Acetaminophen nasal spray nano-agent as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medicines, in particular to an acetaminophen nasal spray nano-agent and a preparation method and application thereof, the nano-agent is acetaminophen poly (lactic-co-glycolic acid) nanoparticles (AAP-PLGA-NPs); the acetaminophen poly (lactic-co-glycolic acid) nanoparticles are spherical in appearance, the particle size is 118 nm, the polydispersity coefficient value is 0.221, and the zeta potential is-23.7 mV. The preparation method of the acetaminophen nasal spray nano-agent comprises the following steps: firstly, taking PLGA as a carrier material, and preparing acetaminophen-loaded PLGA nanoparticles by combining an ultrasonic emulsification method with a solvent evaporation method; after the AAP-PLGA-NPs disclosed by the invention is administrated through a nasal cavity, the accumulation amount of acetaminophen in brain tissues can be remarkably increased and is delayed and eliminated, and the pain threshold value of mice entering plateau rapidly is remarkably increased.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a nasal spray nanometer preparation of acetaminophen and a preparation method and application thereof. Background Art

[0002] With the increase of tourism and scientific research activities in plateau areas, the impact of plateau environment on human health has received increasing attention. Plateau areas usually refer to areas above 3,000 meters above sea level. These areas are characterized by low oxygen, low air pressure, low temperature, dryness and strong ultraviolet radiation, which have significant effects on the human digestive system, respiratory system, circulatory system and nervous system. At present, pain management in plateau environment is an important research topic. Low oxygen partial pressure in plateau areas is one of the main factors causing pain: on the one hand, low oxygen can cause neurohormone release and hemodynamic changes, leading to rapid dilation of cerebral blood vessels and increased cerebral perfusion pressure, which in turn causes acute mountain sickness symptoms such as headache; on the other hand, low oxygen may also activate the microvascular system and aggravate pain perception by inducing the release of vasodilator effectors (such as nitric oxide). In addition, environmental factors such as low pressure, low temperature, dryness and strong ultraviolet rays in plateau areas may also have a comprehensive impact on the human body and aggravate pain perception. Pain in plateau environment will affect the physiological (increased pain sensitivity, prolonged pain duration and expanded pain range), psychological (anxiety and depression, emotional instability) and quality of life (limited daily activities and decreased sleep quality) aspects of people living in the plateau.

[0003] Traditional antipyretic analgesics (such as acetaminophen) play an important role in the management of plateau pain. Acetaminophen can relieve mild to moderate pain by inhibiting prostaglandin synthesis, increasing pain threshold (inhibiting the synthesis and release of bradykinin and histamine), and inhibiting the conduction and perception of pain signals from both central and peripheral pathways. At present, the main administration methods of acetaminophen are oral and intravenous administration. High doses or long-term use may cause liver and kidney damage. In recent years, nasal administration has gradually attracted attention due to its advantages such as non-invasiveness, easy operation, rapid absorption and onset of action, and avoidance of first-pass elimination in the liver. However, nasal administration also has some limitations: 1. Short drug retention time: The mucociliary clearance system in the nasal cavity is the main defense mechanism against foreign bodies, which can quickly remove foreign bodies and pathogens in the nasal cavity. This results in a very limited residence time of nasally applied drugs in the nasal cavity, which affects the absorption and efficacy of the drug. 2. Limited drug bioavailability: Various enzymes in the nasal mucosa, such as aminopeptidases and proteases, can degrade peptide or protein drugs, thereby reducing the bioavailability of the drug. In addition, the enzyme barrier of the nasal mucosa may also produce a "pseudo first-pass effect", which reduces the efficacy of some protein drugs. When studying and applying nasal delivery technology, these limitations need to be fully considered and corresponding measures (development of drug carrier systems such as liposomes, nanoemulsions and nanoparticles) should be taken to overcome and reduce their effects.

[0004] To this end, the inventors of this application, through research, aim to provide a new and efficient drug delivery method and treatment means for pain management in plateau environments, that is, to provide an acetaminophen nasal spray nanoparticle and its preparation method and application, so as to relieve patients' pain and improve their quality of life. Summary of the invention

[0005] The purpose of the present invention is to solve the prior art problems raised in the above background technology and to provide a nasal spray nanometer acetaminophen and its preparation method and application.

[0006] The above-mentioned purpose of the present invention is achieved like this:

[0007] The scheme of the present invention provides an acetaminophen nasal spray nanometer agent, wherein the nanometer agent is acetaminophen polylactic acid glycolic acid copolymer nanoparticles; the acetaminophen polylactic acid glycolic acid copolymer nanoparticles are spherical in appearance, have a particle size of 118nm, a polydispersity coefficient value of 0.221, and a zeta potential of -23.7mV.

[0008] Another aspect of the present invention provides a method for preparing a paracetamol nasal spray nanoparticle, the method comprising: firstly using PLGA as a carrier material, and adopting ultrasonic emulsification combined with solvent volatilization to prepare PLGA nanoparticles loaded with paracetamol, specifically comprising the following steps:

[0009] S1. Forming an oil phase: dissolving 20 mg of PLGA in 1 mL of dichloromethane solution to obtain a polymer solution, and then dissolving 2 mg of acetaminophen in the polymer solution;

[0010] S2, first emulsification: placing a beaker containing the oil phase solution in an ice water bath, and then using an ultrasonic cell disruptor to perform ultrasonic emulsification on the oil phase solution;

[0011] S3, second emulsification and stabilization: adding an appropriate amount of surfactant aqueous solution to the product after the first emulsification in step S2, and performing ultrasonic emulsification treatment again to reduce the particle size of the nanoparticles and improve the stability;

[0012] S4, solvent volatilization: subjecting the liquid subjected to ultrasonic emulsification treatment in step S3 to magnetic low-speed stirring at room temperature to gradually volatilize the organic solvent and gradually solidify and stabilize the PLGA nanoparticles;

[0013] S5, collection and purification: centrifuge the solution treated in step S4 to separate the PLGA nanoparticles loaded with acetaminophen; then high-speed centrifuge at 12000r / min for 15min to collect the nanoparticles, and then repeatedly wash them twice with an appropriate amount of pure water, and redisperse them in a small amount of pure water; freeze-dry the collected nanoparticles, and dry them in a low-temperature freeze dryer for 24h to obtain acetaminophen polylactic acid glycolic acid copolymer nanoparticles.

[0014] Furthermore, the molecular weight of PLGA in step S1 is 13000, and the ratio of lactic acid to hydroxylactic acid is 75:25.

[0015] Furthermore, the ultrasonic cell disruptor in step S2 is set to 60% power, emulsification time is 45s, and runs for 5s with an interval of 5s.

[0016] Furthermore, the surfactant aqueous solution in step S3 is 3 mL of 1 wt % PVA aqueous solution, and the oil phase:water phase ratio is 1:3.

[0017] Furthermore, the ultrasonic emulsification treatment in step S3 is: 60% power, emulsification time is 30s, running is 5s, and interval is 5s.

[0018] Another aspect of the present invention provides an application of a nasal spray nanometer acetaminophen or a preparation method thereof in the preparation of a drug for improving the pain threshold in a plateau environment through nasal administration.

[0019] The difficulty and significance of the technical problem solved by the present invention are:

[0020] The application of PLGA (polylactic-co-glycolic acid) nanoparticles in nasal drug delivery systems has made significant progress. Its unique properties make it have significant advantages in improving drug bioavailability, prolonging drug action time, and reducing drug side effects. PLGA is a copolymer composed of lactic acid and glycolic acid, which has good biocompatibility and degradability. It can be gradually degraded and excreted in the body, reducing the long-term residual problem of drug carriers. PLGA nanoparticles can achieve controlled release of drugs by adjusting the ratio and particle size of its polymers. This controlled release property can prolong the residence time of drugs in the nasal cavity, improve the bioavailability of drugs, and reduce the frequent administration of drugs. Various enzymes in the nasal cavity may degrade certain drugs, and PLGA nanoparticles can protect drugs from enzymolysis to a certain extent, thereby improving the stability of drugs. The applicant of the present invention has designed and provided a nasal nano-agent of acetaminophen using polylactic acid-glycolic acid copolymer (PLGA) nanoparticles as a drug carrier through research, and a preparation method and application thereof, and has experimentally confirmed the effect of nasal use of PLGA nanoparticles loaded with acetaminophen on the pain threshold of mice acutely exposed to high altitude, thus providing a new and efficient drug administration method and treatment means for pain management in high altitude environment, so as to relieve the pain of patients and improve their quality of life.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The scheme of the present invention provides a paracetamol nasal spray nanometer agent and a preparation method and application thereof. The paracetamol nasal spray nanometer agent can significantly increase the accumulation of paracetamol in brain tissue and delay its elimination after nasal administration, and experiments have shown that the paracetamol nasal spray nanometer agent can significantly increase the pain threshold of mice that are rapidly exposed to high altitude, providing a new and efficient drug administration method and treatment means for pain management in high altitude environment, so as to relieve patients' pain and improve their quality of life. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a transmission electron microscopy image of PLGA-NPs and AAP-PLGA-NPs in an embodiment of the present invention;

[0024] Figure 2 is the particle size distribution of AAP-PLGA-NPs in the embodiment of the present invention;

[0025] Figure 3 is the in vitro release curve of AAP-PLGA-NPs in the embodiment of the present invention (n=3);

[0026] Figure 4 This is the CCK-8 experiment in the embodiment of the present invention;

[0027] Figure 5It is the cell death and viability staining detection (X100) in the embodiment of the present invention;

[0028] Figure 6 is a living body fluorescence imaging diagram in an embodiment of the present invention;

[0029] Figure 7 is a blood drug-time curve diagram after nasal administration in an embodiment of the present invention;

[0030] Figure 8 is a brain drug-time curve diagram after nasal administration in an embodiment of the present invention;

[0031] Fig. 9 is a hot plate experiment in an embodiment of the present invention;

[0032] Fig.10 This is the tail flick experiment in the embodiment of the present invention. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0034] The implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0035] The following is a preferred embodiment provided by the present invention: The embodiment of the present invention provides a paracetamol nasal spray nanometer, which is a paracetamol polylactic acid glycolic acid copolymer (PLGA) nanoparticle; the paracetamol polylactic acid glycolic acid copolymer nanoparticle has a spherical appearance, a particle size of 118nm, a polydispersity coefficient value of 0.221, and a zeta potential of -23.7mV. The preparation method thereof is: first, PLGA is used as a carrier material, and PLGA nanoparticles loaded with paracetamol are prepared by ultrasonic emulsification combined with solvent evaporation, specifically:

[0036] Step 1, forming an oil phase: 20 mg of PLGA was dissolved in 1 mL of dichloromethane solution to obtain a polymer solution, and then 2 mg of acetaminophen was dissolved in the polymer solution;

[0037] Step 2, first emulsification: placing a beaker containing the oil phase solution in an ice water bath, and then using an ultrasonic cell disruptor to perform ultrasonic emulsification on the oil phase solution;

[0038] Step 3, second emulsification and stabilization: adding an appropriate amount of surfactant aqueous solution to the product after the first emulsification in step S2, and performing ultrasonic emulsification treatment again to reduce the particle size of the nanoparticles and improve the stability;

[0039] Step 4, solvent volatilization: the particles subjected to ultrasonic emulsification treatment in step S3 are subjected to magnetic low-speed stirring at room temperature to gradually volatilize the organic solvent and gradually solidify and stabilize the PLGA nanoparticles;

[0040] Step 5, collection and purification: Use a centrifuge to centrifuge the solution treated in step S4 to separate the PLGA nanoparticles loaded with acetaminophen; then collect the nanoparticles at 12000r / min high-speed centrifugation for 15min, wash them repeatedly with an appropriate amount of pure water twice, and redisperse them in a small amount of pure water; freeze-dry the collected nanoparticles, and dry them in a low-temperature freeze dryer for 24h to obtain acetaminophen polylactic acid glycolic acid copolymer nanoparticles. The above-mentioned acetaminophen nasal spray nanoparticle or its preparation method according to the embodiment of the present invention can be used to prepare a drug for nasal use to improve the pain threshold of the plateau environment, and provide a new and efficient administration method and treatment method for pain management in the plateau environment to relieve the pain of patients and improve their quality of life.

[0041] The following is an experiment of the above embodiment of the present invention:

[0042] 1. Instruments and Materials:

[0043] NanoBrook Omni particle size potential analyzer (Brookhaven, USA); water purifier (Sichuan Youpu Ultrapure Technology Co., Ltd.); DHG-9070A electric constant temperature blast drying oven (Shanghai Jinghong Experimental Equipment Co., Ltd.); S10-3 constant temperature magnetic stirrer (Shanghai Si Le Instrument Co., Ltd.); ZWY-103D constant temperature oscillator (Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.); LA310s precision electronic balance (Mettler Toledo); UV-visible spectrophotometer (Lambda25, PerkinElmer, USA); JEM-1200EX transmission electron microscope (Jeol, Japan); S-450D ultrasonic cell disruptor (Branson, USA); DMIL-LED fluorescence inverted microscope (Leica, Germany); TDL-60B desktop centrifuge (Shanghai Anting Scientific Instrument Factory); Newton 7.0FT-500 three-dimensional small animal living imaging system (Vilber, France); Multiskan FC-type microplate reader (Thermo Fisher Instrument Co., Ltd., USA); cell culture incubator (Thermo Fisher Instrument Co., Ltd., USA); hypobaric oxygen chamber (Shanghai Tawang Intelligent Technology Co., Ltd.); rat tail light analgesia meter (Jiangsu Saiangs Biotechnology Co., Ltd.); hot plate analgesia meter (Shanghai Xinxin Information Technology Co., Ltd.).

[0044] Acetaminophen API (Shanghai Aladdin Biochemical Technology Co., Ltd.); polylactic acid-glycolic acid copolymer (Shanghai Yuanye Biotechnology Co., Ltd.); indocyanine green (ICG, MedChemExpress, USA); artificial cerebrospinal fluid, artificial nasal fluid (Dongguan Chuangfeng Automation Technology Co., Ltd.); dialysis bag (Sigma-Aldrich, USA); DMEM culture medium, trypsin, fetal bovine serum, PBS buffer (Suzhou Thermo Fisher Instrument Co., Ltd.); cell culture consumables (Costar, USA); CCK-8 detection kit, CalceinAM / PI kit (Beijing Lanjieke Technology Co., Ltd.); mouse hippocampal neuron HT22 cells (Wuhan Pronosai Life Science Technology Co., Ltd.); C57 / BL6 mice (Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.). All experiments were carried out in strict accordance with the 3R principles of experimental animals and provided humane care.

[0045] 2. Experimental Methods

[0046] 1. Preparation of PLGA Nanoparticles

[0047] 1.1. Forming oil phase:

[0048] 20 mg of PLGA (molecular weight 13000, lactic acid: hydroxylactic acid = 75:25) was dissolved in 1 mL of dichloromethane solution, and then 2 mg of acetaminophen was dissolved in the polymer solution.

[0049] 1.2. First emulsification:

[0050] The beaker containing the oil phase solution was placed in an ice water bath to reduce heat generation during the emulsification process. The oil phase solution was ultrasonically emulsified using an ultrasonic cell disruptor (60% power, emulsification time of 45 s, running for 5 s, and intervals of 5 s).

[0051] 1.3. Second emulsification and stabilization:

[0052] An appropriate amount of surfactant aqueous solution (3 mL of 1 wt% PVA aqueous solution, oil phase: water phase = 1:3) was added to the product after the first emulsification, and ultrasonic emulsification treatment was performed again (60% power, emulsification time of 30 s, operation of 5 s, interval of 5 s) to further reduce the particle size of the nanoparticles and improve the stability.

[0053] 1.4. Solvent evaporation:

[0054] The mixture was stirred at low speed by magnetic force for a period of time at room temperature to allow the organic solvent to evaporate gradually and the PLGA nanoparticles to solidify and stabilize gradually.

[0055] 1.5. Collection and purification:

[0056] The emulsified solution was centrifuged using a centrifuge to separate the PLGA nanoparticles loaded with acetaminophen. The nanoparticles were collected by high-speed centrifugation at 12000r / min for 15min, and then washed twice with an appropriate amount of pure water (to remove residual surfactants and organic solvents), and redispersed in a small amount of pure water. The collected nanoparticles were freeze-dried and dried in a low-temperature freeze dryer for 24h to obtain AAP-PLGA-NPs.

[0057] 2. Characterization of Nanoparticles

[0058] 2.1. Determination of drug loading and encapsulation efficiency:

[0059] The developed AAP-PLGA-NPs were centrifuged at 12000rpm for 20 minutes at 4°C, washed twice with pure water, and the supernatant was collected in different vials. The drug loading and encapsulation efficiency of the nanoparticles were determined by ultraviolet visible spectroscopy (UV) at 257nm. The encapsulation efficiency (EE) of the drug-loaded nanoparticles was calculated according to the formula: encapsulation efficiency (EE) = m packages / m drugs x 100% and the drug loading (DL%) of the drug-loaded nanoparticles was calculated according to the formula: drug loading (DL) = m packages / m total x 100%. In the formula, m packages are the mass of drugs encapsulated in the nanoparticles (mg), m drugs are the total mass of drugs put in (mg), and m total is the total mass of drug-loaded nanoparticles (mg).

[0060] 2.2. Particle size and zeta potential determination:

[0061] Take an appropriate amount of PLGA nanoparticle solution, dilute it with pure water to an appropriate concentration, and measure the particle size, PDI and zeta potential of the nanoparticles at 25°C using a nanoparticle size and zeta potential instrument.

[0062] 2.3 Morphological investigation (transmission electron microscopy):

[0063] Take an appropriate amount of PLGA nanoparticle solution, dilute it with pure water to an appropriate concentration, take 1 or 2 drops of the nanoparticle solution and place it on a copper grid, stain it with 10.0 g / L phosphotungstic acid, dry it at room temperature, observe the morphology of the nanoparticles under a transmission electron microscope and take pictures.

[0064] 3. In vitro release experiment of drug-loaded nanoparticles

[0065] Since AAP-PLGA-NPs are prepared for nasal administration and the target of drug action is the brain, three different media were selected to observe its release in vitro, such as artificial cerebrospinal fluid, simulated nasal fluid and phosphate buffered saline (PBS). One side of the dialysis bag was first tied and immersed in 20 mL of release medium overnight. The acetaminophen API and AAP-PLGA-NPs (4 mg in terms of acetaminophen) were accurately weighed and loaded into the dialysis bag. 5 mL of release medium was added. The other end of the dialysis bag was tied and immersed in the release medium. It was placed in a constant temperature oscillator to start dynamic dialysis. The temperature was set to (37±0.5)℃, the oscillation frequency was 100r / min, and the oscillation was 72h. 1 mL was sampled at 0, 2, 4, 6, 8, 12, 24, 48 and 72h, and 1 mL of fresh release medium was added at the same time. After the obtained samples were filtered through a 0.45 μm microporous filter membrane, UV analysis was performed on the samples at 257 nm to determine the concentration of the drug, calculate the cumulative release rate of the drug, and draw an in vitro release curve. The release rate of the drug from the nanoparticles was calculated by using the following equation:

[0066] % release = ((drug concentration × buffer volume / concentration of active ingredient in nanodrug) × 100).

[0067] 4. Determination of toxicity of nanomedicine to HT22 cells

[0068] 4.1、CCK8 cell activity detection:

[0069] HT22 cells were cultured at 3x10 4 The cells were seeded in a 96-well plate at a density of , and complete medium (89% H-DMEM medium, 10% FBS, 1% AB / AM) was added and placed in an incubator at 37°C and 5% CO2 for 24 hours. Then, different concentrations (0.1mM, 1mM, 10mM and 100mM) of Placebo NPs, AAP-soul and AAP-PLGA-NPs were added and incubated with the cells for 24 hours. After removing the original medium, the cells were washed twice with PBS, and 10μl CCK-8 reagent was mixed with 90μl basal medium and added to the well plate. After incubation for another 2 hours in the dark, the absorbance of each well was detected at a wavelength of 450nm using an enzyme reader to calculate the cell survival rate. Cell survival rate (%) = (OD of the drug group-OD of the blank group) / (OD of the control group-OD of the blank group) x 100%.

[0070] 4.2 Live / dead cell staining:

[0071] A live / dead cell assay was used to verify the cell biocompatibility. HT22 cells were cultured at 6x10 cells per well. 4The cells were seeded in a 48-well plate at a density of 100 cells and incubated in complete medium for 24 hours. Then 10 mM AAP-PLGA-NPs was added and cultured for 24 hours. After removing the original medium, the cells were washed twice with PBS. Then, live and dead cell double staining reagents (calcein-AM and PI) were added and incubated in a 37°C incubator in the dark for 20 minutes. After the incubation was completed, the fluorescent staining solution was aspirated and washed three times with PBS. The cell staining was observed under a fluorescence microscope. Live cells were green and dead cells were red. Randomly selected areas were photographed and recorded. Three replicate controls were set for each group.

[0072] 5. In vivo fluorescence imaging

[0073] The target organs of nasal drug administration in vivo were observed using an in vivo fluorescence imaging system. Eighteen healthy C57 / BL6 male mice aged 8 weeks, weighing (21±2) g, were selected. After fasting for 12 hours, 20 μL CCG-labeled AAP-PLGA-NPs were administered nasally. The circulation of the drug in the mice was monitored using a small animal in vivo imaging system, and fluorescence imaging (Ex / Em: 785nm / 810nm) was performed at different time points (0h, 0.5h, 1.5h, 3h, 6h and 24h).

[0074] 6. Determination of blood and brain drug concentrations of AAP-PLGA-NPs in mice

[0075] 48 8-week-old C57 / BL6 male mice with good mental state and body weight of (21±2)g were taken and fasted for 12h. They were randomly divided into 2 groups and given 20ul of AAP-PLGA-NPs and AAP-soul intranasally, respectively, with a dose of 1mg / kg (in terms of acetaminophen). About 0.5mL of blood was collected from the eye sockets at 0.5h, 1h, 1.5h, 2h, 4h, 6h, 12h and 24h. The blood samples were placed in heparin-rinsed centrifuge tubes and centrifuged at 4000r / min for 3min to obtain the upper plasma. After blood collection, the mice were quickly killed by cervical dislocation, brain tissue was removed, rinsed with saline, dried with filter paper, accurately weighed, homogenized with saline, and the supernatant was taken. The sample absorbance was determined by ultraviolet visible spectroscopy (UV), and the acetaminophen concentration in plasma samples and brain tissue was calculated by the working curve method.

[0076] 7. Pharmacodynamic study of AAP-PLGA-NPs

[0077] 7.1. Mouse hot plate licking test:

[0078] At room temperature of 22°C, adjust the temperature of the smart hot plate instrument to (55±0.5)°C and place it on the smart hot plate instrument. During the test, place the soles of the left and right hind paws of 8-week-old healthy C57 / BL6 male mice flat on the center of the hot plate to ensure that the soles are in full contact with the hot plate. Start timing after the mouse's hind paws are placed on the hot plate. The time (s) required for the first licking of the hind paws is used as the reaction latency (pain threshold). The second measurement is performed after an interval of 5 minutes, and the average of the two reaction latencies is used as the basal pain threshold. In order to avoid scalding the animal's hind paw tissue, the test is cut off after 60 seconds. Qualified mice with a basal pain threshold of 5 to 30 seconds and no jumping reaction are screened.

[0079] 7.2. Mouse tail flick experiment:

[0080] During the experiment, the room temperature was controlled at about 22°C, and the radiation power was set to 34W. Healthy C57 / BL6 male mice aged 8 weeks were placed in a mouse holder, and the tails were exposed for light irradiation. The irradiation site was fixed at the middle and lower 1 / 3 of the tail of each mouse, and the pain experiment could be carried out after the mice were calm. The latency from the start of irradiation to the appearance of the tail-flicking reaction was recorded as its pain threshold, and the second measurement was made at an interval of 5 minutes. The average of the two reaction latencies was taken as the basic pain threshold. In order to protect the mouse tail from injury, those that did not flick their tails for more than 10 seconds were recorded as 10 seconds. Qualified mice with a basic pain threshold of 3 to 6 seconds were screened.

[0081] 7.3 Model establishment:

[0082] After the hot plate licking test and tail flick test, 40 qualified mice were screened and randomly divided into 4 groups, 10 mice / group. Control group (C group): mice were placed under normal pressure and normoxic conditions and raised without any treatment; Rapid plateau group (H group): mice were placed in a plateau simulated hypobaric oxygen chamber to establish a model, set at an altitude of 6000 meters, and placed for 7 days; AAP-soul treatment group (AH group): mice were placed in a plateau simulated hypobaric oxygen chamber for 7 consecutive days and given 20ulAAP-soul at 8:00-9:00 am every day, set at an altitude of 6000 meters, and placed for 7 days; AAP-PLGA-NPs treatment group (ANH group): mice were placed in a plateau simulated hypobaric oxygen chamber for 7 consecutive days and given 20ulAAP-PLGA-NPs at 8:00-9:00 am every day, set at an altitude of 6000 meters, and placed for 7 days.

[0083] All mice were subjected to hot plate licking test and tail flicking test at the same time period (14:00-18:00 in the afternoon) 1 day (T1), 2 days (T2), 3 days (T3), 4 days (T4), 5 days (T5), 6 days (T6) and 7 days (T7) after modeling, and the pain threshold was recorded.

[0084] 3. Experimental Results

[0085] 1. Properties of AAP-PLGA-NPs:

[0086] PLGA-NPs and AAP-PLGA-NPs are round and have uniform particle size distribution. Figure 1 The average particle size of PLGA-NPs is 106nm, PDI is 0.243, the average particle size of AAP-PLGA-NPs is 118nm, PDI is 0.221, see Figure 2 As shown. The zeta potential of PLGA-NPs is -24.1mV, and the zeta potential of AAP-PLGA-NPs is -23.7mV. Generally, when the Zeta potential value is greater than or equal to 20mV and PDI <0.3, it indicates that the formulation system is stable and is not prone to precipitation and flocculation. AAP-PLGA-NPs meets this condition, indicating that the formulation has excellent stability. The encapsulation efficiency of AAP-PLGA-NPs is 42.53%, and the drug loading is 3.87%.

[0087] 2. In vitro release:

[0088] In vitro release results (such as Figure 3 As shown in Figure 3, the results showed that the release of AAP-PLGA-NPs in SNF was less than that in PBS and CSF in the first 2 hours. The 12-h cumulative release rates were observed in PBS, SNF, and CSF, respectively, and the release remained constant until 72 hours. The initial slow release of the drug may be due to the slow diffusion of the drug through the PLGA polymer matrix. The results indicate that AAP-PLGA-NPs exhibited better release characteristics in SNF and CSF compared with PBS, so the formulation can be effectively delivered to the brain via the intranasal route. In addition, the sustained release of the drug for 72 hours can maintain the effectiveness of the drug for a longer period of time.

[0089] 3. Cytotoxicity test results (CCK8-HT22):

[0090] Mouse hippocampal neuron HT22 cells were used to detect the cytotoxicity of AAP-PLGA-NPs. Figure 4 shown. Figure 4The results showed that with the increase of treatment concentration, there was no significant change in the cell viability of the Placebo NPs, AAP-PLGA-NPs and AAP-Sol groups, indicating that the concentration had no significant effect on the viability of HT22 cells. Finally, AAP-PLGA-NPs 10mM was selected as the subsequent experimental concentration; compared with the Placebo NPs group at the same concentration, there was no significant decrease in cell viability in the AAP-PLGA-NPs and AAP-Sol groups, and both were higher than 97%, indicating that the effects of nanomaterials and drugs on cells were very small, that is, the drug delivery system had high biocompatibility.

[0091] 4. Live and dead staining:

[0092] The results of cell death and viability staining are shown in Figure 5 As shown in (X100), the green signal represents live cells and the red signal represents dead cells. The number of dead cells stained red by PI in the AAP-PLGA-NPs group was not significantly different from that in the control group, and the field of view was basically green fluorescence signals representing live cells.

[0093] 5. In vivo fluorescence:

[0094] After intranasal administration of ICG-labeled AAP-PLGA-NPs, it was observed that the target organ of the drug action was mainly the brain.

[0095] 6. Blood and brain concentrations of AAP-PLGA-NPs in mice:

[0096] Compared with the AAP-Sol group, the blood and brain drug concentrations of the AAP-PLGA-NPs group showed a trend of decreasing and then increasing as time went on after nasal administration. As time went on, the blood and brain drug concentrations of both groups of mice showed a trend of increasing first and then decreasing. The results showed that AAP-PLGA-NPs had a slow release effect after nasal administration. Figure 7 and Figure 8 shown.

[0097] 7. Pharmacodynamic observation:

[0098] like Fig. 9 and Fig.10 As shown, compared with the AH group (AAP-Sol), the ANH group (AAP-PLGA-NPs) could significantly increase the pain threshold of the hot plate test and the tail-flick latency of mice subjected to acute plateau stress.

[0099] The above-mentioned embodiments of the present invention and their experiments confirm that: by characterizing AAP-PLGA-NPs: using a transmission electron microscope to observe the morphology of AAP-PLGA-NPs; and using a nanoparticle size potentiometer to measure the particle size, polydispersity coefficient and zeta potential of AAP-PLGA-NPs; using ultracentrifugation to determine the encapsulation efficiency and drug loading of AAP-PLGA-NPs; using equilibrium dialysis to evaluate the release of AAP-PLGA-NPs in in vitro media (artificial cerebrospinal fluid, artificial nasal fluid and PBS solution). Then, CCK8 experiment and live-dead staining were used to observe the effect of AAP-PLGA-NPs on the toxicity of hippocampal neuronal cells (HT22). The intensity of the fluorescent signal in the mouse brain at different time points after intranasal administration of ICG-labeled acetaminophen suspension (AAP-Sol) was observed using in vivo fluorescence imaging technology. Then, AAP-Sol was used as the control group to investigate the concentration in brain tissue and blood after intranasal administration of AAP-PLGA-NPs in mice. Finally, the acute plateau mice were used as a model, and AAP-Sol was used as the control group. The hot plate method and tail flick method were used to evaluate the effect of AAP-PLGA-NPs on the pain threshold. Results: The prepared AAP-PLGA-NPs were spherical in appearance, with a particle size of 118nm, a polydispersity coefficient of 0.221, and a zeta potential of -23.7mV; the encapsulation efficiency and drug loading were 42.53% and 3.87%, respectively; the release in vitro (PBS solution, artificial nasal fluid and artificial cerebrospinal fluid) could last up to 72 hours, and the release rate was about 82%. CCK8 results showed that the cell viability of the AAP-PLGA-NPs group was about 97%, and the number of dead cells observed by live-dead staining was no significant difference from that of the control group, indicating that AAP-PLGA-NPs would not cause cytotoxicity and had good biocompatibility. The results of in vivo fluorescence imaging showed that after nasal administration, it was mainly distributed in the animal brain. The in vivo pharmacokinetic results showed that AAP-PLGA-NPs had a slow release effect after nasal administration. Pharmacodynamic studies showed that compared with the AAP-Sol group, the AAP-PLGA-NPs group could significantly increase the pain threshold of the hot plate test and the tail-flick latency of the mice who were acutely exposed to high altitude. Conclusion: AAP-PLGA-NPs can significantly increase the accumulation of acetaminophen in the brain tissue and delay its elimination after nasal administration, and significantly increase the pain threshold of the mice who were acutely exposed to high altitude.

[0100] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A paracetamol nasal spray nanometer, characterized in that: The nano agent is acetaminophen polylactic acid glycolic acid copolymer nanoparticles; the acetaminophen polylactic acid glycolic acid copolymer nanoparticles are spherical in appearance, with a particle size of 118nm, a polydispersity coefficient value of 0.221, and a zeta potential of -23.7mV.

2. The method for preparing a nasal spray nanometer of acetaminophen according to claim 1, characterized in that: The preparation method is: firstly, PLGA is used as a carrier material, and PLGA nanoparticles loaded with acetaminophen are prepared by ultrasonic emulsification combined with solvent volatilization, which specifically includes the following steps: S1. Forming an oil phase: dissolving 20 mg of PLGA in 1 mL of dichloromethane solution to obtain a polymer solution, and then dissolving 2 mg of acetaminophen in the polymer solution; S2, first emulsification: placing a beaker containing the oil phase solution in an ice water bath, and then using an ultrasonic cell disruptor to perform ultrasonic emulsification on the oil phase solution; S3, second emulsification and stabilization: adding an appropriate amount of surfactant aqueous solution to the product after the first emulsification in step S2, and performing ultrasonic emulsification treatment again to reduce the particle size of the nanoparticles and improve the stability; S4, solvent volatilization: subjecting the liquid subjected to ultrasonic emulsification treatment in step S3 to magnetic low-speed stirring at room temperature to gradually volatilize the organic solvent and gradually solidify and stabilize the PLGA nanoparticles; S5, collection and purification: centrifuge the solution treated in step S4 to separate the PLGA nanoparticles loaded with acetaminophen; then high-speed centrifuge at 12000r / min for 15min to collect the nanoparticles, and then repeatedly wash them twice with an appropriate amount of pure water, and redisperse them in a small amount of pure water; freeze-dry the collected nanoparticles, and dry them in a low-temperature freeze dryer for 24h to obtain acetaminophen polylactic acid glycolic acid copolymer nanoparticles.

3. The method for preparing a nasal spray nanometer of acetaminophen according to claim 2, characterized in that: The molecular weight of PLGA in step S1 is 13,000, and the ratio of lactic acid to hydroxylactic acid is 75:

25.

4. The method for preparing a paracetamol nasal spray nanometer according to claim 3, characterized in that: The ultrasonic cell disruptor in step S2 was set to 60% power, emulsification time was 45 s, and run for 5 s with an interval of 5 s.

5. The method for preparing a paracetamol nasal spray nanometer according to claim 4, characterized in that: The surfactant aqueous solution in step S3 is 3 mL of 1 wt % PVA aqueous solution, and the oil phase:water phase ratio is 1:

3.

6. The method for preparing a nasal spray nanometer of acetaminophen according to claim 5, characterized in that: The ultrasonic emulsification treatment in step S3 is: 60% power, emulsification time is 30s, running is 5s, and interval is 5s.

7. Use of the acetaminophen nasal spray nanoparticle according to claim 1 or the preparation method according to any one of claims 2 to 6 in the preparation of a drug for improving the pain threshold in a plateau environment through nasal administration.

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