A food-grade nanoliposome and its preparation method
Food-grade nanoliposomes were prepared using microfluidic methods and high-pressure homogenization, solving the problem of excessive cholesterol caused by cholesterol addition. This method achieved stable and highly encapsulated liposomes suitable for the food industry.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHIHE BIOTECHNOLOGY (CHANGZHOU) CO LTD
- Filing Date
- 2026-03-30
- Publication Date
- 2026-06-30
AI Technical Summary
In the food industry, adding too much cholesterol to the prepared liposomes can lead to excessive cholesterol levels in the human body. Existing technologies make it difficult to construct stable food-grade nanoliposomes without adding cholesterol.
Food-grade nanoliposomes were prepared using microfluidic and high-pressure homogenization methods. By controlling the ratio and flow rate of the organic and aqueous phases and avoiding the use of cholesterol, liposomes with a particle size of less than 200 nm and a dispersion of 0.3 < PDI < 0.45 were prepared.
Stable food-grade nanoliposomes were constructed, which improved the encapsulation efficiency of nutrients, met the market demand for low-cholesterol and cholesterol-free health products, and the preparation method was simple.
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Figure CN122297394A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of liposome preparation, specifically relating to a food-grade nanoliposome and its preparation method. Background Technology
[0002] Liposomes are microvesicles composed of a phospholipid bilayer. Their core structure consists of bilayer vesicles formed by the self-assembly of amphiphilic phospholipid molecules, ranging in size from 20 nanometers to 10 micrometers. This structure is very similar to the cell membrane, hence it is often called an "artificial biomembrane." The hydrophobic regions of the phospholipid bilayer of liposomes can encapsulate lipid-soluble substances, while the internal hydrophilic cavities can hold water-soluble components. This characteristic gives them several key advantages: ① Good biocompatibility and safety: Their components (phospholipids, cholesterol) can be degraded by the body, are non-toxic, and non-immunogenic. ② Outstanding targeting and sustained-release properties: After intravenous injection, liposomes can naturally accumulate in organs such as the liver and spleen (passive targeting); after modification with antibodies and ligands (such as folic acid), they can also precisely recognize specific cells (active targeting), achieving precise drug delivery and slow release. ③ Effective protection and synergistic effects: Liposome encapsulation protects drugs from damage in the body's internal environment, significantly improving drug stability. Simultaneously, it can alter the distribution of drugs in the body, reduce toxicity to normal tissues such as the heart and kidneys, and improve the therapeutic index. Based on these advantages, liposome technology is rapidly expanding from the pharmaceutical field to multiple industries such as cosmetics and food.
[0003] Cholesterol is a lipid substance and an essential raw material for the synthesis of human cell membranes, hormones, and vitamin D. However, when cholesterol levels in the blood are imbalanced and too high, the risk of cardiovascular and cerebrovascular diseases increases. Liposomes, as "artificial biological membranes," often require cholesterol in their construction, especially in the pharmaceutical field. Cholesterol is added to stabilize the structure of liposomes, reduce their particle size and dispersion, and facilitate drug delivery.
[0004] In the pharmaceutical field, even with added cholesterol, the total cholesterol level is generally kept within a certain range due to the small dosage typically used. However, in the food industry, food intake is generally much greater than the dosage required for medication. If excessive cholesterol is added to the prepared liposomes, it can lead to excessive cholesterol levels in the human body. Summary of the Invention
[0005] Purpose of the invention: The purpose of this invention is to provide a food-grade nanoliposome and its preparation method.
[0006] Technical Solution: To solve the above technical problems, the present invention provides a method for preparing food-grade nanoliposomes, comprising the following steps:
[0007] (1) Preparation of organic phase: Dissolve phosphatidylcholine in anhydrous ethanol to obtain a phosphatidylcholine solution as the organic phase;
[0008] (2) Preparation of aqueous phase: The aqueous phase is a 5(6)-carboxyfluorescein solution or ultrapure water;
[0009] (3) Preparation of drug-loaded liposomes: The organic phase and the aqueous phase are mixed to prepare drug-loaded liposomes.
[0010] In step (1), the phosphatidylcholine solution is a Lipoid H 85 solution.
[0011] The concentration of the Lipoid H 85 solution is 10~20 mg / mL.
[0012] Among them, the molecular weight of 5(6)-carboxyfluorescein is 376.32 g / mol.
[0013] In step (3), the volume ratio of the organic phase to the aqueous phase is 1:5 to 1:9.
[0014] In step (3), the flow rates of the organic phase and the aqueous phase are 3-12 ml / min.
[0015] In step (3), the organic phase and the aqueous phase are mixed using a microfluidic method.
[0016] In step (3), crude drug-loaded liposomes are prepared by ethanol injection.
[0017] The method further includes step (4) subjecting the prepared crude drug-loaded liposomes to high-pressure homogenization to obtain the target drug-loaded liposomes.
[0018] The present invention also provides food-grade nanoliposomes prepared by the aforementioned preparation method.
[0019] The food-grade nanoliposomes have a particle size of <200nm and a particle size distribution of 0.3 < PDI < 0.45.
[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The preparation method of the present invention is simple, and no membrane material such as cholesterol is added when constructing the liposome model, yet liposomes can still be constructed with good encapsulation efficiency. Using the liposomes constructed by the present invention to encapsulate nutrients can not only improve the absorption of nutrients by humans, but also meet the current market demand for low-cholesterol and cholesterol-free health products. Attached Figure Description
[0021] Figure 1 This is a schematic diagram of the standard curve for the ultraviolet absorbance of substance 5(6)-carboxyfluorescein at 444 nm as a function of concentration.
[0022] Figure 2 The graph shows the results of ten tests on the particle size distribution and the distribution ratio (%) of scattered light intensity of the liposomes prepared in Example 6.
[0023] Figure 3 The graph shows the results of ten tests on the particle size distribution and the distribution ratio (%) of scattered light intensity of the liposomes prepared in Example 26.
[0024] Figure 4 The results of testing the particle size distribution and scattered light intensity distribution (%) of the liposomes prepared in Comparative Example 9 ten times are shown in the figure.
[0025] Figure 5 The results of testing the particle size distribution and the distribution percentage (%) of scattered light intensity of the liposomes prepared in Comparative Example 10 ten times are shown in the figure. Detailed Implementation
[0026] The technical solution of the present invention will be further described below with reference to the accompanying drawings.
[0027] Example 1: Preparation of a drug-loaded liposome applicable to food
[0028] The formulation of the drug-loaded liposomes included Lipoid H 85 (purchased from Lipoid, catalog number: 97281-49-7), 5(6)-carboxyfluorescein (purchased from Aladdin, catalog number: C107883), dimethyl sulfoxide (Aladdin, catalog number: D103272) and anhydrous ethanol.
[0029] A method for preparing drug-loaded liposomes applicable to food includes the following steps:
[0030] (1) Preparation of organic phase: Lipoid H 85 was dissolved in anhydrous ethanol as the organic phase, and the concentration of Lipoid H 85 was 10 mg / mL;
[0031] (2) Preparation of aqueous phase: 5(6)-carboxyfluorescein was dissolved in dimethyl sulfoxide to prepare 100 μM 5(6)-carboxyfluorescein as aqueous phase;
[0032] (3) Preparation of drug-loaded liposomes: Drug-loaded liposomes were prepared using microfluidics at room temperature with a volume ratio of organic phase to aqueous phase of 1:5 and a flow rate of 3 ml / min. The specific steps were as follows: The prepared organic phase and aqueous phase were respectively aspirated into two clean 10 ml disposable syringes and fixed into the corresponding two inlet ports inside the microfluidic instrument (Precision NanoSystems, NanoAssemblr® Ignite+™ Nanomedicine Delivery System, model: 1 mL~60 mL). Two clean centrifuge tubes were installed and fixed at the outlet of the microfluidic instrument (one as a waste liquid tube and the other as a centrifuge tube for loading drug-loaded liposomes). The volume ratio of organic phase to aqueous phase was set to 1:5 and the flow rate was set to 3 ml / min. The first 3 ml of liquid at the outlet was set as waste liquid and the instrument automatically discarded the first 3 ml of solution. After 3 ml, the instrument automatically moved to another clean centrifuge tube to start collecting the prepared liposome solution. This operation was used to reduce errors and obtain drug-loaded liposomes.
[0033] Example 2
[0034] A method for preparing drug-loaded liposomes applicable to food, the formula and preparation method are the same as in Example 1, except that the volume ratio of organic phase to aqueous phase in step (3) is 1:7.
[0035] Example 3
[0036] A method for preparing drug-loaded liposomes applicable to food, the formula and preparation method are the same as in Example 1, except that the volume ratio of organic phase to aqueous phase in step (3) is 1:9.
[0037] Example 4
[0038] A method for preparing drug-loaded liposomes applicable to food, the formulation and preparation method are the same as in Example 1, except that the flow rate of the organic phase and the aqueous phase in step (3) is 6 ml / min.
[0039] Example 5
[0040] A method for preparing drug-loaded liposomes applicable to food, the formula and preparation method are the same as in Example 1, except that in step (3), the volume ratio of organic phase to aqueous phase is 1:7 and the flow rate is 6 ml / min.
[0041] Example 6
[0042] A method for preparing drug-loaded liposomes applicable to food, the formula and preparation method are the same as in Example 1, except that in step (3), the volume ratio of organic phase to aqueous phase is 1:9 and the flow rate is 6 ml / min.
[0043] Example 7
[0044] A method for preparing drug-loaded liposomes applicable to food, the formulation and preparation method are the same as in Example 1, except that the concentration of Lipoid H 85 in step (1) is 20 mg / mL.
[0045] Example 8
[0046] A method for preparing drug-loaded liposomes applicable to food, the formulation and preparation method are the same as in Example 1, except that in step (1) the concentration of Lipoid H 85 is 20 mg / mL, and in step (3) the volume ratio of organic phase to aqueous phase is 1:7 and the flow rate is 3 ml / min.
[0047] Example 9
[0048] A method for preparing drug-loaded liposomes applicable to food, the formulation and preparation method are the same as in Example 1, except that in step (1) the concentration of Lipoid H 85 is 20 mg / mL, and in step (3) the volume ratio of organic phase to aqueous phase is 1:9 and the flow rate is 3 ml / min.
[0049] Example 10
[0050] A method for preparing drug-loaded liposomes applicable to food, the formulation and preparation method are the same as in Example 1, except that in step (1) the concentration of Lipoid H 85 is 20 mg / mL, and in step (3) the volume ratio of organic phase to aqueous phase is 1:5 and the flow rate is 6 ml / min.
[0051] Example 11
[0052] A method for preparing drug-loaded liposomes applicable to food, the formulation and preparation method are the same as in Example 1, except that in step (1) the concentration of Lipoid H 85 is 20 mg / mL, and in step (3) the volume ratio of organic phase to aqueous phase is 1:7, and the flow rate is 6 ml / min.
[0053] Example 12
[0054] A method for preparing drug-loaded liposomes applicable to food, the formulation and preparation method are the same as in Example 1, except that in step (1) the concentration of Lipoid H 85 is 20 mg / mL, and in step (3) the volume ratio of organic phase to aqueous phase is 1:9, and the flow rate is 6 ml / min.
[0055] Comparative Example 1
[0056] A method for preparing drug-loaded liposomes applicable to food, the formulation and preparation method are the same as in Example 1, except that in step (1) the concentration of Lipoid H 85 is 20 mg / mL, and in step (3) the volume ratio of organic phase to aqueous phase is 1:5 and the flow rate is 8 ml / min.
[0057] Comparative Example 2
[0058] A method for preparing drug-loaded liposomes applicable to food, the formulation and preparation method are the same as in Example 1, except that in step (1) the concentration of Lipoid H 85 is 20 mg / mL, and in step (3) the volume ratio of organic phase to aqueous phase is 1:9 and the flow rate is 8 ml / min.
[0059] Comparative Example 3
[0060] A method for preparing drug-loaded liposomes applicable to food, the formulation and preparation method are the same as in Example 1, except that in step (1) the concentration of Lipoid H 85 is 20 mg / mL, and in step (3) the volume ratio of organic phase to aqueous phase is 1:9 and the flow rate is 10 ml / min.
[0061] Results: The drug-loaded liposomes prepared in the above examples and comparative examples were characterized, and the results are shown in Table 1.
[0062] Table 1. Particle size and dispersity of liposomes encapsulating water-soluble substances prepared by microfluidic method under different parameters.
[0063]
[0064] As shown in Table 1, from Comparative Examples 1, 2, and 3, it can be seen that when the flow rate is ≥8 ml / min, the excessively fast flow rate will make the prepared liposomes more prone to abnormal particle size (particle size >200, standard deviation even greater than 1500) and more uneven dispersion (PDI greater than 0.45). Therefore, the flow rate parameter of the instrument should not exceed 6 ml / min when preparing liposomes using this method.
[0065] In Examples 1-12, as shown in Examples 1 and 7, when the concentration of Lipoid H 85 was doubled while other conditions remained unchanged, the particle size of the prepared liposomes also essentially doubled. Examples 2 and 8 also illustrate this point. However, when the organic phase:water phase volume ratio was 1:9, the particle size of the prepared liposomes did not double after the concentration was doubled, and the particle size increase was much less than 2 times. Examples 3 and 9, and Examples 6 and 12 illustrate this point, demonstrating that when the organic phase:water phase volume ratio was 1:9, the change in particle size of the prepared liposomes with concentration was much smaller than that with other volume ratios, exhibiting excellent stability and showing a highly significant difference. Therefore, an organic phase:water phase volume ratio of 1:9 is the optimal preparation volume ratio for this method.
[0066] After determining that an organic phase to aqueous phase volume ratio of 1:9 is the optimal preparation volume ratio for this method, in the data generated from Examples 3, 6, 9, and 12 above, when the flow rate is 6 ml / min, the prepared liposomes may still exhibit abnormalities. For example, the particle size in Example 6 (with a very large standard deviation, indicating low data repeatability) is shown in the following example. Figure 2 Although its particle size distribution is relatively uniform, the distribution ratio (%) of its scattered light intensity fluctuates greatly. Even if the liposomes prepared in Example 12 are normal, in order to avoid repeating the situation in Example 6, the flow rate of the liposomes prepared by this method should be 3 ml / min.
[0067] In summary, the liposomes prepared using this method in the examples all met the requirements of particle size (<100) and PDI (<0.45). Among them, the liposomes with the organic phase:water volume ratio set at 1:9 and the flow rate at 3 ml / min were the best. Under these parameters, not only were the prepared liposomes more stable (smaller standard deviation and higher data repeatability), but they could also withstand the preparation of a higher concentration of liposomes (the organic phase:water volume ratio of 1:9 can withstand the preparation of a higher concentration of liposomes than other volume ratios), making them suitable for more environments.
[0068] Example 14: Preparation of drug-loaded liposomes applicable to food
[0069] Another drug-loaded liposome formulation applicable to food includes Lipoid H 85, anhydrous ethanol, and ultrapure water; a method for preparing drug-loaded liposomes applicable to food comprises the following steps:
[0070] (1) Preparation of organic phase: Lipoid H 85 was dissolved in anhydrous ethanol to prepare an organic phase with a concentration of 20 mg / mL and a total volume of 10 mL.
[0071] (2) Preparation of aqueous phase: 100 μM 5(6)-carboxyfluorescein + anhydrous ethanol, volume ratio 1:9, total volume 90 mL;
[0072] (3) Preparation of crude drug-loaded liposomes: Mix the organic phase and the aqueous phase, and use magnetic stirring to generate a vortex in the aqueous phase; use a burette to slowly and evenly drop the organic phase into the vortex, controlling the dropping rate at one drop every 2-3 seconds, until all the organic phase is dropped in to prepare crude liposomes.
[0073] (4) Purification: The prepared crude liposomes were subjected to a high-pressure homogenizer (model: Ningbo Xinzhi Experimental-150) with the following parameters: pressure of 30 MPa and 1 pass through the instrument to obtain the target liposomes.
[0074] Example 15
[0075] Another method for preparing drug-loaded liposomes that can be applied to food is the same as in Example 14, except that step (4) is performed twice.
[0076] Example 16
[0077] Another method for preparing drug-loaded liposomes that can be applied to food is the same as in Example 14, except that step (4) is performed 3 times.
[0078] Example 17
[0079] Another method for preparing drug-loaded liposomes that can be applied to food is the same as in Example 14, except that step (4) is performed 4 times.
[0080] Example 18
[0081] Another method for preparing drug-loaded liposomes that can be applied to food is the same as in Example 14, except that step (4) is performed 5 times.
[0082] Example 19
[0083] Another method for preparing drug-loaded liposomes that can be applied to food is the same as in Example 14, except that step (4) is performed 6 times.
[0084] Example 20
[0085] Another method for preparing drug-loaded liposomes that can be applied to food is the same as in Example 14, except that step (4) is performed 7 times.
[0086] Example 21
[0087] Another method for preparing drug-loaded liposomes that can be applied to food is the same as in Example 14, except that the pressure in step (4) is 45 MPa.
[0088] Example 22
[0089] Another method for preparing drug-loaded liposomes that can be applied to food is the same as in Example 14 in terms of formulation and preparation method, except that the pressure in step (4) is 45 MPa and the number of times is 2.
[0090] Example 23
[0091] Another method for preparing drug-loaded liposomes that can be applied to food is the same as in Example 14 in terms of formulation and preparation method, except that the pressure in step (4) is 45 MPa and the number of times is 3.
[0092] Example 24
[0093] Another method for preparing drug-loaded liposomes that can be applied to food is the same as in Example 14 in terms of formulation and preparation method, except that the pressure in step (4) is 45 MPa and the number of times is 4.
[0094] Example 25
[0095] Another method for preparing drug-loaded liposomes that can be applied to food is the same as in Example 14 in terms of formulation and preparation method, except that the pressure in step (4) is 45 MPa and the number of times is 5.
[0096] Example 26
[0097] Another method for preparing drug-loaded liposomes that can be applied to food is the same as in Example 14 in terms of formulation and preparation method, except that the pressure in step (4) is 45 MPa and the number of times is 6.
[0098] Example 27
[0099] Another method for preparing drug-loaded liposomes that can be applied to food is the same as in Example 14 in terms of formulation and preparation method, except that the pressure in step (4) is 45 MPa and the number of times is 7.
[0100] Example 28
[0101] Another method for preparing drug-loaded liposomes that can be applied to food is the same as in Example 14 in terms of formulation and preparation method, except that the pressure in step (4) is 60 MPa and the number of times is 1.
[0102] Example 29
[0103] Another method for preparing drug-loaded liposomes that can be applied to food is the same as in Example 14, except that the pressure in step (4) is 60 MPa and the number of times is 2.
[0104] Comparative Example 3
[0105] Another method for preparing drug-loaded liposomes that can be applied to food is the same as in Example 14, except that the pressure in step (4) is 60 MPa and the number of times is 3.
[0106] Comparative Example 4
[0107] Another method for preparing drug-loaded liposomes that can be applied to food is the same as in Example 14, except that the pressure in step (4) is 60 MPa once + 90 MPa once.
[0108] Comparative Example 5
[0109] Another method for preparing drug-loaded liposomes that can be applied to food is the same as in Example 14, except that the pressure in step (4) is 60 MPa once + 90 MPa twice.
[0110] Comparative Example 6
[0111] Another method for preparing drug-loaded liposomes that can be applied to food is the same as in Example 14, except that the pressure in step (4) is 60 MPa once + 90 MPa four times.
[0112] Comparative Example 7
[0113] Another method for preparing drug-loaded liposomes that can be applied to food is the same as in Example 14, except that the pressure in step (4) is 60 MPa once + 120 MPa once.
[0114] Comparative Example 8
[0115] Another method for preparing drug-loaded liposomes that can be applied to food is the same as in Example 14, except that the pressure in step (4) is 60 MPa once + 120 MPa twice.
[0116] Comparative Example 9
[0117] Another method for preparing drug-loaded liposomes that can be applied to food is the same as in Example 14, except that the pressure in step (4) is 60 MPa once + 120 MPa four times.
[0118] Results: The liposomes prepared in Examples 14-29 and Comparative Examples 3-9 were characterized, and the results are shown in Table 2.
[0119] Table 2. Particle size and dispersity of liposomes prepared by the ethanol injection method combined with high-pressure homogenization.
[0120]
[0121] Table 2 shows that, based on the test results of Examples 14-29, it was found that, while maintaining a constant pressure, increasing the number of passes through the high-pressure homogenizer resulted in smaller liposome particle sizes, as expected. For example, in Examples 14-20, under the same pressure of 30 MPa, each pass through the high-pressure homogenizer reduced the particle size of the prepared liposomes. Similar conclusions were reached in Examples 21-27. Figure 3 It can be seen that the liposomes prepared in Example 26 have uniform particle size, and the distribution ratio (%) of scattered light intensity is relatively stable. In contrast, the results of the liposomes prepared in Example 6... Figure 2Although the liposomes have uniform particle size, the distribution ratio (%) of their scattered light intensity fluctuates considerably. Furthermore, increasing the number of times the homogenizer is used does not significantly reduce the dispersion of the prepared liposomes, and the reduction in particle size is not more than an order of magnitude, remaining between 100 and 200. Within this range, the application of the prepared liposomes will not show any difference.
[0122] While maintaining the same number of high-pressure homogenization cycles, increasing the instrument pressure resulted in smaller liposome particle sizes, as expected. However, the test results of Comparative Examples 4-10 showed that at high pressures, such as 60 MPa, the liposomes prepared in this invention reached their tolerance limit. At this pressure, when the number of cycles exceeded 2, each additional 60 MPa cycle could potentially cause abnormalities in the liposomes, as seen in Comparative Example 4. Furthermore, excessively high pressures can also directly lead to abnormalities in the liposomes, as seen in Comparative Examples 7, 9, and 10. Comparative Examples 9 and 10 each tested the prepared liposomes 10 times; the results are detailed below. Figure 4 and Figure 5 ,from Figure 4 and Figure 5 It can be seen that, due to the non-uniform size of the prepared liposome particles, the distribution ratio (%) of both particle size and scattered light intensity in the test results fluctuates greatly.
[0123] Therefore, we conclude that blindly increasing the pressure and the number of cycles will not cause a magnitude change in the liposomes prepared in this invention; on the contrary, it may cause abnormalities. Testing revealed that the liposomes prepared under the conditions of Example 27 had the best performance parameters, i.e., a pressure of 45 MPa and a cycle count of 7. However, since it did not lead by a magnitude change, and for cost considerations, we subsequently used the conditions of Example 28 for subsequent experiments.
[0124] Application example:
[0125] The encapsulation efficiency of liposomes prepared by microfluidic method in Example 9 and liposomes prepared by ethanol injection method in Example 28 was determined, and 5(6)-carboxyfluorescein with ultraviolet absorption was selected as the standard substance.
[0126] (1) Prepare a concentration-absorbance standard curve for 5(6)-carboxyfluorescein;
[0127] Preparation method:
[0128] A 10 mM 5(6)-carboxyfluorescein solution was prepared by dissolving 5(6)-carboxyfluorescein in dimethyl sulfoxide.
[0129] Take 1 mL of the above solution and add it to a system of 90 mL of water and 10 mL of anhydrous ethanol. At this time, the concentration of 5(6)-carboxyfluorescein is 100 μM.
[0130] The prepared sample was diluted to prepare samples with concentrations of 75 μM, 50 μM, 25 μM, 12.5 μM, 5 μM, and 1 μM, respectively.
[0131] Full-band scanning showed that the UV absorption peak of 5(6)-carboxyfluorescein was at 444 nm. The absorbance of each sample was measured at this wavelength, and a standard curve was plotted using the obtained data. The results are as follows. Figure 1 As shown.
[0132] (2) Preparation of liposomes loaded with 5(6)-carboxyfluorescein by ethanol injection method
[0133] Take the drug-loaded liposomes prepared in Example 28, pour them into an ultrafiltration tube, centrifuge at 5000 rpm / min for 10 min, take the filtrate, measure its absorbance at 444 nm, prepare three samples, obtain three sets of data, take the average value, and calculate the concentration of 5(6)-carboxyfluorescein in the filtrate according to the standard curve.
[0134] (3) Preparation of liposomes loaded with 5(6)-carboxyfluorescein by microfluidic method
[0135] Take the drug-loaded liposomes prepared in Example 9, pour them into an ultrafiltration tube, centrifuge at 5000 rpm / min for 10 min, take the filtrate, measure its absorbance at 444 nm, prepare three samples, obtain three sets of data, take the average value, and calculate the concentration of 5(6)-carboxyfluorescein in the filtrate according to the standard curve.
[0136] (4) Take the two drug-loaded liposomes prepared in steps (2) and (3) above, and combine them with... Figure 1 The ultraviolet absorbance was measured, and the results are shown in Table 3:
[0137] Table 3 Absorbance of liposomes loaded with 5(6)-carboxyfluorescein prepared by ethanol injection and microfluidic control
[0138]
[0139] (5) Calculation of encapsulation efficiency: Encapsulation efficiency = ((concentration before filtration - concentration after filtration) / concentration before filtration) × 100%
[0140] Encapsulation efficiency of ethanol injection method = ((90-22.715) / 90)×100% = 74.76%
[0141] The encapsulation efficiency of the microfluidic method = ((90-11.105) / 90)×100% = 87.67%.
[0142] In summary, the encapsulation efficiency of liposomes prepared by the microfluidic method and the ethanol injection method is still excellent without the addition of other membrane materials such as cholesterol or the use of organic solvents such as chloroform. In particular, the encapsulation efficiency of liposomes prepared by the microfluidic method is even better. Furthermore, the experiment showed that 5(6)-carboxyfluorescein is feasible as a substance for determining the encapsulation efficiency of liposomes.
Claims
1. A method for preparing food-grade nanoliposomes, characterized by, Includes the following steps: (1) Preparation of organic phase: Dissolve phosphatidylcholine in anhydrous ethanol to obtain a phosphatidylcholine solution as the organic phase; (2) Preparation of aqueous phase: The aqueous phase is a 5(6)-carboxyfluorescein solution or ultrapure water; (3) Preparation of drug-loaded liposomes: The organic phase and the aqueous phase are mixed to prepare drug-loaded liposomes.
2. The method for preparing food-grade nanoliposomes according to claim 1, characterized in that, The phosphatidylcholine solution in step (1) is a Lipoid H 85 solution.
3. The method for preparing food-grade nanoliposomes according to claim 2, characterized in that, The concentration of the Lipoid H85 solution is 10~20 mg / mL.
4. The method for preparing food-grade nanoliposomes according to claim 1, characterized in that, The volume ratio of the organic phase to the aqueous phase in step (3) is 1:5 to 1:
9.
5. The method for preparing food-grade nanoliposomes according to claim 1, characterized in that, The flow rates of the organic and aqueous phases in step (3) are 3-12 ml / min.
6. The method of claim 1, wherein the food-grade nanoliposomes are prepared by the method comprising the steps of: In step (3), the organic phase and the aqueous phase are mixed using a microfluidic method.
7. The method for preparing food-grade nanoliposomes according to claim 1, characterized in that, In step (3), crude drug-loaded liposomes are prepared by ethanol injection.
8. The method of claim 7, wherein the food-grade nanoliposomes are prepared by the method comprising the steps of: The method further includes step (4) subjecting the prepared crude drug-loaded liposomes to high-pressure homogenization to obtain the target drug-loaded liposomes.
9. Food-grade nanoliposomes prepared by the preparation method according to any one of claims 1 to 8.
10. The food-grade nanoliposome of claim 9, wherein, The food-grade nanoliposomes have a particle size of <200nm and a particle size distribution of 0.3 < PDI < 0.45.