Preparation method of drug-loaded membrane protein bionic vesicle based on microfluidic technology and application of drug-loaded membrane protein bionic vesicle in targeted melanocyte delivery of drug

Abstract

The invention discloses a preparation method of drug-loaded membrane protein bionic vesicles based on a microfluidic technology and application of the drug-loaded membrane protein bionic vesicles to targeted melanocyte delivery drugs, and belongs to the technical field of pharmaceutical preparations. The method is easy and convenient to operate, assembly of the phospholipid bilayer and insertion of the membrane protein are accurately controlled through the laminar focusing technology, and the stable and efficient drug-loaded membrane protein bionic vesicle is formed. The drug-loaded membrane protein bionic vesicles can effectively target melanocytes, promote the uptake of the vesicles, significantly improve the accumulation of drugs in the melanocytes, and enhance the treatment effect on melanocyte related diseases (such as hyperpigmentation, melanoma, vitiligo and the like). Meanwhile, the drug-loaded bionic vesicles constructed by the invention have relatively low keratinocyte uptake rate, drug uptake of non-targeted tissues is effectively reduced, and side effects of drugs are reduced, so that the drug-loaded membrane protein bionic vesicle system constructed by the invention has great prospects in the aspect of treating melanocyte related diseases.

Description

Technical Field

[0001] The present invention relates to a method for preparing a drug-loaded membrane protein biomimetic vesicle based on microfluidic technology and its application in targeted drug delivery to melanocytes, belonging to the technical field of pharmaceutical preparations. Background Art

[0002] In recent years, skin melanocyte-related diseases, such as hyperpigmentation, vitiligo, senile plaques, melasma, and melanoma, have attracted wide attention. These diseases are often closely related to disorders in melanin synthesis, functional decline, or abnormal proliferation of melanocytes. With the in-depth study of skin melanocyte biology, delivering appropriate active ingredients or drugs to melanocytes has become a key strategy for treating these diseases.

[0003] However, in the epidermis, melanocytes are located in the basal layer and are relatively few in number, accounting for only about 1%-2% of the total number of epidermal cells. This distribution characteristic poses significant challenges for efficient drug delivery to melanocytes. First, conventional non-targeted topical drug delivery systems result in uneven drug distribution in the skin, and most of the drugs are taken up by keratinocytes, failing to effectively concentrate on melanocytes, thus significantly reducing the therapeutic effect of the drugs. Especially when treating skin melanocyte-related diseases, it is often difficult to achieve the desired therapeutic effect. Second, non-targeted topical drug delivery systems do not specifically deliver drugs to melanocytes, and the drugs often act on other skin cells, especially keratinocytes, which may cause a series of side effects. For example, in the treatment of hyperpigmentation diseases, ingredients such as hydroquinone and retinoic acid may cause excessive exfoliation of keratinocytes, leading to adverse reactions such as skin dryness, irritation, and even peeling. Currently, drug delivery systems for melanocytes focus on modifying melanocyte-targeted ligands (such as α-MSH analogs, tyrosine analogs, or CD44 receptor ligands) on the surface of vesicles to enhance melanocyte uptake. However, the modification of these ligands does not specifically recognize keratinocytes, resulting in non-specific uptake of some vesicles by keratinocytes and reducing the targeting efficiency. Therefore, developing a specific drug targeted delivery system that can target drugs to skin melanocytes and reduce the uptake of drugs by keratinocytes, significantly improving the therapeutic effect and side effects of skin melanocyte-related diseases, has important research and application value. However, there is still a lack of a solution for this targeted delivery system. Summary of the Invention

[0004] In view of the above and / or existing problems, the present invention is proposed.

[0005] Based on the intercellular communication mechanism mediated by extracellular vesicles between keratinocytes and melanocytes in the skin, the present invention uses microfluidic technology to prepare drug-loaded keratinocyte membrane protein biomimetic vesicles. The vesicles have a phospholipid bilayer structure, and keratinocyte membrane proteins are embedded in the phospholipid bilayer. At the same time, water-soluble drugs are loaded in the aqueous core of the vesicles, lipid-soluble drugs are loaded in the phospholipid bilayer, or both water-soluble and lipid-soluble drugs are loaded, enabling targeted delivery to melanocytes. The present invention uses a three-channel microfluidic chip to precisely control the insertion of membrane proteins and the formation of vesicles, ensuring the smooth embedding and uniform distribution of keratinocyte membrane proteins in the phospholipid bilayer of the vesicles. At the same time, it avoids the mutual influence between drugs and membrane proteins, improving the stability and loading capacity of the vesicles. The phospholipid bilayer of the vesicles is composed of membrane proteins, phospholipids, and cholesterol, simulating the characteristics of natural keratinocyte cell membranes, enhancing the interaction with melanocytes, enabling efficient drug delivery to melanocytes, and reducing non-specific uptake by keratinocytes. The drug-loaded membrane protein biomimetic vesicles prepared by the present invention can bind to receptors on the surface of melanocytes through specific recognition mediated by membrane proteins, enhancing targeting, reducing non-specific uptake, and improving drug utilization. At the same time, due to the similarity of its cell membrane, the drug-loaded membrane protein biomimetic vesicles have a cell membrane similarity, and the recognition signals between homologous cells are not conducive to uptake, resulting in a decrease in the uptake of keratinocytes. The present invention uses glutathione (GSH) as a model drug and loads it into the aqueous core of the biomimetic vesicles. Glutathione can inhibit the production of melanin in melanocytes by regulating the redox state and inhibiting tyrosinase activity. The drug-loaded biomimetic vesicles can efficiently deliver glutathione to melanocytes, increase the intracellular drug concentration, enhance its effect of inhibiting melanin production, while reducing the uptake of drugs by keratinocytes, reducing non-target effects, and improving the therapeutic effect. The present invention provides an efficient and precise drug delivery platform, providing new strategies and technical solutions for the treatment targeting melanocytes, and having important clinical application value and industrialization prospects.

[0006] The present invention provides a method for preparing drug-loaded membrane protein biomimetic vesicles. The drug-loaded membrane protein biomimetic vesicles include keratinocyte membrane proteins, a phospholipid bilayer, and drugs. In a three-channel laminar flow focusing microfluidic chip, an ethanol solution of the phospholipid bilayer is used as the left flow channel phase, a PBS buffer solution of keratinocyte membrane proteins is used as the middle flow channel phase, and a PBS buffer solution of water-soluble drugs is used as the right flow channel phase. Among them, lipid-soluble drugs can be loaded in the ethanol solution of the left flow channel phase.

[0007] In one embodiment, the left flow channel phase is an organic phase, the phospholipid bilayer includes soybean lecithin and cholesterol, the molar ratio of soybean lecithin to cholesterol is (9-5):3; the total concentration of soybean lecithin and cholesterol is 2.5-25 mg / ml.

[0008] In one embodiment, the PBS buffer solution of the keratinocyte membrane protein is the aqueous phase 1; the concentration of the keratinocyte membrane protein in the aqueous phase 1 is 10 to 100 μg / ml.

[0009] In one embodiment, the keratinocytes include, but are not limited to, human immortalized keratinocytes.

[0010] In one embodiment, the membrane protein includes but is not limited to membrane proteins extracted from cell membranes and artificially synthesized membrane proteins.

[0011] In one embodiment, the method for preparing human keratinocyte membrane protein is as follows: 40 million human immortalized keratinocytes are cultured, the cells are separated from the bottom of the culture dish with a cell scraper, centrifuged, and the cells are washed with ice-cold PBS. Using a cell membrane protein and plasma protein extraction kit (Biyuntian), 1 ml of membrane protein extraction reagent A, which has been supplemented with PMSF just before use, is added to approximately 40 million human immortalized keratinocytes, the cells are fully suspended, and then ice-bathed for 10 minutes. The cell suspension is transferred to a 2 ml ice-bathed pre-cooled glass homogenizer, homogenized until the cells are fully broken, centrifuged at 700g for 10 minutes at 4°C, and the supernatant is collected. Centrifuge at 14,000 rpm at 4°C for 30 minutes, collect the precipitate, add 400 μl of membrane protein extraction reagent B, vortex for 5 seconds, and then ice-bath for 10 minutes. Repeat this process three times, then centrifuge at 14,000g at 4°C for 5 minutes, collect the supernatant, which is the membrane protein solution, and store at -80°C.

[0012] In one embodiment, the membrane protein solution is assayed using a BCA protein concentration assay kit (Beyotime).

[0013] In one embodiment, the right flow channel phase is the aqueous phase 2; the drugs include but are not limited to water-soluble drugs and fat-soluble drugs.

[0014] In one embodiment, the water-soluble drug comprises glutathione, vitamin C, or kojic acid.

[0015] In one embodiment, the concentration of the water-soluble drug in the aqueous phase 2 is 1-30 mg / mL.

[0016] In one embodiment, a three-channel microfluidic chip is passed through at a flow rate ratio FRR of (1:1:1) to (1:3:3) and a total flow rate TFR of 100 to 2000 μl / min; the flow rate ratio is the volume flow rate of the left flow channel phase: the volume flow rate of the middle flow channel phase: the volume flow rate of the right flow channel phase.

[0017] The present invention also provides a drug-loaded membrane protein biomimetic vesicle prepared by the above method.

[0018] In one embodiment, the biomimetic vesicle contains two structures, an outer layer and a core; the outer layer is a phospholipid bilayer embedded with membrane proteins, and the core is a water-soluble drug solution.

[0019] In one embodiment, the water-soluble drug includes glutathione, vitamin C or kojic acid.

[0020] The present invention also provides the application of the drug-loaded membrane protein biomimetic vesicle in the preparation of a drug targeting melanocytes, and the drug includes a drug for hyperpigmentation, a drug for vitiligo, a drug for melanoma or a drug for melasma.

[0021] The present invention uses microfluidic technology to embed human keratinocyte membrane proteins into the phospholipid bilayer of vesicles and load water-soluble drugs into the aqueous core of the vesicles, constructing a drug-loaded membrane protein biomimetic vesicle system aimed at achieving targeted drug delivery to melanocytes. The drug-loaded biomimetic vesicle can significantly enhance its residence time in the living epidermal layer by utilizing the intercellular adhesion proteins present on the phospholipid bilayer, thereby increasing the local concentration of the drug in the epidermis. In addition, due to the characteristics of the membrane proteins on the surface of the biomimetic vesicle, the system can effectively target melanocytes, promote their uptake of the drug-loaded membrane protein biomimetic vesicle, significantly increase the accumulation of the drug in melanocytes, and thus enhance the therapeutic effect of treating melanocyte-related diseases (such as melanoma, vitiligo, etc.). At the same time, the drug-loaded biomimetic vesicle constructed by the present invention has a low uptake rate by keratinocytes, effectively reducing the drug uptake by non-target tissues and reducing the side effects of the drug.

[0022] Beneficial effects:

[0023] The present invention provides a preparation method of a drug-loaded membrane protein biomimetic vesicle based on microfluidic technology and its application in targeted drug delivery to melanocytes. The preparation process is simple and clear, does not require large-scale equipment, and is environmentally friendly.

[0024] The present invention adopts the three-channel fluid laminar flow focusing microfluidic chip technology to precisely control the mixing moment of the membrane protein solution and the drug solution, enabling the two to be mixed instantaneously before assembly, thereby effectively avoiding the mutual interference between the membrane protein and the drug. This design not only ensures that the membrane protein can be completely embedded in the vesicle membrane, inheriting the biological characteristics of the source cells, but also successfully realizes the efficient loading of the drug, guaranteeing the functionality and stability of the drug delivery system.

[0025] The drug-loaded biomimetic vesicles in the present invention can bind to the surface receptors of melanocytes through the specific recognition mediated by membrane proteins, enhancing the targeting to melanocytes and reducing non-specific uptake: compared with the drug-loaded vesicles without embedded membrane proteins, the uptake of the drug-loaded biomimetic vesicles by melanocytes increased by about 3.94 times. In addition, due to the similarity between the surface protein composition of the drug-loaded biomimetic vesicles in the present invention and the cell membrane of keratinocytes itself, the recognition signals between homologous cells are not conducive to uptake, resulting in a decrease in the uptake of the drug-loaded biomimetic vesicles by keratinocytes: compared with the drug-loaded vesicles without embedded membrane proteins, the uptake of the drug-loaded biomimetic vesicles by keratinocytes decreased by about 1.81 times.

[0026] The drug-loaded biomimetic vesicles in the present invention select glutathione as the model water-soluble drug. In in vitro cell experiments, the biomimetic vesicles can significantly increase the glutathione content in melanocytes (increased by about 1.99 times), while the drug-loaded vesicles without embedded membrane proteins only increase the glutathione content in melanocytes by about 1.38 times; the biomimetic vesicles can also slightly increase the glutathione content in keratinocytes (increased by about 1.74 times), while the drug-loaded vesicles without embedded membrane proteins increase the glutathione content in keratinocytes by about 2.62 times.

[0027] Taking hyperpigmentation as the model disease, in in vitro cell experiments, the drug-loaded biomimetic vesicles in the present invention have a significantly better inhibitory effect on melanogenesis (the melanin content is 46.03% of the control group) than the drug-loaded vesicles without embedded membrane proteins (the melanin content is 72.77% of the control group), and are better than the positive control group kojic acid (the melanin content is 56.97% of the control group), and have no cytotoxicity. Description of the Drawings

[0028] Figure 1 It is a structural diagram of a PDMS microfluidic chip.

[0029] Figure 2 It is a DLS data diagram of the drug-loaded membrane protein biomimetic vesicles of the present invention.

[0030] Figure 3 It is a SEM electron micrograph of the drug-loaded membrane protein biomimetic vesicles of the present invention.

[0031] Figure 4The figure is a standard curve of glutathione measured by high performance liquid chromatography in the present invention.

[0032] Figure 5 This is a diagram of the encapsulation efficiency of the drug-loaded membrane protein biomimetic vesicles of the present invention.

[0033] Figure 6 This is a diagram showing the cytotoxicity of the drug-loaded membrane protein biomimetic vesicles of the present invention.

[0034] Figure 7 This is a fluorescence quantification graph of the drug-loaded membrane protein biomimetic vesicles of the present invention characterized by confocal microscopy for HaCaT cell uptake.

[0035] Figure 8 This is a fluorescence quantification graph of the drug-loaded membrane protein biomimetic vesicles of the present invention characterized by confocal microscopy for B16-F10 cell uptake.

[0036] Figure 9 This is a diagram showing the relative intracellular melanin content of B16-F10 cells after treatment with the drug-loaded membrane protein biomimetic vesicles of the present invention. DETAILED DESCRIPTION

[0037] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0038] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0039] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0040] The "drug targeting melanocytes" involved in the present invention refers to the use of biomimetic vesicles to specifically bind to melanocytes to deliver drugs in a targeted manner and increase the uptake of drugs by melanocytes.

[0041] Example 1-3 Extraction of human immortalized keratinocyte membrane proteins

[0042] Take well - growing human immortalized keratinocytes HaCaT, scrape the cells from the bottom of the culture dish with a cell scraper, collect by centrifugation, and wash the cells with ice - cold PBS. Use a cell membrane protein and cytoplasmic protein extraction kit (Beyotime). Add membrane protein extraction reagent A with phenylmethylsulfonyl fluoride added just before use to about 40 million human immortalized keratinocytes, fully suspend the cells and incubate on ice for 10 minutes. Transfer the cell suspension to an ice - cold glass homogenizer, homogenize until the cells are fully disrupted, then centrifuge at 4°C and 700g for 10 minutes, and collect the supernatant. Centrifuge at 4°C and 13000 rpm for 30 minutes, collect the precipitate, wash it twice with PBS, then add membrane protein extraction reagent B, vortex for 5 seconds and incubate on ice for 10 minutes, repeat this process three times. Subsequently, centrifuge at 4°C and 14000g for 5 minutes, and the collected supernatant is the membrane protein solution, which is stored at - 80°C. Based on the membrane protein concentration, Example 2 is used as the preferred process for membrane protein extraction.

[0043] The extraction process of human immortalized keratinocyte membrane protein in the examples is shown in Table 1:

[0044] Table 1 Optimization of the extraction process of human immortalized keratinocyte membrane protein

[0045] Process conditions Example 1 Example 2 Example 3 Homogenization times 10 30 50 Membrane protein extraction reagent B (ml) 0.8 0.8 0.8 Membrane protein concentration (mg / ml) 0.46 1.24 1.19

[0046] Preparation of drug - free membrane protein biomimetic vesicles in Examples 4 - 9

[0047] Dissolve a certain amount of soybean lecithin and cholesterol in absolute ethanol, with a total lipid concentration of 10 mg / ml, to prepare the left - hand flow channel phase. Dissolve a certain amount of keratinocyte membrane protein solution (prepared in Example 2) in 1×PBS buffer to prepare the middle - hand flow channel phase, with the mass concentration of the membrane protein being 10 - 100 μg / ml. Since the aim is to prepare drug - free biomimetic vesicles, 1×PBS buffer is used as the right - hand flow channel phase. Add the above solutions to the three - channel fluid laminar flow focusing microfluidic chip (Laminar - 04, Wuhan Jianmi Intelligent Control Technology Co., Ltd., specific dimensions are as Figure 1 shown) in the three channel phases, then pass through the chip at a certain total flow rate (TFR) and flow rate ratio (left - hand flow channel phase volume flow rate: middle - hand flow channel phase volume flow rate: right - hand flow channel phase volume flow rate = FRR). The obtained solution is dialyzed overnight with a 300 kDa dialysis bag to obtain drug - free membrane protein biomimetic vesicles with keratinocyte membrane proteins embedded in the phospholipid bilayer and no drug molecules in the hydrophilic core.

[0048] It can be concluded from Examples 4 - 6 that with the change of FRR, the particle size of the prepared vesicles shows a downward trend, being 167.8 nm, 90.31 nm, and 87.38 nm respectively, and the polydispersity index first decreases and then increases, being 0.183, 0.151, and 0.156 respectively. Based on the smaller particle size and lower polydispersity index, FRR = 1:1.5:1.5 is preferably selected as the preferred process for membrane protein embedding; it can be concluded from Examples 7, 8, and 9 that with the increase of membrane protein concentration, the particle size of the prepared liposomes gradually increases, being 94.21 nm, 101.23 nm, and 111.78 nm respectively, and the Zeta potential gradually decreases, being -4.13 mV, -7.21 mV, and -7.56 mV respectively. Based on the change of Zeta potential and comprehensively considering the membrane protein utilization rate, Example 8 is selected as the preferred process for subsequent preparation of drug-loaded membrane protein biomimetic vesicles.

[0049] Table 2 Preparation process conditions of drug-free membrane protein biomimetic vesicles in Examples 4 - 9

[0050]

[0051] Preparation of drug-loaded membrane protein biomimetic vesicles in Examples 10 - 16

[0052] Refer to the method of Example 8 to prepare biomimetic vesicles, with the difference that glutathione (GSH) is added to the right flow channel phase. A certain amount of soybean lecithin and cholesterol are mixed and dissolved in absolute ethanol, with a total lipid concentration of 10 mg / ml and a molar ratio of soybean lecithin:cholesterol of 7:3, and it is prepared as the left flow channel phase. A certain amount of keratinocyte membrane protein solution (prepared in Example 2) is dissolved in 1×PBS buffer solution to prepare the middle flow channel phase, and the mass concentration of the membrane protein is 60 μg / ml. A certain amount of glutathione is dissolved in 1×PBS buffer solution as the right flow channel phase, and the concentration of glutathione is 1 - 20 mg / ml. With TFR = 1000 μL / min and FRR = 1:1.5:1.5, through a three-channel fluid laminar flow focusing microfluidic chip, the obtained solution is dialyzed overnight with a 300 kDa dialysis bag to obtain drug-loaded membrane protein biomimetic vesicles with membrane proteins embedded in the phospholipid bilayer and a high concentration of GSH loaded in the hydrophilic core.

[0053] Table 3 Preparation process conditions of drug-loaded membrane protein biomimetic vesicles in Examples 10 - 16

[0054]

[0055]

[0056] Preparation of drug-loaded membrane protein biomimetic vesicles carrying other drugs in Examples 17 - 18

[0057] A certain amount of soy lecithin and cholesterol were mixed and dissolved in absolute ethanol to prepare a left flow channel phase with a total lipid concentration of 10 mg / ml and a molar ratio of soy lecithin to cholesterol of 7:3. A certain amount of keratinocyte membrane protein solution (prepared in Example 2) was dissolved in 1×PBS buffer to prepare an intermediate flow channel phase with a mass concentration of the membrane protein of 60 μg / ml. The water-soluble drugs vitamin C or kojic acid were dissolved in 1×PBS buffer as a right flow channel phase with a concentration of 20 mg / ml. With TFR = 1000 μL / min and FRR = 1:1.5:1.5, through a three-channel laminar flow focusing microfluidic chip, the obtained solution was dialyzed overnight using a 300 kDa dialysis bag to obtain drug-loaded membrane protein biomimetic vesicles with membrane proteins embedded in the phospholipid bilayer and hydrophilic cores loaded with high-concentration vitamin C (Example 17) and kojic acid (Example 18). The prepared drug-loaded membrane protein biomimetic vesicle solution was clear. The DLS test results showed that the particle sizes increased from 111.4 nm to 118.7 nm and 122.1 nm respectively before and after drug loading, and the PDI did not change significantly, verifying the universality of the drug-loaded membrane protein biomimetic vesicles based on microfluidic technology in the high-concentration encapsulation of water-soluble drugs.

[0058] Characterization of the properties of the drug-loaded membrane protein biomimetic vesicles in Example 19

[0059] Performance characterization of the drug-loaded membrane protein biomimetic vesicles based on microfluidic technology and its application in targeted drug delivery to melanocytes in the present invention:

[0060] 1. Characterization of the particle size and potential of the drug-loaded membrane protein biomimetic vesicles

[0061] The particle size, polydispersity index, and Zeta potential of the drug-free membrane protein biomimetic vesicles (HCMP) (prepared in Example 8) and the drug-loaded membrane protein biomimetic vesicles (HCMP@GSH) prepared with different glutathione concentrations in the right flow channel phase (prepared in Examples 10 - 13) were measured using a nano laser particle size analyzer. The results are as Figure 2, The results showed that as the glutathione concentration in the right flow channel phase increased, the average particle size of the prepared drug-loaded membrane protein biomimetic vesicles gradually increased (from 111.4 nm to 115.5 nm, 117.5 nm, 121.0 nm, and 127.2 nm), the ZETA potential showed no significant change ( - 9.49 mV, - 9.31 mV, - 9.97 mV, - 10.37 mV, and - 9.77 mV respectively), and the polydispersity index showed no significant change (0.158, 0.161, 0.158, 0.157, and 0.159 respectively). This indicated that by using the three-channel laminar flow focusing microfluidic chip in the above preparation sequence, the mutual interference between the membrane protein and the drug solution could be successfully reduced. As the glutathione concentration in the right flow channel phase increased, the glutathione concentration loaded in the inner cavity of the biomimetic vesicles increased, resulting in a slight increase in particle size from 111.4 nm to 127.2 nm, and the impact was negligible. To maximize the drug loading capacity of the biomimetic vesicles while ensuring the retention of their biological characteristics, the highest glutathione concentration should be selected. Therefore, Example 13 was preferably used as the preferred process for the drug-loaded membrane protein biomimetic vesicles. At this glutathione concentration (20 mg / ml), compared with the drug-free vesicles without embedded membrane proteins (prepared in Example 5), the particle size of the drug-loaded vesicles without embedded membrane proteins (prepared in Example 14) slightly increased (from 100.3 nm to 110.7 nm), and the polydispersity index and Zeta potential showed no significant change (0.168 and - 2.26 mV respectively).

[0062] 2. Characterization of the Scanning Electron Microscope (SEM) of the Drug-Loaded Membrane Protein Biomimetic Vesicles

[0063] The field emission scanning electron microscope was used to observe and characterize the microscopic morphology of the drug-loaded membrane protein biomimetic vesicles (HCMP@GSH) (prepared in Example 13) and the drug-loaded vesicles without embedded membrane proteins (Control@GSH) (prepared in Example 14). A certain amount of trehalose was added to the vesicle preparation as a lyoprotectant to prepare a trehalose vesicle solution with a final concentration of 100 mM. After rapid freezing it with liquid nitrogen, it was placed in a freeze dryer for lyophilization. After lyophilization was completed, the lyophilized sample was smeared on the conductive adhesive and observed under the scanning electron microscope.

[0064] Figure 3 The results showed that both the drug-loaded membrane protein biomimetic vesicles and the drug-loaded vesicles without embedded membrane proteins prepared by the microfluidic technology were spherical with clear edges. Although there were differences in their sizes compared with the results measured by the nanoparticle laser sizer, this might be due to the collapse of the vesicle structure during the rapid freezing process with liquid nitrogen and the lyophilization process.

[0065] 3. Characterization of the Encapsulation Efficiency and Drug Loading Capacity of the Drug-Loaded Membrane Protein Biomimetic Vesicles

[0066] The free glutathione was separated by ultrafiltration, and the encapsulation efficiency and drug loading of glutathione in the drug-loaded membrane protein biomimetic vesicles (HCMP@GSH) (prepared in Example 13) and drug-loaded vesicles without embedded membrane proteins (Control@GSH) (prepared in Example 14) were determined by high performance liquid chromatography. After the vesicles were prepared, without dialysis, the vesicle solution was directly ultrafiltered through a 3 kDa ultrafiltration centrifugal tube to separate the free glutathione solution. High performance liquid chromatography was used for quantitative analysis of the free glutathione solution. An Agilent C18 column was used, and the mobile phase was a water-acetonitrile solution (95:5), in which the aqueous phase contained 0.1% trifluoroacetic acid, and the detection wavelength was 210 nm. During the determination, a linear relationship between the concentration and the peak area was established through the standard solution of glutathione, and a standard curve was plotted. The results are shown in Figure 4 . Then, by comparing the peak area of the sample with the standard curve, the concentration of free glutathione was calculated. The encapsulation efficiency and drug loading were calculated by the following formulas:

[0067]

[0068] where C 总 is the total initial concentration of glutathione, in mg / mL; C 游离 is the concentration of free glutathione, in mg / mL; C 包封 is the concentration of encapsulated glutathione, in mg / mL; C 囊泡溶液 is the total concentration of vesicles and glutathione in the vesicle solution, in mg / mL.

[0069] The results are shown in Figure 5 . The results showed that there was no significant difference in the encapsulation efficiency of glutathione between the drug-loaded membrane protein biomimetic vesicles prepared by the three-channel laminar flow focusing chip and the drug-loaded vesicles without embedded membrane proteins, which were 6.8419% and 6.8746% respectively. This result indicated that the embedding of membrane proteins did not affect the encapsulation of water-soluble drugs by vesicles. The drug loadings calculated by the formula were 29.2024% and 29.1039% respectively, indicating that the drug-loaded membrane protein biomimetic vesicles prepared by the present invention based on the three-channel laminar flow focusing chip could obtain a high drug loading without affecting the membrane proteins, which was helpful to improve the therapeutic effect of drugs.

[0070] 4. Investigation on the cytotoxicity of the drug-loaded membrane protein biomimetic vesicles

[0071] Using the CCK-8 method, the cytotoxicity of drug-loaded membrane protein biomimetic vesicles and drug-loaded vesicles without embedded membrane proteins against human immortalized keratinocytes (HaCaT cells) and mouse skin melanoma cells (B16-F10) was investigated respectively. Logarithmic phase HaCaT cells and B16-F10 cells were taken respectively, counted, diluted with medium and seeded in 96-well plates, and incubated at 37 °C with 5% CO2. After the cells adhered, 100 μl of drug-loaded membrane protein biomimetic vesicles (HCMP@GSH) (prepared in Example 13) and drug-loaded vesicles without embedded membrane proteins (Control@GSH) (prepared in Example 14) with concentration gradients of 0, 100, 300, and 500 μg / ml were added to them respectively. After incubation for 24 hours, 10 μl of CCK-8 solution was added to each well, and after continued culture for 2 h, the absorbance values of each well were measured at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Blank wells and control wells were set, where the blank wells were medium containing CCK-8 but no cells and vesicles, and the control wells were medium containing cells and CCK-8 but no vesicles.

[0072] Cell viability % = [(-experimental well - blank well) / (control well - blank well)] x 100

[0073] The results are as Figure 6 shown. The results showed that compared with the untreated control wells, the cell viability of both the drug-loaded membrane protein biomimetic vesicles and the drug-loaded vesicles without embedded membrane proteins was higher than 80% at each concentration, indicating that neither had cytotoxicity. For B16-F10 cells, an increase in intracellular glutathione concentration would inhibit intracellular tyrosinase activity, thereby affecting the melanin production pathway and reducing the intracellular melanin content, but inhibiting the normal pathway would have a certain impact on cell viability. Since the drug-loaded membrane protein biomimetic vesicles could deliver more glutathione into B16-F10 cells, their impact on the cell viability of B16-F10 cells was relatively greater compared with the drug-loaded vesicles without embedded membrane proteins; while for HaCaT cells, glutathione helped protect cells from oxidative stress and cell damage. Compared with the drug-loaded vesicles without embedded membrane proteins, the drug-loaded membrane protein biomimetic vesicles delivered less glutathione into HaCaT cells, so their promotion of HaCaT cell viability was less. To balance the therapeutic effect and cell viability, a vesicle concentration of 300 μg / ml was preferably selected as the subsequent experimental condition.

[0074] 5. Investigation of cellular uptake of drug-loaded membrane protein biomimetic vesicles

[0075] To determine the uptake differences of drug-loaded membrane protein biomimetic vesicles and drug-loaded vesicles without embedded membrane proteins by HaCaT and MNT-1 cells, HaCaT and MNT-1 cells were respectively seeded at a density of 1×10 5Cells were inoculated in a confocal dish at a density of

[0076] For HaCaT cells, the results are as Figure 7 shown. The results show that compared with the biomimetic vesicles without embedded membrane proteins, the uptake of the drug-loaded biomimetic vesicles with membrane proteins by HaCaT cells was significantly reduced. The relative fluorescence intensity of rhodamine B in the cells decreased from 5.8756 to 3.2425, a decrease of about 1.81 times. For intracellular glutathione, since glutathione is ubiquitously present in cells, after labeling with Chlorobimane, fluorescence representing glutathione also appeared in untreated cells, which was 20.5581. After treatment with the drug-loaded vesicles without embedded membrane proteins, the intracellular glutathione content increased significantly, and its fluorescence intensity increased from 20.5581 to 53.9439, an increase of about 2.62 times. After treatment with the drug-loaded biomimetic vesicles with membrane proteins, the increase in intracellular glutathione content was relatively low, and the fluorescence intensity only increased from 20.5581 to 35.8136, an increase of about 1.74 times, which was much less than the increase multiple after treatment with the drug-loaded vesicles without embedded membrane proteins. This result indicates that the drug-loaded biomimetic vesicles with membrane proteins of the present invention can reduce their uptake by keratinocytes, thereby reducing the drug concentration in keratinocytes, reducing side effects and improving the therapeutic effect.

[0077] For B16-F10 cells, the results are as Figure 8As shown, the results indicate that compared with the biomimetic vesicles without embedded membrane proteins, the uptake of the drug-loaded membrane protein biomimetic vesicles by B16-F10 cells increased significantly. The fluorescence intensity of rhodamine B in the cells increased from 1.4475 to 5.6982, an increase of approximately 3.94 times. After treatment with the drug-loaded vesicles without embedded membrane proteins, the glutathione content in the cells increased slightly, and its fluorescence intensity only increased from 13.8415 to 19.1674, an increase of approximately 1.38 times. After treatment with the drug-loaded membrane protein biomimetic vesicles, the glutathione content in the cells increased more significantly. Its fluorescence intensity increased from 13.8415 to 27.6126, an increase of approximately 1.99 times, which is higher than the increase after treatment with the drug-loaded vesicles without embedded membrane proteins. This result shows that the drug-loaded membrane protein biomimetic vesicles of the present invention can enhance the uptake by melanocytes, increase the intracellular drug concentration, and improve the therapeutic effect.

[0078] 6. Investigation on the effect of drug-loaded membrane protein biomimetic vesicles in the treatment of hyperpigmentation

[0079] The drug-loaded vesicles were prepared by referring to the method of Example 13. To verify the therapeutic effect of the drug-loaded membrane protein vesicles on skin hyperpigmentation, a cell hyperpigmentation model was established using B16-F10 cells. B16-F10 cells were seeded at 1x10 5Cells were seeded in 12-well plates at a density of cells / well and incubated for 12 hours in DMEM medium. Then, the medium was replaced with medium containing 0.5 μg / ml α-melanocyte-stimulating hormone (α-MSH) (control group, control), medium containing 0.5 μg / ml α-MSH and 0.3 mg / ml drug-loaded vesicles (vesicle treatment group, HCMP@GSH), medium containing 0.5 μg / ml α-MSH and 0.3 mg / ml drug-loaded vesicles without embedded membrane proteins (vesicle treatment group, Control@GSH), medium containing 0.5 μg / ml α-MSH and 0.3 mg / ml kojic acid (kojic acid positive control group, Kojic acid), and medium containing 0.5 μg / ml α-MSH and 0.3 mg / ml glutathione (glutathione treatment group, GSH). The cells were treated for 24 hours. After the treatment, the cells were lysed with 1% Triton solution and then centrifuged at 14,000 rpm for 30 min at 4 °C to isolate the melanin precipitate from the dissolved proteins. The melanin precipitate was resuspended in 1 M sodium hydroxide supplemented with 10% dimethyl sulfoxide and dissolved at 95 °C for 2 hours. The spectrophotometric analysis of melanin content was determined by measuring the absorbance at 405 nm and using a calibration curve obtained with synthetic melanin. The protein content of each sample was measured using a BCA protein concentration assay kit (Beyotime). The cellular melanin concentration under each condition was melanin / protein, and the relative melanin content was the ratio of the cellular melanin concentration under each experimental condition to that of the control group.

[0080] The results are shown in Figure 9 As shown, the results showed that the melanin content in B16-F10 cells treated with drug-loaded membrane protein biomimetic vesicles was 46.03% of that in the control group, the melanin content in B16-F10 cells treated with drug-loaded vesicles without embedded membrane proteins was 72.77% of that in the control wells, and the melanin content in B16-F10 cells treated with free glutathione was 91.63% of that in the control group, compared with the control wells without drugs inhibiting melanin production. This indicates that the drug-loaded membrane protein biomimetic vesicles can deliver more glutathione into melanocytes and achieve better therapeutic effects. It is worth noting that the therapeutic effect of the drug-loaded membrane protein biomimetic vesicles is superior to that of the significant kojic acid positive control group (56.97%), indicating that the drug-loaded membrane protein biomimetic vesicles prepared in the present invention have great prospects in the treatment of melanocyte-related diseases.

[0081] Comparative Example 1: Effect of total lipid concentration on drug-loaded membrane protein biomimetic vesicles

[0082] A certain amount of soybean lecithin and cholesterol were mixed and dissolved in absolute ethanol, with a total lipid concentration of 2.5 mg / ml and a molar ratio of soybean lecithin:cholesterol of 7:3, to prepare the left flow channel phase. A certain amount of keratinocyte membrane protein solution (prepared in Example 2) was dissolved in 1×PBS buffer to prepare the middle flow channel phase, with a mass concentration of the membrane protein of 60 μg / ml. A certain amount of glutathione (GSH) was dissolved in 1×PBS buffer as the right flow channel phase, with a concentration of glutathione of 20 mg / ml. With TFR = 1000 μL / min and FRR = 1:1.5:1.5, through a three-channel fluid laminar flow focusing microfluidic chip, the obtained solution was dialyzed overnight with a 300 kDa dialysis bag to obtain drug-loaded membrane protein biomimetic vesicles. Due to the too low lipid concentration, the encapsulation efficiency of the drug-loaded membrane protein biomimetic vesicles for glutathione was too low, being 1.21%, resulting in waste of the drug.

[0083] Comparative Example 2: Influence of total lipid concentration on drug-loaded membrane protein biomimetic vesicles

[0084] A certain amount of soybean lecithin and cholesterol were mixed and dissolved in absolute ethanol, with a total lipid concentration of 25 mg / ml and a molar ratio of soybean lecithin:cholesterol of 7:3, to prepare the left flow channel phase. A certain amount of keratinocyte membrane protein solution (prepared in Example 2) was dissolved in 1×PBS buffer to prepare the middle flow channel phase, with a mass concentration of the membrane protein of 60 μg / ml. A certain amount of glutathione (GSH) was dissolved in 1×PBS buffer as the right flow channel phase, with a concentration of glutathione of 20 mg / ml. With TFR = 1000 μL / min and FRR = 1:1.5:1.5, through a three-channel fluid laminar flow focusing microfluidic chip, the obtained solution was dialyzed overnight with a 300 kDa dialysis bag to obtain drug-loaded membrane protein biomimetic vesicles. Due to the too high lipid concentration, the density of the membrane protein on the obtained drug-loaded membrane protein biomimetic vesicles was too low, and the targeting effect was reduced.

[0085] Comparative Example 3: Influence of glutathione concentration on drug-loaded membrane protein biomimetic vesicles

[0086] A certain amount of soy lecithin and cholesterol were mixed and dissolved in absolute ethanol, with a total lipid concentration of 10 mg / ml and a molar ratio of soy lecithin:cholesterol of 7:3, to prepare the left flow channel phase. A certain amount of keratinocyte membrane protein solution (prepared in Example 2) was dissolved in 1×PBS buffer to prepare the middle flow channel phase, with a mass concentration of the membrane protein of 60 μg / ml. A certain amount of glutathione (GSH) was dissolved in 1×PBS buffer as the right flow channel phase, with a concentration of glutathione of 30 mg / ml. At TFR = 1000 μL / min and FRR = 1:1.5:1.5, through a three-channel fluid laminar flow focusing microfluidic chip, the obtained solution was dialyzed overnight with a 300 kDa dialysis bag to obtain drug-loaded membrane protein biomimetic vesicles. Due to the too high concentration of glutathione, even if they were ensured to be mixed at the moment before vesicle assembly through the chip, they still affected each other, resulting in too large particle size of the drug-loaded membrane protein biomimetic vesicles and the solution being turbid.

[0087] Comparative Example 4: Influence of TFR on drug-loaded membrane protein biomimetic vesicles

[0088] A certain amount of soy lecithin and cholesterol were mixed and dissolved in absolute ethanol, with a total lipid concentration of 10 mg / ml and a molar ratio of soy lecithin:cholesterol of 7:3, to prepare the left flow channel phase. A certain amount of keratinocyte membrane protein solution (prepared in Example 2) was dissolved in 1×PBS buffer to prepare the middle flow channel phase, with a mass concentration of the membrane protein of 60 μg / ml. A certain amount of glutathione (GSH) was dissolved in 1×PBS buffer as the right flow channel phase, with a concentration of glutathione of 20 mg / ml. At TFR = 2000 μL / min and FRR = 1:1.5:1.5, through a three-channel fluid laminar flow focusing microfluidic chip, the obtained solution was dialyzed overnight with a 300 kDa dialysis bag to obtain drug-loaded membrane protein biomimetic vesicles. Due to the too large total flow rate, the chip couldn't withstand the pressure and leaked.

[0089] Comparative Example 5: Influence of chip type on drug-loaded membrane protein biomimetic vesicles

[0090] A dual-channel laminar flow focusing microfluidic chip was used. Soybean lecithin and cholesterol with a molar ratio of 7:3 were mixed and dissolved in absolute ethanol with a total lipid concentration of 10 mg / ml to prepare the organic phase. A 1×PBS buffer solution containing 60 μg / ml of keratinocyte membrane protein and 20 mg / ml of glutathione was added as the aqueous phase. The aqueous phase was used as the outer phase and the organic phase as the inner phase, and passed through a fluid focusing microfluidic chip (Wuhan Jianmi Intelligent Control Technology Co., Ltd.) at a total flow rate (TFR) = 1 ml / min and a flow rate ratio (aqueous phase / organic phase = FRR) = 6:1. The resulting solution was dialyzed overnight using a 300 kDa dialysis bag to obtain drug-loaded membrane protein biomimetic vesicles. Since the membrane protein and glutathione were premixed, they interacted with each other, resulting in too large particle size and PDI of the prepared drug-loaded membrane protein biomimetic vesicles.

[0091] Comparative Example 6: Effect of cell membrane protein concentration on drug-loaded membrane protein biomimetic vesicles

[0092] A certain amount of soybean lecithin and cholesterol were mixed and dissolved in absolute ethanol with a total lipid concentration of 10 mg / ml and a molar ratio of soybean lecithin:cholesterol of 7:3 to prepare the left flow channel phase. A certain amount of keratinocyte membrane protein solution (prepared in Example 2) was dissolved in 1×PBS buffer solution to prepare the middle flow channel phase with a mass concentration of membrane protein of 150 μg / ml. A certain amount of glutathione (GSH) was dissolved in 1×PBS buffer solution as the right flow channel phase with a concentration of glutathione of 20 mg / ml. Passing through a three-channel fluid laminar flow focusing microfluidic chip at TFR = 1000 μL / min and FRR = 1:1.5:1.5, the resulting solution was dialyzed overnight using a 300 kDa dialysis bag to obtain drug-loaded membrane protein biomimetic vesicles. Due to the too high concentration of cell membrane protein, the self-assembly process of phospholipids during diffusion was restricted, resulting in uneven particle size distribution of the obtained keratinocyte membrane protein biomimetic liposomes and a polydispersity index of 0.37.

[0093] Comparative Example 7: Effect of the addition order of each phase (GSH solution was introduced into the middle flow channel phase and membrane protein solution was introduced into the right flow channel phase) on drug-loaded membrane protein biomimetic vesicles

[0094] A certain amount of soybean lecithin and cholesterol were mixed and dissolved in absolute ethanol, with a total lipid concentration of 10 mg / ml and a molar ratio of soybean lecithin:cholesterol of 7:3, to prepare the left flow channel phase. A certain amount of glutathione (GSH) was dissolved in 1×PBS buffer as the middle flow channel phase, with a concentration of glutathione of 20 mg / ml. A certain amount of keratinocyte membrane protein solution (prepared in Example 2) was dissolved in 1×PBS buffer to prepare the right flow channel phase, with a mass concentration of membrane protein of 60 μg / ml. With TFR = 1000 μL / min and FRR = 1:1.5:1.5, through a three-channel fluid laminar flow focusing microfluidic chip, the obtained solution was dialyzed overnight with a 300 kDa dialysis bag to obtain drug-loaded membrane protein biomimetic vesicles. In the three-channel fluid laminar flow focusing microfluidic chip, since the glutathione solution and the ethanol solution containing lipids came into contact first, the formed phospholipid vesicles had the structural advantage of encapsulating GSH in the core region but lacking membrane protein modification, resulting in a significant decrease in the membrane protein embedding efficiency and loss of targeting. In addition, due to the late timing of the membrane protein entering the system, it could not be effectively embedded during the phospholipid self-assembly process, affecting the biocompatibility and targeting effect of the vesicles.

[0095] Comparative Example 8: Influence of the addition order of each phase (the left flow channel phase is introduced with the membrane protein solution, and the middle flow channel phase is introduced with the ethanol solution) on the drug-loaded membrane protein biomimetic vesicles

[0096] A certain amount of keratinocyte membrane protein solution (prepared in Example 2) was dissolved in 1×PBS buffer to prepare the left flow channel phase, with a mass concentration of membrane protein of 60 μg / ml. A certain amount of soybean lecithin and cholesterol were mixed and dissolved in absolute ethanol, with a total lipid concentration of 10 mg / ml and a molar ratio of soybean lecithin:cholesterol of 7:3, to prepare the middle flow channel phase. A certain amount of glutathione (GSH) was dissolved in 1×PBS buffer as the right flow channel phase, with a concentration of glutathione of 20 mg / ml. With TFR = 1000 μL / min and FRR = 1:1.5:1.5, through a three-channel fluid laminar flow focusing microfluidic chip, the obtained solution was dialyzed overnight with a 300 kDa dialysis bag to obtain drug-loaded membrane protein biomimetic vesicles. In the three-channel fluid laminar flow focusing microfluidic chip, since the glutathione solution and the membrane protein solution were separated by the ethanol solution, they could not be mixed during flow focusing, resulting in too low a concentration of glutathione in the hydrophilic core of some biomimetic vesicles with embedded proteins, while the efficiency of membrane protein embedded in the phospholipid bilayer of some vesicles with a higher concentration of glutathione in the hydrophilic core was too low, making the targeting effect of the prepared drug-loaded membrane protein significantly decreased and the effect of inhibiting melanogenesis reduced in in vitro cell experiments.

[0097] Comparative Example 9: Influence of not encapsulating glutathione in the hydrophilic core of the biomimetic vesicles on the treatment of melanocyte-related diseases with drug-loaded membrane protein biomimetic vesicles

[0098] For the specific implementation manner, refer to step 6 of Example 19 for the investigation of the effect of treating hyperpigmentation. The difference is that an equal amount of free GSH (the same content as the GSH encapsulated in the drug-loaded vesicles) is mixed with the drug-free biomimetic vesicles (prepared in Example 8) and then co-incubated with B16-F10 cells, and the effect on melanocytes is investigated by the change in the intracellular melanin content. Since GSH exists in a free form and is not incorporated into the hydrophilic core of the membrane protein biomimetic vesicles, it can only enter the cells by diffusion, with low efficiency and being easily diluted or degraded by the extracellular environment. The limited efficiency of GSH entering the cells results in a relatively weak melanin inhibitory effect (92.36%), which is close to the group that only adds free GSH.

[0099] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A method for preparing a drug-loaded membrane protein biomimetic vesicle, characterized in that, It includes the following steps: (1) Prepare the organic phase: Mix and dissolve soy lecithin and cholesterol to obtain the organic phase; (2) Prepare the aqueous phase 1: Dissolve the membrane protein to obtain the aqueous phase 1; (3) Prepare the aqueous phase 2: Dissolve the water-soluble drug to obtain the aqueous phase 2; (4) Mix the organic phase, the aqueous phase 1 and the aqueous phase 2 through a three-channel microfluidic chip to obtain the bionic vesicles.

2. The method according to claim 1, wherein In step (4), the organic phase is used as the left flow channel phase, the aqueous phase 1 is used as the middle flow channel phase, and the aqueous phase 2 is used as the right flow channel phase.

3. The method according to claim 2, wherein In step (4), pass through the three-channel microfluidic chip at a flow rate ratio FRR of (1:1:1) to (1:3:3) and a total flow rate TFR of 100 to 2000 μl / min; the flow rate ratio is the volume flow rate of the left flow channel phase: the volume flow rate of the middle flow channel phase: the volume flow rate of the right flow channel phase.

4. The method according to claim 3, wherein In the organic phase of step (1), the molar ratio of soy lecithin to cholesterol is (9 to 5):3; the total concentration of soy lecithin and cholesterol is 2.5 to 25 mg / ml; soy lecithin and cholesterol are dissolved in ethanol.

5. The method according to claim 4, characterized in that In the aqueous phase 1 of step (2), the concentration of the membrane protein is 10 to 100 μg / ml.

6. The method according to claim 5, characterized in that In the aqueous phase 2 of step (2), the water-soluble drug includes glutathione, vitamin C or kojic acid; the concentration of the water-soluble drug is 1 to 30 mg / mL.

7. The method according to claim 6, wherein The membrane protein in step (2) includes but is not limited to the membrane protein extracted from the cell membrane and the artificially synthesized membrane protein; the membrane protein extracted from the cell membrane is the keratinocyte membrane protein.

8. The biomimetic vesicles prepared by the method according to any one of claims 1 to 7, characterized in that, The bionic vesicles contain two structures, an outer layer and an inner core; the outer layer is a phospholipid bilayer embedded with membrane proteins, and the inner core is a water-soluble drug solution.

9. The biomimetic vesicle according to claim 8, wherein The water-soluble drug includes glutathione, vitamin C or kojic acid.

10. Use of the biomimetic vesicles according to claim 8 or 9 in the preparation of a drug targeting melanocytes, characterized in that, The drug includes a drug for hyperpigmentation, a drug for vitiligo, a drug for melanoma or a drug for melasma.

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