A keratinocyte membrane protein biomimetic liposome based on microfluidic technology, a preparation method thereof and a whitening skin care application

Abstract

The application discloses a kind of keratinocyte membrane protein liposome based on microfluidic technology and its preparation method and whitening skin care application, belong to cosmetic technical field.The application provides a kind of keratinocyte membrane protein liposome based on microfluidic technology and its preparation method and application, its operation process is simple, without large equipment, less addition, preparation process is simple and environment-friendly.The liposome can significantly inhibit melanin transport, and in the co-culture test of melanocyte and keratinocyte, the fluorescence intensity of melanosome in keratinocyte is reduced by 3.5 times, which shows that the liposome can effectively inhibit the accumulation of melanin in skin keratinocyte, and provides a new solution for achieving skin health targeted whitening.

Description

Technical Field

[0001] This invention relates to a microfluidic biomimetic liposome of keratinocyte membrane protein, its preparation method, and its whitening and skin care application, belonging to the field of cosmetic technology. Background Technology

[0002] Asian skin naturally has a higher level of hydration, making it more prone to pigmentation problems, both hyperpigmentation and hypopigmentation. Furthermore, skin color often becomes uneven with age. It is estimated that about 15% of the world's population invests in skin whitening products, with the vast majority of these being Asian. Skin whitening is not simply about whitening the skin, but about preventing and eliminating excessive pigmentation. Excessive pigmentation, especially in Asian populations with yellow undertones, can directly lead to noticeable changes in skin tone, such as dullness, yellowing, and opacity. Skin pigmentation is primarily due to the accumulation of melanin granules in keratinocytes, a process involving both melanin production and transport. Most existing whitening products focus on inhibiting melanin production, but their effects are often unsatisfactory, and whitening ingredients such as azelaic acid and hydroquinone can cause significant side effects. Addressing the issue of melanin transport is another angle to achieve whitening effects; if existing melanin cannot be transported smoothly, it will not cause a visual "darker" appearance. Therefore, achieving healthy and safe whitening by inhibiting melanin production is essential; however, currently, there is a lack of targeted inhibition solutions for this purpose. Summary of the Invention

[0003] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0004] This invention is based on the fact that keratinocytes are receptor cells for melanin secretion by melanocytes. Therefore, microfluidic technology is used to embed keratinocyte membrane proteins into a phospholipid bilayer to mimic keratinocytes. This biomimetic strategy can maintain the inherent biological characteristics and functions of keratinocytes as receptor cells for melanin transport. Utilizing these biological characteristics and functions, keratinocyte membrane protein biomimetic liposomes can participate in the melanin transport process in the skin. First, due to the presence of adhesion proteins, the keratinocyte membrane protein biomimetic liposomes adhere to the periphery of keratinocytes, forming a protective barrier and reducing direct contact between melanocytes and keratinocytes. Second, when melanocytes begin to transport melanin to keratinocytes, the dendritic processes of melanocytes can recognize the keratinocyte membrane protein biomimetic liposomes adhering to the periphery of keratinocytes and detach vesicles loaded with melanosomes. Because the particle size of keratinocyte membrane protein biomimetic liposomes is much smaller than that of melanosome-loaded vesicles, a ring of keratinocyte membrane protein biomimetic liposomes surrounds the melanosome-loaded vesicles. When these liposomes come into contact with keratinocytes, they are recognized as homologous keratinocyte membrane protein biomimetic liposomes. Based on cellular uptake results, keratinocytes significantly reduce their uptake of keratinocyte membrane protein biomimetic liposomes. Therefore, keratinocytes reduce their uptake of melanosome-loaded vesicles that are recognized as keratinocyte membrane protein biomimetic liposomes, thereby inhibiting melanosome transport.

[0005] This invention provides a biomimetic liposome based on microfluidic technology for keratinocyte membrane proteins. The biomimetic liposome comprises keratinocyte membrane proteins and a phospholipid bilayer; the phospholipid bilayer is used as an ethanol solution as the inner phase and the keratinocyte membrane protein is used as a PBS buffer solution as the outer phase.

[0006] In one embodiment, the phospholipid bilayer comprises soybean lecithin and cholesterol, wherein the molar ratio of soybean lecithin to cholesterol is (3-2):1.

[0007] In one embodiment, the mass ratio of the keratinocyte membrane protein to the total lipids is 1:(50-500).

[0008] In one embodiment, the keratinocytes include, but are not limited to, human immortalized keratinocytes.

[0009] In one embodiment, the preparation method of the human keratinocyte membrane protein is as follows: Culture approximately 20-40 million immortalized keratinocytes, digest the cells with a cell digestion solution containing EDTA but without trypsin, collect by centrifugation, and wash the cells with ice-cold PBS. Using a cell membrane protein and plasma protein extraction kit (Beyotime), add 1 ml of membrane protein extraction reagent A (pre-added with PMSF) to approximately 40 million immortalized keratinocytes, fully suspend the cells, and incubate on ice for 10 minutes. Transfer the cell suspension to a 2 ml ice-cold glass homogenizer, homogenize until the cells are fully lysed, centrifuge at 700g for 10 minutes at 4°C, and collect the supernatant. Centrifuge at 14000rpm for 30 minutes at 4°C, collect the precipitate, add 400 μL of membrane protein extraction reagent B, vortex for 5 seconds, and incubate on ice for 10 minutes. Repeat this process three times. Subsequently, centrifuge at 14000g for 5 minutes at 4°C, collect the supernatant, which is the membrane protein solution, and store at -80°C.

[0010] In one embodiment, the membrane protein solution was measured using a BCA protein concentration assay kit (Beyotime).

[0011] The present invention also provides a method for preparing biomimetic liposomes of keratinocyte membrane proteins based on microfluidic technology.

[0012] In one implementation, the method includes the following steps:

[0013] Extraction of human keratinocyte membrane proteins: The membrane protein solution was extracted from cultured immortalized human keratinocytes according to the protocol of the cell membrane protein and plasma protein extraction kit (Beyotime) and stored at -80℃.

[0014] Preparation of biomimetic liposomes of keratinocyte membrane proteins: Soybean lecithin and cholesterol were dissolved in anhydrous ethanol to form an organic phase; the membrane protein solution was diluted with 1:1 PBS to form an aqueous phase. The organic phase was used as the internal phase and the aqueous phase as the external phase. The solution was passed through a fluid focusing microfluidic chip with specific TFR and FRR. The collected solution was dialyzed overnight at 4°C to obtain biomimetic liposomes of keratinocyte membrane proteins, which were then stored at 4°C.

[0015] In a preferred embodiment of the present invention, the fluid focusing microfluidic chip has a TFR of 100–1000 μl / min and an FRR of 3:1–6:1.

[0016] In a preferred embodiment of the present invention, the dialysis is performed overnight, wherein the molecular weight cutoff of the dialysis bag is 30-300 kDa.

[0017] In one embodiment, the molar ratio of soybean lecithin to cholesterol in step (1) is (3-2):1; and the concentration of the organic phase is 5-10 mg / ml.

[0018] The present invention also provides the application of the biomimetic liposomes in the preparation of products that inhibit melanin transfer in the skin and treat hyperpigmentation of the skin.

[0019] In one embodiment, the product includes cosmetics and pharmaceuticals.

[0020] This invention utilizes microfluidic technology to embed human keratinocyte membrane proteins into a liposome phospholipid bimolecule, forming a biomimetic liposome. This allows for the construction of a biomimetic targeted nanoparticle for inhibiting melanin transfer in the skin and treating hyperpigmentation. The invention constructs a microfluidic-based biomimetic liposome of keratinocyte membrane protein. After being delivered into the living epidermis via microneedles, this biomimetic liposome effectively remains in the living epidermal layer due to the presence of intercellular adhesion proteins, significantly increasing the drug retention rate. Simultaneously, leveraging the inherent biological characteristics of keratinocytes, the biomimetic liposome can preemptively bind to target sites involved in melanin transport within melanocytes, inhibiting melanin transport pathways in keratinocytes through a competitive mechanism. This allows the carrier itself to inhibit melanosome transport. When combined with cosmetic active ingredients such as vitamin C, arbutin, and niacinamide, which treat skin pigmentation through different mechanisms, a synergistic effect can be achieved.

[0021] Beneficial effects:

[0022] This invention provides a microfluidic biomimetic liposome of keratinocyte membrane protein, its preparation method and application. The operation process is simple, requires no large equipment, requires few additives, and the preparation process is simple and environmentally friendly.

[0023] This invention utilizes a fluid focusing microfluidic chip to enable the self-assembly of phospholipids and membrane proteins within the chip channels, thereby obtaining biomimetic liposomes with a particle size of 73.83 nm and a polydispersity index of 0.189. These liposomes exhibit low cytotoxicity and good biocompatibility. In co-culture of keratinocytes and melanocytes, the keratinocyte membrane protein biomimetic liposomes prepared in this invention can inhibit the transport of melanin to keratinocytes, reducing melanin transport by 3.5 times.

[0024] The membrane protein in this invention is a membrane protein from immortalized keratinocytes. It is embedded within a liposome phospholipid bilayer using a fluid focusing microfluidic chip to form biomimetic nanoliposomes. Keratinocytes are receptor cells for melanin transport by melanocytes. Their cell membranes are equipped with a complex series of surface receptors that enable them to respond to melanocyte biological signals. Therefore, the keratinocyte membrane protein biomimetic liposomes can effectively adhere to the vicinity of melanosome-loaded vesicles and bind to ligands associated with melanosome transport, thereby reducing the number of melanosomes entering the keratinocyte. Furthermore, the presence of cell surface adhesion proteins increases the retention rate of the keratinocyte membrane protein biomimetic liposomes in the living epidermis, thus significantly increasing the bioavailability of the keratinocyte membrane protein biomimetic liposomes and reducing in vivo toxicity.

[0025] The keratinocyte membrane protein liposomes prepared in this invention have a melanosome transport blocking effect on their carriers. When combined with melanin production inhibitors and melanin transport inhibitors, they can have a synergistic effect, thereby increasing the therapeutic effect on skin pigmentation. Attached Figure Description

[0026] Figure 1 This is a structural diagram of a PDMS microfluidic chip.

[0027] Figure 2 This is a particle size and potential diagram of the biomimetic liposomes of keratinocyte membrane proteins of the present invention.

[0028] Figure 3 This is a polyacrylamide gel electrophoresis (SDS-PAGE) image of the human immortalized keratinocyte membrane protein and the keratinocyte membrane protein biomimetic liposomes extracted in this invention.

[0029] Figure 4 This is a SEM image of the biomimetic liposomes of keratinocyte membrane proteins of the present invention.

[0030] Figure 5 This is a Cryo-TEM image of the biomimetic liposomes of keratinocyte membrane proteins of the present invention.

[0031] Figure 6 This is a diagram illustrating the cytotoxicity of the biomimetic liposomes containing keratinocyte membrane proteins of the present invention.

[0032] Figure 7 This is a flow cytometry diagram showing the cellular uptake of the keratinocyte membrane protein biomimetic liposomes of the present invention.

[0033] Figure 8 This is an in vitro pharmacodynamic flow cytometry result of the biomimetic liposomes of keratinocyte membrane proteins of the present invention inhibiting melanosome transport in a co-culture model. Detailed Implementation

[0034] 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.

[0035] 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.

[0036] 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.

[0037] Extraction of membrane proteins from immortalized human keratinocytes in Examples 1-3

[0038] Harvested healthy human immortalized keratinocytes (HaCaT) were digested with a cell digestion solution containing EDTA but without trypsin. The cells were collected by centrifugation and washed with ice-cold PBS. Using a cell membrane and plasma protein extraction kit (Beyotime), membrane protein extraction reagent A (contained with benzyl sulfonyl fluoride before use) was added to approximately 40 million human immortalized 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 homogenate was then centrifuged at 700g for 10 minutes at 4°C, and the supernatant was collected. The mixture was then centrifuged at 14000rpm for 30 minutes at 4°C, and the precipitate was collected. 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.

[0039] The extraction process of human immortalized keratinocyte membrane proteins in the embodiments is shown in Table 1:

[0040] Table 1

[0041]

[0042] Example 4-13 Preparation of biomimetic liposomes of keratinocyte membrane proteins

[0043] A certain amount of soybean lecithin and cholesterol were mixed and dissolved in anhydrous ethanol to prepare an organic phase with a total lipid concentration of 10 mg / ml. A certain amount of keratinocyte membrane protein solution (prepared in Example 2) was dissolved in 1×PBS buffer to prepare an aqueous phase with a protein-to-total lipid mass concentration ratio of 1:(100-300). Using the aqueous phase as the external phase and the organic phase as the internal phase, the solution was passed through a fluid focusing microfluidic chip (Wuhan Jianmi Intelligent Control Technology Co., Ltd.) at a certain total flow rate (TFR) and flow rate ratio (aqueous phase flow rate / organic phase flow rate = FRR). The resulting solution was dialyzed overnight using a 300 kDa dialysis bag to obtain keratinocyte membrane protein biomimetic liposomes.

[0044] Examples 4-6 show that as the ratio of soybean lecithin to cholesterol increases, the particle size of the prepared liposomes first decreases and then increases, reaching 181.4 nm, 120.3 nm, and 130.1 nm respectively, while the polydispersity index decreases to 0.192, 0.179, and 0.181 respectively. Examples 5, 7, and 8 show that as the total liposome filtration rate (TFR) increases, the particle size of the prepared liposomes decreases to 120.3 nm, 97.6 nm, and 57.2 nm respectively, while the polydispersity index slightly increases to 0.179, 0.184, and 0.197 respectively. Based on the smaller particle size and lower polydispersity index, Example 8 is preferred as the preferred process for membrane protein intercalation.

[0045] Table 2. Preparation process conditions of biomimetic liposomes for keratinocyte membrane proteins in Examples 4-13.

[0046]

[0047] The performance characterization of keratinocyte membrane protein biomimetic liposomes based on microfluidic technology in this invention:

[0048] 1. Characterization of particle size and potential of biomimetic liposomes of keratinocyte membrane proteins

[0049] The particle size, polydispersity index, and zeta potential of liposomes without membrane proteins (prepared in Example 8) and HCMP-liposomes with different concentrations of membrane proteins (prepared in Examples 9-11) were determined using a zeta potential and nanoparticle size analyzer. The results are as follows: Figure 2The results showed that with the incorporation of membrane proteins and the increase in membrane protein concentration, the average particle size of the prepared keratinocyte membrane protein biomimetic liposomes increased, the Zeta potential first decreased and then increased, and the polydispersity index did not change significantly, indirectly indicating that the membrane proteins were successfully embedded. Since the Zeta potential of the biomimetic liposomes should decrease with increasing membrane protein incorporation, the Zeta potential of the keratinocyte membrane protein biomimetic liposome with a membrane protein concentration of 0.1 mg / ml (prepared in Example 9) was higher than that of the keratinocyte membrane protein biomimetic liposome with a membrane protein concentration of 0.05 mg / ml (prepared in Example 10), indicating that the membrane protein concentration of 0.1 mg / ml was supersaturated. To avoid membrane protein waste, the preferred membrane protein concentration was 0.05 mg / ml keratinocyte membrane protein biomimetic liposomes (prepared in Example 10), with a particle size of 73.83 nm and a polydispersity index of 0.189.

[0050] 2. Characterization of keratinocyte membrane protein biomimetic liposome polyacrylamide gel electrophoresis (SDS-PAGE) images

[0051] SDS-PAGE was used to detect total proteins, plasma proteins, membrane proteins, and biomimetic liposomes containing keratinocyte membrane proteins at different concentrations (prepared in Examples 8-11). Human immortalized keratinocytes and biomimetic liposomes containing keratinocyte membrane proteins at different concentrations were first lysed with cell lysis buffer, quantified using BCA protein assays, and then subjected to gel electrophoresis. The results showed that the bands of human immortalized keratinocyte membrane proteins and cytoplasmic proteins were significantly different, and their superposition resembled the total protein of human immortalized keratinocytes, indicating successful extraction of human immortalized keratinocyte membrane proteins. The protein bands in the biomimetic liposomes containing keratinocyte membrane proteins at different concentrations were basically consistent with the cell membrane protein bands, indicating successful membrane protein embedding. Figure 3 As shown.

[0052] 3. Characterization of keratinocyte membrane protein biomimetic liposomes by scanning electron microscopy (SEM)

[0053] The microstructure of keratinocyte membrane protein biomimetic liposomes (prepared in Example 10) and membrane-free protein liposomes (prepared in Example 8) was characterized using field emission scanning electron microscopy. A certain amount of trehalose was added to the liposome formulation as a lyophilization protectant to prepare a 100 mM trehalose liposome solution. This solution was then rapidly frozen with liquid nitrogen and lyophilized in a freeze dryer. After lyophilization, the lyophilized sample was coated onto a conductive adhesive and observed under a scanning electron microscope.

[0054] See results Figure 4The results showed that the keratinocyte membrane protein biomimetic liposomes and membrane-free protein liposomes prepared by microfluidic technology were spherical and had clear edges. Although their size differed from the results measured by zeta potential and nanoparticle size analyzer, this may be due to the destruction of liposome structure and aggregation during the liquid nitrogen freezing and freeze-drying process.

[0055] 4. Characterization of keratinocyte membrane protein biomimetic liposomes by cryo-transmission electron microscopy (Cryo-TEM)

[0056] The microstructure of keratinocyte membrane protein biomimetic liposomes (prepared in Example 10) and membrane-free protein liposomes (prepared in Example 8) was characterized by cryo-transmission electron microscopy.

[0057] See results Figure 5 The results showed that the biomimetic liposomes of keratinocyte membrane proteins were spherical and all were monolayered vesicles, while the liposomes without membrane proteins were slightly irregularly spherical and contained a small number of multilayered vesicles. The sizes were consistent with the particle size data measured by the zeta potential and nanoparticle size analyzer.

[0058] 5. Cytotoxicity study of biomimetic liposomes containing keratinocyte membrane proteins

[0059] The cytotoxicity of biomimetic liposomes and protein-free liposomes to human immortalized keratinocytes (HaCaT cells), human melanoma cells (MNT-1 cells), and a co-culture model of the two cells (HaCaT and MNT-1 in a 1:1 ratio) was investigated using the CCK-8 assay. HaCaT and MNT-1 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 biomimetic liposomes (prepared in Example 10) and protein-free liposomes (prepared in Example 8) at concentrations of 0, 100, 500, and 1000 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. The blank wells contained CCK-8 but no cells and liposomes / keratinocyte membrane protein biomimetic liposomes. The control wells contained cells and CCK-8 but no liposomes / keratinocyte membrane protein biomimetic liposomes.

[0060] Cell viability % = [(Experimental wells - Blank wells) / (Control wells - Blank wells)] x 100

[0061] The results are as follows Figure 6As shown, the results indicated that, compared with the untreated control wells, neither the keratinocyte membrane protein biomimetic liposomes nor the non-membrane protein liposomes exhibited significant cytotoxicity at any concentration. Furthermore, the keratinocyte membrane protein biomimetic liposomes showed higher proliferative activity in HaCaT cells, MNT-1 cells, and the co-culture model. This may be due to the better biocompatibility of the keratinocyte membrane protein biomimetic liposomes. Since 500 μg / ml of liposomes / keratinocyte membrane protein biomimetic liposomes showed the highest proliferative activity, 500 μg / ml was selected as the concentration for subsequent experiments.

[0062] 6. Investigation on cellular uptake of keratinocyte membrane protein by biomimetic liposomes

[0063] To determine the differences in liposome uptake and keratinocyte membrane protein biomimetic liposome uptake between HaCaT and MNT-1 cells, HaCaT and MNT-1 cells were respectively subjected to 5 × 10⁻⁶ ppm. 5 Cells were seeded at a density of 100 cells / well in 6-well plates. After 24 hours, the cells were washed twice with PBS (pH 7.4) and incubated for 4 hours at 37°C with 1 mL of DMEM solution containing 0.5 mg / mL Rhodamine B-labeled keratinocyte membrane protein biomimetic liposomes / liposomes (prepared in Examples 12 and 13) for keratinocyte membrane protein biomimetic liposomes. The culture medium was removed, and the cells were washed three times with fresh PBS (pH 7.4) and treated with 500 μL of 500 μg / mL trypsin in calcium- and magnesium-free PBS for 2 minutes. DMEM (1 mL) supplemented with 10% FBS was added to the wells to quench the trypsin, and the cells were recovered and centrifuged at 1000 rpm for 5 minutes. The cell pellet was resuspended in PBS (pH 7.4), washed twice with the same buffer, and recovered by centrifugation at 1000 rpm for 5 minutes. The cell sample was resuspended in 300 μL of PBS (pH 7.4) and analyzed by flow cytometry.

[0064] The results are as follows Figure 7 As shown, the results indicated that, compared to liposomes, HaCaT cells significantly reduced their uptake of keratinocyte membrane protein biomimetic liposomes, with fluorescence intensity decreasing from 2578 to 1044, a decrease of 2.47 times; MNT-1 cells significantly increased their uptake of keratinocyte membrane protein biomimetic liposomes, with fluorescence intensity increasing from 1995.67 to 5301.33, an increase of 2.66 times; the uptake (fluorescence intensity) of keratinocyte membrane protein biomimetic liposomes by MNT-1 cells was 5.1 times that of HaCaT cells, demonstrating that the prepared keratinocyte membrane protein biomimetic liposomes have significant targeting ability for melanocytes.

[0065] 7. In vitro efficacy study of keratinocyte membrane protein biomimetic liposomes inhibiting melanosome transport in a co-culture model.

[0066] To quantitatively measure melanosome transport, HaCaT cells and MNT-1 cells were seeded in six-well plates at a ratio of 2:1, with a density of 5 × 10⁶ cells / well. 5 Cells / well. The culture medium consisted of HaCaT medium and MNT-1 medium in a 2:1 ratio. After culturing for 24 hours, the cells were washed twice with PBS at pH 7.4 and incubated at 37°C for 24 hours with 1 mL of 500 μg / ml keratinocyte membrane protein biomimetic liposomes (prepared in Example 10). The co-cultured cells were harvested, washed with cold PBS, fixed in 4% paraformaldehyde for 10 minutes, and washed with PBS containing 0.1% Triton-X100 for 5 minutes. MNT-1 cells were immunostained with PMEL 17 rabbit monoclonal antibody (Beyotime, AG8635) and goat anti-rabbit IgG (H+L) labeled with Alexa Fluor 488 (Beyotime, A0423). HaCaT cells were incubated with anti-pan-cytokeratin mouse recombinant polyclonal antibody (abcam, ab86734) and goat anti-mouse IgG (H+L) labeled with Alexa Fluor 647 (Beyotime, A0473). The stained cells were analyzed by flow cytometry, and a total of 10,000 cells were collected on the flow cytometer.

[0067] The results are as follows Figure 8 As shown, the results indicated that, compared with the untreated control wells, the fluorescence intensity of Alexa Fluor 488, representing melanosomes, in the co-cultured cell model treated with keratinocyte membrane protein biomimetic liposomes was significantly reduced, from 13649 to 3917, a decrease of 3.5 times, indicating that keratinocyte membrane protein biomimetic liposomes can inhibit the transport of melanin to skin cells.

[0068] Comparative Example 1: Effect of total lipid concentration on keratinocyte membrane protein biomimetic liposomes

[0069] Soybean lecithin and cholesterol were mixed and dissolved in anhydrous ethanol at a molar ratio of 7:3 to prepare the organic phase, with a total lipid concentration of 5 mg / ml. A 1:1 PBS buffer without added keratinocyte membrane proteins was used 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 a 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 liposomes. Due to the low lipid concentration, the obtained liposomes had a small particle size of 57.6 nm and an excessively high polydispersity index of 0.271.

[0070] Comparative Example 2: Effect of total lipid concentration on keratinocyte membrane protein biomimetic liposomes

[0071] Soybean lecithin and cholesterol were mixed and dissolved in anhydrous ethanol at a molar ratio of 7:3, resulting in a total lipid concentration of 20 mg / ml, which served as the organic phase. A 1:1 PBS buffer without added keratinocyte membrane proteins was used 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 a 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 liposomes. Due to the excessively high lipid concentration, the obtained liposomes had an excessively large particle size of 207.4 nm, a significantly increased polydispersity index of 0.352, and exhibited aggregation.

[0072] Comparative Example 3: Effect of total lipid concentration on keratinocyte membrane protein biomimetic liposomes

[0073] Soybean lecithin and cholesterol were mixed and dissolved in anhydrous ethanol at a molar ratio of 7:3 to prepare the organic phase, with a total lipid concentration of 10 mg / ml. A 1:1 PBS buffer without added keratinocyte membrane proteins was used 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 a 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 8:1. The resulting solution was dialyzed overnight using a 300 kDa dialysis bag to obtain liposomes. Due to the excessively high flow rate ratio, laminar diffusion could not be formed within the chip channels, making it difficult to prepare liposomes.

[0074] Comparative Example 4: Effects of TFR on biomimetic liposomes of keratinocyte membrane proteins

[0075] Soybean lecithin and cholesterol were mixed and dissolved in anhydrous ethanol at a molar ratio of 7:3 to prepare the organic phase, with a total lipid concentration of 10 mg / ml. A 1:1 PBS buffer without added keratinocyte membrane proteins was used 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 a fluid focusing microfluidic chip (Wuhan Jianmi Intelligent Control Technology Co., Ltd.) at a total flow rate (TFR) of 2 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 liposomes. Due to the excessively high total flow rate, the chip could not withstand the pressure and leaked.

[0076] Comparative Example 5: Effects of lipid types on biomimetic liposomes of keratinocyte membrane proteins

[0077] Soybean lecithin, cholesterol, and dioleoylphosphatidylethanolamine (DOPE) in a molar ratio of 7:3:1 were dissolved in anhydrous ethanol to prepare an organic phase with a total lipid concentration of 10 mg / ml. A 1:1 PBS buffer with a keratinocyte membrane protein concentration of 0.05 mg / ml was added as the aqueous phase. Using the aqueous phase as the external phase and the organic phase as the internal phase, the solution was passed through a 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 biomimetic liposomes of keratinocyte membrane proteins. Because DOPE is a cationic lipid, its self-assembly during diffusion is restricted by binding to negatively charged groups in the membrane protein, leading to aggregation of the resulting biomimetic liposomes of keratinocyte membrane proteins, with a polydispersity index of 0.41.

[0078] Comparative Example 6: Effects of lipid types on biomimetic liposomes of keratinocyte membrane proteins

[0079] Soybean lecithin, cholesterol, and dioleoylphosphatidylcholine (DOPC) in a molar ratio of 7:3:1 were dissolved in anhydrous ethanol to prepare an organic phase with a total lipid concentration of 10 mg / ml. A 1:1 PBS buffer with a keratinocyte membrane protein concentration of 0.05 mg / ml was added to prepare an aqueous phase. Using the aqueous phase as the outer phase and the organic phase as the inner phase, the solution was passed through a 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 biomimetic liposomes of keratinocyte membrane proteins. The obtained biomimetic liposomes of keratinocyte membrane proteins had a high zeta potential of -16.9 mV, which is unfavorable for membrane protein intercalation into the phospholipid bilayer.

[0080] Comparative Example 7: Effect of cell membrane protein concentration on keratinocyte membrane protein biomimetic liposomes

[0081] Soybean 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, forming the organic phase. A 1:1 PBS buffer solution with a keratinocyte membrane protein concentration of 0.2 mg / ml was added to form the aqueous phase. Using the aqueous phase as the external phase and the organic phase as the internal phase, the solution was passed through a 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 keratinocyte membrane protein biomimetic liposomes. Due to the excessively 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.

[0082] Comparative Example 8: Effect of cell membrane protein concentration on keratinocyte membrane protein biomimetic liposomes

[0083] Soybean 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, forming the organic phase. A 1:1 PBS buffer solution with a keratinocyte membrane protein concentration of 0.01 mg / ml was added to form the aqueous phase. Using the aqueous phase as the external phase and the organic phase as the internal phase, the solution was passed through a 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 keratinocyte membrane protein biomimetic liposomes. Due to the low concentration of cell membrane proteins, the zeta potential of the prepared keratinocyte membrane protein biomimetic liposomes was very similar to that of liposomes without membrane proteins, indicating low surface protein density and poor biological activity.

[0084] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone 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 biomimetic liposome, characterized in that, The biomimetic liposome comprises keratinocyte membrane proteins and a phospholipid bilayer; with the phospholipid bilayer as the inner phase and the keratinocyte membrane proteins as the outer phase; The method for preparing the biomimetic liposomes 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: Dissolve the keratinocyte membrane proteins to obtain an aqueous phase; (3) Using the aqueous phase obtained in step (2) as the external phase and the organic phase obtained in step (1) as the internal phase, biomimetic liposomes are obtained through a fluid focusing microfluidic chip; The total flow rate (TFR) through the fluid focusing microfluidic chip is 0.1~1 ml / min, and the flow rate ratio (FRR) of the aqueous phase / organic phase is (3~6):1; In step (1), the molar ratio of soybean lecithin to cholesterol is (3~2):1; the concentration of the organic phase is 10 mg / ml. The concentration of the aqueous phase in step (2) is 0.033~0.1 mg / ml.

2. The biomimetic liposome as described in claim 1, characterized in that, The mass ratio of the keratinocyte membrane protein to the phospholipid bilayer is 1:(50~500).

3. The biomimetic liposome as described in claim 2, characterized in that, The phospholipid bilayer comprises soybean lecithin and cholesterol, with a molar ratio of soybean lecithin to cholesterol of (3~2):

1.

4. The biomimetic liposome as described in claim 3, characterized in that, The keratinocytes include, but are not limited to, human immortalized keratinocytes.

5. The use of the biomimetic liposomes according to any one of claims 1 to 4 in the preparation of products that inhibit the transfer of melanin in the skin.

6. The application as described in claim 5, characterized in that, The products include cosmetics and pharmaceuticals.

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