Multi-chamber vesicle and preparation method and application thereof

Abstract

The invention discloses a multi-chamber vesicle as well as a preparation method and application thereof. Each multi-chamber vesicle is an anionic liposome, and a cavity of the anionic liposome contains a plurality of cationic liposomes and a buffer solution containing Mg < 2 + >. According to the preparation method of the multi-chamber vesicle, a special oil phase and a water phase with different charges are introduced, so that the multi-chamber vesicle in which a plurality of cationic liposomes are wrapped by a single anionic liposome is successfully prepared. The multi-chamber vesicle is good in biocompatibility, controllable in particle size and stable in mechanical property, and provides technical support for preparation of artificial cells and novel drug carriers in the future.

Description

Technical Field

[0001] This invention relates to a multi-chambered vesicle, its preparation method, and its application. Background Technology

[0002] In recent years, researchers have developed many novel lipid carriers, most of which are small unilamellar vesicles (SUVs) with a diameter of 100 nm. Due to their good biocompatibility, they can serve as both lipid-soluble and water-soluble drug carriers and are widely used in nanomedicine, including as nanosensors, transdermal delivery carriers, and targeted drug delivery carriers. Currently, macromolecular drugs, including supramolecular DNA, RNA complexes, and enzyme-linked drugs, are expected to be part of future therapeutic approaches. However, the low trapping efficiency of liposomes for macromolecular drugs, the complex and time-consuming activity loading process, limits their further clinical application. In contrast, giant unilamellar vesicles (GUVs) with a radius exceeding 1 μm may offer enormous potential for drug delivery applications. Because of their large internal volume, GUVs can carry large quantities of active compounds and particles as small as micrometers. GUVs were initially used to mimic cells and organelles, reconstructing the behavior, morphology, or metabolic characteristics of living cells by constructing artificial compartmentalized systems. By using compartmentalization, GUVs can spatially separate drugs with different pathways of action, thereby avoiding interference with metabolic processes and protecting drugs from degradation. Therefore, with the rise of bottom-up synthetic biology, GUVs have gradually become popular in the field of drug delivery.

[0003] Traditional methods for preparing GUVs include the double emulsion method and octanol-assisted liposome assembly, but these methods have many limitations: (1) low yield of giant vesicles; (2) non-uniform microcapsule size; and (3) inability to prepare multi-chambered vesicles with multiple chambers. Therefore, microfluidics (MF) has been developed for high-throughput production of monodisperse multi-chambered GUVs and vesicles. Microfluidics refers to the technology of manipulating microfluidics within micrometer-sized channels. Due to its advantages of low cost, simple operation, high controllability, and strong reproducibility, this technology has been widely used in the biomedical field, such as the preparation of nanocarriers, drug screening, and tissue engineering. MF technology can precisely control the degree of mixing of fluid molecules by changing factors such as chip shape, fluid velocity, flow rate, and mixing sequence, to obtain highly stable, uniformly dispersed, and better encapsulation monodisperse lipid systems. However, current common microfluidic preparation methods still cannot remove residual oil and organic solvents in vesicles, nor can they prepare multi-chambered vesicle structures in which a single anionic liposome encapsulates several cationic liposomes. This situation urgently needs to be solved. Summary of the Invention

[0004] The purpose of this invention is to fill the gap in the existing technology regarding multi-compartment vesicle structures where a single anionic liposome encapsulates several cationic liposomes, overcoming the shortcomings of traditional microfluidic technology in the preparation of multi-compartment vesicles, and providing a multi-compartment vesicle, its preparation method, and its applications. The preparation method of this invention successfully prepares a multi-compartment vesicle where a single anionic liposome encapsulates several cationic liposomes by introducing a special oil phase and an aqueous phase with different charges, achieving surfactant-free preparation. This multi-compartment vesicle exhibits good biocompatibility, controllable particle size, and stable mechanical properties, providing technical support for the future preparation of artificial cells and novel drug carriers.

[0005] This invention provides a multi-chambered vesicle, each of which is an anionic liposome, and the chamber of the anionic liposome contains a plurality of cationic liposomes and contains Mg. 2+ Buffer solution.

[0006] Preferably, the multi-chambered vesicles satisfy one or more of the following conditions:

[0007] a. The particle size of the multi-chambered vesicles is ≤5μm, preferably, the particle size of the multi-chambered vesicles is 0.9μm-5μm;

[0008] b. The potential of the anionic liposome is -20mV to -40mV, preferably -28.38±1.56mV;

[0009] c. The particle size of the cationic liposomes is 80-120 nm, preferably 96.89 ± 8.41 nm;

[0010] d. The potential of the cationic liposome is 20 mV to 40 mV, preferably, the potential of the cationic liposome is 34.54 ± 1.28 mV;

[0011] e. The amount of lipid phase of the cationic liposomes, based on the molar ratio of all lipid phases of all cationic liposomes to the lipid phase of the anionic liposomes, is (2-5):(1-4).

[0012] Preferably, the multi-chambered vesicles satisfy one or more of the following conditions:

[0013] a. The lipid phase of the anionic liposomes comprises negatively charged lipids, neutral lipids, and cholesterol;

[0014] Preferably, the negatively charged lipid is one or more of dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylglycerol (DOPG), and phosphatidylglycerol (POPG);

[0015] Preferably, the neutral lipid is one or more of dioleoyl lecithin (DOPC), dipalmitoyl phosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl lecithin (POPC), and dioleoyl phosphatidylethanolamine (DOPE).

[0016] Preferably, the molar ratio of the negatively charged lipid to the neutral lipid is 1:(1-5), for example 1:(1-4);

[0017] Preferably, the cholesterol content is 5%-20%, more preferably 5%-15%, for example 10%, where the percentage is the molar percentage of cholesterol relative to the sum of positively charged lipids, neutral lipids, and cholesterol.

[0018] b. The lipid phase of the cationic liposomes includes positively charged lipids, neutral lipids, and cholesterol;

[0019] Preferably, the positively charged lipid is (2,3-dioleoyl-propyl)-trimethylammonium chloride DOTAP and / or dioleoylpropyltrimethylammonium chloride DOTMA;

[0020] Preferably, the neutral lipid is one or more of dioleoyl lecithin (DOPC), dipalmitoyl phosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl lecithin (POPC), and dioleoyl phosphatidylethanolamine (DOPE).

[0021] Preferably, the molar ratio of the positively charged lipid to the neutral lipid is 1:(1-5), for example 1:(3-4);

[0022] Preferably, the cholesterol content is 5%-20%, more preferably 5%-15%, for example 10%, where the percentage is the molar percentage of cholesterol relative to the sum of positively charged lipids, neutral lipids, and cholesterol.

[0023] c. The buffer solution is a tris(hydroxymethyl)aminomethane buffer (Tris buffer) or a phosphate buffer solution;

[0024] Preferably, the concentration of tris(hydroxymethyl)aminomethane or phosphate in the buffer solution is 5 mM-30 mM;

[0025] d. Mg in the buffer solution 2+ The concentration is 8-15 mM, for example, 10 mM;

[0026] e. The particle size of the multi-chambered vesicles is 1.23 μm-3.384 μm.

[0027] More preferably, the multi-chambered vesicle satisfies one or more of the following conditions:

[0028] a. The lipid phase of the anionic liposome is composed of dioleoylphosphatidylserine (DOPS), dioleoyllecithin (DOPC), and cholesterol;

[0029] b. The molar ratio of the negatively charged lipid to the neutral lipid is 1:(1-3), for example 1:2;

[0030] c. The lipid phase of the cationic liposomes consists of (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP), dioleoylphosphatidylethanolamine (DOPE), and cholesterol;

[0031] d. The molar ratio of the positively charged lipid to the neutral lipid is 2:7;

[0032] e. The particle size of the multi-chambered vesicles is 1.658 μm-1.686 μm.

[0033] Preferably, the multi-chambered vesicle is a blank multi-chambered vesicle or a multi-chambered vesicle containing an active drug.

[0034] More preferably, the active pharmaceutical ingredient is a cascade enzyme and / or a hydrophilic chemotherapeutic drug, for example, the cascade enzyme includes glucose oxidase GO. X And catalase (CAT); the hydrophilic chemotherapeutic drug is doxorubicin (DOX) and / or paclitaxel.

[0035] More preferably, the mass ratio of the cascade enzyme to the lipid phase of the anionic liposome is 1:(4-6), for example, 1:4.28;

[0036] More preferably, the amount of the hydrophilic chemotherapeutic drug used is 1:(8-10) based on the mass ratio of the hydrophilic chemotherapeutic drug to the lipid phase of a single cationic liposome.

[0037] The present invention also provides a method for preparing the above-mentioned multi-chambered vesicles, which uses microfluidic technology to prepare water-in-oil droplets from a first aqueous solution, a second aqueous solution and fluorinated oil containing a fluorinated surfactant, and then treats the droplets with a demulsifier and centrifuges them.

[0038] The first aqueous solution includes the anionic liposomes and the solution containing Mg. 2+ Buffer solution;

[0039] The second aqueous solution comprises the cationic liposomes and the solution containing Mg 2+ Buffer solution;

[0040] In the first aqueous solution, the concentration of the lipid phase of the anionic liposome is ≤5 mM;

[0041] In the second aqueous solution, the concentration of the lipid phase of the cationic liposomes is ≤5 mM;

[0042] The fluorinated surfactant is PFPE-PEG-PFPE and / or perfluoropolyether carboxylic acid, wherein PFPE-PEG-PFPE is, for example, PFPE7000-PEG1500-PFPE7000; and the perfluoropolyether carboxylic acid can be PFPE-COOH, for example, PFPE7000-COOH.

[0043] The flow rate ratio of the first aqueous solution and the second aqueous solution to the fluorinated oil containing the fluorinated surfactant is 1:(3-6).

[0044] Preferably, the preparation method meets one or more of the following conditions:

[0045] a. When the fluorinated surfactant contains PFPE7000-PEG1500-PFPE7000, the concentration of PFPE7000-PEG1500-PFPE7000 is ≤2%, where % is the percentage of the mass of PFPE7000-PEG1500-PFPE7000 to the total mass of the fluorinated oil containing the fluorinated surfactant;

[0046] When the fluorinated surfactant contains perfluoropolyether carboxylic acid, the concentration of the perfluoropolyether carboxylic acid in the fluorinated oil containing the fluorinated surfactant is ≤10mM;

[0047] Preferably, the fluorinated surfactant is PFPE7000-PEG1500-PFPE7000 or PFPE7000-COOH;

[0048] b. The flow rate of the first aqueous phase is 200-300 μL / h, preferably 240 μL / h;

[0049] c. The flow rate of the second aqueous phase is 200-300 μL / h, preferably 240 μL / h;

[0050] d. The flow rate of the fluorinated oil containing the fluorinated surfactant is 1000-2000 μL / h, preferably 1200 μL / h;

[0051] e. In the first aqueous solution, the concentration of the lipid phase of the anionic liposome is 1-4 mM, preferably 2-4 mM, for example 3 mM;

[0052] f. In the second aqueous solution, the concentration of the lipid phase of the cationic liposomes is 2-5 mM, preferably 1.5-3 mM;

[0053] g. The fluorinated oil is FC-40 and / or HFE7500;

[0054] h. The volume ratio of the first aqueous solution, the second aqueous solution, and the fluorinated oil containing the fluorinated surfactant is 1:(4-6):1, for example, 1:5:1;

[0055] i. The demulsifier comprises 1H,1H,2H,2H-perfluoro-1-octanol;

[0056] Preferably, the concentration of 1H,1H,2H,2H-perfluoro-1-octanol in the demulsifier is ≤20 v / v%.

[0057] Preferably, the demulsifier is HFE7500 containing 20 v / v% 1H,1H,2H,2H-perfluoro-1-octanol;

[0058] Preferably, the release time of the demulsifier is ≥30 min;

[0059] Preferably, the volume ratio of the demulsifier to the water-in-oil droplet is 1:1;

[0060] Preferably, during the demulsifier treatment, 30 mM Tris buffer solution is also added;

[0061] j. The centrifugation speed is ≥10000 r / min, and the centrifugation time is 10-15 min.

[0062] Preferably, the microfluidic technology is performed in a microfluidic chip;

[0063] The microfluidic chip can be any microfluidic chip conventionally referred to in the art. For example, the microfluidic chip can include an aqueous phase mixing section, an oil-in-water droplet forming section, and a droplet stabilizing section connected in sequence.

[0064] The aqueous phase mixing section is provided with a first aqueous phase inlet and a second aqueous phase inlet;

[0065] The water-in-oil droplet forming section is also provided with an oil phase injection port;

[0066] The droplet stabilization section is equipped with an outlet.

[0067] More preferably, the microfluidic chip satisfies one or more of the following conditions:

[0068] a. The aqueous mixing section has a "Y" shaped structure and an "S" shaped flow channel. Preferably, the "S" shaped flow channel is provided with staggered herringbone grooves.

[0069] b. The water-in-oil droplet forming section is a focused flow pattern structure with an "S"-shaped flow channel. Preferably, the included angle of the focused flow pattern structure is 120°. Preferably, the "S"-shaped flow channel is provided with staggered herringbone grooves, the width of the staggered herringbone grooves is 50μm and the depth is 31μm.

[0070] c. The droplet stabilization section is a spiral-shaped flow channel;

[0071] d. The widths of the first aqueous phase inlet, the second aqueous phase inlet, and the outlet are 100 μm;

[0072] e. The depths of the first aqueous phase inlet, the second aqueous phase inlet, and the outlet are 79 μm;

[0073] f. The main channel of the microfluidic chip is rectangular, with a width of 200 μm and a depth of 79 μm.

[0074] In this invention, the preparation methods of the cationic liposomes and the anionic liposomes are known to those skilled in the art and can be carried out according to conventional liposome preparation methods, such as the thin-film dispersion method described above. For example, the lipid material is dissolved in a solvent, dried into a lipid film under a nitrogen stream, the dried lipid film is hydrated in a buffer solution, and the resulting lipid solution is squeezed multiple times through a polycarbonate filter with a certain pore size to finally obtain a liposome solution.

[0075] The present invention also provides a multi-chambered vesicle prepared by the above-described method for preparing multi-chambered vesicles.

[0076] The present invention also provides the application of the above-described multi-chambered vesicles in the preparation of targeted drug delivery carriers or transdermal delivery carriers.

[0077] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0078] The reagents and raw materials used in this invention are all commercially available.

[0079] The positive and progressive effects of this invention are as follows:

[0080] 1. The multi-chambered vesicles of the present invention achieve precise control of the internal and external components of multi-chambered vesicles, the particle size of multi-chambered vesicles is ≤5μm, and there is no excess oil phase and surfactant, good biocompatibility, stable mechanical properties, and simulates cell characteristics to the greatest extent, providing technical support for the construction of artificial cells and novel drug carriers in the future.

[0081] 2. The multi-chambered vesicles of the present invention were successfully prepared using the preparation method of the present invention by setting up lipid materials and oil phase, which is highly efficient and effectively removes excess oil phase and surfactant, resulting in excellent effect. Attached Figure Description

[0082] Figure 1 This is a schematic diagram of the microfluidic chip structure used in the embodiment.

[0083] Figure 2 This is a particle size distribution diagram of the multi-chambered vesicles in Example 1.

[0084] Figure 3 This is a particle size distribution diagram of the multi-chambered vesicles in Comparative Example 1.

[0085] Figure 4 This is a transmission electron microscope image of a multi-chambered vesicle from Example 1.

[0086] Figure 5 This is a fluorescence microscope image of a multi-chambered vesicle from Example 1.

[0087] Figure 6 Fluorescence colocalization maps of multi-chambered vesicles in Example 6 were captured using confocal microscopy.

[0088] Explanation of reference numerals in the attached figures:

[0089] First aqueous phase inlet 1

[0090] Second aqueous phase inlet 2

[0091] Oil phase injection port 3

[0092] Export 4

[0093] Interlaced herringbone groove 5

[0094] Aqueous mixing section 6

[0095] Oil-in-water droplet formation segment 7

[0096] Droplet stable section 8

[0097] Main Channel 9 Detailed Implementation

[0098] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0099] The main reagents used in the following examples are shown in Table 1:

[0100] Table 1

[0101]

[0102] The multi-chamber vesicles of this invention can be prepared using commercially available microfluidic chips. The embodiments of this application use the following microfluidic chip devices for the preparation of the multi-chamber vesicles; however, those skilled in the art should understand that the use of this chip device does not mean that this application is only applicable to the following microfluidic chip devices.

[0103] The structure of the microfluidic chip is as follows: Figure 1 As shown, it includes a sequentially connected aqueous mixing section 6, an oil-in-water droplet forming section 7, and a droplet stabilizing section 8. The aqueous mixing section 6 has a "Y"-shaped structure with an "S"-shaped flow channel, and the "S"-shaped flow channel contains staggered herringbone mixer (SHM) grooves 5. The oil-in-water droplet forming section 7 has a focusing flow structure with an "S"-shaped flow channel, the included angle of the focusing flow structure is 120°, and the "S"-shaped flow channel contains staggered herringbone mixer (SHM) grooves 5. The staggered herringbone mixer (SHM) grooves 5 have a width of 50 μm and a depth of 31 μm. The droplet stabilizing section 8 has a spiral structure. The microfluidic chip material is polydimethylsiloxane (PDMS), and the main channel 9 is rectangular, with a width of 200 μm and a depth of 79 μm. The microfluidic chip includes a first aqueous phase inlet 1, a second aqueous phase inlet 2, an oil phase inlet 3, and an outlet 4, with each inlet and outlet having a width of 100 μm and a depth of 79 μm.

[0104] Example 1

[0105] This embodiment provides a method for preparing multi-compartment vesicles. The prepared multi-compartment vesicle is an anionic liposome, whose lipid phase consists of DOPC, DOPS, and cholesterol. The compartment of the anionic liposome contains several cationic liposomes and a Tris buffer containing magnesium chloride. The lipid phase of the cationic liposome consists of DOPC, DOTAP, and cholesterol. Specific steps include:

[0106] (1) DOPC, DOPS, and cholesterol dissolved in chloroform were mixed in a glass bottle at a molar ratio of 6:3:1, and DOPC, DOTAP, and cholesterol dissolved in chloroform were mixed in a glass bottle at a molar ratio of 4:4:2. Both were dried separately under a gentle nitrogen stream to form lipid films. The dried DOPC / DOPS / cholesterol lipid films and DOPC / DOTAP / cholesterol lipid films were hydrated separately in a 30 mM Tris buffer containing 10 mM magnesium chloride. The DOPC / DOPS / cholesterol lipid film was hydrated to a final lipid phase concentration of 5 mM, and the DOPC / DOTAP / cholesterol lipid film was hydrated to a final lipid phase concentration of 1.5 mM. The two lipid solutions were squeezed nine times through a 50 nm pore size polycarbonate filter to obtain magnesium chloride-containing Tris buffer containing initial anionic liposomes and magnesium chloride-containing Tris buffer containing cationic liposomes, i.e., the first aqueous phase and the second aqueous phase.

[0107] (2) Add PFPE7000-COOH to HFE7500 containing 2% PFPE7000-PEG1500-PFPE7000 until the concentration of PFPE7000-COOH in HFE7500 is 10mM to form an oil phase;

[0108] (3) Use a disposable syringe to draw up the first aqueous phase, the second aqueous phase and the oil phase respectively. The volume ratio of the first aqueous phase: oil phase: second aqueous phase is 1:5:1. Install them at the corresponding positions of the first aqueous phase injection port 1, the second aqueous phase injection port 2 and the oil phase injection port 3 on the microfluidic device respectively.

[0109] (4) Set the flow rate to 200 μL / h for the first and second aqueous phases and 1200 μL / h for the oil phase, and begin the preparation of multi-chamber vesicles;

[0110] (5) Droplets are formed at the focusing flow pattern structure of the water-in-oil droplet forming section 7 of the microfluidic chip and collected from the outlet 4 of the microfluidic chip into an epoxy resin EP tube. After collection, the droplets are equilibrated at 4°C for 2 hours and then released;

[0111] (6) Remove excess oil phase from the EP tube. The droplet layer was prepared by mixing droplets, 30 mM Tris buffer, and HFE7500 containing 20 v / v% 1H,1H,2H,2H-perfluoro-1-octanol at a volume ratio of 1:1:1. After equilibration for 30 min, the supernatant was transferred to a 2 mL EP tube and centrifuged at 10000 r / min for 15 min. The supernatant was discarded, and the mixture was resuspended in 200 μL PBS to form the final multicompartmental vesicles.

[0112] Example 2

[0113] This embodiment investigates the effect of lipid phase concentration of different anionic liposomes on the particle size and PDI of multicompartmental vesicles, and screens the optimal outer lipid membrane concentration.

[0114] The experimental steps in this embodiment are the same as those in Example 1. The particle size and PDI of multi-chamber vesicles in Tris buffer containing 10mM magnesium chloride at lipid phase concentrations of 5mM, 3mM and 2mM were investigated respectively.

[0115] Example 3

[0116] This embodiment examines the effects of different flow rate ratios on the particle size and PDI of multi-chambered vesicles, and screens for the optimal flow rate.

[0117] The experimental steps in this embodiment are the same as those in Example 1. The oil phase flow rate is fixed at 1200 μL / h, and multi-chamber vesicles are prepared under flow rate ratios (oil phase:water phase) of 6:1, 5:1, 3:1 and 2:1 respectively.

[0118] Example 4

[0119] This embodiment examines the effects of different DOPC:DOPS molar ratios on the potential and PDI of anionic liposomes.

[0120] The experimental steps in this embodiment are the same as in Example 1. The obtained lipid solution was squeezed nine times through a polycarbonate filter with a pore size of 50 nm to obtain anionic liposomes in the initial state. The potential and PDI of the anionic liposomes were measured when the molar ratio of DOPC:DOPS was 1:1, 2:1 and 3:1 respectively. In the whole DOPC / DOPS / cholesterol system, the amount of cholesterol accounted for 10%.

[0121] Example 5

[0122] This embodiment examines the effect of different DOPC:DOTAP molar ratios on the potential and PDI of cationic liposomes.

[0123] The experimental procedures in this embodiment are the same as in Example 1. The particle size, potential, and PDI of the cationic liposomes were measured when the DOPC:DOTAP molar ratio was 4:1, 7:2, and 3:1, respectively. In the entire DOPC / DOTAP / cholesterol system, cholesterol accounted for 10% of the total mass.

[0124] Example 6

[0125] This embodiment provides a glucose oxidase GO-loaded enzyme. XA method for preparing multicompartmental vesicles containing catalase (CAT) and doxorubicin (DOX) was described. The prepared multicompartmental vesicles were anionic liposomes, whose lipid phase consisted of DOPC, DOPS, and cholesterol. The compartments of the anionic liposomes contained GO. X The reaction mixture consists of CAT, several cationic liposomes, and a Tris buffer containing magnesium chloride. The lipid phase of the cationic liposomes is composed of DOPC, DOTAP, and cholesterol. The chambers of the cationic liposomes contain Tris buffer and DOX. The specific steps are as follows:

[0126] (1) DOPC, DOPS, and cholesterol dissolved in chloroform were mixed in a glass bottle at a molar ratio of 6:3:1, and DOPC, DOTAP, and cholesterol dissolved in chloroform were mixed in a glass bottle at a molar ratio of 7:2:1. Both were dried separately under a gentle nitrogen stream to form lipid films. The dried DOPC / DOPS / cholesterol lipid films and DOPC / DOTAP / cholesterol lipid films were hydrated in a 30 mM Tris buffer containing 10 mM magnesium chloride, hydrating the DOPC / DOPS / cholesterol lipid film to a final lipid concentration of 3 mM. The DOPC / DOTAP / cholesterol lipid film was hydrated in a 250 mM ammonium sulfate buffer, hydrating the DOPC / DOTAP / cholesterol lipid film to a final lipid concentration of 2.5 mM. The two lipid solutions were squeezed nine times through a 50 nm pore size polycarbonate filter to obtain magnesium chloride-containing Tris buffer containing initial anionic liposomes and ammonium sulfate buffer containing cationic liposomes, respectively.

[0127] For Tris buffer containing magnesium chloride and naïve anionic liposomes, mix it with GO X When mixed with CAT, the lipid phase of the initial anionic liposomes was mixed with GO. X The mass ratio of CAT to CAT was 4.28:1, resulting in the first aqueous phase.

[0128] For the ammonium sulfate buffer solution containing cationic liposomes, after squeezing, the ammonium sulfate in the aqueous phase of the cationic liposomes is removed by dialysis. Then, 0.5 mg / mL of DOX aqueous solution is added to the liposome solution to allow it to permeate into the lumen of the cationic liposomes. The mass ratio of DOX drug to DOPE / DOTAP / cholesterol lipids is 1:10, thus obtaining an aqueous solution of DOX-loaded cationic liposomes, i.e., the second aqueous phase.

[0129] (2) Add PFPE7000-COOH to HFE7500 containing 2% PFPE7000-PEG1500-PFPE7000 until the concentration of PFPE7000-COOH in HFE7500 is 10mM to form an oil phase;

[0130] (3) Use a disposable syringe to draw up the first aqueous phase, the second aqueous phase and the oil phase respectively. The volume ratio of the first aqueous phase: oil phase: second aqueous phase is 1:5:1. Install them at the corresponding positions of the first aqueous phase injection port 1, the second aqueous phase injection port 2 and the oil phase injection port 3 on the microfluidic device respectively.

[0131] (4) Set the flow rate to 240 μL / h for the first and second aqueous phases and 1200 μL / h for the oil phase, and begin the preparation of multi-chamber vesicles;

[0132] (5) Droplets are formed at the focusing flow pattern structure of the water-in-oil droplet forming section 7 of the microfluidic chip and collected from the outlet 4 of the microfluidic chip into an EP tube. After collection, the droplets are equilibrated at 4°C for 2 hours and then released;

[0133] (6) Remove excess oil phase from the EP tube. The droplet layer was prepared by mixing droplets, 30 mM Tris buffer, and HFE7500 containing 20 v / v% 1H,1H,2H,2H-perfluoro-1-octanol at a volume ratio of 1:1:1. After equilibration for 30 min, the supernatant was transferred to a 2 mL EP tube and centrifuged at 11000 r / min for 15 min. The supernatant was discarded, and the mixture was resuspended in 200 μL PBS to form the final multicompartmental vesicles.

[0134] The lipid phase and GO of multi-compartment vesicle anionic liposomes were labeled with fluorescent dyes NBD and Cy5, respectively. X Fluorescence co-localization was observed using confocal laser scanning microscopy. Results are shown below. Figure 6 GO X Due to Mg 2+ Due to the charge adsorption effect, most of the DOX is adsorbed in the cavity of the anionic liposome, near the anionic lipid phase. DOX is successfully encapsulated in the cavity of the cationic liposome (if it is not successfully encapsulated, DOX will be lost during demulsification and there will be no fluorescence reaction). This indicates that the method of the present invention has successfully prepared multi-chamber vesicles and achieved precise control of the internal and external components of multi-chamber vesicles.

[0135] Comparative Example 1

[0136] This comparative example modifies the first aqueous phase, the second aqueous phase, and the oil phase to prepare multi-chamber vesicles.

[0137] First aqueous phase: 1 mL of glucose solution was added to the lipid membrane of DOPC / DOPS / cholesterol and vortexed for 10 min to obtain a turbid mixture, which was then sonicated for about 20 min to obtain the first aqueous phase.

[0138] Second aqueous phase: 1 mL of sucrose solution was added to the lipid membrane of DOPC / DOTAP / cholesterol and vortexed for 10 min to obtain a turbid mixture, which was then sonicated for about 20 min to obtain the second aqueous phase.

[0139] Oil phase: squalene.

[0140] The remaining experimental steps are the same as in Example 1.

[0141] Effect Example

[0142] 1. Characterization of multi-compartment vesicles

[0143] To observe the morphology of the multi-chambered vesicles, the samples prepared in Example 1 and Comparative Example 1 were observed under a microscope. The multi-chambered vesicles prepared in Example 1 were soluble in water and did not exhibit stratification, proving that excess oil phase had been removed. The results are as follows: Figure 2 and Figure 3 As shown. The results show that the multi-chambered vesicles prepared by the preparation method of Example 1 of the present invention not only have a smaller particle size, but also a more uniform morphology. Figure 2 The multi-chambered vesicles prepared using conventional methods in Comparative Example 1 had larger particle sizes, and most of the vesicles were aggregated. Figure 3 ).

[0144] 2. Appearance and morphology characterization

[0145] To observe the morphology of GUVs, the multi-chambered vesicles prepared in Example 1 were stained with 1% (w / w) uranyl acetate, and their morphology was observed using transmission electron microscopy. The results are as follows: Figure 4 As shown, the multi-chambered vesicles are spherical with a particle size of about 900 nm.

[0146] 3. Concentration screening of the lipid phase of anionic liposomes

[0147] The samples prepared in Example 2 were divided into groups of 50 μL each and placed in glass dishes. The particle size and polydispersity index (PDI) of the prepared samples at different lipid phase concentrations were measured using a Zetasizer Nano ZS (Malvern, UK) instrument. The results are shown in Table 2. The results indicate that in a 30 mM Tris buffer containing 10 mM magnesium chloride, the concentration of the lipid phase for anionic liposomes was 3 mM, resulting in the lowest particle size and PDI. 3 mM is considered the optimal concentration of the lipid phase for anionic liposomes.

[0148] Table 2. Particle size and PDI of multicompartment vesicles at different concentrations

[0149]

[0150] Among multiple products, the best multi-chamber vesicles were prepared when the lipid phase concentration of anionic liposomes was 3 mM and the lipid phase concentration of cationic liposomes was 1.5 mM, the aqueous phase flow rate was 240 μL / h, and the oil phase flow rate was 1200 μL / h, in a 30 mM Tris buffer containing 10 mM magnesium chloride.

[0151] 4. Flow rate ratio (oil phase:water phase) screening

[0152] The samples prepared in Example 3 were divided into two groups, each containing 50 μL, and the particle size and polydispersity index (PDI) were measured at different flow rates using a Zetasizer Nano ZS (Malvern, UK) instrument. The results are shown in Table 3. The results indicate that the particle size and PDI were minimized when the flow rate ratio (Oil:Water) was 5:1, which is the preferred flow rate ratio.

[0153] Table 3. Vesicle particle size and PDI at different flow rates (Oil:Water)

[0154]

[0155] 5. Uniformity of multi-chambered vesicles

[0156] To observe the uniformity of GUVs, 1% NBD-PC fluorescent lipid was added to the anionic liposome preparation process. The remaining steps were the same as in Example 1. Finally, the vesicles were resuspended in 200 μL PBS, and the final product was collected in EP tubes and observed using a fluorescence microscope. The results are as follows: Figure 5 As shown, the size distribution of each multi-chambered vesicle is relatively uniform, and the shape is round.

[0157] 6. Screening of different DOPC:DOPS molar ratios

[0158] The PDI and Zeta potentials of the samples prepared in Example 4 were measured using a laser particle size analyzer. The results are shown in Table 4. The results indicate that the dispersion coefficient is smallest and the potential meets the requirements when the molar ratio of DOPC:DOPS is 2:1, making a DOPC:DOPS molar ratio of 2:1 the optimal solution.

[0159] Table 4. Molar ratio screening of DOPC:DOPS

[0160]

[0161] 6. Screening of different DOPC:DOPS molar ratios

[0162] The particle size, PDI, and Zeta potential of the samples prepared in Example 5 were determined using a laser particle size analyzer. The results are shown in Table 5. The results indicate that the particle size and dispersion coefficient are minimized when the DOPC:DOTAP molar ratio is 7:2, and the potential meets the requirements. Therefore, a DOPC:DOTAP molar ratio of 7:2 is the optimal solution.

[0163] Table 5. DOPC:DOTAP Molar Ratio Screening

[0164]

Claims

1. A multi-chambered vesicle, characterized in that, Each of the aforementioned multi-chambered vesicles is an anionic liposome, and the chamber of the anionic liposome contains a plurality of cationic liposomes and Mg... 2+ Buffer solution.

2. The multi-chambered vesicle according to claim 1, characterized in that, The multi-chambered vesicles satisfy one or more of the following conditions: a. The particle size of the multi-chambered vesicles is ≤5μm; Preferably, the particle size of the multi-chambered vesicles is 0.9 μm-5 μm; b. The potential of the anionic liposomes is -20mV to -40mV; Preferably, the potential of the anionic liposome is -28.38±1.56mV; c. The particle size of the cationic liposomes is 80-120 nm; Preferably, the particle size of the cationic liposomes is 96.89±8.41 nm; d. The potential of the cationic liposomes is 20 mV to 40 mV; Preferably, the potential of the cationic liposome is 34.54 ± 1.28 mV; e. The amount of lipid phase used in the cationic liposomes, based on the molar ratio of all lipid phases of all the cationic liposomes to the lipid phase of the anionic liposomes, is (2-5):(1-4).

3. The multi-chambered vesicle according to claim 1, characterized in that, The multi-chambered vesicles satisfy one or more of the following conditions: a. The lipid phase of the anionic liposomes comprises negatively charged lipids, neutral lipids, and cholesterol; Preferably, the negatively charged lipid is one or more of dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylglycerol (DOPG), and phosphatidylglycerol (POPG); Preferably, the neutral lipid is one or more of the following: dioleoyl lecithin (DOPC), dipalmitoyl phosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl lecithin (POPC), and dioleoyl phosphatidylethanolamine (DOPE); Preferably, the molar ratio of the negatively charged lipid to the neutral lipid is 1:(1-5), for example 1:(1-4); Preferably, the cholesterol content is 5%-20%, more preferably 5%-15%, for example 10%, the percentage being the molar percentage of cholesterol relative to the sum of positively charged lipids, neutral lipids and cholesterol; b. The lipid phase of the cationic liposomes includes positively charged lipids, neutral lipids, and cholesterol; Preferably, the positively charged lipid is (2,3-dioleoyl-propyl)-trimethylammonium chloride DOTAP and / or dioleoylpropyltrimethylammonium chloride DOTMA; Preferably, the neutral lipid is one or more of the following: dioleoyl lecithin (DOPC), dipalmitoyl phosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl lecithin (POPC), and dioleoyl phosphatidylethanolamine (DOPE); Preferably, the molar ratio of the positively charged lipid to the neutral lipid is 1:(1-5), for example 1:(3-4); Preferably, the cholesterol content is 5%-20%, more preferably 5%-15%, for example 10%, the percentage being the molar percentage of cholesterol relative to the sum of positively charged lipids, neutral lipids and cholesterol; c. The buffer solution is tris(hydroxymethyl)aminomethane buffer solution or phosphate buffer solution (PBS); Preferably, the concentration of tris(hydroxymethyl)aminomethane or phosphate in the buffer solution is 5 mM-30 mM; d. Mg in the buffer solution 2+ The concentration is 8-15 mM, for example, 10 mM; e. The particle size of the multi-chambered vesicles is 1.23 μm-3.384 μm.

4. The multi-chambered vesicle according to claim 3, characterized in that, The multi-chambered vesicles satisfy one or more of the following conditions: a. The lipid phase of the anionic liposome is composed of dioleoylphosphatidylserine (DOPS), dioleoyllecithin (DOPC), and cholesterol; b. The molar ratio of the negatively charged lipid to the neutral lipid is 1:(1-3), for example 1:2; c. The lipid phase of the cationic liposomes consists of (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP), dioleoylphosphatidylethanolamine (DOPE), and cholesterol; d. The molar ratio of the positively charged lipid to the neutral lipid is 2:7; e. The particle size of the multi-chambered vesicles is 1.658 μm-1.686 μm.

5. The multi-chambered vesicle according to claim 1, characterized in that, The multi-chambered vesicles are either blank multi-chambered vesicles or multi-chambered vesicles containing active drugs; Preferably, the active pharmaceutical ingredient is a cascade enzyme and / or a hydrophilic chemotherapeutic agent, for example, the cascade enzyme includes glucose oxidase GO. X and catalase (CAT); the hydrophilic chemotherapeutic drug is doxorubicin (DOX) and / or paclitaxel; More preferably, the mass ratio of the cascade enzyme to the lipid phase of the anionic liposome is 1:(4-6), for example, 1:4.28; More preferably, the amount of the hydrophilic chemotherapeutic drug used is 1:(8-10) based on the mass ratio of the hydrophilic chemotherapeutic drug to the lipid phase of a single cationic liposome.

6. A method for preparing a multi-chambered vesicle as described in any one of claims 1-5, characterized in that, Water-in-oil droplets were prepared by using microfluidic technology to combine a first aqueous solution, a second aqueous solution, and fluorinated oil containing a fluorinated surfactant. After treatment with a demulsifier, the droplets were centrifuged. The first aqueous solution includes the anionic liposomes and the solution containing Mg. 2+ Buffer solution; The second aqueous solution comprises the cationic liposomes and the solution containing Mg 2+ Buffer solution; In the first aqueous solution, the concentration of the lipid phase of the anionic liposome is ≤5 mM; In the second aqueous solution, the concentration of the lipid phase of the cationic liposomes is ≤5 mM; The fluorinated surfactant is PFPE-PEG-PFPE and / or perfluoropolyether carboxylic acid, wherein PFPE-PEG-PFPE is, for example, PFPE7000-PEG1500-PFPE7000; and the perfluoropolyether carboxylic acid can be PFPE-COOH, for example, PFPE7000-COOH. The flow rate ratio of the first aqueous solution and the second aqueous solution to the fluorinated oil containing the fluorinated surfactant is 1:(3-6).

7. The method for preparing multi-chambered vesicles according to claim 6, characterized in that, The preparation method described herein meets one or more of the following conditions: a. When the fluorinated surfactant contains PFPE7000-PEG1500-PFPE7000, the concentration of PFPE7000-PEG1500-PFPE7000 is ≤2%, where % is the percentage of the mass of PFPE7000-PEG1500-PFPE7000 to the total mass of the fluorinated oil containing the fluorinated surfactant; When the fluorinated surfactant contains perfluoropolyether carboxylic acid, the concentration of the perfluoropolyether carboxylic acid in the fluorinated oil containing the fluorinated surfactant is ≤10mM; Preferably, the fluorinated surfactant is PFPE7000-PEG1500-PFPE7000 or PFPE7000-COOH; b. The flow rate of the first aqueous phase is 200-300 μL / h; Preferably, the flow rate of the first aqueous phase is 240 μL / h; c. The flow rate of the second aqueous phase is 200-300 μL / h; Preferably, the flow rate of the second aqueous phase is 240 μL / h; d. The flow rate of the fluorinated oil containing the fluorinated surfactant is 1000-2000 μL / h; Preferably, the flow rate of the fluorinated oil containing the fluorinated surfactant is 1200 μL / h; e. In the first aqueous solution, the concentration of the lipid phase of the anionic liposome is 1-4 mM; Preferably, in the first aqueous solution, the concentration of the lipid phase of the anionic liposome is 2-4 mM, for example, 3 mM; f. In the second aqueous solution, the concentration of the lipid phase of the cationic liposomes is 2-5 mM; Preferably, in the second aqueous solution, the concentration of the lipid phase of the cationic liposomes is 1.5-3 mM; g. The fluorinated oil is FC-40 and / or HFE7500; h. The volume ratio of the first aqueous solution, the second aqueous solution, and the fluorinated oil containing the fluorinated surfactant is 1:(4-6):1, for example, 1:5:1; i. The demulsifier comprises 1H,1H,2H,2H-perfluoro-1-octanol; Preferably, the concentration of 1H,1H,2H,2H-perfluoro-1-octanol in the demulsifier is ≤20 v / v%. Preferably, the demulsifier is HFE7500 containing 20 v / v% of 1H,1H,2H,2H-perfluoro-1-octanol; Preferably, the release time of the demulsifier is ≥30 min; Preferably, the volume ratio of the demulsifier to the water-in-oil droplet is 1:1; Preferably, during the demulsifier treatment, 30 mM Tris buffer is also added; j. The centrifugation speed is ≥10000 r / min, and the centrifugation time is 10-15 min.

8. The method for preparing multi-chambered vesicles according to claim 6, characterized in that, The microfluidic technology described herein is performed within a microfluidic chip; The microfluidic chip includes an aqueous phase mixing section, an oil-in-water droplet forming section, and a droplet stabilizing section connected in sequence. The aqueous phase mixing section is provided with a first aqueous phase inlet and a second aqueous phase inlet; The water-in-oil droplet forming section is also provided with an oil phase injection port; The droplet stabilization section is equipped with an outlet; Preferably, the microfluidic chip satisfies one or more of the following conditions: a. The aqueous phase mixing section has a "Y" shaped structure and an "S" shaped flow channel. Preferably, the "S" shaped flow channel is provided with staggered herringbone-shaped grooves. b. The water-in-oil droplet forming section is a focused flow pattern structure with an "S"-shaped flow channel. Preferably, the included angle of the focused flow pattern structure is 120°. Preferably, the "S"-shaped flow channel is provided with staggered herringbone grooves, the width of the staggered herringbone grooves is 50μm and the depth is 31μm. c. The droplet stabilization section is a spiral-shaped flow channel; d. The widths of the first aqueous phase inlet, the second aqueous phase inlet, and the outlet are 100 μm; e. The depths of the first aqueous phase inlet, the second aqueous phase inlet, and the outlet are 79 μm; f. The main channel of the microfluidic chip is rectangular, with a width of 200 μm and a depth of 79 μm.

9. A multi-chambered vesicle, characterized in that, It is prepared using the method for preparing multi-chambered vesicles as described in any one of claims 6-8.

10. The use of a multi-chambered vesicle as described in any one of claims 1-5 or claim 9 in the preparation of a targeted drug delivery carrier or a transdermal delivery carrier.

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