W / O / W type nano-emulsion loaded with cocaine esterase as well as preparation method and application of W / O / W type nano-emulsion

Abstract

The invention discloses a W / O / W type nano-emulsion loaded with cocaine esterase as well as a preparation method and application of the W / O / W type nano-emulsion. The nano-emulsion is prepared from cocaine esterase, a phosphate buffer solution, soybean phospholipid, vitamin E polyethylene glycol succinate, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 and medium chain triglyceride. The preparation method comprises the following steps: preparing the inner water phase; preparing an oil phase; slowly injecting the internal water phase into the oil phase under shearing to obtain W / O type primary emulsion; slowly adding the primary emulsion into the external water phase under shearing, and performing high-speed shearing to obtain a secondary emulsion; and performing high-pressure homogenization on the secondary emulsion to obtain the final W / O / W type nano-emulsion. The cocaine esterase is combined with the nano-emulsion for the first time, and the nano-emulsion prepared by the method has the effects of improving the temperature stability of the cocaine esterase and prolonging the in-vivo half-life period, and can be applied to rescue or prevention of cocaine acute poisoning.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a W / O / W type nanoemulsion loaded with cocaine esterase, a preparation method thereof, and an application thereof. Background Art

[0002] As a potent central nervous system stimulant, cocaine has extremely strong hallucinogenic and addictive properties. Taking a large dose of cocaine can cause heart failure and severely inhibit the respiratory center in the brain, thereby leading to death. According to statistics, between 2020 and 2021, the number of people who died from overdose of cocaine in the United States exceeded 20,000, and the number of drug overdose deaths involving cocaine increased by 22% compared with the previous year (Spencer, M.R, A.M, & Garnett, M.F (2023). Co-involvement of Opioids in Drug Overdose Deaths Involving Cocaine and Psychostimulants, 2011 - 2021. NCHS databrief, (474), 1 - 8.). Acute cocaine poisoning has become a global problem endangering human health. Currently, the clinical treatment methods for acute cocaine poisoning mainly rely on symptomatic treatment, such as using benzodiazepines, short-acting barbiturates, etc., which can only relieve symptoms and there is no specific drug that can directly eliminate cocaine in the body.

[0003] Effective enzyme therapy is considered the most promising strategy for treating cocaine overdose. Bacterial cocaine esterase (CocE) exhibits high catalytic activity towards cocaine and can catalyze the hydrolysis of cocaine to produce non-toxic metabolite benzoic acid. Its quadruple mutant (T172R / G173Q / L196C / I301C, hereinafter referred to as E196 - 301, K M = 1.5 μM, k cat = 3450 min -1 ) can rapidly clear cocaine in the body and is a highly potential cocaine antidote drug. However, as an exogenous protein drug, cocaine esterase has defects such as being sensitive to temperature and having a short half-life in the body, which limits its clinical application. Therefore, preparing a preparation for loading and delivering unstable protein drugs to improve the thermal stability and in vivo half-life of cocaine esterase, thereby enhancing the drug efficacy, is of great significance for the application of cocaine esterase in the rescue or prevention of acute cocaine poisoning.

[0004] Lipid nanoparticles are drug delivery carriers composed of materials such as phospholipids, with particle sizes ranging from dozens to hundreds of nanometers, mainly including the following dosage forms: liposomes, solid lipid nanoparticles, nanostructured lipid carriers, microemulsions, and nanoemulsions. In the study of loading enzymes through lipid nanoparticles, liposomes and nanoparticles are usually used. This is because liposomes are composed of a phospholipid bilayer, similar to the cell membrane structure, with high biocompatibility, and their closed structure can effectively protect enzymes from degradation by body fluids (such as proteases, extreme pH). Polymer nanoparticles (such as PLGA, chitosan) can immobilize enzymes through physical entrapment or chemical bonding, providing a mechanical barrier, prolonging the circulation time, and achieving targeted delivery through surface modification. However, nanoemulsions often require high-energy homogenization, ultrasound, or high-temperature and high-pressure treatment during preparation, which may cause enzyme denaturation and inactivation due to excessive temperature, so they are generally not used for enzyme loading, which leads to certain limitations in the administration of enzyme substances.

[0005] Therefore, developing a water-in-oil-in-water nanoemulsion based on cocaine esterase E196-301 for the rescue or prevention of acute cocaine poisoning is of great significance in the delivery of unstable protein drugs for treating diseases. Summary of the Invention

[0006] The purpose of the present invention is to solve the deficiencies of the existing technology. Aiming at the deficiencies of cocaine esterase in acute cocaine poisoning and prevention, a W / O / W nanoemulsion containing cocaine esterase and its preparation method are provided. For the first time, cocaine esterase is combined with nanoemulsion, and the thermal stability of cocaine esterase is improved through the special structure of the water-in-oil-in-water nanoemulsion.

[0007] In order to achieve the above invention purpose, the present invention provides the following technical solutions:

[0008] In the first aspect, the present invention provides a preparation method of a W / O / W nanoemulsion loaded with cocaine esterase, which greatly reduces the influence of high temperature on thermally unstable enzymes. The preparation method includes the following steps:

[0009] Step (1), prepare the inner aqueous phase: Dilute cocaine esterase with phosphate buffer as the inner aqueous phase;

[0010] Step (2), prepare the oil phase: Add soybean phospholipid, vitamin E polyethylene glycol succinate, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 to medium-chain triglycerides and melt at high temperature to obtain the oil phase;

[0011] Step (3), prepare the primary emulsion: After the oil phase is cooled under high-speed shearing, slowly inject the inner aqueous phase into the oil phase and continue high-speed shearing to obtain a W / O primary emulsion;

[0012] Step (4), preparing a secondary emulsion: Using phosphate buffer as the external aqueous phase, slowly add the W / O primary emulsion into the external aqueous phase under high-speed shearing, and continue high-speed shearing under an ice bath to obtain a secondary emulsion;

[0013] Step (5), preparing a final emulsion: Subject the secondary emulsion to high-pressure homogenization at 4°C to obtain the final W / O / W type nanoemulsion;

[0014] Step (6), ultrafiltration concentration: Centrifuge and concentrate the nanoemulsion through an ultrafiltration tube at 4000 r / min, and simultaneously separate the enzyme-containing nanoemulsion from the unencapsulated enzyme.

[0015] Preferably, in the step (1), the dosage of cocaine esterase (E196-301) is 0.5-1 mg / mL based on the total volume of the W / O / W type nanoemulsion.

[0016] Preferably, in the step (2), the mass ratio of the total mass of soybean phospholipid, vitamin E polyethylene glycol succinate, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 to the mass of cocaine esterase is (10-30):1, and the more preferred mass ratio is 20:1. The method of the present invention prolongs the in vivo half-life of the enzyme by surface-modifying the nanoemulsion with distearoyl phosphatidylethanolamine-polyethylene glycol 2000.

[0017] Preferably, in the step (2), the mass ratio of soybean phospholipid to vitamin E polyethylene glycol succinate is (4-5):1, and the more preferred ratio is 4.4:1; the mass ratio of soybean phospholipid to distearoyl phosphatidylethanolamine-polyethylene glycol 2000 is 40:1.

[0018] Preferably, in the step (2), the melting temperature of the oil phase is 60-80°C.

[0019] Preferably, in the steps (3) and (4), the shearing speed is 8000-12000 r / min, and the shearing time is 10-15 min.

[0020] Preferably, in the step (3), the cooling temperature is 20-30°C, and the more preferred temperature is 25°C.

[0021] Preferably, in the step (5), the high-pressure homogenization pressure is 1200-1500 bar, and the number of homogenization times is 3-6 times.

[0022] Preferably, in the step (6), the molecular weight cut-off of the ultrafiltration tube is 100 kDa, and the centrifugation time is 2-4 h.

[0023] Preferably, in the W / O / W type nanoemulsion, the volume ratio of the oil phase to the internal aqueous phase is (10-12):1, and the more preferred ratio is 10:1; the volume ratio of the external aqueous phase to the oil phase is (10-12):1, and the more preferred ratio is 10:1.

[0024] Preferably, the purity of the soybean phospholipid is not less than 95%.

[0025] Preferably, the pH value of the phosphate buffer solution is 7.2 - 7.6.

[0026] In a second aspect, the present invention provides a cocaine esterase W / O / W type nanoemulsion for the rescue or prevention of cocaine poisoning, which is prepared by the above method.

[0027] Specifically, the nanoemulsion consists of three phases: an inner aqueous phase, an oil phase, and an outer aqueous phase. The inner aqueous phase includes cocaine esterase (E196 - 301) and a phosphate buffer solution; the oil phase includes soybean phospholipid, vitamin E polyethylene glycol succinate (TPGs), distearoyl phosphatidylethanolamine - polyethylene glycol 2000 (DSPE - MPEG2000), and medium - chain triglycerides (MCT); the outer aqueous phase is a phosphate buffer solution.

[0028] In a third aspect, the present invention provides the application of the above - mentioned W / O / W type nanoemulsion loaded with cocaine esterase in the preparation of a preparation for the rescue or prevention of acute cocaine poisoning.

[0029] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0030] The present invention selects soybean phospholipid as the main material and encapsulates the enzyme in the form of a nanoemulsion. E196 - 301 is encapsulated in a nanoemulsion (CocE NEs) composed of soybean phospholipid, vitamin E polyethylene glycol succinate (TPGs), medium - chain triglycerides (MCT), and distearoyl phosphatidylethanolamine - polyethylene glycol 2000 (DSPE - MPEG2000). The present invention optimizes the preparation method of the nanoemulsion. For the first time, the temperature for preparing the primary emulsion is controlled around room temperature, overcoming the technical prejudice in the prior art that the primary emulsion can only be prepared at high temperatures, and making the remaining enzyme activity of the prepared enzyme - containing nanoemulsion higher than 90%. The nanoemulsion is in the form of a water - in - oil - in - water emulsion. E196 - 301 is encapsulated in the innermost aqueous phase. The oil phase plays a role in isolating the external environment, enhancing the stability of the enzyme, and there is MPEG2000 modification on the surface of the nanoparticles, enabling the nanoemulsion to have the ability to extend the in - vivo half - life. The present invention combines cocaine esterase with the nanoemulsion for the first time through a unique preparation method, improving the thermal stability while extending the in - vivo half - life. Aiming at the deficiencies of existing cocaine esterase in storage and treatment, a more stable detoxifying enzyme preparation with a long in - vivo half - life is prepared by pharmaceutical means, providing a basis for the further clinical application of cocaine esterase and a new idea for the research and development of long - acting enzymes.

[0031] In summary, the beneficial effects of the present invention are specifically as follows:

[0032] (1) The nanoemulsion prepared by the present invention can improve the stability of E196-301 at 37 °C and 40 °C, and can maintain more than 95% activity within 100 h at 37 °C and more than 90% activity within 720 min at 40 °C.

[0033] (2) The nanoemulsion prepared by the present invention can improve the in vivo half-life of E196-301. When the dose is 3 mg / kg, the improvement effect is 3-4 times.

[0034] (3) The nanoemulsion prepared by the present invention can effectively degrade the subsequently injected cocaine within 60 minutes after intravenous injection (1 mg / kg), greatly reduce the cocaine content in the blood, and at the same time weaken the influence of cocaine on the excited state of the mouse brain. Description of the Drawings

[0035] Figure 1 It is the process flow chart of the preparation method of the cocaine esterase nanoemulsion in Examples 1-7 of the present invention.

[0036] Figure 2 It is the transmission electron microscope image of the cocaine esterase nanoemulsion in Example 1 of the present invention.

[0037] Figure 3 It is the graph of the change of particle size (A) and PDI (B) of the cocaine esterase nanoemulsion (CocE NEs) in Example 1 of the present invention when placed at 4 °C in different media for 28 days.

[0038] Figure 4 It is the graph of the change of enzyme activity with time (A, B, C) of the cocaine esterase nanoemulsion (CocE NEs) and E196-301 in Example 1 of the present invention at different temperatures and the thermal drift result graph (D, E, F) of E196-301 in three states.

[0039] Figure 5 It is the graph of the change process of the concentration in vivo with time after the cocaine esterase nanoemulsion (CocE NEs) and E196-301 in Example 1 of the present invention are injected at a dose of 1 mg / kg (A) and 3 mg / kg (B).

[0040] Figure 6 It is the graph of the change of the concentrations of cocaine (A) and benzoic acid (B) in vivo when the cocaine esterase nanoemulsion (CocE NEs) and E196-301 in Example 1 of the present invention are injected with cocaine 1 h after injection.

[0041] Figure 7The distance traveled by the mice during the entire process when CocE NEs and E196-301 in Example 1 of the present invention were intravenously injected into the mouse tail vein at a dose of 3 mg / kg and then cocaine at a dose of 25 mg / kg was intraperitoneally injected 45 min later. (A) Total distance traveled by the mice; (B) Time course of the distance traveled by the mice, with the injection of cocaine being 0 min. Detailed implementation mode

[0042] The technical solution of the present invention will be further specifically described below with reference to the accompanying drawings and through specific embodiments, but the protection scope of the present invention is not limited thereby.

[0043] The preparation method of the W / O / W type nanoemulsion loaded with cocaine esterase provided by the present invention includes the following steps:

[0044] Step (1), preparing the internal aqueous phase: Dilute cocaine esterase with phosphate buffer solution as the internal aqueous phase; wherein, the pH value of the phosphate buffer solution is 7.2-7.6;

[0045] Step (2), preparing the oil phase: Add soybean phospholipid, vitamin E polyethylene glycol succinate, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 to medium-chain triglyceride and melt at 60-80 °C to obtain the oil phase; wherein, the mass ratio of soybean phospholipid to vitamin E polyethylene glycol succinate is (4-5):1, the mass ratio of soybean phospholipid to distearoyl phosphatidylethanolamine-polyethylene glycol 2000 is 40:1, and the total mass ratio of soybean phospholipid, vitamin E polyethylene glycol succinate, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 to cocaine esterase is (10-30):1; the purity of the soybean phospholipid is not less than 95%;

[0046] Step (3), preparing the primary emulsion: Shear the oil phase at a high speed of 8000-12000 r / min for 10-15 min and cool to 20 °C-30 °C, slowly inject the internal aqueous phase into the oil phase through a 1 mL syringe, and continue high-speed shearing to obtain a W / O type primary emulsion; the volume ratio of the oil phase to the internal aqueous phase is 10:1;

[0047] Step (4), preparing the secondary emulsion: Use phosphate buffer solution as the external aqueous phase, and slowly transfer the W / O type primary emulsion into the external aqueous phase through a pipette under high-speed shearing, and continue high-speed shearing in an ice bath to obtain the secondary emulsion; the volume ratio of the external aqueous phase to the oil phase is 10:1;

[0048] Step (5), preparing the final emulsion: Subject the secondary emulsion to high-pressure homogenization at 4 °C and 1200-1500 bar for 3-6 times to obtain the final W / O / W type nanoemulsion;

[0049] Step (6), ultrafiltration concentration: Concentrate the nanoemulsion by centrifuging through an ultrafiltration tube at 4000 r / min for 2 - 4 h, while separating the enzyme-containing nanoemulsion from the unencapsulated enzyme; the molecular weight cut-off of the ultrafiltration tube is 100 kDa.

[0050] The following further describes the present invention.

[0051] Example 1:

[0052] (1) Prepare the inner aqueous phase: Dilute 50 mg of E196 - 301 with 0.5 mL of phosphate buffer as the inner aqueous phase;

[0053] (2) Prepare the oil phase: Add 0.8 g of soy lecithin, 0.18 g of vitamin E polyethylene glycol succinate, and 0.02 g of distearoyl phosphatidylethanolamine - polyethylene glycol 2000 to 5 g of medium-chain triglycerides, and melt at 80 °C to obtain the oil phase;

[0054] (3) Prepare the primary emulsion: Cool the oil phase to 25 °C under high-speed shearing at 8000 r / min, and slowly inject the inner aqueous phase into the oil phase using a syringe, and continue high-speed shearing at 10000 r / min for 10 min to obtain a W / O type primary emulsion;

[0055] (4) Prepare the secondary emulsion: Slowly add the primary emulsion to 50 mL of the outer aqueous phase phosphate buffer under high-speed shearing at 12000 r / min, and continue high-speed shearing for 10 min under ice bath to obtain the secondary emulsion;

[0056] (5) Prepare the final emulsion: Subject the secondary emulsion to high-pressure homogenization at 4 °C, 1200 bar, and homogenize repeatedly 5 times to obtain the final W / O / W type nanoemulsion.

[0057] (6) Concentrate the enzyme-containing nanoemulsion by centrifuging through a 100 kDa ultrafiltration tube at 4000 r / min for 2 h.

[0058] Example 2:

[0059] (1) Prepare the inner aqueous phase: Dilute 50 mg of E196 - 301 with 0.5 mL of phosphate buffer as the inner aqueous phase;

[0060] (2) Prepare the oil phase: Add 0.4 g of soy lecithin, 0.09 g of vitamin E polyethylene glycol succinate, and 0.01 g of distearoyl phosphatidylethanolamine - polyethylene glycol 2000 to 5 g of medium-chain triglycerides, and melt at 80 °C to obtain the oil phase;

[0061] (3) Prepare the primary emulsion: Cool the oil phase to 25 °C under high-speed shearing at 8000 r / min, and slowly inject the inner aqueous phase into the oil phase using a syringe, and continue high-speed shearing at 10000 r / min for 10 min to obtain a W / O type primary emulsion;

[0062] (4) Preparation of secondary emulsion: Slowly add the primary emulsion into 50 mL of external aqueous phase phosphate buffer under high-speed shearing at 12000 r / min, and continue high-speed shearing for 10 min under ice bath to obtain the secondary emulsion;

[0063] (5) Preparation of final emulsion: Subject the secondary emulsion to high-pressure homogenization at 4 °C, 1200 bar, and homogenize repeatedly for 5 times to obtain the final W / O / W type nanoemulsion.

[0064] (6) Concentrate the enzyme-containing nanoemulsion by centrifugation at 4000 r / min for 2 h through a 100 kDa ultrafiltration tube.

[0065] Example 3:

[0066] (1) Preparation of internal aqueous phase: Dilute 50 mg of E196-301 with 0.5 mL of phosphate buffer as the internal aqueous phase;

[0067] (2) Preparation of oil phase: Add 1.2 g of soy lecithin, 0.27 g of vitamin E polyethylene glycol succinate, and 0.03 g of distearoyl phosphatidylethanolamine-polyethylene glycol 2000 to 5 g of medium-chain triglycerides, and melt at 80 °C to obtain the oil phase;

[0068] (3) Preparation of primary emulsion: Cool the oil phase to 25 °C under high-speed shearing at 8000 r / min, and slowly inject the internal aqueous phase into the oil phase using a syringe, and continue high-speed shearing at 10000 r / min for 10 min to obtain the W / O type primary emulsion;

[0069] (4) Preparation of secondary emulsion: Slowly add the primary emulsion into 50 mL of external aqueous phase phosphate buffer under high-speed shearing at 12000 r / min, and continue high-speed shearing for 10 min under ice bath to obtain the secondary emulsion;

[0070] (5) Preparation of final emulsion: Subject the secondary emulsion to high-pressure homogenization at 4 °C, 1200 bar, and homogenize repeatedly for 5 times to obtain the final W / O / W type nanoemulsion.

[0071] (6) Concentrate the enzyme-containing nanoemulsion by centrifugation at 4000 r / min for 2 h through a 100 kDa ultrafiltration tube.

[0072] Example 4:

[0073] (1) Preparation of internal aqueous phase: Dilute 50 mg of E196-301 with 0.5 mL of phosphate buffer as the internal aqueous phase;

[0074] (2) Preparation of oil phase: Add 0.8 g of soy lecithin, 0.18 g of vitamin E polyethylene glycol succinate, and 0.02 g of distearoyl phosphatidylethanolamine-polyethylene glycol 2000 to 5 g of medium-chain triglycerides, and melt at 80 °C to obtain the oil phase;

[0075] (3) Preparation of primary emulsion: The oil phase was cooled to 4 °C under high-speed shearing at 8000 r / min, and the inner aqueous phase was slowly injected into the oil phase using a syringe, followed by continuous high-speed shearing at 10000 r / min for 10 min to obtain a W / O type primary emulsion;

[0076] (4) Preparation of secondary emulsion: The primary emulsion was slowly added to 50 mL of the outer aqueous phase phosphate buffer under high-speed shearing at 12000 r / min, and high-speed shearing was continued for 10 min under ice bath to obtain a secondary emulsion;

[0077] (5) Preparation of final emulsion: The secondary emulsion was subjected to high-pressure homogenization at 4 °C, 1200 bar, and homogenized repeatedly 5 times to obtain the final W / O / W type nanoemulsion.

[0078] (6) Concentrate the enzyme-containing nanoemulsion by centrifugation at 4000 r / min for 2 h through a 100 kDa ultrafiltration tube.

[0079] Example 5:

[0080] (1) Preparation of inner aqueous phase: 50 mg of E196-301 was diluted with 0.5 mL of phosphate buffer as the inner aqueous phase;

[0081] (2) Preparation of oil phase: 0.8 g of soy lecithin, 0.18 g of vitamin E polyethylene glycol succinate, and 0.02 g of distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were added to 5 g of medium-chain triglycerides and melted at 80 °C to obtain the oil phase;

[0082] (3) Preparation of primary emulsion: The oil phase was cooled to 40 °C under high-speed shearing at 8000 r / min, and the inner aqueous phase was slowly injected into the oil phase using a syringe, followed by continuous high-speed shearing at 10000 r / min for 10 min to obtain a W / O type primary emulsion;

[0083] (4) Preparation of secondary emulsion: The primary emulsion was slowly added to 50 mL of the outer aqueous phase phosphate buffer under high-speed shearing at 12000 r / min, and high-speed shearing was continued for 10 min under ice bath to obtain a secondary emulsion;

[0084] (5) Preparation of final emulsion: The secondary emulsion was subjected to high-pressure homogenization at 4 °C, 1200 bar, and homogenized repeatedly 5 times to obtain the final W / O / W type nanoemulsion.

[0085] (6) Concentrate the enzyme-containing nanoemulsion by centrifugation at 4000 r / min for 2 h through a 100 kDa ultrafiltration tube.

[0086] Example 6:

[0087] (1) Preparation of inner aqueous phase: 50 mg of E196-301 was diluted with 0.5 mL of phosphate buffer as the inner aqueous phase;

[0088] (2) Preparation of the oil phase: 0.8 g of soy lecithin, 0.18 g of vitamin E polyethylene glycol succinate, and 0.02 g of distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were added to 5 g of medium-chain triglycerides and melted at 80 °C to obtain the oil phase;

[0089] (3) Preparation of the primary emulsion: The oil phase was cooled to 60 °C under high-speed shearing at 8000 r / min, and the inner aqueous phase was slowly injected into the oil phase using a syringe, followed by continuous high-speed shearing at 10000 r / min for 10 min to obtain a W / O type primary emulsion;

[0090] (4) Preparation of the secondary emulsion: The primary emulsion was slowly added to 50 mL of the outer aqueous phase phosphate buffer under high-speed shearing at 12000 r / min, and high-speed shearing was continued for 10 min under an ice bath to obtain the secondary emulsion;

[0091] (5) Preparation of the final emulsion: The secondary emulsion was subjected to high-pressure homogenization at 4 °C, 1200 bar, and homogenized repeatedly 5 times to obtain the final W / O / W type nanoemulsion.

[0092] (6) The enzyme-containing nanoemulsion was concentrated by centrifugation at 4000 r / min for 2 h through a 100 kDa ultrafiltration tube.

[0093] Example 7:

[0094] (1) Preparation of the inner aqueous phase: 50 mg of E196-301 was diluted with 0.5 mL of phosphate buffer as the inner aqueous phase;

[0095] (2) Preparation of the oil phase: 0.8 g of soy lecithin, 0.18 g of vitamin E polyethylene glycol succinate, and 0.02 g of distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were added to 5 g of medium-chain triglycerides and melted at 80 °C to obtain the oil phase;

[0096] (3) Preparation of the primary emulsion: Under high-speed shearing at 80 °C and 8000 r / min, the inner aqueous phase was slowly injected into the oil phase using a syringe, followed by continuous high-speed shearing at 10000 r / min for 10 min to obtain a W / O type primary emulsion;

[0097] (4) Preparation of the secondary emulsion: The primary emulsion was slowly added to 50 mL of the outer aqueous phase phosphate buffer under high-speed shearing at 12000 r / min, and high-speed shearing was continued for 10 min under an ice bath to obtain the secondary emulsion;

[0098] (5) Preparation of the final emulsion: The secondary emulsion was subjected to high-pressure homogenization at 4 °C, 1200 bar, and homogenized repeatedly 5 times to obtain the final W / O / W type nanoemulsion.

[0099] (6) Concentrate the enzyme-containing nanoemulsion by centrifuging at 4000 r / min for 2 h through a 100 kDa ultrafiltration tube.

[0100] Perform performance tests on the nanoemulsions prepared in Examples 1-7 as follows:

[0101] I. Parameter characterization of cocaine esterase nanoemulsion

[0102] Detect the particle size, PDI, encapsulation efficiency, and relative enzyme activity of the cocaine esterase nanoemulsion prepared in the above examples. The remaining enzyme activity of the nanoemulsion is detected by measuring the amount of benzoic acid generated after reacting with cocaine. Relative enzyme activity = (benzoic acid concentration / reaction time / enzyme concentration) 纳米乳液 / (benzoic acid concentration / reaction time / enzyme concentration) E196-301 × 100%. Detect the unencapsulated enzyme concentration by BCA kit. Encapsulation efficiency = (total enzyme concentration - unencapsulated enzyme concentration) / total enzyme concentration × 100%. The results are shown in Table 1. It can be seen from Table 1 that when the mass ratio of the total mass of soybean phospholipid, vitamin E polyethylene glycol succinate, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 to the mass of E196-301 is 20:1 and the cooling temperature is 25 °C (Example 1), the particle size and PDI (Polymer dispersity index) of the nanoemulsion are optimal, and the encapsulation efficiency and enzyme activity are relatively high.

[0103] Table 1: Results of parameter characterization of cocaine esterase nanoemulsion (mean ± SD)

[0104]

[0105] II. Morphology observation of cocaine esterase nanoemulsion

[0106] Figure 2 This is the transmission electron microscopy image of the cocaine esterase nanoemulsion of Example 1 of the present invention. To more intuitively observe the morphology of the nanoemulsion, it was observed by transmission electron microscopy. As shown in the figure, the morphology of this nanoemulsion presents uniform spherical shapes. There is a light-colored oil phase wrapping a light-transmitting aqueous phase in the large spherical particles, indicating that a water-in-oil-in-water nano-system has been formed. The diameter of the large particles is between 60 - 100 nm, and the particles are relatively uniform.

[0107] III. Storage stability of cocaine esterase nanoemulsion

[0108] Figure 3Particle size and PDI change diagram of cocaine esterase nanoemulsion (CocE NEs) in Example 1 of the present invention stored at 4 °C for 28 days in different media (values are expressed as mean ± SD). The storage stability of the nanoemulsion was evaluated by measuring the particle size and PDI of the nanoemulsion during storage at 4 °C for 28 days in PBS, 10% FBS / PBS, and 10% FBS / DMEM. As shown in the figure, the particle size ( Figure 3 A in Figure 3 ) and PDI ( Figure 3 B in

[0109] ) of the cocaine esterase nanoemulsion did not change significantly during storage at 4 °C for 28 days in the above three media, indicating that the cocaine esterase nanoemulsion in Example 1 had excellent storage stability under this condition. IV. Temperature stability of cocaine esterase nanoemulsion

[0110] Figure 4 Graphs showing the changes in enzyme activity over time (A, B, C) of cocaine esterase nanoemulsion (CocE NEs) and E196-301 in Example 1 of the present invention at different temperatures, and the thermal drift results graphs (D, E, F) of E196-301 in three states, with values expressed as mean ± SD. To demonstrate the improvement in the temperature tolerance of E196-301 by the nanoemulsion, the stability of E196-301 and the nanoemulsion at 37 °C, 40 °C, and 45 °C was compared. The method was as follows: 500 μL of E196-301 (0.2 mg / ml, in 0.1M PB / in 0.1M PB + 0.5 mg / ml BSA) and cocaine esterase nanoemulsion (0.2 mg / ml, in 0.1M PB) were placed in glass tubes and incubated in an oven at 37 °C / a metal bath at 40 °C / a metal bath at 45 °C. Samples of 50 μL were taken at regular intervals and diluted for enzyme activity detection. Additionally, the melting temperature (Tm) of E196-301 (PB / PB with BSA) and CocE NEs was detected by a thermal drift experiment.

[0111] Figure 4As shown in (A), (B), and (C), E196-301 is relatively sensitive to temperature changes, and when diluted with 0.1M PB containing 0.5mg / ml BSA, the stability of E196-301 at 37°C and 40°C is better than that when diluted with 0.1M PB. When diluted with 0.1M PB, the remaining enzyme activity after 100h at 37°C is about 70%, and the enzyme activity only remains about 40% after 720min at 40°C; while when diluted with 0.1M PB containing 0.5mg / ml BSA, the remaining enzyme activity after 100h at 37°C is about 80%, and the remaining enzyme activity after 720min at 40°C is about 50%. When the temperature is raised to 45°C, the remaining enzyme activity of both is below 10% after 120min. However, the cocaine esterase nanoemulsion can maintain more than 95% activity within 100h at 37°C, more than 90% activity within 720min at 40°C, and more than 30% activity within 120min at 45°C. The cocaine esterase nanoemulsion improves the temperature stability of E196-301, enabling it to maintain more than 90% activity for a long time at 37°C - 40°C. Figure 4 As shown in the results of (D), (E), and (F), the Tm value of E196-301 diluted with PB is 44°C, while the Tm value of E196-301 diluted with PB containing BSA increases to 50°C. Most importantly, CocE NEs provides good thermal protection for this enzyme, increasing its Tm value to 73°C.

[0112] V. In vivo half-life of cocaine esterase nanoemulsion

[0113] Figure 5 This is the change process of the concentration in vivo over time after injecting the cocaine esterase nanoemulsion (CocE NEs) and E196-301 in Example 1 of the present invention (the values are expressed as mean ± SEM). To verify the effect of the nanoemulsion of the present invention in enhancing the in vivo half-life of E196-301, the cocaine esterase nanoemulsion and E196-301 were injected into rats via the tail vein at doses of 1mg / kg and 3mg / kg. Blood samples were taken at 2, 5, 10, 15, 30, 45, 60, 90, and 120min after injection. After dilution, the blood samples were reacted with cocaine to obtain the reaction rate, and the enzyme concentration at each time point was deduced by the reaction rate. The results are as Figure 5 shown in (A). When the dose is 1mg / kg, the in vivo half-life of E196-301 is 6.076 ± 1.2min, while that of the cocaine esterase nanoemulsion is 13.56 ± 4.8min; as Figure 5As shown in (B) of , when the dose was 3 mg / kg, the in vivo half-life of E196-301 was 16.26 ± 1.9 min, while that of cocaine esterase nanoemulsion was 57.25 ± 14.7 min. The results showed that the nanoemulsion could effectively prolong the in vivo half-life of E196-301 and increase the in vivo retention time.

[0114] VI. Effect of Cocaine Esterase Nanoemulsion on Eliminating Cocaine in Vivo

[0115] Figure 6 This is the effect of the cocaine esterase nanoemulsion (CocE NEs) in Example 1 of the present invention and E196-301 on clearing cocaine subsequently injected into the blood 1 h after injection (the values are expressed as mean ± SEM). To prove that the cocaine esterase nanoemulsion has the ability to metabolize cocaine in vivo for a longer time than E196-301 and can achieve a better preventive effect, 1 h after intravenous tail vein injection of the cocaine esterase nanoemulsion and E196-301 at a dose of 1 mg / kg, cocaine at a dose of 5 mg / kg was injected into the tail vein. Blood samples were taken at the 5th, 10th, 15th, 30th, 45th, and 60th min after the injection of cocaine to detect the concentrations of cocaine and its metabolite benzoic acid in the blood, and to compare the ability of the cocaine esterase nanoemulsion and E196-301 to metabolize cocaine in vivo after 1 h. As Figure 6 shown in (A) of , the cocaine concentration in the cocaine esterase nanoemulsion group was significantly lower than that in the E196-301 group; as Figure 6 shown in (B) of , the content of benzoic acid in the blood samples of the cocaine esterase nanoemulsion group was significantly higher than that in the E196-301 group. The results indicated that 1 h after injection at a dose of 1 mg / kg, the content of the cocaine esterase nanoemulsion in the blood was still sufficient to metabolize 5 mg / kg of cocaine, and it could convert cocaine into benzoic acid in a short time, showing a significant improvement compared with E196-301.

[0116] The pharmacokinetic parameters (mean ± SD) of cocaine in this example are shown in Table 2.

[0117] Table 2: Comparison of Pharmacokinetic Parameters of Cocaine Esterase Nanoemulsion and E196-301 in Rats (Mean ± SD)

[0118]

[0119]

[0120] (where λz is the terminal elimination rate constant, t 1 / 2z is the half-life, C max is the maximum concentration, AUC 0-t is the area under the plasma concentration-time curve over a certain time, AUC 0-∞is the area under the plasma concentration-time curve for infinite time, AUC ％Extrap is the percentage of the extrapolated area in the whole AUC, MRT 0-∞ is the mean residence time, Vz is the apparent volume of distribution, and CL is the clearance rate.)

[0121] VII. Effects of Cocaine Esterase Nanoemulsion on the Open-Field Activity of ICR Mice after Cocaine Injection

[0122] Figure 7 This is the graph of the distance traveled by the mice during the whole process (values are expressed as mean ± SEM) when CocE NEs and E196-301 in Example 1 of the present invention were intravenously injected into the tail veins of mice at a dose of 3 mg / kg and then 25 mg / kg of cocaine was intraperitoneally injected 45 min later. After cocaine enters the body, it will quickly reach the brain and stimulate the mice to produce an excited state. An index characterizing the degree of excitement can be obtained by recording the distance traveled by the mice in the open field. To verify that CocE NEs can rapidly metabolize cocaine entering the body within a relatively long time after injection and weaken or completely eliminate the effects of cocaine on the body, the present invention set up four groups, namely the Control group (intraperitoneal injection of PBS 45 min after intravenous injection of PBS), the Cocaine group (intraperitoneal injection of cocaine 45 min after intravenous injection of PBS), the E196-301 group (intraperitoneal injection of cocaine 45 min after intravenous injection of E196-301), and the CocENEs group (intraperitoneal injection of cocaine 45 min after intravenous injection of CocE NEs). The doses of E196-301 and CocE NEs were 3 mg / kg, and the dose of cocaine was 25 mg / kg. The distance traveled by the mice during the whole process was recorded and comparative analysis was carried out. The results showed ( Figure 7 A in Figure 7 B in), E196-301 had basically lost the effect of rapidly degrading cocaine at 45 min, and the degree of excitement of the mice was comparable to that of the cocaine control group; while CocE NEs could rapidly clear the cocaine entering the body within 45 min after injection and prevent the excitatory effect caused by cocaine. The results indicate that CocE NEs can significantly increase the action time of cocaine esterase in the body and prevent the effects of cocaine on the behavior of mice for a relatively long time (45 min). (Compared with the Cocaine group, P < 0.0001, **** in the CocE NEs group; compared with the E196-301 group, P < 0.001, *** in the CocE NEs group.).

[0123] The above are only the preferred embodiments of the present invention and do not impose any form of limitation on the present invention. There are other variations and modifications without exceeding the technical solutions recorded in the claims.

Claims

1. A method for preparing a W / O / W type nanoemulsion loaded with cocaine esterase, characterized in that: The preparation method comprises the following steps: Step (1), preparing an internal aqueous phase: diluting cocaine esterase with phosphate buffer as the internal aqueous phase; Step (2), preparing an oil phase: adding soybean lecithin, vitamin E polyethylene glycol succinate, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 to medium chain triglycerides, and melting to obtain an oil phase; Step (3), preparing colostrum: cooling the oil phase to 20°C-30°C under high-speed shearing, slowly injecting the inner water phase into the oil phase, and continuing high-speed shearing to obtain W / O type colostrum; Step (4), preparing a secondary emulsion: using phosphate buffer as the external aqueous phase, adding the W / O type colostrum to the external aqueous phase under high-speed shearing, and continuing high-speed shearing under an ice bath to obtain a secondary emulsion; Step (5), preparing the final emulsion: repeatedly subjecting the secondary emulsion to high pressure homogenization to obtain a final W / O / W nanoemulsion, and concentrating it by ultrafiltration tube centrifugation.

2. The preparation method according to claim 1, characterized in that: The amount of cocaine esterase used in step (1) is 0.5-1 mg / mL based on the total volume of the W / O / W nanoemulsion.

3. The preparation method according to claim 1, characterized in that: In step (2), the mass ratio of the total mass of soybean lecithin, vitamin E polyethylene glycol succinate, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 to cocaine esterase is (10-30):

1.

4. The preparation method according to claim 3, characterized in that: In step (2), the mass ratio of soybean lecithin to vitamin E polyethylene glycol succinate is (4-5):1; the mass ratio of soybean lecithin to distearoyl phosphatidylethanolamine-polyethylene glycol 2000 is 40:

1.

5. The preparation method according to claim 1, characterized in that: The melting temperature of the oil phase in step (2) is 60-80°C.

6. The preparation method according to claim 1, characterized in that: The shearing speed in steps (3) and (4) is 8000-12000 r / min, and the shearing time is 10-15 min.

7. The preparation method according to claim 1, characterized in that: In step (5), the pressure of high pressure homogenization is 1200-1500 bar, and the number of homogenization times is 3-6 times.

8. The preparation method according to claim 1, characterized in that: In the W / O / W type nanoemulsion, the volume ratio of the oil phase to the inner water phase is (10-12):1, and the volume ratio of the outer water phase to the oil phase is (10-12):

1.

9. A W / O / W type nanoemulsion loaded with cocaine esterase, characterized in that: The method is prepared by any one of claims 1 to 8.

10. Use of the W / O / W nanoemulsion loaded with cocaine esterase as claimed in claim 9 in the preparation of a rescue preparation or a preventive preparation for acute cocaine poisoning.

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