Lipid thin film, liposome, and preparation method therefor and use thereof

Abstract

A lipid thin film, a liposome, and a preparation method therefor and a use thereof, wherein the liposome has an average particle size of 75-110 nm, preferably 85-110 nm, and more preferably 88-99 nm; and a polydispersity index of not greater than 0.2, preferably 0.06-0.08. The liposome particles have good uniformity and stability. The particle uniformity is significantly improved compared with commercially available liposomes. The liposome solution can maintain a clear state for a long time, and remains free from sedimentation for up to 3 years, which greatly improves the stability of the liposome solution, reduces batch defective rates, lowers the production cost, and is suitable for large-scale production.

Description

A lipid film, liposomes, their preparation method and uses

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese patent application No. 202411634820.6, filed on November 15, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of biopharmaceuticals. Specifically, this invention relates to a lipid film, liposomes, their preparation methods, and their uses. Background Technology

[0004] Liposomes are ultramicroscopic spherical carrier formulations formed from a lipid bilayer. Their structure is similar to that of a cell membrane. When amphipathic molecules such as phospholipids are dispersed in an aqueous phase, the hydrophobic tails of the molecules aggregate, while the hydrophilic heads are exposed in the aqueous phase, forming closed vesicles with a bilayer structure. The aqueous phase and bilayer within the vesicles can encapsulate various drugs of different polarities. Liposomes function as both adjuvants and carriers, serving as carriers for antigens that induce hapten-specific immune responses. Encapsulating antigens in liposomes protects them from degradation, thereby reducing the dosage and number of inoculations, decreasing the toxicity of the encapsulated antigen, enhancing the animal's tolerance to high doses of pathogens or their toxins, and improving antigen stability.

[0005] An ideal liposome should have the following characteristics: (1) The liposome is round and does not aggregate, and the particle size range can be effectively controlled by the preparation method to achieve the purpose of sustained and controlled release of drugs and targeted delivery; (2) The liposome has high stability and can be stored for a long time; (3) The liposome has a high encapsulation rate and drug loading capacity, especially for drugs with poor thermal stability or water-soluble biological macromolecules; (4) The liposome is easy to sterilize or aseptically handle.

[0006] There are various methods for preparing liposomes, such as thin-film dispersion, mechanical dispersion, reverse-phase evaporation, double emulsion, melt deposition, injection, freeze-drying, surfactant treatment, calcium fusion, and carrier deposition. Among these, reverse-phase evaporation and freeze-drying combined with repeated thaw cycles are good methods for encapsulating water-soluble drugs. However, reverse-phase evaporation requires a long heat treatment process, making it unsuitable for proteins and peptides with poor thermal stability. Furthermore, organic solvents can easily remain during the preparation process, posing a significant potential hazard. Freeze-drying combined with repeated thaw cycles facilitates aseptic operation and yields lyophilized liposome powders with good stability. However, when these lyophilized powders react with water to form a liposome solution, the particle size typically increases several times, resulting in non-spherical morphology and a tendency to aggregate. This makes it difficult to effectively control the particle size range, raising safety concerns.

[0007] Traditional thin-film dispersion methods for liposome preparation have a simple process, but require further treatment such as sonication or homogenization to ensure uniform liposome particle size.

[0008] Chinese patent application 201080024992.1 discloses a method for preparing liposome formulations by homogenizing a lipid suspension with a high-shear mixer to obtain liposome particles. However, this method has high requirements for equipment and operation, and the microstructure of liposomes has many irregular shapes.

[0009] In addition, liposomes currently have a wide range of applications, such as drug carriers, microencapsulated enzyme carriers, and active ingredient microparticles in skincare products, showing great potential for development. However, problems such as poor uniformity, instability, and easy sedimentation still exist in the preparation of liposome particles.

[0010] Therefore, improving the uniformity, stability, and batch consistency of liposome particles remains an urgent problem to be solved.

[0011] Invention Summary

[0012] To address the above problems, the present invention aims to provide a lipid film, liposomes, a method for preparing the same, and their applications. The lipid film prepared by the method of the present invention is uniform, bubble-free, crack-free, and wrinkle-free, and the liposomes prepared from this lipid film exhibit good uniformity and stability.

[0013] definition:

[0014] Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art. For definitions of terms in this field, those skilled in the art may refer to Current Protocols in Molecular Biology (Ausubel).

[0015] Although this invention displays numerical ranges and parameter approximations in a wide range, the values ​​shown in the specific embodiments are described as accurately as possible. However, any value inherently contains a certain degree of error due to the standard deviation present in their respective measurements. Furthermore, all ranges disclosed herein should be understood to encompass any and all subranges contained therein. For example, the range “2 to 40” should be considered to include any and all subranges between the minimum value of 2 and the maximum value of 40 (inclusive), that is, all subranges starting with a minimum value of 2 or greater, such as 2 to 6.1, and subranges ending with a maximum value of 40 or less, such as 5.5 to 40. Additionally, any references marked “incorporated herein” should be understood to be incorporated herein in their entirety.

[0016] The term “or” as used herein may be used interchangeably with the terms “and / or” unless the context clearly indicates otherwise.

[0017] The term "average particle size" as used in this paper refers to the equivalent diameter of the largest particle when the cumulative distribution in the particle size distribution curve is 50%. For a real particle swarm consisting of particles of different sizes and shapes, compared with a hypothetical particle swarm consisting of uniform spherical particles, if the total length of the particle diameters of the two is the same, then the diameter of the spherical particles is called the average particle size of the real particle swarm.

[0018] The above-mentioned objective of the present invention is achieved by providing the following technical solution:

[0019] In a first aspect, the present invention provides a liposome with an average particle size of 75-110 nm and a particle size index (PDI) of no more than 0.2.

[0020] In this invention, the average particle size of the liposomes can be selected from any two values ​​within the range of 75nm, 76nm, 77nm, 78nm, 79nm, 80nm, 81nm, 82nm, 83nm, 84nm, 85nm, 86nm, 87nm, 88nm, 89nm, 90nm, 91nm, 92nm, 93nm, 94nm, 95nm, 96nm, 97nm, 98nm, 99nm, 100nm, 101nm, 102nm, 103nm, 104nm, 105nm, 106nm, 107nm, 108nm, 109nm, 110nm or above, preferably 85-110nm, more preferably 88-99nm.

[0021] Preferably, the liposomes have a particle size of 0.06-0.08, more preferably 0.06, 0.07 or 0.08.

[0022] Secondly, the present invention provides a method for preparing a lipid film, comprising the following steps: dissolving lipids in an organic solvent, and then removing the organic solvent by rotary evaporation to obtain a lipid film; wherein the rotary evaporation comprises performing two rotary evaporations sequentially at a first rotation speed and a second rotation speed, wherein the first rotation speed is kept constant or decreased stepwise, preferably decreased stepwise, and the second rotation speed is kept constant or increased stepwise, preferably increased stepwise; the first rotation speed and the second rotation speed are not kept constant simultaneously.

[0023] Preferably, the first rotational speed is 10-220 rpm; the second rotational speed is 10-200 rpm.

[0024] Preferably, the rotary evaporation time at the first rotation speed is 1-90 min; the rotary evaporation time at the second rotation speed is 1-90 min.

[0025] Preferably, the single decrease in the first stepwise reduction in speed is 20-210 rpm, and more preferably 40-200 rpm.

[0026] Preferably, the frequency of the first stepwise reduction in rotational speed is 1-10 times, and more preferably 3-5 times.

[0027] Preferably, the time interval for the first stepwise reduction of rotational speed is 1-60 min / time, and more preferably 5-50 min / time.

[0028] Preferably, the single increase in the second stepwise speed increase is 5-60 rpm, more preferably 5-40 rpm.

[0029] Preferably, the frequency of the second stepwise increase in rotational speed is 1-10 times, more preferably 3-5 times.

[0030] Preferably, the time interval for the stepwise increase in the second rotational speed is 1-60 min / time, and more preferably 5-50 min / time.

[0031] According to some embodiments of the present invention, the vacuum level is reduced in a stepwise manner during the rotary evaporation.

[0032] Preferably, the stepwise reduction of vacuum degree is carried out under the following conditions: initial vacuum degree is 100-220 hPa; second vacuum degree is 80-100 hPa; final vacuum degree is 10-40 hPa; single reduction is 10-140 hPa; reduction frequency is 2-10 times; time interval is 1-90 min / time.

[0033] Preferably, the stepwise reduction of vacuum degree is carried out under the following conditions: initial vacuum degree is 100-150 hPa; second vacuum degree is 80-95 hPa; final vacuum degree is 10-20 hPa; single reduction is 10-80 hPa; reduction frequency is 3-5 times; time interval is 1-60 min / time.

[0034] Preferably, the rotary evaporation further includes the following conditions: the rotary evaporation temperature is 37-80℃, preferably 40-65℃; the total rotary evaporation time is 50-180min, preferably 60min; and the low-temperature cycling temperature is -10℃ to -4℃, preferably -10℃.

[0035] According to some embodiments of the present invention, the lipid comprises phospholipids and sterols.

[0036] Preferably, the weight ratio of phospholipids to sterols is 20-4000:50-1000, more preferably 20-2000:50-500.

[0037] Preferably, the phospholipid is selected from one or more of dioleoylphosphatidyl base (DOPC), egg yolk phosphatidylcholine, phosphocholine and natural phospholipid derivatives, and is preferably dioleoylphosphatidyl base.

[0038] Preferably, the sterol is selected from one or more of cholesterol, stigmasterol and ergosterol, with cholesterol being the most preferred.

[0039] Preferably, the organic solvent is selected from one or more of anhydrous ethanol, chloroform and diethyl ether, with anhydrous ethanol being the most preferred.

[0040] Thirdly, the present invention provides a lipid film prepared by the preparation method described in the second aspect of the present invention.

[0041] Preferably, the lipid film has a thickness of 0.01-0.1 μm and a water content of 1-10% by weight, preferably 1-5% by weight.

[0042] Fourthly, the present invention provides a method for preparing liposomes according to the first aspect of the present invention, comprising the following steps:

[0043] (1) The lipid film according to the third aspect of the present invention is dispersed in an aqueous solution and hydrated to form a lipid suspension;

[0044] (2) The lipid suspension is homogenized to obtain a homogenized solution;

[0045] (3) The homogenized solution is extruded through a filter membrane to obtain liposomes.

[0046] According to some embodiments of the present invention, in step (1), the aqueous solution contains phosphate and sodium chloride.

[0047] Preferably, the concentration of phosphate in the aqueous solution is 10-50 mmol / L, more preferably 20 mmol / L.

[0048] Preferably, the concentration of sodium chloride in the aqueous solution is 100-350 mmol / L, and more preferably 150 mmol / L.

[0049] Preferably, the water bath is carried out under the following conditions: water bath temperature is 40-60℃, and rotation speed is 50-100rpm.

[0050] According to some embodiments of the present invention, in step (2), the homogenization process is carried out under the following conditions: the homogenization pressure is 80-150 bar, preferably 80-120 bar; the number of homogenizations is 3-9 times, preferably 3-6 times, more preferably 3 times; the homogenization frequency is 30-50 Hz; and the water bath temperature is 40-60°C.

[0051] According to some embodiments of the present invention, in step (3), the extrusion is carried out under the following conditions: the homogeneous solution is extruded sequentially through a first filter membrane and a second filter membrane, wherein the pore size of the first filter membrane is larger than the pore size of the second filter membrane.

[0052] Preferably, the pore size of the first filter membrane is 100-800nm, more preferably 150-800nm, further preferably 150-500nm, and even more preferably 150-300nm; the pore size of the second filter membrane is 50-100nm, more preferably 90-100nm.

[0053] Preferably, the material is extruded through the first filter membrane 1-12 times, more preferably 1-5 times, and through the second filter membrane 1-12 times, more preferably 1-9 times.

[0054] Preferably, the first filter membrane and / or the second filter membrane are polycarbonate filter membranes.

[0055] Fifthly, the present invention provides an immune composition comprising liposomes and an immune adjuvant as described in the first aspect of the present invention.

[0056] Preferably, the encapsulation efficiency of the liposomes for the immune adjuvant is not less than 85%, and more preferably 89.19%-93.97%.

[0057] Preferably, the immune adjuvant is selected from one or more of saponins, CpG oligodeoxynucleotides, and monophosphoryl lipids; more preferably, the saponins are selected from one or more of saponins from saponins, ginsenosides, platycodon saponins, astragalus saponins, notoginseng saponins, glycyrrhiza saponins, albizia bark saponins, ophiopogon saponins, bupleurum saponins, or bamboo rhizome saponins; more preferably, the saponins from saponins are selected from one or more of QS-7, QS-17, QS-18, or QS-21, preferably QS-21.

[0058] According to some embodiments of the present invention, the immune composition further comprises an antigen.

[0059] Preferably, the antigen is selected from one of the following: human immunodeficiency virus, human herpesvirus, varicella-zoster virus, human mast virus, hepatitis A, B, C or E virus, respiratory syncytial virus, human papillomavirus, influenza virus, Mycobacterium tuberculosis, Salmonella, Neisseria such as Neisseria meningitidis or Neisseria gonorrhoeae, Treponema spp. such as Treponema relapsing fever or Treponema dutoni, Chlamydia spp. such as Chlamydia trachomatis, Bordetella spp. such as Bordetella pertussis, Plasmodium spp. such as Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax or Plasmodium norotri, or Toxoplasma spp. such as Toxoplasma gondii.

[0060] In a sixth aspect, the present invention provides the use of liposomes according to the first aspect of the invention or immune compositions according to the fifth aspect of the invention in the preparation of vaccines.

[0061] Preferably, the vaccine is a vaccine for preventing viral, bacterial and / or parasitic infections, or the vaccine is a vaccine for treating viral, bacterial and / or parasitic infections with immunotherapy.

[0062] The present invention has at least the following beneficial effects:

[0063] The lipid films prepared by the method of the present invention are uniform, bubble-free, crack-free, and wrinkle-free, and their thickness meets the requirements.

[0064] The liposome particles prepared by the method of this invention exhibit good uniformity, with an average particle size concentrated in the range of 88-99 nm, a PDI concentrated in the range of 0.06-0.08, and an encapsulation efficiency concentrated in the range of 89.19%-93.97% when loaded with drug. Compared with commercially available liposomes, particle uniformity is significantly improved. The liposome solution obtained by the method of this invention can maintain a clear state for a long time, avoiding sedimentation for up to 3 years, greatly improving the stability of the liposome solution, reducing batch defect rate, saving production costs, and making it suitable for large-scale production.

[0065] Brief description of the attached figures

[0066] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0067] Figure 1 shows the lipid films prepared in Example 1 and Comparative Example 1;

[0068] Figure 2 shows the detection results of liposomes obtained in group 3 of Table 5 in Example 5;

[0069] Figure 3 shows the microscopic morphology of liposomes under cryo-electron microscopy.

[0070] The best way to implement an invention

[0071] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0072] Example 1: Preparation of lipid films

[0073] Before membrane formation, remove the rotary flask from the rotary evaporator and add 0.5 mol / L sodium hydroxide solution to the flask, soaking for 30 minutes. Manually drain the sodium hydroxide solution, then rinse with water for injection until neutral, and rinse 2-3 times with anhydrous ethanol. Preheat the rotary evaporator's water bath for 20 minutes.

[0074] Add DOPC and cholesterol to anhydrous ethanol at a weight ratio of 1000:250, stir until fully dissolved, and then transfer the solution to a rotary evaporator flask.

[0075] Open the inlet and outlet pipes, introduce nitrogen for 5 minutes, then turn off the nitrogen and close the exhaust valve. Turn on the condenser, set the low-temperature circulation device temperature to -10℃, and begin rotary evaporation for film formation. Set the water bath temperature to 37℃, set the initial vacuum to 120 hPa, and run for 35 minutes. Then adjust to 80 hPa and run for 15 minutes, then adjust to 40 hPa and run for 20 minutes, and finally adjust to 20 hPa and run for 30 minutes. During rotary evaporation, the rotation speed is adjusted as follows: 100 rpm for 45 minutes, then 40 rpm for 20 minutes, then 80 rpm for 20 minutes, and finally 100 rpm for 15 minutes. After rotary evaporation, a lipid film is formed on the wall of the rotary evaporation flask. Observation of the lipid film morphology shows that the film is uniform and free of bubbles, as shown in the left image of Figure 1.

[0076] Example 2: Preparation of lipid films by rotary evaporation at multiple different vacuum levels

[0077] The film-forming method in this embodiment is the same as in Embodiment 1, except that the vacuum degree adjustment method is different, as shown in the table below. The vacuum degree decreases in a stepwise manner. When the initial vacuum degree decreases stepwise from 100-150 hPa, the film is uniform, free of bubbles, and has good film-forming properties. When the vacuum degree decreases stepwise from 220 hPa, a few bubbles appear in the lipid film. When the vacuum degree decreases stepwise from 225 hPa, a large number of bubbles are generated in the lipid film, and the film is relatively wet, resulting in poor film-forming properties. Therefore, the initial vacuum degree is 100-220 hPa, preferably 100-150 hPa.

[0078] Table 1. Lipid films prepared by rotary evaporation under different vacuum conditions.

[0079] Example 3: Preparation of lipid films by rotary evaporation at multiple different rotation speeds

[0080] The film-forming method in this embodiment is the same as in Embodiment 1, except that the rotation speed adjustment method is different, as shown in the table below. The rotation speed is divided into a first rotation speed and a second rotation speed, with the first rotation speed gradually decreasing and the second rotation speed gradually increasing. Studies in groups 1 to 5 show that when the first rotation speed decreases from ≤220 rpm to ≥10 rpm with a single decrease of ≥20 rpm, and the second rotation speed increases from ≥10 rpm to ≤200 rpm with a single increase of ≤60 rpm, the lipid film has a uniform texture, is free of bubbles and ruptures, and exhibits good film-forming properties. In group 6, when the first rotation speed decreases by 10 rpm in a single increment, the lipid film shows wrinkles and is relatively thick, resulting in poor film-forming properties. It is evident that the decrease in the first rotation speed has a certain impact on the film-forming properties of the lipid film. This may be because a slow decrease in rotation speed prevents the lipid solution from spreading in time, leading to wrinkles and a thicker film. Therefore, a single decrease in the first rotation speed of 20 rpm or more helps improve the uniformity and stability of the lipid film. When the second rotation speed increase in Group 7 was above 70 rpm, the lipid film was severely ruptured and the film-forming properties were poor. This may be because the lipid solution could not be spread evenly due to the excessively rapid increase in rotation speed, resulting in obvious cracks and affecting the stable formation of lipid films. Therefore, when the second rotation speed increase was ≤60 rpm, it helped to improve the uniformity and stability of the lipid film.

[0081] Table 2. Lipid films prepared by rotary evaporation at different rotation speeds.

[0082] Comparative Example 1

[0083] Lipid films were prepared under the conditions of Example 1, the difference being that a single vacuum level was used. The states of the resulting lipid films are shown in the table below. As can be seen from the table, when using a single vacuum level of 90-220 hPa for rotary evaporation, the film was relatively wet and liquid, failing to achieve the ideal state of a dry lipid film. The liquid on the film may be due to the incomplete evaporation of the solvent ethanol. When the vacuum level was reduced to 20 hPa, the lipid film showed wrinkles and significant bubbles, as shown in the right image of Figure 1. It is evident that using a single vacuum level cannot achieve a satisfactory film-forming state. Compared to film formation under a single vacuum level, using multiple different vacuum levels allows the lipids to exhibit better film-forming properties. A slow decrease in vacuum level helps to improve the uniformity of the lipid-formed film.

[0084] Table 3. Lipid film states under different single vacuum conditions

[0085] Comparative Example 2: Preparation of lipid films by single-speed rotary evaporation

[0086] The film-forming method for this comparative example is the same as that in Example 1, except that different single rotation speeds are used for rotary evaporation film formation. The specific single rotation speeds are shown in the table below.

[0087] When the rotation speed is 40 rpm, the resulting lipid films are relatively thick. When the rotation speed is increased to 100-220 rpm, although the lipid films become thinner, they rupture. This shows that using a single rotation speed for film formation cannot achieve a satisfactory film formation.

[0088] Table 4. Lipid films obtained by rotary evaporation at different single rotation speeds.

[0089] Example 4 Hydration

[0090] After obtaining the lipid film according to the film-forming method of Example 1, an aqueous solution containing disodium hydrogen phosphate, potassium dihydrogen phosphate, and sodium chloride was added to a rotary evaporator containing the lipid film. The total concentration of disodium hydrogen phosphate and potassium dihydrogen phosphate in the aqueous solution was 20 mmol / L, and the concentration of sodium chloride was 150 mmol / L. Hydration was initiated, nitrogen gas was introduced, and the water bath temperature was set to 50°C and the rotation speed was 70 rpm. The lipid film on the wall of the rotary evaporator was washed off and placed into a beaker that was sterilized by dry heat to obtain a lipid suspension.

[0091] Example 5 Homogenization and Extrusion

[0092] Step 1: First, drain any residual ethanol from the tubing. Place an alkaline solution (0.5 mol / L sodium hydroxide solution) into the sample container. Start the homogenizer / extruder to fill the entire tubing with the alkaline solution and soak for 30 minutes. Next, flush the tubing with water for injection until neutral. Finally, balance the tubing with an aqueous solution containing disodium hydrogen phosphate, potassium dihydrogen phosphate, and sodium chloride (solution composition as in Example 4) to bring the pH of the discharged liquid to 6.0–6.3. Close the extrusion tubing valve and open the homogenization tubing valve. Pour the lipid suspension into the homogenizer and adjust the homogenization valve to maintain the homogenization pressure at 80–150 bar. Collect the sample after homogenization. Repeat this process several times, closing the homogenization tubing valve and opening the extrusion tubing valve.

[0093] Step Two: Remove the two extrusion valves. First, install the drain disc, then install the 150-800nm ​​filter membrane. Loosen the extrusion line valve and extrude the sample through the 150-800nm ​​filter membrane 1-5 times. Then close the extrusion line valve. After removing the 150-800nm ​​filter membrane, install the 50-100nm filter membrane. Loosen the extrusion line valve and extrude the sample through the 50-100nm filter membrane 1-9 times. The filter membrane used should have uniform pore size and straight pore diameter. In this embodiment, a polycarbonate filter membrane is used as an example.

[0094] Step 3: The sample is extruded again through the internal extrusion pipe of the homogenizing extruder to obtain the extruded sample. The sample is collected in a sterilized blue-capped bottle.

[0095] The average particle size and PDI of liposomes were determined by laser dynamic light scattering, and the stability of the samples was observed.

[0096] Laser dynamic light scattering detection method: First, rinse the measuring cell once with filtered double-distilled water. Then, sonicate the sample in water (100W, 5-7 times) and filter it through a 0.22μm needle filter (Pall Corporation). Place 1 ml of sample into the measuring cell (sample height in the measuring cell should be 10mm-15mm). Following the instrument instructions, open the sample cell cover, place the sample into the measuring cell (with the V-shaped side facing the user), and click Start to begin the measurement.

[0097] Stability test: The liposome solution was placed at 2-8℃ for 6 months, 12 months, 18 months and 36 months and the appearance of the liposome solution was observed.

[0098] When the homogenization pressure is 80-150 bar and the homogenization cycles are 3-9, the resulting liposome solution is a clear, opalescent solution with relatively spherical liposome particles and a relatively uniform particle size. The average particle size ranges from a minimum of 86.19 nm to a maximum of 113.25 nm, and the PDI ranges from a minimum of 0.09 to a maximum of 0.19. Therefore, the preferred homogenization pressure is 80-150 bar, and the preferred number of homogenization cycles is 3-9.

[0099] When liposome particles are extruded using a polycarbonate filter membrane, if the pore size of the polycarbonate filter membrane extruded in the first extrusion is 150-800 nm, and the pore size of the second extrusion is 50-100 nm, and the number of extrusions is 2-9, the resulting liposome solution appears as a clear solution with a milky sheen, and the particles are relatively spherical. As can be seen from groups 1-4 in Table 5, the particle size is relatively uniform, with an average particle size ranging from a minimum of 88.36 nm to a maximum of 109.92 nm, concentrated in the 88-99 nm range. The PDI ranges from a minimum of 0.06 to a maximum of 0.18, concentrated in the 0.06-0.08 range.

[0100] Taking the liposome solution obtained in group 3 of Table 5 as an example, its stability was tested, and the test results are shown in Table 6. As can be seen from Table 6, when stored for 6 months, 12 months, 18 months, and 36 months, the liposome solution of the present invention was a clear, opalescent solution that could remain clear for up to 3 years without sedimentation. This indicates that the liposome solution containing liposomes with an average particle size of 85-110 nm and a PDI of 0.06-0.08 is relatively uniform and can maintain good stability over a long period of time.

[0101] Table 5. Liposomes prepared under different extrusion conditions

[0102] Table 6. Stability of liposome solutions under different conditions

[0103] Example 6: Encapsulated Drug

[0104] Procedure: Treat the reactor with an alkaline solution (0.5M sodium hydroxide) for 30 minutes, then rinse thoroughly with water for injection. Connect a nitrogen cylinder to the double-walled glass reactor's piping using a silicone tube. Slightly purge the nitrogen until a weak airflow can be felt at the top of the reactor's condenser tube, and purge for 10 minutes. Add adjuvants (saponin QS21 and CpG oligodeoxynucleotides) to the double-walled glass reactor. Specifically, dissolve QS21 in an aqueous solution (solution composition as in Example 4), add blank liposomes, stir until homogeneous, and control the stirring speed at 60–100 rpm to form QS21 liposomes in the aqueous solution. Then add CpG oligodeoxynucleotides, stir until homogeneous, and control the stirring speed at 60–100 rpm for 30 minutes to obtain CpG-QS21 liposomes. Dry heat sterilize the three-way cap of the glass bottle, including the silicone tube and filter. Transfer the sterilized glass bottle to a laminar flow hood, unpack, and self-clean for 15 minutes. Nitrogen gas is introduced into sterilized glass bottles in a laminar flow hood. The semi-finished product is then filtered into the glass bottles through a Millipak 20 filter at a speed of 150–350 rpm. After filtration, the QS-21 encapsulation rate is measured.

[0105] Encapsulation efficiency determination: The encapsulation efficiency of QS21 was determined using high performance liquid chromatography (HPLC) with octadecylsilane-bonded silica gel as the packing material; linear gradient elution was performed using formic acid-water (1:1000 v / v) as mobile phase A and formic acid-acetonitrile (1:1000 v / v) as mobile phase B; the flow rate was 1.0 mL / min; the column temperature was 40 °C; the injection volume was 100 μL; the detector was CAD (nebulization temperature: 50 °C; acquisition frequency: 10 Hz).

[0106] The encapsulation ratio is calculated as follows:

[0107] In the formula: A L The sum of the peak areas of QS-21a and QS-21b encapsulated in liposomes; where QS-21a is the main peak of QS-21 and QS-21b is an isomer of QS-21, both of which have adjuvant activity.

[0108] A F This is the sum of the peak areas of free QS-21a and QS-21b;

[0109] 100 represents the dilution factor of the encapsulated portion of the solution;

[0110] 20 represents the dilution factor for the free portion of the solution.

[0111] Results: As shown in Table 5, after loading the blank liposomes with saponin QS21 and CpG oligodeoxynucleotides of the present invention, the encapsulation efficiency of QS21 was the lowest at 86.19% and the highest at 93.97%, concentrated between 89.19% and 93.97%.

[0112] Figure 3 shows the microscopic characterization of liposomes as detected by cryo-electron microscopy.

[0113] In summary, the liposomes prepared by the method of this invention are uniform, with an average particle size concentrated in the range of 88-99 nm, a particle size concentrated in the range of 0.06-0.08 nm, and an encapsulation efficiency concentrated in the range of 89.19-93.97%. Compared with commercially available liposomes, this method significantly improves particle uniformity, maintains the clarity of the liposome solution, prevents sedimentation for up to 3 years, greatly improves the stability of the liposome solution, reduces batch defect rate, saves production costs, and is suitable for large-scale production.

[0114] The above descriptions are merely several exemplary embodiments of the present invention and are not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any equivalent or related embodiments obtained by those skilled in the art through some modifications or variations made to the above-disclosed technical content without departing from the scope of the present invention are all within the scope of the present invention.

Claims

1. A liposome having an average particle size of 75-110 nm, preferably 85-110 nm, more preferably 88-99 nm; and a particle size not exceeding 0.2, preferably 0.06-0.

08.

2. A method of preparing a lipid film comprising the steps of: The lipids were dissolved in an organic solvent, and then the organic solvent was removed by rotary evaporation to obtain a lipid film. The rotary evaporation includes performing two rotary evaporations sequentially at a first rotational speed and a second rotational speed. The first rotational speed is kept constant or decreases in a stepwise manner, preferably decreasing in a stepwise manner. The second rotational speed is kept constant or increases in a stepwise manner, preferably increasing in a stepwise manner. The first rotational speed and the second rotational speed are not kept constant at the same time.

3. The production method according to claim 2, wherein, The first rotational speed is 10-220 rpm; the second rotational speed is 10-200 rpm; Preferably, the rotary evaporation time at the first rotation speed is 1-90 min; the rotary evaporation time at the second rotation speed is 1-90 min; Preferably, the single decrease in the first stepwise reduction in speed is 20-210 rpm, more preferably 40-200 rpm; Preferably, the frequency of the first stepwise reduction in rotational speed is 1-10 times, more preferably 3-5 times; Preferably, the time interval for the first stepwise reduction of rotational speed is 1-60 min / time, and more preferably 5-50 min / time; Preferably, the single increase in the second stepwise increase in speed is 5-60 rpm, more preferably 5-40 rpm; Preferably, the frequency of the second stepwise increase in rotational speed is 1-10 times, more preferably 3-5 times; Preferably, the time interval for the stepwise increase in the second rotational speed is 1-60 min / time, and more preferably 5-50 min / time.

4. The production method according to claim 2 or 3, wherein The vacuum level decreases in a stepwise manner during the rotary evaporation process; Preferably, the stepwise reduction of vacuum degree is carried out under the following conditions: initial vacuum degree is 100-220 hPa; second vacuum degree is 80-100 hPa; final vacuum degree is 10-40 hPa; single reduction is 10-140 hPa; reduction frequency is 2-10 times; time interval is 1-90 min / time. Preferably, the stepwise reduction of vacuum degree is carried out under the following conditions: initial vacuum degree is 100-150 hPa; second vacuum degree is 80-95 hPa; final vacuum degree is 10-20 hPa; single reduction is 10-80 hPa; reduction frequency is 3-5 times; time interval is 1-60 min / time.

5. The production method according to any one of claims 2 to 4, wherein, The rotary evaporation also includes the following conditions: the rotary evaporation temperature is 37-80℃, preferably 40-65℃; the total rotary evaporation time is 50-180min, preferably 60min; and the low-temperature cycling temperature is -10℃ to -4℃, preferably -10℃.

6. The production method according to any one of claims 2 to 5, wherein, The lipids comprise phospholipids and sterols; Preferably, the weight ratio of the phospholipids to sterols is 20-4000:50-1000, more preferably 20-2000:50-500; Preferably, the phospholipid is selected from one or more of dioleoylphosphatidyl base, egg yolk phosphatidylcholine, phosphocholine and natural phospholipid derivatives, and is preferably dioleoylphosphatidyl base; Preferably, the sterol is selected from one or more of cholesterol, stigmasterol, and ergosterol, with cholesterol being the most preferred; Preferably, the organic solvent is selected from one or more of anhydrous ethanol, chloroform and diethyl ether, with anhydrous ethanol being the most preferred.

7. A lipid film prepared by any one of claims 2 to 6; Preferably, the lipid film has a thickness of 0.01-0.1 μm and a water content of 1-10% by weight, preferably 1-5% by weight.

8. A method for preparing liposomes according to claim 1, comprising the following steps: (1) The lipid film according to claim 7 is dispersed in an aqueous solution and hydrated to form a lipid suspension; (2) The lipid suspension is homogenized to obtain a homogenized solution; (3) The homogenized solution is extruded through a filter membrane to obtain liposomes.

9. The production method according to claim 8, wherein In step (1), the aqueous solution contains phosphate and sodium chloride; Preferably, the concentration of phosphate in the aqueous solution is 10-50 mmol / L, more preferably 20 mmol / L; Preferably, the concentration of sodium chloride in the aqueous solution is 100-350 mmol / L, more preferably 150 mmol / L; Preferably, the water bath is carried out under the following conditions: water bath temperature is 40-60℃, and rotation speed is 50-100rpm.

10. The production method according to claim 8 or 9, wherein In step (2), the homogenization process is carried out under the following conditions: the homogenization pressure is 80-150 bar, preferably 80-120 bar; the number of homogenizations is 3-9 times, preferably 3-6 times, more preferably 3 times; the homogenization frequency is 30-50 Hz; and the water bath temperature is 40-60℃.

11. The production method according to any one of claims 8 to 10, wherein, In step (3), the extrusion is carried out under the following conditions: the homogeneous solution is extruded sequentially through a first filter membrane and a second filter membrane, wherein the pore size of the first filter membrane is larger than the pore size of the second filter membrane; Preferably, the pore size of the first filter membrane is 100-800 nm, more preferably 150-800 nm, further preferably 150-500 nm, and even more preferably 150-300 nm; the pore size of the second filter membrane is 50-100 nm, more preferably 90-100 nm. Preferably, the material is extruded through the first filter membrane 1-12 times, more preferably 1-5 times, and through the second filter membrane 1-12 times, more preferably 1-9 times. Preferably, the first filter membrane and / or the second filter membrane are polycarbonate filter membranes.

12. An immune composition comprising the liposomes and an immune adjuvant according to claim 1; Preferably, the encapsulation efficiency of the liposomes for the immune adjuvant is not less than 85%, and more preferably 89.19%-93.97%. Preferably, the immune adjuvant is selected from one or more of saponins, CpG oligodeoxynucleotides, and monophosphoryl lipids; more preferably, the saponins are selected from one or more of saponins from saponins, ginsenosides, platycodon saponins, astragalus saponins, notoginseng saponins, glycyrrhiza saponins, albizia bark saponins, ophiopogon saponins, bupleurum saponins, and bamboo rhizome saponins; more preferably, the saponins from saponins are selected from one or more of QS-7, QS-17, QS-18, and QS-21, preferably QS-21.

13. The immunological composition of claim 12, wherein, The immune composition further comprises an antigen; Preferably, the antigen is selected from one of the following: human immunodeficiency virus, human herpesvirus, varicella-zoster virus, human mast virus, hepatitis A, B, C or E virus, respiratory syncytial virus, human papillomavirus, influenza virus, Mycobacterium tuberculosis, Salmonella, Neisseria such as Neisseria meningitidis or Neisseria gonorrhoeae, Treponema spp. such as Treponema relapsing fever or Treponema dutoni, Chlamydia spp. such as Chlamydia trachomatis, Bordetella spp. such as Bordetella pertussis, Plasmodium spp. such as Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax or Plasmodium norotri, or Toxoplasma spp. such as Toxoplasma gondii.

14. Use of the liposomes according to claim 1 or the immune composition according to claim 12 or 13 in the preparation of a vaccine; Preferably, the vaccine is a vaccine for preventing viral, bacterial and / or parasitic infections, or the vaccine is a vaccine for treating viral, bacterial and / or parasitic infections with immunotherapy.

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