Nano-selenium modified liposome as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines and vaccines, in particular to a nano-selenium modified liposome as well as a preparation method and application thereof. The nano-selenium modified lipidosome disclosed by the invention comprises a lipidosome and nano-selenium. Wherein the nano-selenium is modified on the surface of the liposome; the liposome is composed of cholesterol, phospholipid, and distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000. The nano-selenium modified liposome can be suitable for different treatment scenes, becomes a multifunctional drug delivery platform, has the characteristics of long circulation, good biocompatibility, high drug loading rate, good stability, increase of retention time of a treatment drug in a body and the like, can be directly entrapped with an antigen, can also be cooperatively combined with a drug and a vaccine, and has a wide application prospect. The defects of low bioavailability, poor stability, toxic and side effects and the like of the existing medicinal preparation are overcome, and the immunotherapy effect is remarkably enhanced while the treatment medicine in the liposome is promoted to achieve a remarkable slow release effect.

Description

Technical Field

[0001] The present invention relates to the fields of biomedicine technology and vaccine technology, and in particular to a nano-selenium modified liposome and a preparation method and application thereof. Background Art

[0002] Foot-and-mouth disease (FMD) is a highly contagious disease with an incubation period of up to 2-4 days. Typical symptoms include high fever (40-41°C), oral and hoof blister ulcers, salivation, lameness, and sudden death in young animals due to myocarditis (mortality rate exceeding 50%). Prevalence and outbreaks of FMD can lead to significant direct and indirect economic losses, including decreased animal performance, increased costs of prevention and control measures, and international trade restrictions. To address the challenges posed by the FMD virus, vaccine development is crucial to ensuring the sustainable development of the livestock industry and public health safety.

[0003] Vaccines can effectively prevent or even eliminate a wide range of diseases, and adjuvants play a crucial role in this process. While widely used, conventional adjuvants (such as oil and aluminum adjuvants) are ineffective in protecting antigens from the internal environment, leading to antigen degradation during transport and compromising vaccine stability and immune efficacy. Advances in nanotechnology have fueled the rapid development of adjuvants, with nanoadjuvants becoming a research hotspot.

[0004] As a potential delivery vehicle, liposomes can theoretically encapsulate or carry a variety of active pharmaceutical ingredients (such as viral antigens, anti-tumor drugs, insulin, etc.). However, they still have significant limitations in vaccine applications, especially for major epidemic diseases such as FMD: (1) The antigen / drug encapsulation efficiency and controlled release performance are not ideal. It is difficult to achieve high and stable encapsulation and precise and controlled release at the target site for active ingredients with different physical and chemical properties (such as proteins and nucleic acids). (2) The delivery efficiency and immunomodulatory activity are insufficient. Unmodified conventional liposomes have limited delivery efficiency, lack the ability to actively target immune cells, and have weak immune co-stimulatory function, making it difficult to effectively activate strong and long-lasting specific immune responses (especially cellular immunity). (3) Stability is affected by various factors such as environmental factors, physical factors, and chemical factors.

[0005] The biological activity of selenium (Se) has attracted attention in the search for immune-enhancing strategies. However, the use of free nano-Se in delivery systems has drawbacks: poor stability and easy aggregation, insufficient targeting leading to low bioavailability, and potential dose-dependent toxicity, making it difficult to safely and effectively apply to vaccine systems.

[0006] Therefore, while liposomes have the potential to carry a variety of active ingredients (including FMD antigens), traditional liposomes themselves have key drawbacks, such as difficulty in encapsulation and controlled release, insufficient delivery efficiency and immunogenicity, and poor stability. Furthermore, selenium, an element with potential immunomodulatory functions, lacks a stable and controllable delivery vehicle. A new modified liposome that combines efficient delivery with safe immune-enhancing properties is urgently needed to overcome these bottlenecks. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to address the deficiencies of the prior art. The first aspect is to provide a nano-selenium modified liposome.

[0008] The second aspect is to provide a method for preparing nano-selenium modified liposomes.

[0009] The third aspect is to provide a nano-selenium modified liposome loaded with embedded material.

[0010] The fourth aspect is to provide a method for preparing nano-selenium modified liposomes loaded with embedded substances.

[0011] The fifth aspect is to provide a pharmaceutical composition.

[0012] The sixth aspect is to provide a foot-and-mouth disease vaccine adjuvant.

[0013] The fifth aspect is to provide the use of nano-selenium modified liposomes, or nano-selenium modified liposomes loaded with embedded materials, or pharmaceutical compositions, or foot-and-mouth disease vaccine adjuvants in the preparation of drugs.

[0014] The sixth aspect is to provide a foot-and-mouth disease vaccine.

[0015] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0016] The invention discloses a nano-selenium modified liposome, which comprises liposome and nano-selenium.

[0017] Wherein, the nano-selenium is modified on the surface of the liposome.

[0018] The liposome is composed of cholesterol, phospholipids, and distearoylphosphatidylethanolamine-polyethylene glycol 2000.

[0019] The nano-selenium is obtained by reacting sodium selenite and reduced glutathione.

[0020] The cholesterol and phospholipids are modified with distearoylphosphatidylethanolamine-polyethylene glycol 2000 to prepare liposomes; and the liposomes are modified with nano-selenium to prepare nano-selenium modified liposomes.

[0021] The cholesterol and phospholipids account for 90-99 wt% of the total mass of the liposomes. Specifically, the mass ratio of cholesterol to phospholipids is 1:2-12; the distearoyl phosphatidylethanolamine-polyethylene glycol 2000 accounts for 1-10 wt% of the total mass of the liposomes.

[0022] In some embodiments of the present invention, the cholesterol and phospholipids account for 95wt% of the total mass of the liposomes; the mass ratio of cholesterol to phospholipids is 1:7.5; the distearoyl phosphatidylethanolamine-polyethylene glycol 2000 accounts for 5wt% of the total mass of the liposomes

[0023] The phospholipids include any one of neutral phospholipids, anionic phospholipids and cationic phospholipids.

[0024] Specifically, the neutral phospholipid is sphingomyelin (SM), phosphatidylcholine (phosphatidylcholine, PC) or soybean lecithin; the anionic phospholipid is phosphatidylserine (PS), phosphatidylglycerol (PG) or phosphatidic acid (PA); and the cationic phospholipid is dioleoyltrimethylammonium propane (DOTAP).

[0025] A method for preparing nano-selenium modified liposomes comprises the following steps:

[0026] (A) Cholesterol, phospholipids, and distearoylphosphatidylethanolamine-polyethylene glycol 2000 were sonicated in an organic solvent and then evaporated to prepare a lipid film.

[0027] (B) adding a buffer solution to the lipid membrane of step (A), subjecting the membrane to ultrasonic disruption, and then passing the membrane to obtain liposomes;

[0028] (C) Adding glutathione solution to the liposomes of step (B), stirring, adding sodium selenite solution to reduce with glutathione to obtain nano-selenium, continuing stirring, and dialyzing at low temperature to obtain nano-selenium modified liposomes.

[0029] Among them, in step (A), the organic solvent includes but is not limited to methanol, ethanol and other organic solvents. The organic solvents in the prior art that can dissolve phospholipids, cholesterol and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 are all within the scope of protection of the present invention, and there is no requirement for the amount of organic solvent used, as long as it can dissolve phospholipids, cholesterol and distearoyl phosphatidylethanolamine-polyethylene glycol 2000.

[0030] Wherein, in step (B), the buffer includes but is not limited to PBS buffer, citric acid buffer, and ammonium sulfate solution. Buffers that can be used as lipid membrane solvents in the prior art are within the scope of protection of the present invention. In some embodiments of the present invention, the buffer is PBS buffer (pH 7.8), citric acid solution (pH 7.2), and 250mM ammonium sulfate solution.

[0031] Wherein, in step (B), the ultrasonic crushing treatment is carried out under the following conditions: 20-50 kHz, 50-150 W, and ultrasonic treatment for 10-30 min at room temperature.

[0032] In some embodiments of the present invention, the sonication is performed at 70W and room temperature for 10 to 30 minutes. The sonicated solution is transferred to a serum bottle and further sonicated using an ultrasonic cell disruptor at 30kHz and 100W for 10 minutes at room temperature.

[0033] Wherein, the membrane is a 200-400nm polycarbonate membrane.

[0034] In some embodiments of the present invention, 400 nm and 200 nm polycarbonate membranes are used and each is passed through the membrane 3 to 5 times.

[0035] Wherein, in step (C), the concentration of the glutathione solution is 2.49-3.55 mg / mL, preferably 3.02 mg / mL; the concentration of the sodium selenite solution is 0.35-0.5 mg / mL, preferably 0.425 mg / mL.

[0036] The invention discloses a nano-selenium modified liposome loaded with an embedding substance, wherein the embedding substance is embedded in the nano-selenium modified liposome.

[0037] Wherein, the embedded material is a pharmaceutical active ingredient; preferably, the pharmaceutical active ingredient is a biological pharmaceutical active ingredient and / or an organic compound pharmaceutical active ingredient.

[0038] In some embodiments of the present invention, the biological drug active ingredients include foot-and-mouth disease virus antigen and insulin, and the organic compound drug active ingredients include the anti-tumor drug - doxorubicin hydrochloride.

[0039] The drug-lipid ratio of the liposome to the embedded material in the nano-selenium modified liposome needs to be screened according to the different embedded materials.

[0040] A method for preparing nano-selenium-modified liposomes loaded with embedded materials comprises the following steps:

[0041] (a) Using an organic solvent as a solvent, cholesterol, phospholipids, and distearoylphosphatidylethanolamine-polyethylene glycol 2000 are dissolved by ultrasonication, and then a lipid film is prepared by evaporation;

[0042] (b) adding a buffer containing the embedded substance to the lipid membrane of step (b), subjecting the membrane to ultrasonic disruption, and then passing the membrane to obtain liposomes;

[0043] (c) adding glutathione solution to the liposomes of step (c), stirring, adding sodium selenite solution to reduce with glutathione to obtain nano-selenium, continuing stirring, and dialyzing at low temperature to obtain nano-selenium modified liposomes.

[0044] A pharmaceutical composition comprising the nano-selenium modified liposome and a pharmaceutically active ingredient; preferably, the pharmaceutically active ingredient is a biological pharmaceutically active ingredient and / or an organic compound pharmaceutically active ingredient.

[0045] A foot-and-mouth disease vaccine adjuvant contains the nano-selenium modified liposome.

[0046] The use of the nano-selenium modified liposome, or the nano-selenium modified liposome loaded with embedded material, or the pharmaceutical composition, or the foot-and-mouth disease vaccine adjuvant in the preparation of medicines is also within the scope of protection of the present invention.

[0047] The use of the drug is determined by the use of the specific encapsulated active pharmaceutical ingredient or the use of the active pharmaceutical ingredient in the pharmaceutical composition.

[0048] Wherein, the drug is a drug for humans or animals.

[0049] Specifically, the drug is a foot-and-mouth disease vaccine, an immune enhancer, a hypoglycemic drug, or an anti-tumor drug.

[0050] A foot-and-mouth disease vaccine is prepared by embedding foot-and-mouth disease virus antigen in the nano-selenium modified liposome; or combining the foot-and-mouth disease virus antigen with the nano-selenium modified liposome.

[0051] Wherein, the foot-and-mouth disease virus antigen is an inactivated foot-and-mouth disease virus antigen.

[0052] The mass ratio of the nano-selenium modified liposome to the foot-and-mouth disease virus antigen is 1:0.0005-0.015, preferably 1:0.01.

[0053] Beneficial effects:

[0054] (1) The present invention optimizes different process parameters of liposomes to prepare nano-selenium-modified liposomes with excellent physical and chemical properties. The liposomes can be used as a new type of adjuvant, and have the characteristics of long circulation, good biocompatibility, high drug loading rate, high stability and good dispersibility. They can not only embed sufficient amounts of therapeutic drugs with different physical and chemical properties, but also be used in combination with therapeutic drugs. They overcome the shortcomings of existing drug preparations such as low bioavailability, poor stability and toxic side effects. While promoting the significant sustained release effect of therapeutic drugs in liposomes, they also significantly enhance the immunotherapy effect.

[0055] (2) The nano-selenium modified liposomes prepared by the present invention can be applied to different treatment scenarios by adjusting the composition, particle size and surface properties, becoming a multifunctional drug delivery platform.

[0056] (3) The present invention can achieve a higher selenium encapsulation efficiency by reducing selenium to nano-selenium and further modifying it on the surface of liposomes. Nano-selenium not only performs well in terms of bioavailability, biological activity and safety. As a "therapeutic drug carrier", nano-selenium provides a feasible method for enhancing the immunogenicity and effectiveness of therapeutic drugs with different physical and chemical properties. After being modified with nano-selenium, liposomes not only disguise themselves, but also evade the phagocytosis of the body's own surveillance system after entering the body, thereby greatly increasing the retention time of therapeutic drugs in the body.

[0057] (4) The present invention takes foot-and-mouth disease as the prevention and treatment target, uses optimized liposomes to encapsulate foot-and-mouth disease virus antigens, and further coats the liposomes with nano-selenium, thereby improving the stability of liposome transportation in the body, slowing down the release of foot-and-mouth disease antigens, and increasing the antigen encapsulation rate. At the same time, the prepared nano-selenium-modified liposomes can also synergistically enhance the immune effect with other foot-and-mouth disease vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] The present invention will be further described below in detail with reference to the accompanying drawings, and the above and / or other advantages of the present invention will become more apparent.

[0059] Figure 1 These are nano-selenium modified liposomes prepared under different concentrations of sodium selenite.

[0060] Figure 2 The average particle size of the antigen-loaded nano-selenium modified liposomes stored at 4°C for 1 to 7 weeks.

[0061] Figure 3 Transmission electron microscopy image of nano-selenium modified liposomes loaded with antigen.

[0062] Figure 4 This is the ICP-MS graph of selenium in nano-selenium-modified liposomes loaded with antigens.

[0063] Figure 5 This is the antigen release curve in liposomes.

[0064] Figure 6 The antibody titers of each group at different times after immunization.

[0065] Figure 7 The figure shows the dynamic changes of interleukin-6 (IL-6) in the serum of mice after immunization.

[0066] Figure 8 The figure shows the dynamic changes of interferon-γ (IFN-γ) in the serum of mice after immunization. DETAILED DESCRIPTION

[0067] The present invention will be further described below in conjunction with specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0068] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0069] In the following examples, the encapsulation efficiency is determined by:

[0070] Use ultrafiltration tubes to remove free antigens, and measure the content using a UV-visible spectrophotometer. Use a 10kD ultrafiltration tube to perform ultrafiltration at a certain speed, add PBS buffer to wash away free antigens, collect the filtrate and liposome suspension, and measure the antigen concentration in the filtrate and the original concentration of the added antigen solution by UV absorption. Calculate the drug encapsulation efficiency according to the formula:

[0071] Encapsulation efficiency % = (W total - W free) / W total × 100%

[0072] Wherein Wfree is the free antigen content; Wtotal represents the total antigen content.

[0073] Example 1: Optimization of liposome membrane materials

[0074] 1. Effects of different membrane materials on the physicochemical parameters of nano-selenium-modified liposomes

[0075] Liposomes were prepared using a thin film dispersion method. Different membrane materials (①100 mg of lecithin, ②16.6 mg of cholesterol (CH) and 83.3 mg of lecithin, and ③16.6 mg of cholesterol (CH), 83.3 mg of lecithin, and 5 mg of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000)) were placed in an eggplant-shaped flask and sonicated using an organic solvent as the solvent. A rotary evaporator was set at 37°C and 170 rpm to remove the organic solvent while simultaneously rotary evaporating the solution to form a film. Foot-and-mouth disease antigen was diluted in 10 mL of PBS buffer (pH 7.8) to a final concentration of 100 μg / mL. This solution was added to the lipid film as a hydrating solution and sonicated at 70 W at room temperature for 10 to 30 minutes. The sonicated solution was transferred to a serum bottle and sonicated using an ultrasonic cell disruptor at 30 kHz, 100 W, and room temperature for 10 minutes. Liposomes loaded with FMD antigen were obtained by passing the mixture through a 400nm and 200nm polycarbonate membranes five times each using a liposome extruder. At 4°C, 0.425mg / mL reduced glutathione was added to the FMD antigen-loaded liposome solution and slowly stirred for 20 minutes. Then, 3mg / mL sodium selenite was added and stirred for 12 hours. The mixture was dialyzed at low temperature for 24 hours to obtain nano-selenium-modified liposomes loaded with FMD antigen. The liposome encapsulation efficiency, particle size, dispersibility, and potential were used as evaluation criteria to investigate the physicochemical properties of nano-selenium-modified liposomes loaded with FMD antigen prepared using different membrane materials, as well as their stability after storage at 25°C for one week.

[0076] The results are shown in Table 1. Different membrane materials used to prepare liposomes will affect the physicochemical properties of liposomes, the process efficiency of drug encapsulation and the stability. (1) The particle size of liposomes prepared with lecithin alone was too large, and obvious flocculation occurred after being placed at 25°C for one week. The encapsulation efficiency of the encapsulated antigen was only 56.7%, the lowest among the three groups. (2) The particle size of the nano-selenium liposomes prepared with lecithin and cholesterol was 145nm, which was relatively small and uniform in dispersion. The encapsulation efficiency of the antigen was also improved, and the stability of the liposome was significantly improved. (3) The particle size of the nano-selenium liposomes prepared with lecithin, cholesterol and DSPE-PEG2000 was 132nm, which was smaller in size and more uniform in dispersion. The encapsulation efficiency was 84.5%, the highest among the three groups. The physicochemical properties of the liposomes were better than those of the liposomes prepared with the other two groups of lipid materials, and the stability of the liposomes was further improved. Therefore, lecithin, cholesterol and DSPE-PEG2000 were subsequently selected as lipid materials to further optimize the formulation of nano-selenium modified liposomes.

[0077] Table 1 Effects of different membrane materials on the physicochemical properties of nano-selenium modified liposomes

[0078]

[0079] 2. Effects of different phospholipids on the physicochemical parameters of nano-selenium-modified liposomes

[0080] Phospholipids are an important class of amphiphilic molecules that are the main components of biological membranes and are also widely used in drug delivery systems (such as liposomes) and industrial fields. They can be classified into the following three types based on their charge properties: ① Neutral phospholipids: those with uncharged head groups, such as sphingomyelin (SM), phosphatidylcholine (PC), and soybean lecithin. ② Anionic phospholipids: those with negatively charged head groups, such as phosphatidylserine (PS), phosphatidylglycerol (PG), and phosphatidic acid (PA). ③ Cationic phospholipids: those with positively charged groups introduced through chemical modification, such as dioleoyltrimethylammonium propane (DOTAP).

[0081] Representative substances among neutral phospholipids, anionic phospholipids and cationic phospholipids were selected to prepare liposomes, and then modified with nano-selenium to investigate the changes in the physicochemical parameters of liposomes prepared from different phospholipids after being functionalized with nano-selenium.

[0082] Liposomes were prepared using a thin film dispersion method. Different membrane materials (① 16.6 mg cholesterol (CH), 83.3 mg phosphatidylcholine (PC), and 5 mg DSPE-PEG2000; ② 16.6 mg cholesterol (CH), 83.3 mg phosphatidylglycerol (PG), and 5 mg DSPE-PEG2000; ③ 16.6 mg cholesterol (CH), 83.3 mg dioleoyltrimethylammonium propane (DOTAP), and 5 mg DSPE-PEG2000) were placed in an eggplant-shaped flask and sonicated using an organic solvent as the solvent. A rotary evaporator was set at 37°C and 170 rpm to remove the organic solvent while simultaneously rotary evaporating the solution to form a film. Foot-and-mouth disease antigen was diluted in 10 mL of PBS buffer (pH 7.8) to a final concentration of 100 μg / mL. This solution was added to the lipid film as a hydrating solution and sonicated at 70 W at room temperature for 10 to 30 minutes. The sonicated solution was transferred to a serum bottle and sonicated for 10 minutes using an ultrasonic cell disruptor at 30kHz, 100W, and room temperature. A liposome extruder was used to pass through 400nm and 200nm polycarbonate membranes five times each to obtain liposomes loaded with foot-and-mouth disease antigen. At 4°C, 0.425mg / mL reduced glutathione was added to the liposome liquid loaded with foot-and-mouth disease antigen, and the mixture was slowly stirred for 20 minutes. Then, 3mg / mL sodium selenite was added, and stirring was continued for 12 hours. The mixture was dialyzed at low temperature for 24 hours to obtain nano-selenium-modified liposomes loaded with foot-and-mouth disease antigen. The liposome encapsulation efficiency, particle size, dispersibility, and potential were used as evaluation indicators to investigate the physicochemical properties of nano-selenium-modified liposomes prepared with different membrane materials and their stability after being stored at 25°C for one week.

[0083] As shown in Table 2, after the liposomes prepared by phospholipids of different charge types were modified by nano-selenium, there were differences in the physicochemical properties such as particle size, dispersibility and encapsulation efficiency of the liposomes. The absolute value of the charge of the anionic and cationic liposomes modified by nano-selenium is significantly greater than the charge value of the neutral liposomes. The liposomes with a charge can make the stability and dispersibility of the liposomes better due to the electrostatic repulsion of the surface charge. The experimental results also found that the stability and dispersibility of the liposomes prepared by anions and cations are better than those prepared by neutral charge. Because the charge characteristics of nano-selenium are mainly affected by its surface chemical state, in the liquid environment of pH>7 of this patent, nano-selenium presents a negative charge and can be adsorbed on the positively charged cationic liposomes by electrostatic action. Therefore, the process optimization of subsequent nano-selenium liposomes can select cationic liposomes, which can not only effectively adsorb nano-selenium, but also the liposomes after nano-selenium modification are negatively charged, which can better maintain the stability of the liposomes.

[0084] Table 2 Effects of different phospholipid types on the physicochemical properties of nano-selenium modified liposomes

[0085]

[0086] Example 2: Optimization of the mass ratio of liposome membrane components

[0087] In this example, the mass ratio of the liposome membrane materials (CH, DOTAP, DSPE-PEG2000) was optimized.

[0088] In the preparation of liposomes, DSPE-PEG2000 is mainly used to improve the stability of liposomes. The amount of DSPE-PEG2000 usually accounts for 1 to 10 wt% of the total mass of the liposomes. As the amount of DSPE-PEG2000 increases, the stability of the liposomes improves. However, considering the cost of subsequent process scale-up, this patent selects the condition where DSPE-PEG2000 accounts for 5 wt% of the total mass of the liposomes and optimizes the other two membrane materials (CH and DOTAP) in the liposomes.

[0089] DSPE-PEG2000, representing 5 wt% of the total liposome mass, and CH and DOTAP in a mass ratio of 1:1-12 were placed in an eggplant-shaped flask. The membrane material was sonicated using methanol as the solvent. A rotary evaporator was set at 37°C and 170 rpm to remove the organic solvent while simultaneously rotary evaporating the solution to form a membrane. Foot-and-mouth disease antigen was diluted in 10 mL of PBS buffer (pH 7.8) to a final concentration of 100 μg / mL (at a drug-to-lipid ratio of 1:0.01 for liposomes:FMD antigen). This solution was added to the lipid membrane as a hydrating solution and sonicated at 70 W at room temperature for 10-30 minutes. The sonicated solution was transferred to a serum bottle and sonicated using an ultrasonic cell disruptor at 30 kHz, 100 W, and room temperature for 10 minutes. The membrane was then passed through a 400 nm and 200 nm polycarbonate membrane using a liposome extruder three times each to obtain liposomes loaded with FMD antigen. The effects of different mass ratios of CH and DOTAP in the membrane material on the antigen encapsulation efficiency were investigated, and the particle size of the liposomes and the particle size of the liposomes after being stored at 25°C for one week were detected.

[0090] The results are shown in Table 3. By testing the particle size and encapsulation efficiency of the liposomes, it was found that the liposome encapsulation efficiency increased with the increase in the amount of DOTAP. When the mass ratio of CH to DOTAP ranged from 1:7.5 to 10, the particle size and encapsulation efficiency of the liposomes were better than those of the other test groups. When the CH to DOTAP ratio was 1:7.5, the particle size and encapsulation efficiency of the liposomes were the best. However, when the CH to DOTAP ratio was 1:12, the liposome film-forming property deteriorated and the antigen encapsulation efficiency decreased. Therefore, a CH to DOTAP mass ratio of 1:7.5 was selected for further optimization.

[0091] Table 3 Effect of different CH and DOTAP mass ratios on encapsulation efficiency

[0092]

[0093] Example 3: Optimization of the mass ratio of foot-and-mouth disease antigen to liposomes

[0094] Fix the quality (CH:DOTAP=1:7.5, DSPE-PEG2000 accounts for the 5wt% of liposome total mass) of liposome membrane material, take the encapsulation efficiency of antigen as index, the dosage of foot-and-mouth disease disease antigen is screened.Concrete, investigate when the mass ratio (abbreviation medicine-lipid ratio) of liposome and foot-and-mouth disease disease antigen is 1:0.0005 (antigen concentration 5 μ g / mL), 1:0.001 (antigen concentration 10 μ g / mL), 1:0.005 (antigen concentration 50 μ g / mL), 1:0.01 (antigen concentration 100 μ g / mL), 1:0.015 (antigen concentration 150 μ g / mL), 1:0.02 (antigen concentration 200 μ g / mL), 1:0.025 (antigen concentration 250 μ g / mL), on the impact of antigen encapsulation efficiency, and detect the particle diameter of liposome and place the particle diameter of liposome after one week at 25 ℃.The preparation process of liposome with reference to the step among the embodiment 2

[0095] The results, shown in Table 4, show that the liposome encapsulation efficiency gradually increased with increasing FMD antigen concentration. However, the finite internal cavity of the liposomes limits the amount of antigen encapsulated. When the drug-to-lipid ratio ranged from 1:0.0005 to 0.015, the liposome particle size and encapsulation efficiency were superior to those of the other experimental groups, with the highest encapsulation efficiency achieved at a drug-to-lipid ratio of 1:0.01.

[0096] Table 4 Effect of different drug-to-lipid ratios on encapsulation efficiency

[0097]

[0098] Example 4: Effects of different sodium selenite concentrations on nano-selenium modified liposomes

[0099] CH, DOTAP, and DSPE-PEG2000 (5 wt% of the total liposome mass) in a 1:7.5 mass ratio were placed in an eggplant-shaped flask. Methanol was used as the solvent and the membrane material was sonicated. A rotary evaporator was set at 37°C and 170 rpm to remove the organic solvent while simultaneously rotary evaporating the solution to form a membrane. Foot-and-mouth disease antigen was diluted in 10 mL of PBS buffer (pH 7.8) to a final concentration of 100 μg / mL (drug-to-lipid ratio of 1:0.01). This solution was added to the lipid membrane as a hydrating solution and sonicated at 70 W at room temperature for 10-30 minutes. The sonicated solution was transferred to a serum bottle and sonicated using an ultrasonic cell disruptor at 30 kHz, 100 W, and room temperature for 10 minutes. The membrane was then passed through a 400 nm and 200 nm polycarbonate membrane using a liposome extruder five times each to obtain liposomes loaded with foot-and-mouth disease antigen.

[0100] Under 4 ° C conditions, reduced glutathione and sodium selenite were added in a molar ratio of 4:1. Specifically, reduced glutathione (0.35-4.62 mg / mL) was added to 10 mL of liposome liquid loaded with foot-and-mouth disease antigen, and slowly stirred for 20 minutes. Then, different concentrations of sodium selenite (0.05-0.65 mg / mL) were added, and stirring was continued for 12 hours. Dialysis was performed at low temperature for 24 hours to obtain nano-selenium-modified liposomes loaded with foot-and-mouth disease antigen. The particle size, polydispersity coefficient and potential of the liposomes were measured using a Malvern laser particle size analyzer. The particle size, dispersibility and potential of the liposomes were used as the evaluation indicators to investigate the stability of nano-selenium-modified liposomes loaded with foot-and-mouth disease antigen under different concentrations of sodium selenite, and repeated 3 times.

[0101] The results are as follows Figure 1 As shown in Table 5, the liposomes loaded with FMD antigen appeared as a white liquid, while the nano-selenium-modified liposomes loaded with FMD antigen, under the action of reduced glutathione, reduced the selenium ions in sodium selenite to nano-selenium, resulting in an orange color. As the concentration of sodium selenite increased, the color of the solution gradually deepened to orange-red. However, when the liposome surface reached saturation and could no longer adsorb more nano-selenium, the color of the nano-selenium-modified liposomes loaded with FMD antigen stopped changing. Specifically, when the concentration of sodium selenite was between 0.35 and 0.5 mg / mL, the physicochemical properties of the nano-selenium-modified liposomes loaded with FMD antigen, including particle size, dispersibility, potential, and encapsulation efficiency, were superior to those of the other experimental groups. Specifically, when the concentration of sodium selenite was 0.425 mg / mL, the nano-selenium-modified liposomes loaded with FMD antigen had the optimal particle size and the highest encapsulation efficiency, both of which were significantly different from the other groups when compared pairwise.

[0102] Table 5 Particle size, dispersibility and potential of nano-selenium modified liposomes under different concentrations of sodium selenite (n=3)

[0103]

[0104] Example 5 Evaluation of the stability of nano-selenium modified liposomes loaded with foot-and-mouth disease antigen

[0105] 1. Observation at 4°C and 25°C

[0106] Nano-selenium modified liposomes loaded with foot-and-mouth disease antigen (wherein the concentration of sodium selenite is 0.425 mg / mL) were placed at 4°C and 25°C for stability observation. Samples were taken at different time points to determine the encapsulation efficiency of the antigen. The experiment was repeated 3 times, and the average encapsulation efficiency was taken. The results are shown in Tables 6 and 7. At a storage temperature of 4°C, the encapsulation efficiency of the nano-selenium modified liposomes was relatively stable over 6 weeks, with no flocculation. Compared with the results in Table 7, the encapsulation efficiency of the nano-selenium modified liposomes at 25°C decreased slightly, and slight flocculation occurred at 5 weeks.

[0107] Table 6 Stability of antigen-loaded nanoselenium-modified liposomes at 4°C (n=3)

[0108]

[0109]

[0110] Table 7 Stability of antigen-loaded nanoselenium modified liposomes at 25°C (n=3)

[0111]

[0112] Furthermore, the average particle size, polydispersity index, and potential of the antigen-loaded nano-selenium modified liposomes (wherein the concentration of sodium selenite was 0.425 mg / mL) were measured using a Malvern laser particle size analyzer at 4°C. The average particle size was 108.56±0.77nm, with uniform size and a small standard deviation, indicating that the liposome particle size distribution was relatively uniform; the polydispersity index (PDI) was 0.154±0.024, indicating that the liposome particle size distribution was relatively concentrated and had good monodispersity; and the zeta potential was -7.29±1.39mV, indicating that the liposomes had moderate electrostatic repulsion and were not prone to aggregation. This shows that the antigen-loaded nano-selenium modified liposomes had good particle size uniformity and stability during storage. Figure 2 The average particle size of antigen-loaded nano-selenium modified liposomes (wherein the sodium selenite concentration is 0.425 mg / mL) stored at 4°C for 1 to 7 weeks.

[0113] 2. Morphological observation

[0114] After the antigen-loaded nano-selenium modified liposome suspension in step 1 was diluted 10 times with deionized water as the dispersion medium, the copper mesh was immersed in the suspension and removed after about 1 to 2 minutes. Then, the copper mesh was placed in a 3% phosphotungstic acid solution and stained for 2 to 3 minutes. After that, the mesh was removed and the unbound dye was washed with clean water for three times. Then, the mesh was observed using a transmission electron microscope. Figure 3 It can be seen that the nano-selenium modified liposomes loaded with antigens appear as hollow circles with a particle size of about 100 nm and uniform particle size.

[0115] 3. Determination of selenium content by inductively coupled plasma mass spectrometry

[0116] The concentration of selenium in the above-mentioned antigen-loaded nano-selenium modified liposomes was measured using an inductively coupled plasma mass spectrometer (ICP-MS). The selenium concentration measured by ICP-MS in the antigen-loaded nano-selenium modified liposomes was 132.1 mg / L. Using the formula selenium yield = measured selenium content / theoretical selenium content × 100%, the selenium yield in the antigen-loaded nano-selenium modified liposomes was 70.01%. This indicates that during the synthesis of the antigen-loaded nano-selenium modified liposomes, 70.01% of the theoretical selenium was successfully encapsulated into the liposomes, indicating that the method for synthesizing nano-selenium in this experiment is good and can achieve a higher selenium encapsulation efficiency. Figure 4 This is the ICP-MS graph of selenium in nano-selenium-modified liposomes loaded with antigens.

[0117] 4. Investigation of in vitro drug release behavior of antigen-loaded nano-selenium modified liposomes

[0118] By simulating the in vivo environment, we investigated how the antigens loaded in nano-selenium modified liposomes are released over time when used as drug carriers. The specific process is as follows:

[0119] A vertical diffusion cell was used to investigate the drug release behavior of antigen-loaded nano-selenium modified liposomes. 4 mL of antigen-loaded nano-selenium modified liposome suspension was precisely measured and placed in the supply cell. The receiving cell contained 50 times the release medium (PBS buffer) to receive the drug diffused from the supply cell. At 37°C, samples were taken at the set sampling time points to determine the antigen concentration. After each sampling, an equal volume of release medium was immediately added to the receiving cell to maintain the volume of the medium in the receiving cell unchanged and ensure consistency of the experimental conditions. An ultraviolet spectrophotometer was used to detect the antigen concentration in the removed samples. Finally, the antigen release curve was plotted with different sampling time points as the horizontal axis and the cumulative drug release rate as the vertical axis. Free antigen and antigen-loaded liposomes were used as controls.

[0120] The results are as follows Figure 5 As shown in the figure, free antigen showed a burst release within 4 hours, with a release rate significantly faster than that of the liposome group, indicating that antigen not encapsulated in liposomes was more easily released and that liposomes significantly enhanced the sustained release of antigens. However, the antigen release rate in liposomes modified with nanoselenium was further reduced, indicating that nanoselenium modification of liposomes can further significantly enhance the sustained release effect of liposomes.

[0121] Example 6: Evaluation of the immune effect of foot-and-mouth disease vaccine adjuvants

[0122] Thirty-two female Balb / c mice weighing 18-22 g were randomly divided into four groups of eight mice each. Each group received subcutaneous immunizations at a dose of 0.5 mL per mouse. Group 1 received normal saline; Group 2 received a conventionally prepared foot-and-mouth disease vaccine adjuvanted with ISA206; Group 3 received a combination of nanoselenium-modified liposomes and an ISA206-adjuvanted foot-and-mouth disease vaccine. The nanoselenium-modified liposomes were antigen-free and prepared using the same method as the antigen-loaded nanoselenium-modified liposomes, with PBS buffer as the hydration medium; and Group 4 received nanoselenium-modified liposomes loaded with foot-and-mouth disease antigen. The antigen content of the vaccine was 20 μg / mL, with the same antigen content in each group. A second immunization was performed two weeks later using the same procedure.

[0123] 1. Antibody titer detection

[0124] At 0, 1, 2, 3, 4, 5, 6 and 7 weeks after immunization, blood was collected from mice in each experimental group, and the antibody level was determined by liquid ELISA. The dynamic changes in the antibody level were plotted in a line graph.

[0125] The results are as follows Figure 6 As shown in the figure, with the exception of the PBS negative control group, FMDV-specific antibody levels in the serum of mice in groups 2 to 4 gradually increased with increasing days of immunization. After 4 weeks, antibody levels in group 2 began to decline and maintained a downward trend. Although antibody levels in group 3 also began to decline after 4 weeks, they remained higher than those in group 2, indicating that nanoselenium-modified liposomes can enhance immunity and serve as a potential adjuvant for foot-and-mouth disease virus vaccines. After 4 weeks, antibody levels in group 4 were higher than those in groups 2 and 3, with no significant downward trend, remaining at a high level. This indicates that antigen-loaded nanoselenium-modified liposomes are more effective than nanoselenium-modified liposomes when used in conjunction with ISA206 adjuvant. Their immune response and sustained antigen release were superior to those of ISA206-adjuvanted vaccines, highlighting their advantages as a novel adjuvant.

[0126] 2. Determination of cytokines

[0127] In order to confirm whether the nano-selenium modified liposomes induce the body to produce pro-inflammatory factors, this example uses IL-6 and IFN-γ ELISA diagnostic kits to detect the levels of IL-6 and IFN-γ in the serum of the four experimental groups of mice. This further evaluates the effects of different vaccines on the expression and secretion levels of cytokines in mice. The results are shown in Figure 2. Figure 7 and Figure 8 shown.

[0128] Depend on Figure 7It can be seen that all three vaccine groups can induce the secretion of IL-6 inflammatory factors by mouse dendritic cells. The level of pro-inflammatory factors in Group 3 was significantly higher than that in Group 2 (***, P<0.001), indicating that increased selenium concentration in mice can promote the increase of IL-6 levels. The combination of nano-selenium modified liposomes and ISA206 vaccine can enhance the production of IL-6. The level of pro-IL-6 in Group 4 was significantly higher than that in other groups (****, P<0.0001), showing the strongest IL-6 induction ability, indicating that directly loading the antigen into nano-selenium modified liposomes can significantly induce the production of more cytokines.

[0129] Depend on Figure 8 It can be seen that, except for Group 1 (control group), other groups can induce mice to secrete IFN-γ inflammatory factors, and as the immunization time prolongs, the IFN-γ in the experimental group mice gradually increases. After the second immunization, the IFN-γ levels in Groups 3 and 4 increased significantly, gradually reaching a peak, among which Group 4 increased faster, and the difference was significant compared with Group 2 (***, P<0.001). Specifically, compared with Group 1 (control group), Group 2 was able to induce a certain degree of IFN-γ production, but the overall level was relatively low. The IFN-γ concentration in Group 3 was higher than that in the ISA206 vaccine group alone, indicating that the combination of nano-selenium modified liposomes and ISA206 vaccine can enhance the production of IFN-γ. The IFN-γ concentration in Group 4 was the highest, showing the strongest IFN-γ induction ability, indicating that nano-selenium modified liposomes loaded with antigens can significantly promote the production of cytokines.

[0130] Example 7: Preparation of Nano-Selenium-Modified Liposomes Loaded with Insulin

[0131] CH, DOTAP, and DSPE-PEG2000 (5 wt% of the total liposome mass) in a mass ratio of 1:7.5:0.2 were placed in an eggplant-shaped flask. Methanol was used as the solvent and the membrane material was sonicated. A rotary evaporator was set at 37°C and 170 rpm to remove the organic solvent while simultaneously rotary evaporating the solution to form a membrane. Insulin was dissolved in 10 mL of citric acid solution (pH 7.2) (final insulin concentration of 1-5 mg / mL). This solution was added to the lipid membrane as a hydrating solution and sonicated at 70 W at room temperature for 10-30 minutes. The sonicated solution was transferred to a serum bottle and sonicated at 30 kHz, 100 W, and room temperature for 10 minutes using an ultrasonic cell disruptor. The insulin-loaded liposomes were then passed through 400 nm and 200 nm polycarbonate membranes three times each using a liposome extruder to obtain the insulin-loaded liposomes.

[0132] At 4°C, 0.425 mg / mL reduced glutathione was added to 10 mL of insulin-loaded liposomes. After slow stirring for 20 minutes, 0.4 mg / mL sodium selenite was added. Stirring was continued for 12 hours. The solution was dialyzed at low temperature for 24 hours to obtain insulin-loaded nanoselenium-modified liposomes. The physicochemical parameters of the insulin-encapsulated nanoselenium liposomes were then measured.

[0133] The results are shown in Table 8. The nano-selenium liposomes loaded with insulin have uniform particle size and good dispersion. The liposomes are weakly electronegative and are not prone to aggregation. The encapsulation efficiency of insulin in the nano-selenium liposomes is 90.7%, indicating that the preparation process is stable and reliable.

[0134] Table 8 Analysis results of the physicochemical properties of nano-selenium liposomes loaded with insulin

[0135]

[0136] Example 8: Preparation of Nano-Selenium-Modified Liposomes Loaded with Doxorubicin Hydrochloride

[0137] CH, DOTAP, and DSPE-PEG2000 (5 wt% of the total liposome mass) in a 1:7.5 mass ratio were placed in an eggplant-shaped flask. Methanol was used as the solvent and the membrane material was sonicated. A rotary evaporator was set at 37°C and 170 rpm to remove the organic solvent while simultaneously evaporating the solution to form a membrane. Doxorubicin hydrochloride was dissolved in 10 mL of PBS buffer (pH 7.2) (final concentration of 50 mg / mL) and sonicated at 70 W at room temperature for 10-30 minutes. The sonicated solution was transferred to a serum bottle and sonicated at 30 kHz, 100 W, and room temperature for 10 minutes using an ultrasonic cell disruptor. The liposomes were then passed through 400 nm and 200 nm polycarbonate membranes three times each using a liposome extruder. Free doxorubicin hydrochloride was removed by dialysis to obtain doxorubicin hydrochloride-loaded liposomes.

[0138] At 4°C, 0.425 mg / mL reduced glutathione was added to 10 mL of doxorubicin hydrochloride-loaded liposomes. After slow stirring for 20 minutes, 0.4 mg / mL sodium selenite was added. Stirring was continued for 12 hours. The solution was dialyzed at low temperature for 24 hours to obtain nano-selenium-modified liposomes loaded with doxorubicin hydrochloride. The physicochemical parameters of the nano-selenium-modified liposomes loaded with doxorubicin hydrochloride were measured.

[0139] Table 9 Analysis results of physicochemical parameters of nano-selenium modified liposomes loaded with doxorubicin hydrochloride

[0140]

[0141] As can be seen from Table 9, the particle size of the prepared nano-selenium modified liposomes loaded with doxorubicin hydrochloride is 118 nm, the polydispersity coefficient is 0.137, the Zeta potential is -6.4, and the nano-selenium liposomes are slightly negatively charged, indicating that the liposomes have moderate electrostatic repulsion, are not easy to aggregate, and have better storage stability. At the same time, the nano-selenium liposomes can highly encapsulate doxorubicin hydrochloride, and the encapsulation efficiency reaches 97.7%, indicating that the preparation process of the nano-selenium liposomes is efficient.

[0142] In summary, the nano-selenium-modified liposomes developed in this patent can encapsulate drugs with various physical and chemical properties due to their unique amphiphilic structure and high modifiability, and can adapt to different treatment scenarios by adjusting the composition, particle size and surface properties, becoming a multifunctional drug delivery platform.

[0143] The present invention provides a method and concept for preparing and applying nano-selenium-modified liposomes. There are many methods and approaches for implementing this technical solution. The above is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention. Any components not specified in this embodiment can be implemented using existing technologies.

Claims

1. A nano-selenium modified liposome, characterized in that: The nano-selenium modified liposomes include liposomes and nano-selenium; The nano-selenium is modified on the surface of the liposome; the liposome is composed of cholesterol, phospholipids, and distearoylphosphatidylethanolamine-polyethylene glycol 2000.

2. The nano-selenium modified liposome according to claim 1, characterized in that The cholesterol and phospholipids account for 90-99 wt% of the total mass of the liposomes, wherein the mass ratio of cholesterol to phospholipids is 1:2-12; the distearoyl phosphatidylethanolamine-polyethylene glycol 2000 accounts for 1-10 wt% of the total mass of the liposomes.

3. The nano-selenium modified liposome according to claim 1, characterized in that The phospholipids include any one of neutral phospholipids, anionic phospholipids, and cationic phospholipids; The neutral phospholipid is sphingomyelin, phosphatidylcholine or soybean lecithin; the anionic phospholipid is phosphatidylserine, phosphatidylglycerol or phosphatidic acid; and the cationic phospholipid is dioleoyltrimethylammonium propane.

4. The method for preparing the nano-selenium modified liposome according to claim 1, characterized in that: The steps include: (A) Cholesterol, phospholipids, and distearoylphosphatidylethanolamine-polyethylene glycol 2000 were sonicated in an organic solvent and then evaporated to prepare a lipid film. (B) adding a buffer solution to the lipid membrane of step (A), subjecting the membrane to ultrasonic disruption, and then passing the membrane to obtain liposomes; (C) adding the glutathione solution to the liposomes of step (B), stirring, adding the sodium selenite solution to reduce the liposomes with glutathione to obtain nano-selenium, continuing stirring, and dialyzing at low temperature to obtain nano-selenium modified liposomes; Preferably, in step (C), the concentration of the glutathione solution is 2.49-3.55 mg / mL, and the concentration of the sodium selenite solution is 0.35-0.5 mg / mL; more preferably, the concentration of the glutathione solution is 3.02 mg / mL; and the concentration of the sodium selenite solution is 0.425 mg / mL.

5. A nano-selenium modified liposome loaded with an embedded substance, characterized in that: embedding the embedded material in the nano-selenium modified liposome according to claim 1; Wherein, the embedded material is a pharmaceutical active ingredient; preferably, the pharmaceutical active ingredient is a biological pharmaceutical active ingredient and / or an organic compound pharmaceutical active ingredient.

6. The method for preparing the embedded material-carrying nano-selenium modified liposomes according to claim 5, characterized in that: The steps include: (a) Using an organic solvent as a solvent, cholesterol, phospholipids, and distearoylphosphatidylethanolamine-polyethylene glycol 2000 are dissolved by ultrasonication, and then a lipid film is prepared by evaporation; (b) adding a buffer containing the embedded substance to the lipid membrane of step (b), subjecting the membrane to ultrasonic disruption, and then passing the membrane to obtain liposomes; (c) adding glutathione solution to the liposomes of step (c), stirring, adding sodium selenite solution to reduce with glutathione to obtain nano-selenium, continuing stirring, and dialyzing at low temperature to obtain nano-selenium modified liposomes.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the nano-selenium modified liposomes according to claim 1 and a pharmaceutically active ingredient; preferably, the pharmaceutically active ingredient is a biological pharmaceutically active ingredient and / or an organic compound pharmaceutically active ingredient.

8. A foot-and-mouth disease vaccine adjuvant, characterized in that Contains the nano-selenium modified liposome according to claim 1.

9. Use of the nano-selenium modified liposome according to claim 1, or the nano-selenium modified liposome carrying an embedded substance according to claim 5, or the pharmaceutical composition according to claim 7, or the foot-and-mouth disease vaccine adjuvant according to claim 8 in the preparation of a medicine; in, The drug is a drug for humans or animals; preferably, the drug is any one of a foot-and-mouth disease vaccine, an immunopotentiator, a hypoglycemic drug, and an anti-tumor drug.

10. A foot-and-mouth disease vaccine, characterized in that: The foot-and-mouth disease virus antigen is embedded in the nano-selenium modified liposome according to claim 1; or the foot-and-mouth disease virus antigen is combined with the nano-selenium modified liposome according to claim 1; Wherein, the foot-and-mouth disease virus antigen is an inactivated foot-and-mouth disease virus antigen, and the mass ratio of the nano-selenium modified liposome to the foot-and-mouth disease virus antigen is 1:0.0005-0.015; preferably, the mass ratio of the nano-selenium modified liposome to the foot-and-mouth disease virus antigen is 1:0.01.

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