Preparation method of lipid-like vesicles

Abstract

The invention discloses a method for preparing lipoid vesicles by a T-type mixing method, which specifically comprises the following steps: respectively dissolving a nonionic surfactant and cholesterol in absolute ethyl alcohol, and carrying out ultrasonic treatment to completely dissolve the nonionic surfactant and cholesterol; calculating the volume of the required solution according to the mass ratio of the nonionic surfactant to the cholesterol, and uniformly mixing the nonionic surfactant and the cholesterol to obtain a mixed solution; and the mixed solution and distilled water are simultaneously extruded through two inlets in the upper end of the T-shaped mixing pipe respectively, an organic phase is diluted into a water phase, and the polarity of the solvent is increased, so that the nonionic surfactant is aggregated and precipitated, and the lipoid vesicles are obtained at the output end. And ultrasonically reducing the particle size of the obtained lipoid vesicles through an ultrasonic cell crusher. And extruding by using a liposome extruder to obtain the lipoid vesicles with uniform particle size. The preparation method of the lipid vesicles is simple, the requirement on equipment is low, and conditions are easy to control; the obtained lipoid vesicles are small in particle size and good in monodispersity and stability, and can be used for preparing drug-loaded lipoid vesicles.

Description

Technical Field

[0001] The invention relates to the field of pharmaceutical preparations, and in particular to a method for preparing lipid vesicles. The lipid vesicles with small particle size and narrow particle size distribution are prepared by a T-type mixing method. Background Art

[0002] In the nano drug delivery system, both liposomes and lipid vesicles play an important role in reducing drug toxicity and improving bioavailability. However, since liposomes are prepared from phospholipids, separation and purification are difficult, the cost is relatively high, and the encapsulation rate and stability are not very high, so there are certain application limitations. The preparation cost of lipid vesicles is lower than that of liposomes, and they have good stability and a relatively simple preparation method, which has great development prospects.

[0003] Lipid vesicles are a single-layer or multi-layer drug carrier formed by non-ionic surfactants and cholesterol. They are similar to liposomes in structure and composition, so they have the advantages of low toxicity and biodegradability of liposomes. The water phase core similar to liposomes can encapsulate water-soluble and fat-soluble drug molecules, improve drug solubility, control drug release, maintain effective blood drug concentration, and this drug carrier can overcome the problem of phospholipid instability. At present, lipid vesicles have become a new carrier for systemic and transdermal drug delivery, and are a hot topic in the field of pharmaceutical research.

[0004] Lipid vesicles are single-layer or multi-layer vesicles prepared from cholesterol and nonionic surfactants, so they can also be called nonionic surfactant vesicles (NSVs). They are similar to liposomes in terms of structural composition and physical properties, so lipid vesicles have some similar properties to liposomes, such as increasing the stability of drugs, changing the distribution of drugs in the body, improving drug efficacy, and reducing drug toxicity and side effects. Since nonionic surfactants are non-immunogenic, low-toxic, and have good biocompatibility, they are more stable than phospholipids, are not easily oxidized, and do not require special conditions for storage. Therefore, lipid vesicles are not only less toxic and low-cost, but also biodegradable.

[0005] The conventional methods for preparing lipid vesicles include thin film dispersion method, ether injection method and reverse phase evaporation method, etc. The lipid vesicles prepared by these methods often have certain shortcomings, such as incomplete hydration of surfactants during the hydration process, or due to the dispersed form of the vesicles, some drugs may precipitate, accumulate, fuse or leak during storage, and some preparation techniques are time-consuming and require special equipment. In order to solve the shortcomings of traditional preparation methods such as thin film hydration method, such as difficulty in large-scale production, large amount of organic reagent residues, time-consuming and labor-intensive, the present invention uses a relatively controllable T-tube mixing method to prepare lipid vesicles. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing lipid vesicles by T-type mixing method.

[0007] To achieve the above object, the present invention provides the following solutions: The present invention provides a method for preparing lipid vesicles, which specifically comprises the following steps: Dissolve the nonionic surfactant and cholesterol in anhydrous ethanol respectively, and completely dissolve them by ultrasonication. Calculate the required solution volume according to the mass ratio of the nonionic surfactant to cholesterol, and mix them evenly to obtain a mixed solution as the organic phase; Using water as the aqueous phase, or an aqueous solution of the drug as the aqueous phase; The organic phase and the aqueous phase are connected to the two input ends of the upper end of the T-shaped mixing tube respectively, and the organic phase and the aqueous phase are extruded into the T-shaped mixing tube at the same time. The two phases are mixed in the mixing area in the middle of the T-shaped mixing tube. The organic phase is diluted into the aqueous phase, and the polarity of the solvent increases, so that the non-ionic surfactant aggregates and precipitates, thereby obtaining the primary product of lipid vesicles at the output end; The obtained lipid vesicle primary product is ultrasonically crushed by an ultrasonic cell disruptor to reduce the particle size, and then extruded by a liposome extruder to obtain lipid vesicles with uniform particle size.

[0008] Preferably, in the above steps, the nonionic surfactant is selected from Span 80, Span 40, Tween 80, Brij S100, more preferably Span 80 or Span 40, and even more preferably Span 80.

[0009] Preferably, in the above steps, the mass ratio of the nonionic surfactant to cholesterol is 1-5:1, such as 1:1, 2:1, 3:1, 4:1, 5:1; more preferably 2-4:1, and even more preferably 2:1.

[0010] Preferably, in the above steps, the organic phase further comprises an organic solvent, and the organic solvent is anhydrous ethanol.

[0011] Preferably, in the above steps, the flow rate of the organic phase is 0.1-0.5 mL / s, and the flow rate of the aqueous phase is 0.1-0.5 mL / s. In some exemplary embodiments of the present invention, the flow rates of the organic phase and the aqueous phase are the same, and the flow rates of the organic phase and the aqueous phase are both 0.1-0.5 mL / s. Further preferably, the flow rates of the organic phase and the aqueous phase are both 0.1 mL / s, and the mixing is more uniform and sufficient at this flow rate.

[0012] Preferably, in the above steps, the ultrasonic disruption is intermittent ultrasonic treatment, and the number of ultrasonic treatments is not less than 10. For example, in some exemplary embodiments of the present invention, ultrasonic cell disruptor is used for 6S, intermittently for 6S, for a total of 11 times to reduce the particle size of lipid vesicles.

[0013] Preferably, in the above steps, the number of cycles of extrusion through the liposome extruder is not less than 10 times. Specifically, the initial product is extruded repeatedly through the liposome extruder. In some exemplary embodiments of the present invention, the number of cycles of extrusion is 10 times to obtain small and uniform lipid vesicles.

[0014] The present invention also provides lipid vesicles prepared by the method for preparing lipid vesicles by the T-type mixing method as described above.

[0015] The present invention also provides the application of lipid vesicles prepared by the T-type mixing method as described above in drug loading.

[0016] In one embodiment, the drug loaded is doxorubicin.

[0017] The particle size of the lipid vesicles prepared by the method of the invention is between 110 and 135 nm, and the particle size is uniform.

[0018] The particle size of the drug-loaded lipid vesicles prepared by the present invention has an encapsulation rate of more than 65%.

[0019] Compared with the prior art, the present invention has at least one of the following beneficial effects: The present invention uses a T-shaped mixing tube to prepare lipid vesicles, and controls the process conditions of T-tube mixing and the formula composition of lipid vesicles to prepare more stable and more uniform lipid vesicles. The particle size is further reduced and made uniform by combining ultrasonic crushing with extrusion process.

[0020] The preparation method of the lipid vesicles of the present invention is simple, the conditions are easy to control, and it is suitable for large-scale industrial production; in addition, the amount of raw materials such as non-ionic surfactants is small, which saves costs; experiments have found that the lipid vesicles prepared by this method have small particle size, good monodispersity and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1It is a schematic diagram of the structure of a T-type mixer and a physical diagram of the T-type mixing device used in the embodiment, in which 1 is the input end, 2 is the mixing zone, and 3 is the output end.

[0023] Figure 2 The graph is a graph showing the particle size results of lipid vesicles prepared using different non-ionic surfactants and mass ratios in the examples.

[0024] Figure 3 This is a graph showing the particle size results of whether a liposome extruder is used to prepare lipid vesicles in the examples.

[0025] Figure 4 The figure is a comparison of the results of preparing lipid vesicles using the T-tube method and the ethanol injection method in the examples.

[0026] Figure 5 The figures are comparison results of preparing lipid vesicles using T-tubes at different flow rates in the examples.

[0027] Figure 6 The figure is a comparison of the results of preparing drug-loaded lipid vesicles using the T-tube method and the ethanol injection method in the examples.

[0028] Figure 7 It is the standard curve diagram of DOX in the embodiment. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] The first aspect of the present invention provides a method for preparing lipid vesicles by T-type mixing method, comprising the following steps: Dissolve the nonionic surfactant and cholesterol in anhydrous ethanol respectively, and dissolve them completely by ultrasound. Calculate the required solution volume according to the mass ratio of nonionic surfactant to cholesterol, and mix them evenly to obtain a mixed solution. Take a 1mL syringe from a T-type mixing tube to absorb the above-mentioned mixed solution according to the volume of the above ratio. If the mixed solution is less than 1mL, add anhydrous ethanol to 1mL, and another 1mL syringe absorbs 1mL of distilled water. Install the two syringes that have absorbed the solution on the T-type mixing tube. After installation, squeeze out the contents of the two syringes at the same time, dilute the organic phase into the aqueous phase, increase the polarity of the solvent, and cause the nonionic surfactant to aggregate and precipitate, thereby obtaining lipid vesicles at the output end. The obtained lipid vesicles are reduced in particle size by ultrasound (working 6S, intermittent 6S, a total of 11 times) of an ultrasonic cell disruptor. Then extrude it using a liposome extruder (reciprocating 10 times) to obtain lipid vesicles with uniform particle size.

[0035] In one embodiment, in the above steps, the nonionic surfactant is Span 80, Span 40, Tween 80, Brij S100.

[0036] In one embodiment, in the above steps, the mass ratio of the nonionic surfactant to cholesterol is 1:1, 2:1, 3:1, 4:1, or 5:1.

[0037] In one embodiment, in the above steps, the lipid vesicles with uniform particle size are prepared by extrusion through a liposome extruder.

[0038] The second aspect of the present invention provides lipid vesicles prepared by the method for preparing lipid vesicles by the T-type mixing method as described above.

[0039] The third aspect of the present invention provides the use of lipid vesicles prepared by the T-type mixing method as described above in drug delivery.

[0040] In one embodiment, the drug loaded is doxorubicin.

[0041] The following is further described in conjunction with specific embodiments. The raw materials involved in the following specific embodiments, unless otherwise specified, can all be commercially available; the instruments used, unless otherwise specified, can all be commercially available; the processes involved, unless otherwise specified, are all routinely selected by those skilled in the art.

[0042] The schematic diagram of the structure of the T-type mixing tube used in the following embodiments is shown in Figure 1 In the left figure, the organic phase and the aqueous phase are respectively input into the T-type mixing tube from the input ends 1 at both ends, mixed, aggregated and precipitated in the mixing zone 2, and output from the output end 3. See the specific physical picture of the T-type mixing device for details. Figure 1 Right picture. Example 1

[0043] This example provides a method for preparing lipid vesicles by T-type mixing method, and compares the particle sizes of lipid vesicles prepared by different mass ratios of surfactant Span80, Span 40, Tween 80, Brij S100 and cholesterol. Specifically, the method comprises the following steps: Weigh 85.7 mg of surfactant and 63.9 mg of cholesterol, dissolve them in 5 mL of anhydrous ethanol respectively, and dissolve them completely by ultrasound. According to the mass ratio of non-ionic surfactant to cholesterol of 1:1, 2:1, 3:1, 4:1, and 5:1, determine the required volume of non-ionic surfactant solution and cholesterol solution. Accurately measure the required volume of solution, mix it evenly, mark it, and obtain a mixed solution. Take a 1mL syringe from a T-type mixing tube to absorb the above mixed solution according to the volume of the above ratio. If the mixed solution is less than 1mL, add anhydrous ethanol to 1mL, and another 1mL syringe absorbs 1mL of distilled water. Install the two syringes that have absorbed the solution on the T-type mixing tube. After installation, squeeze out the contents of the two syringes at the same time, dilute the organic phase into the aqueous phase, increase the polarity of the solvent, and cause the non-ionic surfactant to aggregate and precipitate, thereby obtaining lipid vesicles at the output end. The sample obtained after mixing through the T tube is the blank lipid vesicle, which is placed in a 5mL centrifuge tube and marked. The obtained lipid vesicles were ultrasonicated by an ultrasonic cell disruptor (working 6S, resting 6S, a total of 11 times) to reduce the particle size.

[0044] The particle size determination results are shown in Table 1 and Figure 2. The particle size and PDI results of blank lipid vesicles prepared by T-tube mixing method with nonionic surfactants tween80 and brijS100 and cholesterol were not ideal. The particle size results showed that the particle size was large and the dispersion was poor. Analysis of the results showed that the reason why the particle size results of blank vesicles prepared by Tween80 and Brij S100 were not ideal was that lipid vesicles were closed structures composed of nonionic surfactants and cholesterol. In the molecular structure of nonionic surfactants, the hydrophilic groups were mainly a certain number of oxygen-containing groups, generally ether groups and hydroxyl groups; the lipophilic groups were long-chain fatty acids or long-chain fatty alcohols, etc., which were combined with the hydrophilic groups by ester bonds or ether bonds. The hydrophilic groups of tween80 were composed of long chains of polyoxyethylene, so that the hydrophilic groups of tween80 occupied the main position in the whole molecule; the molecular structure of brijS100 was composed of a long alkyl chain, dominated by lipophilic groups. The measurement results of span (Span 80, Span 40) are relatively good, with relatively uniform particle size and good dispersibility. The experimental results can be screened, and span80 and span40 can be used to synthesize lipid vesicles for subsequent experimental condition screening.

[0045] Table 1 Results of preparing lipid vesicles with different nonionic surfactants and different mass ratios Example 2

[0046] This example provides a method for preparing lipid vesicles by T-type mixing. On the basis of Example 1, surfactants Span 80 and Span 40 are mixed with cholesterol to prepare lipid vesicles, which are then extruded through a liposome extruder.

[0047] The particle size determination results are as follows Figure 3 As shown. The synthesized lipid vesicles were extruded through a liposome extruder and then measured, where Span80 is a lipid vesicle synthesized using a nonionic surfactant Span80, Span80-2 is a lipid vesicle extruded through a liposome extruder after synthesis, and Span 40 has the same meaning as above. The results showed that the particle size results of blank vesicles prepared by mixing Span 80 and Span 40T tubes and the particle size results of lipid vesicles prepared using a liposome extruder were compared, and it was found that the blank lipid vesicles prepared with span80 had better measurement results, with a particle size of about 180 nm, and the particle size of the blank vesicles extruded using a liposome extruder was reduced to about 130 nm. Therefore, the lipid vesicles prepared by T-tube mixing in subsequent experiments were assumed to have been prepared using a liposome extruder.

[0048] Comparative Example 1 This comparative example provides an ethanol injection method for preparing lipid vesicles, wherein the surfactants used are Span 80 and Span 40, and specifically comprises the following steps: Weigh 59.6 mg of surfactant and 15.1 mg of cholesterol, dissolve them in 5 mL of anhydrous ethanol respectively, and dissolve them completely by ultrasound. Determine the required volume of nonionic surfactant solution and cholesterol solution according to the mass ratio of nonionic surfactant to cholesterol of 1:1, 2:1, 3:1, 4:1, and 5:1. After accurately measuring the required volume, place it in a 2mL centrifuge tube, mix it evenly, mark it, and obtain a mixed solution. Select a clean vial of suitable size, put a stirrer in it, and use a pipette to accurately measure 5mL of distilled water and place it in the vial. Stir at a speed of 600rpm. Use a 2.5 mL syringe to absorb 2mL of the above mixed solution. If the mixed solution is less than 2mL, use anhydrous ethanol to make up to 2mL. Suspend the syringe above the vial, about 2 cm from the liquid surface, and slowly inject the mixed solution into the stirring aqueous solution through the needle. It takes 12 minutes for the mixed solution to be injected into the aqueous solution, and continue stirring for 60 to 90 minutes to evaporate the residual ethanol. After the stirring is completed, the sample is placed in a centrifuge tube and marked to obtain blank lipid vesicles.

[0049] The particle size results of different lipid vesicles prepared in this comparative example are shown in Table 2. The lipid vesicles prepared by the ethanol injection method all have good particle size results and dispersibility. Among them, the blank vesicles prepared with a mass ratio of Span 80 to cholesterol of 5:1 are the best. The blank vesicles prepared with a mass ratio of Span 40 of 4:1 are the best.

[0050] Table 2 Results of lipid vesicles prepared using ethanol injection method

[0051] Compared with lipid vesicles prepared by T-tube mixing method, Figure 4 As shown in the figure, the results of lipid vesicles prepared by using Span 40 are not as good as those prepared by using Span 80, and the particle size results of blank vesicles prepared by Span 80 are very similar, whether it is the ethanol injection method or the T-tube mixing method, which shows that the increase in the specific gravity of Span 80 has little effect on the particle size of lipid vesicles. Compared with the ethanol injection method, the T-tube mixing method is simpler, less time-consuming, and more efficient. Example 3

[0052] This example compares the effect of flow rate on the preparation of lipid vesicles in the T-type mixing method. The specific steps are as follows: Weigh 55.3 mg of Span 80 and 17.2 mg of cholesterol, dissolve them in 5 mL of anhydrous ethanol respectively, and dissolve them completely by ultrasound. According to the mass ratio of nonionic surfactant to cholesterol of 2:1, determine the required volume of nonionic surfactant solution and cholesterol solution. Accurately measure the required volume of solution, mix them evenly, mark them well, and obtain a mixed solution. Take a 1mL syringe from a T-type mixing tube to absorb the above mixed solution according to the volume of the above ratio. If the mixed solution is less than 1mL, add anhydrous ethanol to 1mL. The other 1mL syringe absorbs 1mL of distilled water. Install the two syringes that have absorbed the solution on the T-type mixing tube. After installation, squeeze out the contents of the two syringes at a flow rate of 0.5 mL / s, 0.2 mL / s, or 0.1mL / s at the same time. The organic phase is diluted into the aqueous phase, and the polarity of the solvent increases, causing the nonionic surfactant to aggregate and precipitate, thereby obtaining lipid vesicles at the output end. The sample obtained after mixing through the T tube is the blank lipid vesicle, which is placed in a 5mL centrifuge tube and marked well. The obtained lipid vesicles were ultrasonicated by an ultrasonic cell disruptor (working 6S, resting 6S, a total of 11 times) to reduce the particle size.

[0053] According to the above method, lipid vesicles at different flow rates were prepared by Figure 5 As shown, the particle size of the lipid vesicles becomes better as the flow rate decreases. The reason may be that the slow flow rate allows for a more complete reaction. When the flow rate is 0.1 mL / s, the result is the best. Subsequent experiments will use this as a condition to prepare lipid vesicles. Example 4

[0054] This embodiment provides a method for preparing lipid vesicles for drug loading by T-type mixing method, and the specific steps are as follows: Weigh 55.3 mg of Span 80 and 17.2 mg of cholesterol, dissolve them in 5 mL of anhydrous ethanol respectively, and sonicate to completely dissolve them. Determine the required volume of nonionic surfactant solution and cholesterol solution according to the mass ratio of nonionic surfactant to cholesterol of 1:1, 2:1, 3:1, 4:1, and 5:1. Dilute 1 mg / mL of doxorubicin aqueous solution with distilled water to 0.1 mg / mL of DOX aqueous solution. Accurately measure the nonionic surfactant solution and cholesterol solution, mix them evenly, mark them, and obtain a mixed solution. Take a 1mL syringe from a T-type mixing tube and absorb the above-mentioned mixed solution according to the above-mentioned volume. If the mixed solution is less than 1mL, use anhydrous ethanol to make up to 1mL. Use another 1mL syringe to absorb 1mL of 0.1mg / mL DOX aqueous solution, install the syringe of the T-type mixing tube, mix and squeeze out the contents of the two syringes at the same time, place them in a 5mL centrifuge tube, mark them, and the obtained sample is the drug-loaded lipid vesicles. Take the sample obtained after mixing in the T tube, extrude it using a liposome extruder (reciprocating 10 times) to reduce the particle size of the lipid vesicles, and place the extruded sample in a centrifuge tube and mark it.

[0055] Comparative Example 2 This comparative example provides an ethanol injection method for preparing drug-loaded lipid vesicles, which specifically includes the following steps: Weigh 55.3 mg of Span 80 and 17.2 mg of cholesterol, dissolve them in 5 mL of anhydrous ethanol respectively, and sonicate to completely dissolve them. Determine the required volume of nonionic surfactant solution and cholesterol solution according to the mass ratio of nonionic surfactant to cholesterol of 1:1, 2:1, 3:1, 4:1, and 5:1. After accurately measuring the required volume, place it in a 2mL centrifuge tube, mix it evenly, mark it, and obtain a mixed solution. Select a clean vial of suitable size, put a stirring bar in it, and use a pipette to accurately measure 5mL of 0.1 mg / mL DOX aqueous solution and place it in the vial. Stir at a speed of 600rpm. Use a 2.5 mL syringe to draw 2mL of the above mixture. If the mixture is less than 2mL, use anhydrous ethanol to make up to 2mL. Suspend the syringe above the vial, about 2 cm from the liquid surface, and slowly inject the mixture into the stirring 0.1 mg / mL DOX aqueous solution through the needle. The mixture was injected into the aqueous solution in 12 minutes and stirred for 60 to 90 minutes to evaporate the residual ethanol. After stirring, the sample was placed in a centrifuge tube and marked to obtain drug-loaded lipid vesicles.

[0056] Drug-loaded lipid vesicles were prepared according to Example 4 and Comparative Example 2, and their physicochemical properties were measured. The results are as follows: Figure 6As shown in the figure, it was found that the particle sizes of DOX-lipid vesicles prepared by Span 80 under the T-tube mixing method at different mass ratios were relatively close. And the results were better than those of the ethanol injection method. During the preparation process, the lipid vesicles prepared by the ethanol injection method could still smell the odor of ethanol after 60 minutes of continuous stirring, indicating that there was still residual ethanol that was not completely volatilized after long-term stirring. It further shows the advantage of using the T-tube mixing method to prepare lipid vesicles. Therefore, the use of Span 80 is more suitable for preparing lipid vesicles by the T-type mixing method, and the optimal ratio of Span 80 to prepare DOX-lipid vesicles is a span80 to cholesterol mass ratio of 2:1, which greatly reduces the amount of pharmaceutical excipients. Example 5

[0057] The quality of drug-loaded lipid vesicles was evaluated. The specific methods and results are as follows: (1) Determine the encapsulation efficiency of the drug-loaded lipid vesicles. The formula for determining the liposome encapsulation efficiency is: EE% = [C total -C free ) / C total 】*100% 1. Weigh DOX and prepare different concentration gradients using PBS buffer. Use PBS as the blank control group and measure its fluorescence absorption at 480nm.

[0058] 2. Measure the fluorescence value of free drug in the synthesized sample, and then use Triton to destroy the lipid vesicle structure to completely release it and measure the fluorescence value of the total drug amount.

[0059] 3. Results Make a standard curve based on the fluorescence value of the standard concentration gradient. Figure 7 As shown, the formula y=67193.96x+1005.43 is obtained, R 2 =0.9998Substitute the measured sample fluorescence value into the calculation and obtain the encapsulation efficiency of DOX by liposomes through the formula as shown in the table.

[0060] Table 3 Fluorescence values ​​and encapsulation efficiency of free and total DOX in lipid vesicles

[0061] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0062] The above-described embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be construed as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent application shall be subject to the attached claims, and the description may be used to interpret the content of the claims.

Claims

1. A method for preparing lipid vesicles, characterized in that: The specific steps include: (1) Use nonionic surfactant and cholesterol as the organic phase and connect them to one input end of a T-type mixing tube. (2) Use water as the aqueous phase and connect it to the other input end of the T-type mixing tube. (3) The organic phase and the aqueous phase are mixed in the middle mixing zone of the T-shaped mixing tube and discharged from the output end to obtain the initial product; (4) The primary product is crushed by ultrasound and then extruded through a liposome extruder to obtain the lipid vesicles.

2. The method for preparing lipid vesicles according to claim 1, characterized in that: The mass ratio of the nonionic surfactant to cholesterol is 1-5:

1.

3. The method for preparing lipid vesicles according to claim 1, characterized in that: The nonionic surfactant is one or more of Span 80, Span 40, Tween 80, and Brij S100.

4. The method for preparing lipid vesicles according to claim 1, characterized in that: The organic phase also includes an organic solvent, and the organic solvent is anhydrous ethanol.

5. The method for preparing lipid vesicles according to claim 1, characterized in that: The aqueous phase also includes drugs.

6. The method for preparing lipid vesicles according to claim 1, characterized in that: The flow rate of the organic phase was 0.1~0.5 mL / s, and the flow rate of the aqueous phase was 0.1~0.5 mL / s.

7. The method for preparing lipid vesicles according to claim 1, characterized in that: The ultrasonic crushing is intermittent ultrasonic treatment, and the number of ultrasonic treatments is not less than 10 times.

8. The method for preparing lipid vesicles according to claim 1, characterized in that: The number of cycles of extrusion through the liposome extruder is not less than 10 times.

9. A lipid vesicle prepared by the method for preparing a lipid vesicle according to any one of claims 1 to 8.

10. Use of the lipid vesicle as claimed in claim 9 in drug delivery.

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