Keratinocyte membrane protein bionic liposome based on microfluidic technology, as well as preparation method therefor and skin whitening and skin care application thereof

Abstract

The present invention provides a keratinocyte membrane protein bionic liposome based on the microfluidic technology, as well as a preparation method therefor and skin whitening and skin care application thereof. The bionic liposome comprises keratinocyte membrane protein and a phospholipid bilayer, with the phospholipid bilayer as the internal phase and the keratinocyte membrane protein as the external phase. The preparation method of the bionic liposome comprises the following steps: (1) preparing an organic phase: mixing and dissolving soybean lecithin and cholesterol to obtain an organic phase; (2) preparing an aqueous phase: dissolving keratinocyte membrane protein to obtain an aqueous phase; (3) taking the aqueous phase obtained in the step (2) as an external phase, taking the organic phase obtained in the step (1) as an internal phase, and focusing a microfluidic chip by means of a fluid to obtain the bionic liposome.

Description

A microfluidic-based keratinocyte membrane protein biomimetic liposome and its preparation method and whitening skin care application Technical Field

[0001] The invention relates to a keratinocyte membrane protein biomimetic liposome based on microfluidic technology, a preparation method thereof, and whitening and skin care applications, belonging to the technical field of cosmetics. Background Art

[0002] Asians have naturally higher levels of skin hydration, making them more susceptible to hyperpigmentation or hypopigmentation. Furthermore, uneven skin coloration is common with aging. It is estimated that approximately 15% of the world's population uses skin-lightening products, with the majority of these individuals in Asia. Skin lightening isn't simply about whitening the skin, but rather preventing and eliminating excessive pigmentation. Excessive pigmentation, particularly in Asians with yellow skin, can directly lead to noticeable skin tone changes such as dullness, yellowing, and opacity. Skin pigmentation is primarily due to the accumulation of melanin granules in keratinocytes, a process that involves melanin production and transport. Existing whitening products mostly focus on inhibiting melanin production, but these results are often unsatisfactory, and whitening ingredients such as rhododendrol and hydroquinone can cause significant side effects. Inhibiting melanin transport offers another approach to achieving whitening results. If existing melanin cannot be transported smoothly, it won't cause visual "darkening." Therefore, achieving healthy and safe skin whitening through melanin inhibition is crucial, but currently, there is a lack of targeted solutions for this purpose.

[0003] Summary of the Invention

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

[0005] The present 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 phospholipid bilayers to simulate keratinocytes. This biomimetic strategy can maintain the biological characteristics and functions of keratinocytes as receptor cells for melanin transport. Utilizing this biological characteristic and function, keratinocyte membrane protein biomimetic liposomes can participate in the melanin transport process in the skin. First, due to the presence of adhesion proteins, keratinocyte membrane protein biomimetic liposomes will adhere to the periphery of keratinocytes, forming a protective barrier that reduces direct contact between melanocytes and keratinocytes; secondly, when melanocytes begin to transport melanin to keratinocytes, the dendritic processes of the melanocytes can recognize the keratinocyte membrane protein biomimetic liposomes adhered to the periphery of the keratinocytes and shed the vesicles loaded with melanosomes. Because the particle size of keratinocyte membrane protein biomimetic liposomes is much smaller than that of melanosome-loaded vesicles, the melanosome-loaded vesicles are surrounded by a ring of keratinocyte membrane protein biomimetic liposomes. When these vesicles come into contact with keratinocytes, they are recognized as homologous keratinocyte membrane protein biomimetic liposomes. Cellular uptake results show that keratinocytes significantly reduce their uptake of keratinocyte membrane protein biomimetic liposomes. Consequently, keratinocytes reduce their uptake of melanosome-loaded vesicles, which are recognized as keratinocyte membrane protein biomimetic liposomes, thereby inhibiting melanosome transport.

[0006] The invention provides a keratinocyte membrane protein biomimetic liposome based on microfluidic technology. The biomimetic liposome comprises keratinocyte membrane protein and a phospholipid bilayer; an ethanol solution of the phospholipid bilayer is used as an inner phase, and a PBS buffer solution of the keratinocyte membrane protein is used as an outer phase.

[0007] In one embodiment, the phospholipid bilayer comprises soybean lecithin and cholesterol, and the molar ratio of soybean lecithin to cholesterol is (3-2):1.

[0008] In one embodiment, the mass ratio of the keratinocyte membrane protein to total lipids is 1:(50-500).

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

[0010] In one embodiment, the method for preparing human keratinocyte membrane protein comprises the following steps: culturing approximately 20-40 million human immortalized keratinocytes, digesting the cells with a cell digestion solution containing EDTA but without trypsin, collecting the cells by centrifugation, and washing the cells with ice-cold PBS. Using a cell membrane protein and plasma protein extraction kit (Biyuntian), 1 ml of Membrane Protein Extraction Reagent A, supplemented with PMSF immediately prior to use, is added to approximately 40 million human immortalized keratinocytes. The cells are thoroughly suspended and then incubated on ice for 10 minutes. The cell suspension is transferred to a 2 ml ice-cold glass homogenizer and homogenized until the cells are fully disrupted. The suspension is then centrifuged at 700 g for 10 minutes at 4°C, and the supernatant is collected. The suspension is then centrifuged at 14,000 rpm 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, the suspension is centrifuged at 14,000 g for 5 minutes at 4°C, and the supernatant is collected as the membrane protein solution and stored at -80°C.

[0011] In one embodiment, the membrane protein solution is assayed using a BCA protein concentration assay kit (Beyotime).

[0012] The present invention also provides a method for preparing keratinocyte membrane protein biomimetic liposomes based on microfluidic technology.

[0013] In one embodiment, the method comprises the following steps:

[0014] 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 (Biyuntian) and stored at -80°C.

[0015] Preparation of keratinocyte membrane protein biomimetic liposomes: Soy lecithin and cholesterol were dissolved in anhydrous ethanol to form an organic phase. The membrane protein solution was diluted with 1× PBS to form an aqueous phase. The organic phase served as the inner phase, and the aqueous phase as the outer phase. The collected solution was dialyzed overnight at 4°C to obtain keratinocyte membrane protein biomimetic liposomes, which were then stored at 4°C.

[0016] As a preferred solution of the present invention, wherein: the fluid focusing microfluidic chip, wherein TFR = 100 ~ 1000 μl / min, FRR = 3:1 ~ 6:1.

[0017] As a preferred embodiment of the present invention, the dialysis is performed overnight, wherein the molecular weight cut-off of the dialysis bag is 30 to 300 kDa.

[0018] 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.

[0019] The present invention also provides the use of the bionic liposome in preparing a product for inhibiting skin melanin transfer and treating skin hyperpigmentation.

[0020] In one embodiment, the product includes cosmetics and pharmaceuticals.

[0021] The present invention utilizes microfluidics to embed human keratinocyte membrane proteins into liposome phospholipid bimolecules to form biomimetic liposomes, thereby constructing a biomimetic targeted nanoparticle for inhibiting skin melanin transfer and treating skin hyperpigmentation. The present invention constructs a keratinocyte membrane protein biomimetic liposome based on microfluidics. After the biomimetic liposome is delivered into the living epidermis via microneedles, it can effectively remain in the living epidermis due to the presence of intercellular adhesion proteins, thereby significantly increasing the retention rate of the drug in the living epidermis. At the same time, utilizing the inherent biological characteristics of keratinocytes, the keratinocyte membrane protein biomimetic liposome can preemptively bind to targets involved in transport in melanocytes, inhibiting the pathway involved in melanin transport in keratinocytes through a competitive mechanism, so that the carrier itself has the effect of inhibiting melanosome transport. If combined with cosmetic active ingredients such as vitamin C, arbutin, and niacinamide that treat skin pigmentation through different mechanisms, a synergistic effect can be achieved. Beneficial effects:

[0022] The present invention provides a keratinocyte membrane protein biomimetic liposome based on microfluidic technology, and a preparation method and application thereof. The operation process is simple, no large-scale equipment is required, little additive is required, and the preparation process is simple and environmentally friendly.

[0023] The present invention utilizes a fluid-focusing microfluidic chip to self-assemble phospholipids and membrane proteins within the chip channels, resulting in membrane protein biomimetic liposomes with a particle size of 73.83 nm and a polydispersity index of 0.189. These liposomes exhibit low cytotoxicity and excellent biocompatibility. In co-cultures of keratinocytes and melanocytes, the keratinocyte membrane protein biomimetic liposomes inhibited melanin transport into keratinocytes, reducing melanin transport by 3.5 times.

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

[0025] The keratinocyte membrane protein liposomes prepared by the present invention have a carrier that itself has a melanosome transport blocking effect. If used with an encapsulated melanin production inhibitor, a melanin transport inhibitor, etc., it can have a synergistic effect, thereby increasing the therapeutic effect on skin pigmentation. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0027] FIG2 is a graph showing the particle size and potential of the keratinocyte membrane protein biomimetic liposomes of the present invention.

[0028] FIG3 is a polyacrylamide gel electrophoresis (SDS-PAGE) diagram of human immortalized keratinocyte membrane proteins extracted in the present invention and keratinocyte membrane protein biomimetic liposomes.

[0029] FIG4 is a SEM image of the keratinocyte membrane protein biomimetic liposome of the present invention.

[0030] FIG5 is a Cryo-TEM image of the keratinocyte membrane protein biomimetic liposome of the present invention.

[0031] FIG6 is a graph showing the cytotoxicity of the keratinocyte membrane protein biomimetic liposomes of the present invention.

[0032] FIG. 7 is a flow cytometric analysis diagram of the cellular uptake of the keratinocyte membrane protein biomimetic liposomes of the present invention.

[0033] FIG8 is a flow cytometric graph showing the in vitro efficacy of the keratinocyte membrane protein biomimetic liposomes of the present invention in inhibiting melanosome transport in a co-culture model. DETAILED DESCRIPTION

[0034] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0035] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0036] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0037] Example 1-3 Extraction of human immortalized keratinocyte membrane proteins

[0038] Well-grown human immortalized keratinocytes (HaCaT) were digested with a cell digestion buffer containing EDTA but without trypsin, collected by centrifugation, and washed with ice-cold PBS. Using a membrane protein and plasma protein extraction kit (Beyotime), Membrane Protein Extraction Reagent A, supplemented with phenylmethylsulfonyl fluoride, was added to approximately 40 million human immortalized keratinocytes. The cells were thoroughly suspended and incubated on ice for 10 minutes. The cell suspension was transferred to an ice-cold glass homogenizer and homogenized until the cells were fully disrupted. The suspension was then centrifuged at 700 g for 10 minutes at 4°C, and the supernatant was collected. The suspension was then centrifuged at 14,000 rpm for 30 minutes at 4°C, and the precipitate was collected. Membrane Protein Extraction Reagent B was added, vortexed for 5 seconds, and then incubated on ice for 10 minutes. This process was repeated three times. The suspension was then centrifuged at 14,000 g for 5 minutes at 4°C, and the supernatant, the membrane protein solution, was collected and stored at -80°C.

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

[0040] Table 1

[0041] Example 4-13 Preparation of keratinocyte membrane protein biomimetic liposomes

[0042] A certain amount of soy lecithin and cholesterol were mixed and dissolved in anhydrous ethanol, and the total lipid concentration was 10 mg / ml to prepare an organic phase. A certain amount of keratinocyte membrane protein solution (prepared in Example 2) was dissolved in 1×PBS buffer to prepare an aqueous phase, and the mass concentration ratio of membrane protein to total lipid was 1: (100-300). The aqueous phase was used as the external phase and the organic phase as the internal phase. The total flow rate (TFR) and flow rate ratio (aqueous phase volume flow rate / organic phase volume flow rate = FRR) were passed through a fluid focusing microfluidic chip (Wuhan Jianmi Intelligent Control Technology Co., Ltd.). The resulting solution was dialyzed overnight with a 300kDa dialysis bag to obtain keratinocyte membrane protein biomimetic liposomes.

[0043] It can be concluded from Examples 4-6 that as the ratio of soy lecithin to cholesterol increases, the particle size of the prepared liposomes first decreases and then increases, and is 181.4 nm, 120.3 nm, and 130.1 nm, respectively, and the polydispersity index decreases, and is 0.192, 0.179, and 0.181, respectively; it can be concluded from Examples 5, 7, and 8 that as TFR increases, the particle size of the prepared liposomes decreases, and is 120.3 nm, 97.6 nm, and 57.2 nm, respectively, and the polydispersity index increases slightly, and is 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 embedding.

[0044] Table 2 Preparation process conditions of keratinocyte membrane protein biomimetic liposomes in Examples 4-13

[0045] The performance characterization of the keratinocyte membrane protein biomimetic liposomes based on microfluidic technology in the present invention is as follows:

[0046] 1. Characterization of particle size and potential of keratinocyte membrane protein biomimetic liposomes

[0047] The particle size, polydispersity index, and zeta potential of membrane protein-free liposomes (prepared in Example 8) and keratinocyte membrane protein-mimetic liposomes (HCMP-liposomes) containing varying membrane protein concentrations (prepared in Examples 9-11) were measured using zeta potential and nanoparticle size analysis. The results, shown in Figure 2, show that with the incorporation of membrane protein and an increase in membrane protein concentration, the average particle size of the prepared keratinocyte membrane protein-mimetic liposomes increased, the zeta potential initially decreased and then increased, and the polydispersity index remained unchanged, indirectly indicating successful membrane protein incorporation. Since the Zeta potential of the biomimetic liposomes should show a downward trend as the embedding of membrane proteins increases, the Zeta potential of the biomimetic liposomes containing keratinocyte membrane proteins (prepared in Example 9) with a membrane protein concentration of 0.1 mg / ml increases compared to the biomimetic liposomes containing keratinocyte membrane proteins (prepared in Example 10) with a membrane protein concentration of 0.05 mg / ml, indicating that the membrane protein concentration is 0.1 mg / ml and is oversaturated. To avoid waste of membrane proteins, the biomimetic liposomes containing keratinocyte membrane proteins (prepared in Example 10) with a membrane protein concentration of 0.05 mg / ml are preferred, and their particle size is 73.83 nm and the polydispersity index is 0.189.

[0048] 2. Characterization of keratinocyte membrane protein biomimetic liposome polyacrylamide gel electrophoresis (SDS-PAGE)

[0049] SDS-PAGE was used to detect human immortalized keratinocyte whole protein, plasma protein, membrane protein, and keratinocyte membrane protein biomimetic liposomes with different membrane protein concentrations (prepared in Examples 8-11). Human immortalized keratinocytes and keratinocyte membrane protein biomimetic liposomes with different membrane protein concentrations were first lysed with cell lysis buffer, and then subjected to gel electrophoresis after BCA protein quantification. The results showed that the human immortalized keratinocyte membrane protein and cytoplasmic protein bands were significantly different, and the superposition of the two was similar to the human immortalized keratinocyte whole protein, indicating that the human immortalized keratinocyte membrane protein was successfully extracted; the protein bands in the keratinocyte membrane protein biomimetic liposomes with different membrane protein concentrations were basically consistent with the cell membrane protein bands, indicating that the membrane protein was successfully embedded, as shown in Figure 3.

[0050] 3. Scanning electron microscopy (SEM) characterization of keratinocyte membrane protein biomimetic liposomes

[0051] The micromorphology 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 preparation as a lyoprotectant to a final concentration of 100 mM. This trehalose liposome solution was then flash-frozen with liquid nitrogen and lyophilized in a freeze dryer. After lyophilization, the lyophilized sample was applied to a conductive adhesive and observed under a scanning electron microscope.

[0052] The results are shown in Figure 4, which show that the keratinocyte membrane protein biomimetic liposomes and membrane-free protein liposomes prepared by microfluidic technology are spherical and have clear edges, although their sizes are different from the results measured by zeta potential and nanoparticle size analyzer. This may be due to the destruction of the liposome structure and aggregation during the liquid nitrogen quick freezing and freeze-drying processes.

[0053] 4. Characterization of Keratinocyte Membrane Protein Biomimetic Liposomes by Cryo-Transmission Electron Microscopy (Cryo-TEM)

[0054] The microscopic morphologies of keratinocyte membrane protein biomimetic liposomes (prepared in Example 10) and membrane-free protein liposomes (prepared in Example 8) were observed and characterized using cryo-transmission electron microscopy.

[0055] The results are shown in Figure 5. The results show that the keratinocyte membrane protein biomimetic liposomes are spherical and unilamellar vesicles, while the non-membrane protein liposomes have a slightly irregular spherical shape with a small number of multilamellar vesicles. The sizes of these liposomes are consistent with the particle size data measured by zeta potential and nanoparticle size analyzer.

[0056] 5. Cytotoxicity Study of Keratinocyte Membrane Protein Biomimetic Liposomes

[0057] The CCK-8 assay was used to investigate the cytotoxicity of keratinocyte membrane protein-mimetic liposomes and protein-free liposomes against immortalized human keratinocytes (HaCaT cells), human melanoma cells (MNT-1 cells), and a co-culture model of the two cells (HaCaT to MNT-1 ratio of 1:1). HaCaT and MNT-1 cells in the logarithmic phase were counted, diluted with culture medium, and plated onto 96-well plates. The cells were incubated at 37°C in a 5% CO2 atmosphere. After cell attachment, 100 μl of keratinocyte membrane protein-mimetic liposomes (prepared in Example 10) or membrane protein-free liposomes (prepared in Example 8) were added to the plates at concentrations of 0, 100, 500, and 1000 μg / ml, respectively. After 24 hours of incubation, 10 μl of CCK-8 solution was added to each well. After an additional 2 hours of incubation, the absorbance of each well was measured at 450 nm using a microplate reader. Set up blank and control wells. The blank wells contain culture medium containing CCK-8 but no cells or liposomes / keratinocyte membrane protein biomimetic liposomes, while the control wells contain culture medium containing cells and CCK-8 but no liposomes / keratinocyte membrane protein biomimetic liposomes. Cell viability % = [(experimental well - blank well) / (control well - blank well)] x 100

[0058] The results are shown in Figure 6, which show that compared with the untreated control wells, the keratinocyte membrane protein biomimetic liposomes and the membrane protein-free liposomes had no significant cytotoxicity at all concentrations, and the keratinocyte membrane protein biomimetic liposomes had a higher proliferation effect on HaCaT cells, MNT-1 cells and co-culture models. 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 had the highest proliferation effect on cells, 500 μg / ml was selected as the concentration for subsequent experiments.

[0059] 6. Investigation of cellular uptake of keratinocyte membrane protein biomimetic liposomes

[0060] In order to determine the difference in the uptake of liposomes and keratinocyte membrane protein biomimetic liposomes between HaCaT and MNT-1 cells, HaCaT and MNT-1 cells were cultured at 5×10 5The cells were seeded in a 6-well plate at a density of 10 cells / well. After 24 hours, the cells were washed twice with PBS at pH = 7.4 and incubated at 37°C for 4 hours 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). The culture medium was removed, the cells were washed three times with fresh PBS at pH = 7.4, and treated with 500 μL of 500 μg / mL trypsin in PBS without calcium and magnesium for 2 minutes. DMEM (1 mL) supplemented with 10% FBS was added to the wells to quench the trypsin, the cells were recovered, and the cells were centrifuged at 1000 rpm for 5 minutes. The cell pellet was resuspended in PBS at 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 at pH = 7.4 and analyzed by flow cytometry using a flow cytometer.

[0061] The results are shown in Figure 7, which show that compared with liposomes, the uptake of keratinocyte membrane protein biomimetic liposomes by HaCaT cells was significantly reduced, and the fluorescence intensity decreased from 2578 to 1044, a decrease of 2.47 times; the uptake of keratinocyte membrane protein biomimetic liposomes by MNT-1 cells was significantly increased, and the fluorescence intensity increased 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, proving that the prepared keratinocyte membrane protein biomimetic liposomes have significant targeting to melanocytes.

[0062] 7. In vitro efficacy study of keratinocyte membrane protein biomimetic liposomes in inhibiting melanosome transport in a co-culture model

[0063] To quantitatively measure melanosome transport, HaCaT cells and MNT-1 cells were seeded in a 2:1 ratio in six-well plates at a density of 5 × 10 5cells / well. The culture medium consisted of HaCaT medium and MNT-1 medium in a 2:1 ratio. After 24 hours of culture, the cells were washed twice with PBS (pH 7.4) and incubated with 1 mL of 500 μg / mL keratinocyte membrane protein biomimetic liposomes (prepared in Example 10) at 37°C for 24 hours. 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 PMEL17 rabbit monoclonal antibody (Beyotime, AG8635) and Alexa Fluor 488-labeled goat anti-rabbit IgG (H+L) (Beyotime, A0423). HaCaT cells were incubated with anti-pan-cytokeratin mouse recombinant polyclonal antibody (abcam, ab86734) and Alexa Fluor 647-labeled goat anti-mouse IgG (H+L) (Beyotime, A0473). The stained cells were analyzed by flow cytometry, and a total of 10,000 cells were collected on the flow cytometer.

[0064] The results are shown in Figure 8. The results show that compared with the untreated control wells, in the co-culture cell model after treatment with keratinocyte membrane protein biomimetic liposomes, the fluorescence intensity of AlexaFluor 488 representing melanosomes in keratinocytes decreased significantly 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.

[0065] Comparative Example 1: Effect of total lipid concentration on keratinocyte membrane protein biomimetic liposomes

[0066] Soybean lecithin and cholesterol in a molar ratio of 7:3 were mixed and dissolved in anhydrous ethanol with a total lipid concentration of 5 mg / ml to prepare an organic phase. 1×PBS buffer without the addition of keratinocyte membrane protein was used as the aqueous phase. The aqueous phase was used as the external phase and the organic phase as the internal phase. The total flow rate (TFR) = 1 ml / min and the flow rate ratio (aqueous phase / organic phase = FRR) = 6:1 were passed through a fluid focusing microfluidic chip (Wuhan Jianmi Intelligent Control Technology Co., Ltd.). The resulting solution was dialyzed overnight with a 300 kDa dialysis bag to obtain liposomes. Due to the low lipid concentration, the obtained liposome particle size was too small, at 57.6 nm, and the polydispersity index was too large, at 0.271.

[0067] Comparative Example 2: Effect of total lipid concentration on keratinocyte membrane protein biomimetic liposomes

[0068] Soybean lecithin and cholesterol in a molar ratio of 7:3 were mixed and dissolved in anhydrous ethanol with a total lipid concentration of 20 mg / ml to prepare an organic phase. 1×PBS buffer without the addition of keratinocyte membrane protein was used as the aqueous phase. The aqueous phase was used as the external phase and the organic phase as the internal phase. The total flow rate (TFR) = 1 ml / min and the flow rate ratio (aqueous phase / organic phase = FRR) = 6:1 were passed through a fluid focusing microfluidic chip (Wuhan Jianmi Intelligent Control Technology Co., Ltd.). The resulting solution was dialyzed overnight with a 300 kDa dialysis bag to obtain liposomes. Due to the high lipid concentration, the obtained liposome particle size was too large, at 207.4 nm, the polydispersity index increased significantly, to 0.352, and aggregation occurred.

[0069] Comparative Example 3: Effect of total lipid concentration on keratinocyte membrane protein biomimetic liposomes

[0070] Soy lecithin and cholesterol in a molar ratio of 7:3 were mixed and dissolved in anhydrous ethanol to a total lipid concentration of 10 mg / ml to prepare the organic phase. 1×PBS buffer without the addition of 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 total flow rate (TFR) was 1 ml / min and the flow rate ratio (aqueous phase / organic phase = FRR) was 8:1. The resulting solution was dialyzed overnight with a 300 kDa dialysis bag to obtain liposomes. Due to the excessive flow rate ratio, laminar diffusion could not be formed in the chip channel, making it difficult to prepare liposomes.

[0071] Comparative Example 4: Effect of TFR on keratinocyte membrane protein biomimetic liposomes

[0072] Soybean lecithin and cholesterol in a molar ratio of 7:3 were mixed and dissolved in anhydrous ethanol with a total lipid concentration of 10 mg / ml to prepare the organic phase. 1×PBS buffer without the addition of keratinocyte membrane protein was used as the aqueous phase. The aqueous phase was used as the external phase and the organic phase as the internal phase. The total flow rate (TFR) was 2 ml / min and the flow rate ratio (aqueous phase / organic phase = FRR) was 6:1. The resulting solution was dialyzed overnight with a 300 kDa dialysis bag to obtain liposomes. Due to the excessive total flow rate, the chip could not withstand the pressure and leaked.

[0073] Comparative Example 5: Effect of lipid types on keratinocyte membrane protein biomimetic liposomes

[0074] Soy lecithin, cholesterol, and dioleoylphosphatidylethanolamine (DOPE) in a molar ratio of 7:3:1 were dissolved in anhydrous ethanol to a total lipid concentration of 10 mg / ml to prepare the organic phase. A 1×PBS buffer solution with a keratinocyte membrane protein concentration of 0.05 mg / ml was added as the aqueous phase. The aqueous phase served 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 with a 300 kDa dialysis bag to obtain keratinocyte membrane protein biomimetic liposomes. Because DOPE is a cationic lipid, it binds to the negatively charged groups in the membrane protein during the diffusion self-assembly process, resulting in a restricted self-assembly process and aggregation of the resulting keratinocyte membrane protein biomimetic liposomes, with a polydispersity index of 0.41.

[0075] Comparative Example 6: Effect of lipid types on keratinocyte membrane protein biomimetic liposomes

[0076] Soy lecithin, cholesterol, and dioleoylphosphatidylcholine (DOPC) in a molar ratio of 7:3:1 were mixed and dissolved in anhydrous ethanol to a total lipid concentration of 10 mg / ml to prepare the organic phase. 1×PBS buffer with a keratinocyte membrane protein concentration of 0.05 mg / ml was added as the aqueous phase. The aqueous phase was used as the external phase and the organic phase as the internal phase. The total flow rate (TFR) was 1 ml / min and the flow rate ratio (aqueous phase / organic phase = FRR) was 6:1. The resulting solution was dialyzed overnight with a 300 kDa dialysis bag to obtain keratinocyte membrane protein biomimetic liposomes. The resulting keratinocyte membrane protein biomimetic liposomes had a high Zeta potential of -16.9 mV, which was not conducive to the embedding of membrane proteins into the phospholipid bilayer.

[0077] Comparative Example 7: Effect of Cell Membrane Protein Concentration on Keratinocyte Membrane Protein Biomimetic Liposomes

[0078] Soybean lecithin and cholesterol in a molar ratio of 7:3 were mixed and dissolved in anhydrous ethanol with a total lipid concentration of 10 mg / ml to prepare an organic phase. 1×PBS buffer with a keratinocyte membrane protein concentration of 0.2 mg / ml was added as the aqueous phase. The aqueous phase was used as the external phase and the organic phase as the internal phase. The total flow rate (TFR) was 1 ml / min and the flow rate ratio (aqueous phase / organic phase = FRR) was 6:1. The obtained solution was dialyzed overnight with a 300 kDa dialysis bag to obtain keratinocyte membrane protein biomimetic liposomes. Due to the high concentration of cell membrane protein, the self-assembly process of phospholipids during diffusion was restricted, resulting in an uneven particle size distribution of the obtained keratinocyte membrane protein biomimetic liposomes with a polydispersity index of 0.37.

[0079] Comparative Example 8: Effect of Cell Membrane Protein Concentration on Keratinocyte Membrane Protein Biomimetic Liposomes

[0080] Soybean lecithin and cholesterol in a molar ratio of 7:3 were mixed and dissolved in anhydrous ethanol with a total lipid concentration of 10 mg / ml to prepare an organic phase. 1×PBS buffer with a keratinocyte membrane protein concentration of 0.01 mg / ml was added as the aqueous phase. The aqueous phase was used as the external phase and the organic phase as the internal phase. The total flow rate (TFR) = 1 ml / min and the flow rate ratio (aqueous phase / organic phase = FRR) = 6:1 were passed through a fluid focusing microfluidic chip (Wuhan Jianmi Intelligent Control Technology Co., Ltd.). The resulting solution was dialyzed overnight with a 300 kDa dialysis bag to obtain keratinocyte membrane protein biomimetic liposomes. Due to the low concentration of cell membrane protein, the Zeta potential of the prepared keratinocyte membrane protein biomimetic liposomes was very similar to that of liposomes without membrane protein, the protein density on the liposome surface was too low, and the biological activity was poor.

[0081] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A biomimetic liposome, It is characterized in that The bionic liposome comprises keratinocyte membrane protein and phospholipid bilayer; the phospholipid bilayer is used as the inner phase and the keratinocyte membrane protein is used as the outer phase.

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

3. The biomimetic liposome according to claim 2, It is characterized in that The phospholipid bilayer comprises soybean lecithin and cholesterol, and the molar ratio of soybean lecithin to cholesterol is (3-2):

1.

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

5. A method for preparing the biomimetic liposome according to any one of claims 1 to 4, It is characterized in that The following steps are involved: (1) preparing an organic phase: mixing and dissolving soybean lecithin and cholesterol to obtain an organic phase; (2) preparing an aqueous phase: dissolving keratinocyte membrane proteins to obtain an aqueous phase; (3) The aqueous phase obtained in step (2) is used as the external phase, and the organic phase obtained in step (1) is used as the internal phase, and a bionic liposome is obtained by using a fluid focusing microfluidic chip.

6. The method according to claim 5, It is characterized in that 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.

7. The method according to claim 6, It is characterized in that In step (1), the molar ratio of soybean lecithin to cholesterol is (3-2):1; and the concentration of the organic phase is 5-10 mg / ml.

8. The method according to claim 7, It is characterized in that The concentration of the aqueous phase in step (2) is 0.033-0.1 mg / ml.

9. Use of the biomimetic liposome according to any one of claims 1 to 4 in the preparation of a product for inhibiting the transfer of skin melanin.

10. The use according to claim 9, It is characterized in that The products include cosmetics and medicines.

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