the copper mesh film. Dry the samples using an infrared baking lamp for half an hour. Place the samples in a TEM (Hitachi HT-7700, Japan), adjust the parameters, and capture images.

[0096] (2) Dynamic light scattering (DLS) was used to measure the hydrodynamic diameter, polydispersity index (PDI), and zeta potential of OM@LD and OM@LDT using a nanoparticle size, potential, and molecular weight analyzer (NanoZS90, Malvern Instruments Ltd., UK). The encapsulation efficiency and drug loading rate were calculated using formulas during the synthesis process. Stability was assessed by observing the appearance of the synthesized samples and re-measuring the hydrodynamic diameter, PDI, and zeta potential to observe their changes.

[0097] (3) For the full spectrum scanning experiment, the OD value was read and the data was analyzed by a multi-functional microplate reader (Platinum Elmer Instruments, Inc., USA).

[0098] (4) To test the cytotoxicity of the drugs, different samples were mixed with cell suspension, and 10 μl of CCK-8 was added to each well. After incubation for 2 hours, the absorbance at 450 nm was measured using a multi-functional microplate reader to assess cell viability. IV. Penetration detection of OM@LD and OM@LDT

[0099] (1) To visualize OM@LD and OM@LDT in different organs, mice were injected intraperitoneally with FITC-labeled OM@LD, OM@LDT and an equal volume of PBS. The mice were sacrificed at 6, 12 and 24 hours, and the tissues of major organs, including brain, liver, kidney and spleen, were collected. Fluorescence imaging was performed on IVIS SPECTRUM (Perkin Elmer, Hopkinton, MA, USA) (excitation: 745 nm; emission: 800 nm), and the fluorescence signal was superimposed on the bright field image.

[0100] (2) In addition, 100 μg / ml of FITC-containing OM@LD and OM@LDT were added to cultured HT22 cells, and images were captured using a medical fluorescence microscope (Olympus, Japan).

[0101] V. Cell Experiments

[0102] (1) Detection of reactive oxygen species (ROS) levels: The ROS levels in HT22 cells were detected using the DCFH-DA probe. HT22 cells were suspended in 0.25% trypsin, centrifuged at 1000g for 5 min at 4°C, and then co-incubated with the DCFH-DA fluorescent probe. ROS levels were detected according to the manufacturer's instructions. HT22 cells were treated with DAPI for 10 min. ROS levels in HT22 cells were detected using a fluorescence microscope (Olympus, Japan).

[0103] (2) Apoptosis assay: Annexin V-FITC and propidium iodide (PI) staining were used to evaluate apoptosis. Due to PI staining, dead and late-stage apoptotic cells appeared red. Annexin V-FITC appeared green and can cross damaged cell membranes and accumulate in the cytoplasm. Cells were observed using a fluorescence microscope (Olympus, Japan). At least three fields of view were observed for each sample, and representative images are shown.

[0104] (3) Enzyme-linked immunosorbent assay (ELISA): HT22 cells were ruptured and the supernatant was collected for cell experiments. Tumor necrosis factors TNF-α, IL-1β and IL-6 were detected using an ELISA kit, and the operation was carried out according to the manufacturer's instructions.

[0105] VI. Animal Experiments

[0106] (1) H&E staining: Soak brain slices in environmentally friendly dewaxing solution for 20 minutes, then in anhydrous ethanol for 5 minutes, then in 75% alcohol for 5 minutes, and finally rinse with tap water. Hematoxylin staining: Soak slices in hematoxylin staining solution for 3-5 minutes, then rinse with tap water, then differentiate with differentiation solution, rinse again with tap water, then use blueing solution to turn the slices blue, and finally rinse thoroughly with running water. Eosin staining: Soak slices in 85% and 95% alcohol solutions for 5 minutes each, then stain with eosin solution for 5 minutes. Dehydration and mounting: Soak slices in anhydrous ethanol for 5 minutes, then in xylene for 5 minutes to clear, and finally mount with neutral resin. Microscopic examination, image acquisition and analysis.

[0107] (2) Nissl staining: Brain sections were dewaxed in water: xylene I and xylene II for 15 minutes each, followed by gradient ethanol dehydration: 100% I, 100% II, 95%, 90%, 80%, 70%, and 50% for 5 minutes each, and washed three times with distilled water for 5 minutes each. Then, the sections were stained with 1% toluidine blue at 60°C for 40 minutes and washed with distilled water. The dyes were then dehydrated with 70%, 80%, 95%, and 100% ethanol, cleared with xylene, and finally sealed with neutral glue. Neurons were observed and photographed under a microscope.

[0108] (3) Open Field Test (DFT): The open field apparatus used for mice (Shanghai Xinruan Technology Co., Ltd., China) measures 50cm x 50cm x 40cm. At the start of the experiment, the mouse is gently placed in the center or a corner of the open field apparatus. The animal's behavior in the open field is then recorded. The observation time is typically 5 minutes. Observation indicators include distance traveled, time spent traveling, and speed of movement. After each test, the bottom of the enclosure is divided into 16 grids, with the four central grids considered the central area. A camera and motion tracking software (Any-maze video analysis system, Shanghai Xinruan Technology Co., Ltd., China) are used to record and analyze the mouse's behavior.

[0109] (4) Y-maze test: The Y-maze test apparatus used for mice (Shanghai Xinruan Technology Co., Ltd., China) has three arms, each measuring 35cm x 5cm x 15cm. At the start of the experiment, the mouse was placed at the junction of the three arms, with only two arms open. The mouse's behavior was recorded and analyzed using a camera and motion tracking software (Any-maze video analysis system, Shanghai Xinruan Technology Co., Ltd., China), typically observed for 5 minutes. After the experiment, after a 30-minute interval, all three arms were opened, and the mouse's behavior was observed and recorded again.

[0110] (5) New object recognition experiment: The apparatus used for mice (Shanghai Xinruan Technology Co., Ltd., China) was 50cm x 50cm x 40cm in size. Before the experiment, the mice were acclimatized to the box for 5 minutes. Then, the mice were placed in the box containing two objects of the same shape and color and allowed to explore freely for 5 minutes. Afterward, the apparatus and objects between the mice were cleaned. After 2 hours of storage, the mice were placed in the same box to explore for 5 minutes (collection phase). In both phases, one object was replaced by a new object. The number of times the mice explored the object in both phases was recorded. The recognition index was calculated as the percentage of the total number of explorations spent exploring the new object in the acquisition phase.

[0111] VII. Experimental Results

[0112] We synthesized two omeprazole nanoliposomes, OM@LD and OM@LDT, using a thin-film hydration method. Both drugs are essentially approximately spherical omeprazole nanoparticles encapsulated within a phospholipid bilayer composed of lecithin, cholesterol, and DSPE-PEG2000-Mal. The difference lies in that OM@LDT has an additional actively targeted peptide, TAT peptide, modified on its surface compared to OM@LD. As... Figure 1 As shown. TAT peptide is a cell-penetrating peptide that has the ability to enter the cell membrane without relying on receptors, without causing permanent damage to the cell membrane, and has very low toxicity. Its modification is formed through a Michael addition reaction between maleimide in DSPE-PEG2000-MAL and the thiol group of the N-terminal cysteine ​​(C) residue of the TAT peptide.

[0113] We later observed the two drugs using transmission electron microscopy, and saw... Figure 2 Both drugs are spherical in shape, with high density and a diameter of approximately 200 nm. OM@LDT has a distinct tail-like structure on its surface, which is likely a surface-modified TAT peptide. Overall, the drugs are uniform in size and orderly distributed.

[0114] We then characterized the two synthesized omeprazole nanoliposomes by Malvern hydrodynamics and their size and polydispersity index (PDI), using a blank liposome carrier without omeprazole as a control, denoted as L. L had an average particle size of 150.1 nm, a PDI of 0.159, and a Zeta potential of -16 ± 0.6 mV. OM@LD had an average particle size of 139.5 nm, a PDI of 0.208, and a Zeta potential of -11.4 ± 0.6 mV. OM@LDT had an average particle size of 166.7 nm, a PDI of 0.169, and a Zeta potential of -17.9 ± 0.4 mV.

[0115] We performed full-spectral scans on OM (omeprazole), L (blank liposome carrier), DSPE-PEG2000-TAT, OM@LD, and OM@LDT. The results showed that the characteristic peaks of our synthesized OM@LD and OM@LDT appeared at 233 nm, 251 nm, 260 nm, and 266 nm, basically including the characteristic peaks of omeprazole (OM), lecithin and cholesterol, and DSPE-PEG2000-TAT, proving that omeprazole nanoliposomes were successfully synthesized.

[0116] We selected HT22 cells (mouse hippocampal neurons) as the test subject for toxicity assays. Using the CCK-8 assay, we found that the cell viability of HT22 cells stimulated with OM was 91.08±93.48%, L-stimulated cells were 91.68±3.11%, OM@LD was 91.67±4.09%, and OM@LDT was 97.24±4.27%. We further tested different concentrations of OM@LDT: 25 μg / ml OM@LDT stimulated cells with 92.85±3.81%, 50 μg / ml OM@LDT with 91.63±5.43%, 100 μg / ml OM@LDT with 97.05±1.96%, and 200 μg / ml OM@LDT with 102±4.71%. Therefore, we believe that the components of the drug synthesis are basically non-toxic, and the drug stimulation concentration of 100ug / ml is the most stable and does not produce cytotoxicity, so it can be used as the standard stimulation concentration for subsequent cell experiments.

[0117] Next, we studied the stability of the drug. Omeprazole is a lipid-soluble drug, soluble in alkaline solutions, and inherently unstable, easily decomposing and turning purple in solution. Therefore, we labeled omeprazole dissolved in 0.1 mmol / L NaOH as O, omeprazole dissolved in PBS as P, L as the blank liposome carrier, OM@LD as D, and OM@LDT as T. After one week of storage at 4°C, we found that P had decomposed and turned pale purple, while O remained stable and clear, L was slightly grayish-white, and OM@LD and OM@LDT were cool, clear, and slightly yellowish, without turning purple. This indicates that the unprotected P decomposed, while O demonstrated that alkalinity protects the stability of omeprazole. This also indirectly proves that we synthesized omeprazole nanoliposomes, successfully encapsulating omeprazole within the phospholipid bilayer for protection, thus achieving drug stability.

[0118] Meanwhile, after storing the effectively synthesized drug for six months, it still showed a cool, transparent, pale yellow color instead of purple. We also compared the drug synthesized six months ago with the drug synthesized now using Malvern hydrodynamics and PDI (polydispersity index) tests, and the results also proved that omeprazole nanoliposomes have stability.

[0119] like Figure 3 We administered 1 ml (0.5 mg / ml) of FITC-containing OM@LD and OM@LDT via intraperitoneal injection 6, 12, and 24 hours before the experiment, respectively. The control group received PBS 6 hours prior. Small animal organ imaging experiments were then conducted. We found that our synthesized OM@LD and OM@LDT could cross the blood-brain barrier and reach the mouse brain. OM@LD reached its peak concentration 12 hours after administration and was metabolized by the liver and kidneys, with some drug remaining in the brain after 24 hours. OM@LDT, due to its TAT ​​peptide properties, also reached its peak concentration at 12 hours, with greater retention after 24 hours, and was more abundant in the liver, kidneys, and spleen. Clearly, we achieved blood-brain barrier crossing through nanoliposomes, enabling the synthesized OM@LD and OM@LDT to successfully reach the brain.

[0120] To further our research on omeprazole nanoliposomes (OM@LD, OM@LDT), we conducted a cell membrane penetration experiment. We labeled the nuclei of HT22 mouse hippocampal neurons with blue DAPI, the cell membrane with red DIL, and the drug with green FITC. The resulting red-green-blue combined image demonstrates that our synthesized drug successfully penetrated the cell membrane of HT22 hippocampal neurons and entered the cytoplasm.

[0121] Figure 4 The results showed that OM@LDT exhibited anti-oxidative stress effects in HT22 cells. The green fluorescence intensity of the OM@LDT group was similar to that of the negative control group and significantly lower than that of the positive control group. Data analysis showed that the mean fluorescence intensity (Mean) of the OM@LDT group was 78.51±5.99, while the mean fluorescence intensity (Mean) of the negative control group was 70.39±4.886, and that of the positive control group was 132.1±16.21. The difference between the negative and positive control groups was statistically significant (P<0.05). The difference between the negative and OM@LDT groups was not statistically significant (P>0.05). The difference between the positive and OM@LDT groups was statistically significant (P<0.05). This indicates that OM@LDT exhibits anti-oxidative stress effects in HT22 cells.

[0122] OM@LDT can alleviate LPS-induced apoptosis in HT22 cells. Similarly, we conducted an anti-apoptotic experiment using HT22 cells to simulate mouse hippocampal neurons. Annexin V was used in green to represent early apoptosis, and PI was used in red to represent late apoptosis. In the control group, the number of cells undergoing early and late apoptosis was very small. LPS induction resulted in a large number of cells undergoing late apoptosis and even death. While apoptosis also occurred in the LPS+OM@LDT group, the number of cells was significantly reduced (P<0.05), demonstrating a statistically significant difference compared to the LPS group. This proves that OM@LDT can alleviate LPS-induced apoptosis in HT22 cells.

[0123] OM@LDT can alleviate LPS-induced inflammatory cytokine production in HT22 cells. We established a cellular inflammation model by stimulating HT22 cells with LPS (100 μg / ml) and then administered OM@LDT (100 μg / ml) to counteract the inflammation. We used ELISA to detect the expression of IL-1β, IL-6, and TNF-α in each group to evaluate the anti-inflammatory effect of OM@LDT. The expression of IL-1β, IL-6, and TNF-α was increased in the LPS group, while it was significantly decreased in the OM@LDT group, with statistically significant differences compared to the LPS group, indicating its anti-inflammatory ability.

[0124] Figure 5 The results showed that intraperitoneal injection of OM@LD and OM@LDT improved the seven-day survival rate of CLP-induced SAE mice. Simultaneously, HE staining revealed a decreased regularity in the arrangement of hippocampal cells in the CLP group, with sparse cell distribution in local areas, damage-related changes such as neuronal loss, and incomplete hippocampal structure, possibly indicating inflammation. In contrast, the Sham group, CLP+OM@LD group, and OM@LDT group showed more regular and dense cell arrangement in the hippocampus, with uniform cell density and normal extracellular structure; no obvious signs of damage were observed. Our drugs reversed the pathological manifestations of CLP-induced hippocampal inflammation in mice without causing damage to liver, kidney, or spleen tissues, thus effectively protecting multiple organs. Nissell staining results showed that the degree of damage in the four groups, from most severe to least severe, was: CLP group > CLP+OM@LD group > CLP+OM@LDT group ≈ Sham group. The damage was mainly concentrated in the pyramidal cells of the CA1 and CA3 regions of the hippocampus and the granular cells of the dentate gyrus, manifested as abnormal neuronal morphology, reduced / dissolved Nissl bodies, disordered cell arrangement and glial cell reaction. The damage was most significant in the CLP model group (CLP+PBS), while the CLP+OM@LDT group could significantly alleviate these damages.

[0125] Figure 6Results showed that, following CLP induction, we routinely administered intraperitoneal injections of 0.5 ml each of OM@LD and OM@LDT at 2 and 6 hours post-surgery. OFT was performed on postoperative day 1, Y-maze on postoperative day 3, and NORT on postoperative day 7. (See attached image) Figure 6 A. In OFT, CLP mice had significantly lower walking distance, average speed, and central region time percentage, while Sham mice had the highest values ​​for all indicators. The OM@LDT group showed statistically significant differences from the CLP group in walking distance and average speed. Figure 6 C), but there appeared to be no significant difference in the proportion of time spent in the central region among the groups. This suggests that OM@LDT appears to improve motor function in mice more effectively, while OM@LD shows only moderate improvement. Neither OM@LD nor OM@LDT significantly reduced anxiety-depressive-like behaviors in SAE mice. In the Y-maze assay, no statistically significant differences were observed among the groups. In the NORT cognitive index, both the OM@LD and OM@LDT groups showed ideal performance compared to the CLP group, with statistically significant differences, indicating that OM@LD and OM@LDT improved cognitive function in mice. CLP-induced cognitive impairment in SAE mice could be reversed by OM@LD and OM@LDT. Based on the above data analysis, OM@LD and OM@LDT can improve CLP-induced motor function decline and cognitive impairment in SAE mice.

[0126] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A nano-liposome of omeprazole, characterized in that, The omeprazole nanoliposome comprises a nanoliposome and omeprazole encapsulated inside the nanoliposome; Preferably, the nanoliposome comprises lecithin, cholesterol, DSPE-PEG2000-Mal; Preferably, the lecithin is soybean lecithin.

2. A modified omeprazole nanoliposome, characterized in that, The modified omeprazole nanoliposome comprises the omeprazole nanoliposome of claim 1, and a targeting peptide connected to the nanoliposome in the omeprazole nanoliposome; Preferably, the targeting peptide is a TAT peptide; Preferably, the nanoliposome comprises lecithin, cholesterol, DSPE-PEG2000-TAT peptide, wherein the DSPE-PEG2000-TAT peptide is formed by Michael addition reaction between the maleimide of the DSPE-PEG2000-Mal and the thiol of the cysteine residue at the N-terminus of the TAT peptide; Preferably, the lecithin is soybean lecithin.

3. The omeprazole nanoliposome according to claim 1, characterized by, The average particle size of the omeprazole nanoliposome is 139.5 nm, the PDI is 0.208, and the Zeta potential is -11.4±0.6 mv.

4. The modified omeprazole nanoliposome according to claim 2, characterized by, The average particle size of the modified omeprazole nanoliposome is 166.7 nm, the PDI is 0.169, and the Zeta potential is -17.9±0.4 mv.

5. A method of preparing the omeprazole nanoliposome of claim 1, characterized by, The preparation method comprises the following steps: The DSPE-PEG2000-Mal, lecithin, and cholesterol dissolved in the organic solvent are rotary evaporated to remove the organic solvent, to obtain a uniform film, which is dried; Omeprazole and a hydration solution are added to the container with the film for hydration, to obtain a suspension; The suspension is ice-bath ultrasonicated, to obtain the omeprazole nanoliposome; Preferably, the mass ratio of DSPE-PEG2000-Mal, lecithin, cholesterol, and omeprazole is 2.5:4:2:2; preferably, the mass of DSPE-PEG2000-Mal, lecithin, cholesterol, and omeprazole is 12.5 mg, 20 mg, 10 mg, and 10 mg, respectively; Preferably, the organic solvent is deuterium chloroform-d; Preferably, the temperature of the rotary evaporation is 37 degrees; Preferably, the drying condition is vacuum drying for 2 hours; Preferably, the hydration solution is a phosphate buffer; Preferably, the amount of the phosphate buffer added is 20 ml; Preferably, the ice-bath ultrasonication condition is: power 80 w, working for 2 s, intermittent for 2 s, for a total of 5 min.

6. The production method according to claim 5, characterized by, The preparation method further comprises a filtration step, preferably, sterilization by a syringe filter to remove unbound active ingredients in the mixture.

7. The production method according to claim 6, characterized by, The preparation method further comprises a centrifugation step, preferably, centrifugation at 4500 rpm, 4℃, for 10 min, in a centrifuge to remove omeprazole that is not encapsulated, and the supernatant is taken as the omeprazole nanoliposome, i.e. OM@L-D.

8. A method of preparing the modified omeprazole nanoliposome of claim 2, characterized by, The preparation method comprises the following steps: The DSPE-PEG2000-TAT peptide, lecithin, and cholesterol dissolved in the organic solvent are rotary evaporated to remove the organic solvent, to obtain a uniform film, which is dried; Omeprazole and a hydration solution are added to the container with the film for hydration, to obtain a suspension; The suspension is ice-bath ultrasonicated to obtain the modified omeprazole nanoliposome; Preferably, the mass ratio of DSPE-PEG2000-TAT peptide, lecithin, cholesterol and omeprazole is 1:10:5:5; preferably, the mass of DSPE-PEG2000-TAT peptide, lecithin, cholesterol and omeprazole is 2 mg, 20 mg, 10 mg and 10 mg respectively; Preferably, the organic solvent is tritiated chloroform-d; Preferably, the temperature of the rotary evaporation is 37 degrees; Preferably, the drying condition is vacuum drying for 2 hours; Preferably, the hydration solution is a phosphate buffer; Preferably, the amount of the phosphate buffer added is 20 ml; Preferably, the ice-bath ultrasonication condition is: power 80w, working for 2s, intermittent for 2s, for a total of 5min; Preferably, the preparation method of the DSPE-PEG2000-TAT peptide comprises the following steps: reacting TAT peptide with the sequence YGRKKRRQRRR with DSPE-PEG2000-Mal; Preferably, the reaction condition is room temperature uniform stirring and standing for 24 hours; Preferably, the TAT peptide is dissolved in an organic solvent; the DSPE-PEG2000-Mal is dissolved in an organic solvent; Preferably, 10 mg of TAT peptide is dissolved in 5 ml of methanol; 12.5 mg of DSPE-PEG2000-Mal is dissolved in 3 ml of tritiated chloroform-d; Preferably, the preparation method further comprises a filtration step; preferably, the mixture is filtered and sterilized by a syringe filter to remove unbound active ingredients; Preferably, the preparation method further comprises a centrifugation step; preferably, the centrifuge is set to 4500 rpm, 4 degrees C, 10 min, and centrifuged to remove omeprazole that is not wrapped, and the supernatant is the modified omeprazole nanoliposome, OM@L-D-T.

9. Use of the omeprazole nanoliposome of claim 1 or the modified omeprazole nanoliposome of claim 2 in the preparation of a drug for treating sepsis-related encephalopathy.

10. Use of the omeprazole nanoliposome of claim 1 or the modified omeprazole nanoliposome of claim 2 in the preparation of an antioxidant, anti-inflammatory and anti-apoptotic drug.

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