A method for preparing drug-loaded membrane protein biomimetic vesicles based on microfluidic technology and its application in targeting melanocytes for drug delivery.

Abstract

This invention discloses a method for preparing drug-loaded membrane protein biomimetic vesicles based on microfluidic technology and their application in targeted drug delivery to melanocytes, belonging to the field of pharmaceutical formulation technology. The method is simple to operate, using laminar flow focusing technology to precisely control the assembly of the phospholipid bilayer and the insertion of membrane proteins, forming stable and efficient drug-loaded membrane protein biomimetic vesicles. These drug-loaded membrane protein biomimetic vesicles can effectively target melanocytes, promote vesicle uptake, significantly increase drug accumulation within melanocytes, and enhance the therapeutic effect on melanocyte-related diseases (such as hyperpigmentation, melanoma, vitiligo, etc.). Simultaneously, the drug-loaded biomimetic vesicles constructed in this invention have a low keratinocyte uptake rate, effectively reducing drug uptake in non-targeted tissues and lowering drug side effects. Therefore, the drug-loaded membrane protein biomimetic vesicle system constructed in this invention has significant potential in the treatment of melanocyte-related diseases.

Description

Technical Field

[0001] This invention relates to a method for preparing drug-loaded membrane protein biomimetic vesicles based on microfluidic technology and its application in targeting melanocytes for drug delivery, belonging to the field of pharmaceutical formulation technology. Background Technology

[0002] In recent years, skin melanocyte-related diseases, such as hyperpigmentation, vitiligo, age spots, melasma, and melanoma, have attracted widespread attention. These diseases are often closely related to disorders of melanin synthesis, functional decline, or abnormal proliferation of melanocytes. With in-depth research into the biology of skin melanocytes, 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 a significant challenge to the efficient delivery of drugs to melanocytes. First, conventional non-targeted topical drug delivery systems result in uneven distribution of drugs in the skin, with most being taken up by keratinocytes and failing to effectively target melanocytes, thus significantly reducing the therapeutic effect, especially in the treatment of melanocyte-related skin diseases, where ideal efficacy is often difficult to achieve. Second, non-targeted topical drug delivery systems fail to deliver drugs specifically to melanocytes, and the drugs often act on other skin cells, especially keratinocytes, potentially causing a series of side effects. For example, in the treatment of hyperpigmentation, ingredients such as hydroquinone and retinoic acid may cause excessive exfoliation of keratinocytes, leading to adverse reactions such as dry skin, irritation, and even peeling. Currently, drug delivery systems targeting melanocytes focus on modifying the vesicle surface with melanocyte-targeting ligands (such as α-MSH analogs, tyrosine analogs, or CD44 receptor ligands) to enhance melanocyte uptake. However, these ligand modifications do not specifically recognize keratinocytes, leading to non-specific uptake of some vesicles by keratinocytes and reducing targeting efficiency. Therefore, developing a specific drug-targeted delivery system capable of delivering drugs directly to skin melanocytes and reducing drug uptake by keratinocytes would significantly improve the treatment efficacy and reduce side effects of melanocyte-related diseases, possessing significant research and application value. However, a suitable solution for such a targeted delivery system is currently lacking. Summary of the Invention

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

[0005] This invention utilizes microfluidic technology to fabricate biomimetic vesicles containing drug-loaded keratinocyte membrane proteins, based on the intercellular communication mechanism between keratinocytes and melanocytes via extracellular vesicles. These vesicles possess a phospholipid bilayer structure, with keratinocyte membrane proteins embedded within the phospholipid bilayer. Simultaneously, the vesicle's water-filled core is loaded with water-soluble drugs, the phospholipid bilayer is loaded with lipid-soluble drugs, or both water-soluble and lipid-soluble drugs are loaded, enabling targeted delivery to melanocytes. This invention employs a three-channel microfluidic chip to precisely control the insertion of membrane proteins and vesicle formation, ensuring smooth embedding and uniform distribution of keratinocyte membrane proteins within the vesicle's phospholipid bilayer. This also avoids interactions between the drug and the membrane proteins, improving vesicle stability and loading capacity. The vesicle's phospholipid bilayer, composed of membrane proteins, phospholipids, and cholesterol, mimics the characteristics of the natural keratinocyte membrane, enhancing interaction with melanocytes and enabling efficient drug delivery to melanocytes while reducing non-specific uptake by keratinocytes. The drug-loaded membrane protein biomimetic vesicles prepared in this invention can bind to receptors on the surface of melanocytes through membrane protein-mediated specific recognition, enhancing targeting, reducing non-specific uptake, and improving drug utilization. Simultaneously, these drug-loaded membrane protein biomimetic vesicles utilize cell membrane similarity; because their surface protein composition is similar to that of keratinocytes, recognition signals between homologous cells are unfavorable for uptake, thus reducing keratinocyte uptake. This invention uses glutathione (GSH) as a model drug loaded into the water core of the biomimetic vesicles. Glutathione can inhibit melanin production in melanocytes by regulating redox state and inhibiting tyrosinase activity. These drug-loaded biomimetic vesicles can efficiently deliver glutathione to melanocytes, increasing intracellular drug concentration, enhancing its inhibitory effect on melanin production, while reducing drug uptake by keratinocytes, minimizing non-targeting effects, and improving therapeutic efficacy. This invention provides an efficient and precise drug delivery platform, offering a new strategy and technical solution for targeted melanocyte therapy, and has significant clinical application value and industrialization prospects.

[0006] This invention provides a method for preparing drug-loaded membrane protein biomimetic vesicles, wherein the biomimetic vesicles comprise keratinocyte membrane proteins, a phospholipid bilayer, and a drug. In a three-channel laminar flow aggregation microfluidic chip, an ethanol solution of the phospholipid bilayer serves as the left-side flow channel phase, a PBS buffer solution of the keratinocyte membrane proteins serves as the middle flow channel phase, and a PBS buffer solution of the water-soluble drug serves as the right-side flow channel phase. The ethanol solution in the left-side flow channel phase can be loaded with a lipid-soluble drug.

[0007] In one embodiment, the left-side flow channel phase is an organic phase, and the phospholipid bilayer includes soybean lecithin and cholesterol, with a molar ratio of soybean lecithin to cholesterol of (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 aqueous phase 1; the concentration of the keratinocyte membrane protein in aqueous phase 1 is 10-100 μg / ml.

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

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

[0011] In one embodiment, the preparation method of the human keratinocyte membrane protein is as follows: 40 million immortalized keratinocytes are cultured, and the cells are separated from the bottom of the culture dish using a cell scraper, collected by centrifugation, and washed with ice-cold PBS. Using a cell membrane protein and plasma protein extraction kit (Beyotime), 1 ml of membrane protein extraction reagent A (pre-added with PMSF) is added to approximately 40 million immortalized keratinocytes, and the cells are fully resuspended and then incubated on ice for 10 minutes. The cell suspension is transferred to a 2 ml ice-cold glass homogenizer, homogenized until the cells are fully lysed, centrifuged at 700g for 10 minutes at 4°C, and the supernatant is collected. Centrifuged at 14000rpm for 30 minutes at 4°C, the precipitate is collected, 400 μL of membrane protein extraction reagent B is added, vortexed for 5 seconds, and then incubated on ice for 10 minutes. This process is repeated three times. Subsequently, centrifuged at 14000g for 5 minutes at 4°C, and the supernatant is collected as the membrane protein solution, which is stored at -80°C.

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

[0013] In one embodiment, the right-side flow channel phase is aqueous phase 2; the drug includes, but is not limited to, water-soluble drugs and lipid-soluble drugs.

[0014] In one embodiment, the water-soluble drug includes 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, the flow rate ratio FRR is (1:1:1) to (1:3:3) and the total flow rate TFR is 100 to 2000 μl / min, passing through a three-channel microfluidic chip; the flow rate ratio is the volumetric flow rate of the left flow channel: the volumetric flow rate of the middle flow channel: the volumetric flow rate of the right flow channel.

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

[0018] In one embodiment, the biomimetic vesicle comprises two structures: an outer layer and a core; the outer layer is a phospholipid bilayer with embedded 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 vesicles in the preparation of drugs targeting melanocytes, the drugs including drugs for hyperpigmentation, vitiligo, melanoma, or chloasma.

[0021] This invention utilizes microfluidic technology to embed human keratinocyte membrane proteins into the phospholipid bilayer of vesicles and load water-soluble drugs into the water core of the vesicles, constructing a drug-loaded membrane protein biomimetic vesicle system aimed at achieving targeted drug delivery to melanocytes. This drug-loaded biomimetic vesicle utilizes intercellular adhesion proteins present on the phospholipid bilayer, significantly enhancing its retention time in the living epidermis, thereby increasing the local drug concentration in the epidermis. Furthermore, due to the characteristics of the surface membrane proteins of the biomimetic vesicles, this system can effectively target melanocytes, promoting their uptake of the drug-loaded membrane protein biomimetic vesicles and significantly increasing drug accumulation within melanocytes, thus enhancing the therapeutic effect on melanocyte-related diseases (such as melanoma, vitiligo, etc.). Simultaneously, the drug-loaded biomimetic vesicles constructed in this invention have a low uptake rate by keratinocytes, effectively reducing drug uptake in non-targeted tissues and lowering drug side effects.

[0022] Beneficial effects:

[0023] This invention provides a method for preparing drug-loaded membrane protein biomimetic vesicles based on microfluidic technology and its application in targeting melanocytes for drug delivery. The preparation process is simple and straightforward, requires no large equipment, and is environmentally friendly.

[0024] This invention employs a three-channel laminar flow focusing microfluidic chip technology to precisely control the mixing of membrane protein and drug solutions, ensuring instantaneous mixing before assembly and effectively preventing interference between the membrane proteins and the drug. This design not only ensures the complete embedding of membrane proteins into the vesicle membrane, inheriting the biological characteristics of the source cell, but also successfully achieves efficient drug loading, guaranteeing the functionality and stability of the drug delivery system.

[0025] The drug-loaded biomimetic vesicles of this invention can bind to receptors on the surface of melanocytes through membrane protein-mediated specific recognition, enhancing targeting to melanocytes and reducing non-specific uptake: compared to drug-loaded vesicles without membrane protein embedding, melanocyte uptake of drug-loaded biomimetic vesicles increased by approximately 3.94 times. Furthermore, because the surface protein composition of the drug-loaded biomimetic vesicles in this invention is similar to that of the keratinocyte's own cell membrane, the recognition signals between homologous cells are unfavorable for uptake, thus reducing keratinocyte uptake: compared to drug-loaded vesicles without membrane protein embedding, keratinocyte uptake of drug-loaded biomimetic vesicles decreased by approximately 1.81 times.

[0026] In this invention, the drug-loaded biomimetic vesicles use glutathione as the model water-soluble drug. In in vitro cell experiments, these biomimetic vesicles significantly increased the glutathione content in melanocytes (by approximately 1.99 times), while drug-loaded vesicles without membrane protein embedding only increased the glutathione content in melanocytes by approximately 1.38 times. Simultaneously, these biomimetic vesicles also slightly increased the glutathione content in keratinocytes (by approximately 1.74 times), while drug-loaded vesicles without membrane protein embedding increased the glutathione content in keratinocytes by approximately 2.62 times.

[0027] The drug-loaded biomimetic vesicles of this invention use hyperpigmentation as a model disease. In in vitro cell experiments, the inhibitory effect on melanin production (melanin content of 46.03% of the control group) is significantly better than that of drug-loaded vesicles that do not embed in membrane proteins (melanin content of 72.77% of the control group), and better than that of the positive control group kojic acid (melanin content of 56.97% of the control group), and there is no cytotoxicity. Attached Figure Description

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

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

[0030] Figure 3 This is a SEM image of the drug-loaded membrane protein biomimetic vesicle of the present invention.

[0031] Figure 4This is a standard curve of glutathione measured by high performance liquid chromatography in this invention.

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

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

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

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

[0036] Figure 9 This is a graph showing the relative intracellular melanin content of B16-F10 cells after treatment with drug-loaded membrane protein biomimetic vesicles according to the present invention. Detailed Implementation

[0037] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0038] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0039] Secondly, the term "one embodiment" or "embodiment" as used 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 different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0040] The "medicine targeting melanocytes" involved in this invention refers to the targeted delivery of drugs by utilizing the specific binding of biomimetic vesicles to melanocytes, thereby increasing the uptake of drugs by melanocytes.

[0041] Extraction of membrane proteins from immortalized human keratinocytes in Examples 1-3

[0042] Well-grown immortalized human keratinocytes (HaCaT) were collected. Cells were scraped from the bottom of the culture dish using a cell scraper, collected by centrifugation, and washed with ice-cold PBS. Using a cell membrane protein and plasma protein extraction kit (Beyotime), membrane protein extraction reagent A (contained with benzyl sulfonyl fluoride before use) was added to approximately 40 million immortalized human keratinocytes. After fully suspending the cells, they were incubated on ice for 10 minutes. The cell suspension was transferred to an ice-cold glass homogenizer and homogenized until the cells were fully dysplastic. The homogenizer was then centrifuged at 700g for 10 minutes at 4°C, and the supernatant was collected. The mixture was then centrifuged at 13000rpm for 30 minutes at 4°C, and the precipitate was collected. After washing twice with PBS, membrane protein extraction reagent B was added, and the mixture was vortexed for 5 seconds and then incubated on ice for 10 minutes. This process was repeated three times. Subsequently, the mixture was centrifuged at 14000g for 5 minutes at 4°C, and the supernatant was collected as the membrane protein solution, which was stored at -80°C. Based on the membrane protein concentration, Example 2 was used as the preferred process for membrane protein extraction.

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

[0044] Table 1. Optimization of extraction process for human immortalized keratinocyte membrane proteins.

[0045] 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] Examples 4-9: Preparation of drug-free membrane protein biomimetic vesicles

[0047] A certain amount of soybean lecithin and cholesterol were dissolved in anhydrous ethanol to prepare a total lipid concentration of 10 mg / ml, forming the left-side 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 membrane protein concentration of 10–100 μg / ml. Since the aim was to prepare drug-free biomimetic vesicles, the 1×PBS buffer was used as the right-side flow channel phase. The above solutions were simultaneously added to a three-channel laminar focusing microfluidic chip (Laminar-04, Wuhan Jianmi Intelligent Control Technology Co., Ltd., specific dimensions as shown). Figure 1 After the three channels shown, the flow rate (TFR) and flow rate ratio (left channel phase volume flow rate: middle channel phase volume flow rate: right channel phase volume flow rate = FRR) are passed through the chip. The resulting solution is dialyzed overnight using a 300kDa dialysis bag to obtain non-drug-loaded membrane protein biomimetic vesicles with keratinocyte membrane proteins embedded in the phospholipid bilayer and hydrophilic cores without drug molecules.

[0048] Examples 4-6 show that the particle size of the prepared vesicles decreases with the change of FRR, reaching 167.8 nm, 90.31 nm, and 87.38 nm, respectively. The polydispersity index first decreases and then increases, reaching 0.183, 0.151, and 0.156, respectively. Based on the smaller particle size and lower polydispersity index, an FRR of 1:1.5:1.5 is the preferred process for membrane protein embedding. Examples 7, 8, and 9 show that the particle size of the prepared liposomes gradually increases with the increase of membrane protein concentration, reaching 94.21 nm, 101.23 nm, and 111.78 nm, respectively. The Zeta potential gradually decreases, reaching -4.13 mV, -7.21 mV, and -7.56 mV, respectively. Based on the change in Zeta potential and 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] Examples 10-16: Preparation of drug-loaded membrane protein biomimetic vesicles

[0052] Bionic vesicles were prepared according to the method in Example 8, except that glutathione (GSH) was added to the right-side flow channel phase. A certain amount of soybean lecithin and cholesterol were dissolved in anhydrous ethanol, with a total lipid concentration of 10 mg / ml and a soybean lecithin:cholesterol molar ratio of 7:3, to prepare the left-side 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 membrane protein concentration of 60 μg / ml. A certain amount of glutathione was dissolved in 1×PBS buffer as the right-side flow channel phase, with a glutathione concentration of 1–20 mg / ml. The solution was dialyzed overnight using a 300 kDa dialysis bag at a TFR of 1000 μL / min and FRR of 1:1.5:1.5 through a three-channel laminar flow focusing microfluidic chip. The resulting solution was then dialyzed overnight using a 300 kDa dialysis bag to obtain drug-loaded membrane protein bionic vesicles with membrane proteins embedded in the phospholipid bilayer and a hydrophilic core loaded with a high concentration of GSH.

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

[0054]

[0055]

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

[0057] A certain amount of soybean lecithin and cholesterol were mixed and dissolved in anhydrous ethanol, with a total lipid concentration of 10 mg / ml and a soybean lecithin:cholesterol molar ratio of 7:3, to prepare the left-side 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 membrane protein mass concentration of 60 μg / ml. Water-soluble drugs vitamin C or kojic acid were dissolved in 1×PBS buffer as the right-side flow channel phase, with a concentration of 20 mg / ml. The solution was dialyzed overnight using a 300 kDa dialysis bag at a TFR of 1000 μL / min and FRR of 1:1.5:1.5 through a three-channel laminar flow focusing microfluidic chip. The resulting solution was then 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 a hydrophilic core loaded with a high concentration of vitamin C (Example 17) and kojic acid (Example 18). The prepared drug-loaded membrane protein biomimetic vesicle solution was clear. DLS test results showed that the particle size increased from 111.4 nm to 118.7 nm and 122.1 nm before and after drug loading, respectively, while the PDI did not change significantly. This verifies that the drug-loaded membrane protein biomimetic vesicle based on microfluidic technology of the present invention has universality for high-concentration encapsulation of water-soluble drugs.

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

[0059] This invention describes the performance characterization of drug-loaded membrane protein biomimetic vesicles based on microfluidic technology and their application in targeting melanocytes for drug delivery.

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

[0061] The particle size, polydispersity index, and zeta potential of drug-free membrane protein biomimetic vesicles (HCMP) (prepared in Example 8) and drug-loaded membrane protein biomimetic vesicles (HCMP@GSH) (prepared in Examples 10-13) prepared with different glutathione concentrations in the right-side flow channel phase were determined using a nanolaser particle size analyzer. The results are as follows: Figure 2The results showed that as the concentration of glutathione in the right-side 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), while the ZETA potentials did not change significantly (-9.49 mV, -9.31 mV, -9.97 mV, -10.37 mV and -9.77 mV, respectively), and the polydispersity index did not change significantly (0.158, 0.161, 0.158, 0.157 and 0.159, respectively). This indicates that using a three-channel fluid laminar flow focusing microfluidic chip according to the above preparation sequence can successfully reduce the mutual interference between membrane proteins and drug solutions. As the concentration of glutathione in the right-side flow channel phase increases, the concentration of glutathione loaded in the lumen of the biomimetic vesicles also increases, resulting in a slight increase in particle size from 111.4 nm to 127.2 nm, but this effect is negligible. To maximize the drug loading capacity of the biomimetic vesicles while preserving their biological characteristics, the highest possible glutathione concentration should be selected. Therefore, Example 13 is preferred as the preferred process for drug-loaded membrane protein biomimetic vesicles. At this glutathione concentration (20 mg / ml), compared to unloaded vesicles without membrane protein embedding (prepared in Example 5), the particle size of the drug-loaded vesicles without membrane protein embedding (prepared in Example 14) is slightly increased (from 100.3 nm to 110.7 nm), while the polydispersity index and Zeta potential show no significant changes (0.168 and -2.26 mV, respectively).

[0062] 2. Characterization of drug-loaded membrane protein biomimetic vesicles by scanning electron microscopy (SEM)

[0063] The microstructure of drug-loaded membrane protein biomimetic vesicles (HCMP@GSH) (prepared in Example 13) and drug-loaded vesicles without membrane protein embedding (Control@GSH) (prepared in Example 14) were characterized using field emission scanning electron microscopy. A certain amount of trehalose was added to the vesicle formulation as a lyophilization protectant to prepare a trehalose vesicle solution with a final concentration of 100 mM. The solution was then rapidly frozen with liquid nitrogen and lyophilized in a freeze dryer. After lyophilization, the lyophilized sample was spread onto a conductive adhesive and observed under a scanning electron microscope.

[0064] Figure 3 The results showed that both the drug-loaded membrane protein biomimetic vesicles prepared by microfluidic technology and the drug-loaded vesicles without embedded membrane proteins were spherical with clear edges, although their size differed from that measured by a nanolaser particle size analyzer. This may be due to the collapse of the vesicle structure during the liquid nitrogen freezing and freeze-drying processes.

[0065] 3. Characterization of drug-loaded membrane protein biomimetic vesicle encapsulation efficiency and drug loading capacity

[0066] Free glutathione was separated using ultrafiltration, and the encapsulation efficiency and drug loading of glutathione in drug-loaded membrane protein-inspired vesicles (HCMP@GSH) (prepared in Example 13) and drug-loaded vesicles without membrane protein embedding (Control@GSH) (prepared in Example 14) were determined by high-performance liquid chromatography (HPLC). After vesicle preparation, the vesicle solution was directly ultrafiltered through a 3 kDa ultrafiltration centrifuge tube to separate the free glutathione solution without dialysis. The free glutathione solution was quantitatively analyzed using HPLC with a C18 column, a water-acetonitrile solution (95:5) as the mobile phase (containing 0.1% trifluoroacetic acid), and a detection wavelength of 210 nm. During the determination, a linear relationship between concentration and peak area was established using a standard solution of glutathione, and a standard curve was plotted. The results are shown in [Figure number missing]. Figure 4 Then, the concentration of free glutathione was calculated by comparing the peak area of ​​the sample with the standard curve. Encapsulation efficiency and loading capacity were calculated using the following formulas:

[0067]

[0068] Among them, C 总 The initial total glutathione concentration is expressed in mg / mL; C 游离 This refers to the concentration of free glutathione, in mg / mL; C 包封 The concentration of encapsulated glutathione is expressed in mg / mL; C 囊泡溶液 This represents the total concentration of vesicles and glutathione in the vesicle solution, expressed in mg / mL.

[0069] See results Figure 5 The results showed that there was no significant difference in the encapsulation efficiency of glutathione between drug-loaded membrane protein biomimetic vesicles prepared by the three-channel laminar flow focusing chip and those without embedded membrane proteins, which were 6.8419% and 6.8746%, respectively. This indicates that the embedding of membrane proteins does not affect the encapsulation of water-soluble drugs by the vesicles. The drug loading amounts calculated by the formula were 29.2024% and 29.1039%, respectively, indicating that the drug-loaded membrane protein biomimetic vesicles prepared by the three-channel laminar flow focusing chip of this invention can achieve a high drug loading amount without affecting the membrane proteins, which helps to improve the therapeutic effect of the drug.

[0070] 4. Cytotoxicity study of drug-loaded membrane protein biomimetic vesicles

[0071] The cytotoxicity of drug-loaded membrane protein biomimetic vesicles and drug-loaded vesicles without membrane protein embedding to human immortalized keratinocytes (HaCaT cells) and mouse skin melanoma cells (B16-F10) was investigated using the CCK-8 assay. HaCaT cells and B16-F10 cells in the logarithmic growth phase were counted, diluted with culture medium, and seeded into 96-well plates. The plates were incubated at 37°C with 5% CO2. After cell attachment, 100 μl of drug-loaded membrane protein biomimetic vesicles (HCMP@GSH) (prepared in Example 13) and drug-loaded vesicles without membrane protein embedding (Control@GSH) (prepared in Example 14) at concentrations of 0, 100, 300, and 500 μg / ml were added to each well. After 24 hours of incubation, 10 μl of CCK-8 solution was added to each well, and the cells were cultured for another 2 hours. The absorbance of each well was then measured at 450 nm using a microplate reader. Blank wells and control wells were set up. Blank wells contained CCK-8 but no cells or vesicles, while control wells contained cells and CCK-8 but no vesicles.

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

[0073] The results are as follows Figure 6 As shown, the results indicated that, compared to the untreated control wells, both drug-loaded membrane protein biomimetic vesicles and drug-loaded vesicles without membrane protein embedding exhibited cell viability above 80% at all concentrations, indicating no cytotoxicity. For B16-F10 cells, increased intracellular glutathione concentration inhibited intracellular tyrosinase activity, thereby affecting the melanin production pathway and reducing intracellular melanin content; however, inhibition of the normal pathway still had some impact on cell viability. Since drug-loaded membrane protein biomimetic vesicles could deliver more glutathione into B16-F10 cells, their effect on B16-F10 cell viability was relatively greater than that of drug-loaded vesicles without membrane protein embedding. For HaCaT cells, glutathione helps protect cells from oxidative stress and cell damage; compared to drug-loaded vesicles without membrane protein embedding, drug-loaded membrane protein biomimetic vesicles delivered less glutathione into HaCaT cells, thus having a less significant effect on HaCaT cell viability. To balance therapeutic efficacy and cell viability, a vesicle concentration of 300 μg / ml was selected as the optimal experimental condition for subsequent experiments.

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

[0075] To determine the differences in drug-loaded membrane protein biomimetic vesicles and drug-loaded vesicles without membrane protein embedding between HaCaT and MNT-1 cells, HaCaT and MNT-1 cells were respectively injected with 1×10⁻⁶ cells. 5Cells were seeded at a density of 100 cells / well in laser confocal microscopy dishes. After cell adhesion, the cells were washed twice with PBS (pH 7.4) and incubated at 37°C for 24 hours with 1 mL of medium containing 300 μg / mL rhodamine B-labeled drug-loaded membrane protein biomimetic vesicles and drug-loaded vesicles without membrane protein embedding (prepared in Examples 15 and 16). After incubation, the original medium was replaced with medium containing chlorpromazine and incubated for another 30 minutes to fluorescently label intracellular glutathione. The fluorescence signals representing vesicles and glutathione were quantified using ImageJ software via laser confocal microscopy imaging.

[0076] For HaCaT cells, the results were as follows: Figure 7 As shown, the results indicated that, compared to biomimetic vesicles without membrane protein embedding, HaCaT cells exhibited significantly reduced uptake of drug-loaded membrane protein biomimetic vesicles. The relative fluorescence intensity of intracellular rhodamine B decreased from 5.8756 to 3.2425, a decrease of approximately 1.81-fold. Regarding intracellular glutathione, since glutathione is ubiquitous in cells, even untreated cells labeled with Chlorobimane showed fluorescence representing glutathione, at 20.5581. After treatment with drug-loaded vesicles without membrane protein embedding, the intracellular glutathione content significantly increased, with its fluorescence intensity rising from 20.5581 to 53.9439, an increase of approximately 2.62-fold. However, after treatment with drug-loaded membrane protein biomimetic vesicles, the increase in intracellular glutathione content was relatively small, with the fluorescence intensity only increasing from 20.5581 to 35.8136, an increase of approximately 1.74-fold, far less than the increase after treatment with drug-loaded vesicles without membrane protein embedding. These results indicate that the drug-loaded membrane protein biomimetic vesicles of the present invention can reduce the uptake of drugs by keratinocytes, thereby reducing the concentration of drugs within keratinocytes, reducing side effects, and improving efficacy.

[0077] For B16-F10 cells, the results were as follows: Figure 8As shown, the results indicate that, compared to biomimetic vesicles without membrane protein embedding, B16-F10 cells showed a significant increase in uptake of drug-loaded membrane protein biomimetic vesicles. The intracellular fluorescence intensity of rhodamine B increased from 1.4475 to 5.6982, an increase of approximately 3.94 times. After treatment with drug-loaded vesicles without membrane protein embedding, the intracellular glutathione content increased slightly, with its fluorescence intensity only increasing from 13.8415 to 19.1674, an increase of approximately 1.38 times. However, after treatment with drug-loaded membrane protein biomimetic vesicles, the intracellular glutathione content increased significantly, with its fluorescence intensity increasing from 13.8415 to 27.6126, an increase of approximately 1.99 times, which is higher than the increase after treatment with drug-loaded vesicles without membrane protein embedding. These results demonstrate that the drug-loaded membrane protein biomimetic vesicles of the present invention can enhance the uptake by melanocytes, increase intracellular drug concentration, and improve therapeutic efficacy.

[0078] 6. Efficacy of drug-loaded membrane protein biomimetic vesicles in treating hyperpigmentation

[0079] Drug-loaded vesicles were prepared according to the method in Example 13. To verify the therapeutic effect of drug-loaded membrane protein vesicles on hyperpigmentation of the skin, a hyperpigmentation model was established using B16-F10 cells. B16-F10 cells were grown at a rate of 1x10⁻¹². 5Cells were seeded at a density of 100 cells / well in 12-well plates and incubated for 12 hours in DMEM medium. The medium was then changed to the following solutions: control group (0.5 μg / ml α-melanocyte-stimulating hormone (α-MSH)); vesicle treatment group (HCMP@GSH) (0.5 μg / mα-MSH and 0.3 mg / ml drug-loaded vesicles); vesicle treatment group (Control@GSH) (0.5 μg / mα-MSH and 0.3 mg / ml drug-loaded vesicles not embedded in membrane proteins); kojic acid positive control group (kojic acid); and glutathione treatment group (GSH) (0.5 μg / mα-MSH and 0.3 mg / ml glutathione). Cells were treated for 24 hours. After treatment, cells were lysed using 1% Triton solution, followed by centrifugation at 14,000 rpm for 30 min at 4°C to separate melanin precipitate from the dissolved protein. The melanin precipitate was resuspended in 1M sodium hydroxide with 10% dimethyl sulfoxide and dissolved at 95°C for 2 h. Spectrophotometric analysis of melanin content was performed by measuring absorbance at 405 nm and using a calibration curve obtained from synthetic melanin. Protein content of each sample was measured using a BCA protein assay kit (Beyotime). Cellular melanin concentration under each condition was expressed as melanin / protein, and relative melanin content was the ratio of cell melanin concentration under each experimental condition to the control group.

[0080] The results are as follows Figure 9 As shown, the results indicated that, compared to the control wells without the addition of a melanin-inhibiting drug, 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 group; and the melanin content in B16-F10 cells treated with free glutathione was 91.63% of that in the control group. This suggests that drug-loaded membrane protein biomimetic vesicles can deliver more glutathione into melanocytes, achieving better therapeutic effects. Notably, the therapeutic effect of drug-loaded membrane protein biomimetic vesicles was superior to that of the significantly kojic acid-positive control group (56.97%), indicating that the drug-loaded membrane protein biomimetic vesicles prepared in this invention have significant potential 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 anhydrous ethanol, with a total lipid concentration of 2.5 mg / ml and a soybean lecithin:cholesterol molar ratio of 7:3, to prepare the left-side 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 membrane protein mass concentration of 60 μg / ml. A certain amount of glutathione (GSH) was dissolved in 1×PBS buffer as the right-side flow channel phase, with a glutathione concentration of 20 mg / ml. The solution was obtained by dialysis overnight using a 300 kDa dialysis bag at a TFR of 1000 μL / min and FRR of 1:1.5:1.5 through a three-channel laminar flow focusing microfluidic chip, yielding drug-loaded membrane protein biomimetic vesicles. Due to the low lipid concentration, the encapsulation efficiency of the drug-loaded membrane protein biomimetic vesicles for glutathione was too low, at 1.21%, resulting in drug waste.

[0083] Comparative Example 2: Effect 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 anhydrous ethanol, with a total lipid concentration of 25 mg / ml and a soybean lecithin:cholesterol molar ratio of 7:3, to prepare the left-side 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 membrane protein concentration of 60 μg / ml. A certain amount of glutathione (GSH) was dissolved in 1×PBS buffer as the right-side flow channel phase, with a glutathione concentration of 20 mg / ml. The solution was obtained by passing the solution through a three-channel laminar flow focusing microfluidic chip at a TFR of 1000 μL / min and FRR of 1:1.5:1.5, and dialyzed overnight using a 300 kDa dialysis bag to obtain drug-loaded membrane protein biomimetic vesicles. Due to the excessively high lipid concentration, the density of membrane proteins on the obtained drug-loaded membrane protein biomimetic vesicles was too low, resulting in reduced targeting effectiveness.

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

[0086] A certain amount of soybean lecithin and cholesterol were dissolved in anhydrous ethanol, with a total lipid concentration of 10 mg / ml and a soybean lecithin:cholesterol molar ratio of 7:3, to prepare the left-side 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 membrane protein mass concentration of 60 μg / ml. A certain amount of glutathione (GSH) was dissolved in 1×PBS buffer as the right-side flow channel phase, with a glutathione concentration of 30 mg / ml. The solution was obtained by passing the solution through a three-channel laminar flow focusing microfluidic chip at a TFR of 1000 μL / min and FRR of 1:1.5:1.5, and dialyzing overnight using a 300 kDa dialysis bag to obtain drug-loaded membrane protein biomimetic vesicles. Due to the excessively high glutathione concentration, even with chip-based mixing to ensure mixing just before vesicle assembly, they still interfered with each other, resulting in excessively large drug-loaded membrane protein biomimetic vesicles and a turbid solution.

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

[0088] A certain amount of soybean lecithin and cholesterol were mixed and dissolved in anhydrous ethanol, with a total lipid concentration of 10 mg / ml and a soybean lecithin:cholesterol molar ratio of 7:3, to prepare the left-side 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 membrane protein mass concentration of 60 μg / ml. A certain amount of glutathione (GSH) was dissolved in 1×PBS buffer as the right-side flow channel phase, with a glutathione concentration of 20 mg / ml. The solution was obtained by passing the solution through a three-channel laminar flow focusing microfluidic chip at a TFR of 2000 μL / min and FRR of 1:1.5:1.5. Dialyzing the resulting solution overnight using a 300 kDa dialysis bag yielded drug-loaded membrane protein biomimetic vesicles. Due to the excessively high total flow rate, the chip could not withstand the pressure and leaked.

[0089] Comparative Example 5: The Influence of Chip Type on Drug-Loaded Membrane Protein Bionic Vesicles

[0090] A dual-channel laminar flow focusing microfluidic chip was used. Soy lecithin and cholesterol were mixed and dissolved in anhydrous ethanol at a molar ratio of 7:3, resulting in a total lipid concentration of 10 mg / ml, to prepare the organic phase. A 1:1 PBS buffer solution containing 60 μg / ml keratinocyte membrane protein and 20 mg / ml glutathione was added as the aqueous phase. The aqueous phase was used as the external phase, and the organic phase as the internal phase. The solution was passed through the fluid focusing microfluidic chip (Wuhan Jianmi Intelligent Control Technology Co., Ltd.) at a total flow rate (TFR) of 1 ml / min and a flow rate ratio (aqueous phase / organic phase = FRR) of 6:1. The resulting solution was dialyzed overnight using a 300 kDa dialysis bag to obtain drug-loaded membrane protein biomimetic vesicles. Due to the pre-mixing of the membrane protein and glutathione, their interaction resulted in excessively large particle size and a high particle size index (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 Bionic Vesicles

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

[0093] Comparative Example 7: Effect of the order of phase addition (GSH solution introduced into the middle flow channel phase, membrane protein solution 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 anhydrous ethanol, with a total lipid concentration of 10 mg / ml and a soybean lecithin:cholesterol molar ratio of 7:3, to prepare the left-side flow channel phase. A certain amount of glutathione (GSH) was dissolved in 1×PBS buffer to prepare the middle flow channel phase, with a glutathione concentration 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-side flow channel phase, with a membrane protein mass concentration of 60 μg / ml. The solution was obtained by dialysis overnight using a 300 kDa dialysis bag at a TFR of 1000 μL / min and FRR of 1:1.5:1.5 through a three-channel laminar flow focusing microfluidic chip, yielding drug-loaded membrane protein biomimetic vesicles. In a three-channel laminar flow focusing microfluidic chip, the glutathione solution initially contacts the lipid-containing ethanol solution, resulting in phospholipid vesicles that encapsulate GSH in the core region but lack the structural advantage of membrane protein modification. This leads to a significant decrease in membrane protein embedding efficiency and loss of targeting. Furthermore, because membrane proteins enter the system relatively late, they cannot effectively embed during phospholipid self-assembly, affecting the biocompatibility and targeting effect of the vesicles.

[0095] Comparative Example 8: Effect of the order of phase addition (membrane protein solution introduced into the left flow channel phase, ethanol solution introduced into the middle flow channel phase) on 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-side flow channel phase, with a membrane protein concentration of 60 μg / ml. A certain amount of soybean lecithin and cholesterol were mixed and dissolved in anhydrous ethanol, with a total lipid concentration of 10 mg / ml and a soybean lecithin:cholesterol molar ratio 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-side flow channel phase, with a glutathione concentration of 20 mg / ml. The solution was dialyzed overnight using a 300 kDa dialysis bag at a TFR of 1000 μL / min and FRR of 1:1.5:1.5 to obtain drug-loaded membrane protein biomimetic vesicles. In a three-channel laminar flow focusing microfluidic chip, the glutathione solution and the membrane protein solution are separated by an ethanol solution, preventing them from mixing during flow focusing. This results in some biomimetic vesicles with embedded proteins having excessively low glutathione concentrations in their hydrophilic cores, while some vesicles with higher glutathione concentrations in their hydrophilic cores have excessively low efficiency in embedding membrane proteins in their phospholipid bilayers. Consequently, the targeting effect of the prepared drug-loaded membrane proteins is significantly reduced, leading to a decrease in the inhibitory effect on melanin production in in vitro cell experiments.

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

[0098] The specific implementation method refers to step 6 of Example 19, and the effect of treating hyperpigmentation is investigated. The difference is that an equal amount of free GSH (with the same GSH content as that encapsulated in drug-loaded vesicles) is mixed with unloaded biomimetic vesicles (prepared in Example 8) and co-incubated with B16-F10 cells. The effect on melanocytes is investigated by observing changes in intracellular melanin content. Since GSH exists in free form and is not loaded with the hydrophilic core of the membrane protein biomimetic vesicles, it can only enter the cell through diffusion, which is inefficient and easily diluted or degraded by the extracellular environment. The limited efficiency of GSH entering the cell results in a weak melanin inhibition effect (92.36%), which is close to that of the group that only added free GSH.

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

Claims

1. A method for preparing drug-loaded membrane protein biomimetic vesicles, characterized in that, Includes the following steps: (1) Preparation of organic phase: Soybean lecithin and cholesterol are mixed and dissolved to obtain organic phase; (2) Preparation of aqueous phase 1: The membrane protein was dissolved in PBS buffer solution to obtain aqueous phase 1; (3) Preparation of aqueous phase 2: Dissolve the water-soluble drug in PBS buffer solution to obtain aqueous phase 2; (4) The organic phase, aqueous phase 1 and aqueous phase 2 are mixed through a three-channel microfluidic chip to obtain the biomimetic vesicle; 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; In step (4), the flow rate ratio FRR is 1:1.5:1.5 and the total flow rate TFR is 100~1000μl / min, which is passed through a three-channel microfluidic chip; the flow rate ratio is the volumetric flow rate of the left flow channel: the volumetric flow rate of the middle flow channel: the volumetric flow rate of the right flow channel. In the organic phase described in step (1), the molar ratio of soybean lecithin to cholesterol is (5~9):3; the total concentration of soybean lecithin and cholesterol is 2.5~25 mg / ml; and soybean lecithin and cholesterol are dissolved in ethanol. In step (2), the concentration of membrane protein in aqueous phase 1 is 60 μg / ml; In step (3), the water-soluble drug in the aqueous phase 2 is glutathione; the concentration of the water-soluble drug is 1~20 mg / mL. The membrane protein mentioned in step (2) is a keratinocyte membrane protein.

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