Method for promoting cells to uptake exogenous substances based on regulation and control of cell membrane tension

Abstract

The invention discloses a method for promoting cells to ingest exogenous substances based on regulation and control of cell membrane tension, the method promotes cells to ingest exogenous substances by regulating and controlling the cell membrane tension, and the method for regulating and controlling the cell membrane tension is to change the elastic modulus of a cell inoculated substrate. Experimental results show that the stiffness of the substrate significantly affects the membrane tension of the cells, and the cell uptake ability is optimal under the moderate membrane tension. For different cell types and exogenous substances, the optimal membrane tension intervals of cellular uptake are different. According to the method, by optimizing the mechanical microenvironment where the cells are located, a new strategy is provided for improving the uptake efficiency of the cells on exogenous substances.

Description

Technical Field

[0001] The present invention relates to the fields of biomaterials and cell mechanobiology, and in particular to a method for promoting cell uptake of exogenous substances based on regulating cell membrane tension. Background Art

[0002] Cellular uptake is a fundamental process for exchanging substances between cells and their environment, crucial for maintaining normal cellular function and executing specific biological tasks. In the biomedical field, efficient and controllable cellular uptake technology is one of the core technical bottlenecks in gene therapy (such as CRISPR-Cas9 system delivery), targeted tumor therapy (such as antibody-drug conjugates), and vaccine development (such as mRNA vaccine delivery).

[0003] However, existing research has mostly focused on optimizing the physical and chemical properties of the delivery system itself (such as size, shape, surface charge and chemical modification), while treating the cell membrane as a static barrier and ignoring its main dynamic response. Cell membrane biomechanics studies have shown that the mechanical properties of the cell membrane (such as membrane tension) play an important role in regulating cellular uptake. Membrane tension reflects the tensile state of the membrane, affects the fluidity and stability of the membrane, and some uptake pathways are extremely sensitive to changes in membrane tension. Therefore, it is of great significance to develop a method to optimize the mechanical properties of cells by regulating the cell mechanical microenvironment to improve the uptake efficiency. While optimizing the delivery system, paying attention to the influence of the mechanical properties of the cell membrane provides important scientific guidance for breaking through the bottleneck of delivery efficiency and developing a delivery system that meets the needs of precise and personalized treatment. Summary of the Invention

[0004] Based on the above problems, the present invention provides a method for regulating cell membrane tension to promote cell uptake of exogenous substances.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] A method for promoting cell uptake of exogenous substances based on regulating cell membrane tension, characterized in that the method promotes cell uptake of exogenous substances by regulating cell membrane tension, and the method for regulating cell membrane tension is to change the elastic modulus of the substrate on which the cells are inoculated.

[0007] Preferably, the cells are at least one of primary cells, stem cells, immortalized cells, and passaged cells.

[0008] Preferably, the cells are mBMSCs cells and / or L929 cells.

[0009] Preferably, the elastic modulus of the substrate is 17-1080 kPa; the membrane tension of the cell membrane is 0.20-0.87 mNm -1 .

[0010] Preferably, the exogenous substance is a bioactive substance delivery carrier, including at least one of nanoparticles, liposomes, polymer microspheres, inorganic nanomaterials and composite carriers thereof.

[0011] Preferably, the bioactive substance delivery carrier is at least one of polystyrene microspheres, lipid nanoparticles, and gold nanoparticles.

[0012] Preferably, the material of the substrate is at least one of polyvinyl alcohol, polydimethylsiloxane, polycaprolactone, gelatin, cellulose, and silicon dioxide.

[0013] Preferably, when the cells are mBMSCs cells, the substrate material is polyvinyl alcohol; the cell membrane tension of the cells is 0.31 to 0.42 mN m -1 The substrate is prepared by dissolving polyvinyl alcohol powder in deionized water to form a 7% to 9% solution, freezing it at -(15 to 25)°C for 20 to 30 hours, and thawing it at room temperature for 10 to 15 hours to obtain a polyvinyl alcohol substrate with an elastic modulus of 24kPa to 33kPa.

[0014] Preferably, when the cells are L929 cells, the substrate material is polydimethylsiloxane; the cell membrane tension is 0.66 to 0.76 mN m -1 ;

[0015] The substrate is prepared by mixing a main agent and a curing agent in a mass ratio of (18-22):1, vacuuming the resulting mixed colloid to remove bubbles, pouring the prepared colloid into a mold and heating and curing it at 100° C. for 2 hours to complete the curing, thereby obtaining a polydimethylsiloxane substrate with an elastic modulus of 730 kPa to 860 kPa;

[0016] The main agent is 184 Silicone Elastomer Base, and the curing agent is 184 Silicone Elastomer Curing Agent.

[0017] The beneficial effects of the present invention are:

[0018] (1) The present invention confirms the regulatory effect of substrate stiffness on cell mechanical properties and uptake behavior, and reveals the "membrane tension window" effect, that is, there is an optimal membrane tension range for cell uptake behavior. Within this "window", moderate membrane tension has the best uptake effect, while excessively high or low membrane tension may limit the cell's uptake ability.

[0019] (2) The present invention promotes cellular uptake by altering the cellular microenvironment. This physical signal can be applied to cells in a timely and localized manner, resulting in greater safety and controllability compared to existing research that optimizes the physical and chemical properties of delivery systems (size, shape, surface charge, and chemical modification). This approach provides a new approach and method for addressing the low cellular uptake efficiency of existing technologies by optimizing the mechanical microenvironment of cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the method of the present invention.

[0021] Figure 2 Cell membrane tension of mBMSCs cells on different PVA substrate surfaces.

[0022] Figure 3 Confocal images of mBMSCs cells grown on polystyrene fluorescent microspheres on different PVA substrates.

[0023] Figure 4 This is a diagram evaluating the uptake effect of polystyrene fluorescent microspheres by mBMSCs cells on different PVA substrate surfaces.

[0024] Figure 5 Cell membrane tension of L929 cells on different PDMS substrate surfaces.

[0025] Figure 6 Confocal images of L929 cells grown on lipid nanoparticles on different PDMS substrates.

[0026] Figure 7 This figure shows the evaluation of the lipid nanoparticle uptake effect of L929 cells on different PDMS substrate surfaces. DETAILED DESCRIPTION

[0027] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0028] Unless otherwise specified, all reagents involved in the embodiments of the present invention are commercially available products and can be purchased through commercial channels.

[0029] Example 1: Regulating the cell membrane tension of mBMSCs by using PVA substrates of different stiffness to promote cell uptake of polystyrene microspheres

[0030] (1) PVA powder was dissolved in deionized water to prepare four different concentrations of PVA solutions: 5%, 7%, 9%, and 15%. The higher the PVA concentration, the greater the elastic modulus of the resulting PVA hydrogel base. The prepared solution was poured into a mold and placed in a -20°C refrigerator for 24 hours. After taking it out, it was thawed at room temperature for 12 hours. The elastic moduli of the four groups of bases measured by mechanical experiments were: 17 kPa for 5% concentration, 24 kPa for 7% concentration, 33 kPa for 9% concentration, and 54 kPa for 15% concentration.

[0031] (2) mBMSCs cells were plated at 5000 cells / cm 2 Cells were seeded at different densities on PVA hydrogel substrates with elastic moduli of 17 kPa, 24 kPa, 33 kPa, and 54 kPa, respectively. The cells were evenly distributed on the substrate and cultured in a 37°C, 5% CO2 incubator for 24 h.

[0032] (3) Discard the supernatant, rinse the cells with PBS, and perform atomic force microscopy experiments to measure the mechanical properties of the cells.

[0033] (4) After culturing the cells in the plate according to step (2), the supernatant was discarded, the cells were rinsed with PBS, 1 mg / mL polystyrene fluorescent microsphere dilution was added, and the cells were incubated in the dark in an incubator for 2.5 h.

[0034] (5) Aspirate the supernatant, rinse the cells three times with PBS, and perform the staining experiment. First, fix, permeabilize, and block the cells. Aspirate the blocking solution and wash twice with PBS. Prepare DAPI staining solution with PBS at a ratio of 5:1, add 200 μL to each well, and incubate at room temperature in the dark for 10 minutes. Finally, aspirate the DAPI staining solution and rinse three times with PBS solution. Use a laser confocal microscope to capture the cell uptake of microspheres.

[0035] Figure 2 The cell membrane tension of mBMSCs cells on different substrates is 0.20±0.04 mN m on PVA-17 kPa substrate. -1 , 0.32 ± 0.05 mN m on PVA-24 kPa substrate -1 , 0.41±0.04 mN m on PVA-33 kPa substrate -1 , 0.57±0.10 mN m on PVA-54 kPa substrate -1 The cell membrane tension of cells on different substrates is ranked as follows: PVA-17kPa <PVA-24kPa<PVA-33kPa<PVA-54kPa。

[0036] Figure 3Confocal images of mBMSCs cultured on different substrates and polystyrene fluorescent microspheres. In these confocal images, the cell nuclei appear blue, and the polystyrene fluorescent microspheres appear red. mBMSCs cultured on PVA-17kPa and PVA-54kPa substrates showed poor uptake of the polystyrene fluorescent microspheres, while those cultured on PVA-24kPa and PVA-33kPa substrates showed significant uptake of the polystyrene fluorescent microspheres.

[0037] Figure 4 The data are as follows: the average fluorescence intensity of polystyrene fluorescent microspheres on different substrates. The greater the fluorescence intensity, the more fluorescent microspheres are taken up by the cells, and the better the uptake effect. The data show that the uptake of mBMSCs cells can be enhanced on PVA-24kPa and PVA-33kPa substrates (where the cell membrane tension is moderate). The specific fluorescence intensity data are as follows: the average fluorescence intensity on the PVA-17kPa substrate is 96.96±50.95, on the PVA-24kPa substrate is 206.45±24.49, on the PVA-33kPa substrate is 256.46±44.39, and on the PVA-54kPa substrate is 124.94±21.73.

[0038] Example 2: Regulating L929 cell membrane tension by using PDMS substrates of different stiffness to promote cellular uptake of lipid nanoparticles

[0039] (1) PDMS substrates with different stiffness were prepared by adjusting the mass ratio of the main agent (184Silicone Elastomer Base) to the curing agent (184Silicone Elastomer Curing Agent) (25:1, 22:1, 18:1, 16:1). Generally, the higher the ratio of the curing agent, the greater the elastic modulus of the obtained PDMS substrate. According to the above ratio, the prepared PDMS mixed colloid was fully stirred to ensure that the main agent and the curing agent were evenly mixed. The mixed colloid was placed in a vacuum pump and vacuumed for 10 minutes to remove bubbles in the mixed colloid. The prepared colloid was poured into a mold, placed on a heating plate, and heated at 100°C for 2 hours to complete the curing. The elastic moduli of the four groups of substrates measured by mechanical experiments were as follows: 540kPa for the ratio of 25:1, 730kPa for the ratio of 22:1, 860kPa for the ratio of 18:1, and 1080kPa for the ratio of 16:1.

[0040] (2) L929 cells were plated at 6000 cells / cm 2Cells were seeded at different densities on PDMS substrates with elastic moduli of 540 kPa, 730 kPa, 860 kPa, and 1080 kPa, respectively. The cells were evenly distributed on the substrates and cultured in a 37°C, 5% CO2 incubator for 24 h.

[0041] (3) Discard the supernatant, rinse the cells with PBS, and perform atomic force microscopy experiments to measure the mechanical properties of the cells.

[0042] (4) After culturing the cells in the plate according to step (1), the supernatant was discarded, the cells were rinsed with PBS, 2 mg / mL of lipid nanoparticle dilution was added, and the cells were incubated in the dark in an incubator for 2.5 h.

[0043] (5) The supernatant was discarded, and the cells were rinsed three times with PBS. The staining experiment was performed according to the staining steps described in Example 1, and the uptake of lipid nanoparticles by the cells was photographed using a laser confocal microscope.

[0044] Figure 5 The cell membrane tension of L929 cells on different substrates is 0.52±0.02 mN m on the PDMS-540 kPa substrate. -1 , 0.67±0.04 mN m on PDMS-730 kPa substrate -1 , 0.75 ± 0.04 mN m on PDMS-860 kPa substrate -1 , 0.87 ± 0.05 mN m on PDMS-1080 kPa substrate -1 The cell membrane tension of cells on different substrates is ranked as follows: PDMS-540kPa <PDMS-730kPa<PDMS-860kPa<PDMS-1080kPa。

[0045] Figure 6 Confocal images of L929 cells coated with lipid nanoparticles on different substrates. Cell nuclei appear blue and lipid nanoparticles appear red in the confocal images.

[0046] Figure 7 Average fluorescence intensity data for lipid nanoparticles on different substrates. A higher fluorescence intensity indicates greater cellular uptake of lipid nanoparticles and better cellular uptake. The average fluorescence intensity on PDMS-540 kPa substrates was 176.97 ± 21.95, on PDMS-730 kPa substrates was 325.21 ± 38.08, on PDMS-860 kPa substrates was 275.20 ± 28.78, and on PDMS-1080 kPa substrates was 154.93 ± 30.73.

[0047] pass Figure 6 and Figure 7It can be seen that on the PDMS-730kPa substrate and the PDMS-860kPa substrate, the membrane tension of the cells is moderate, which can enhance the uptake of lipid nanoparticles by L929 cells.

[0048] Cellular uptake capacity may exist within a "membrane tension window." When membrane tension is too high, the cell membrane's ability to deform is limited, thus impairing endocytosis. Conversely, when membrane tension is too low, the cell membrane's excessive relaxation may lack sufficient dynamic tension, leading to decreased uptake. Therefore, the membrane tension range within which cells maximize their endocytic capacity may be limited, suggesting an ideal "membrane tension window" within which cells can most effectively uptake nanoparticles. The location of this window may vary for different cell types and the substances being taken up.

Claims

1. A method for promoting cell uptake of exogenous substances based on regulating cell membrane tension, characterized in that: The method promotes cells to take up exogenous substances by regulating the tension of cell membranes. The method for regulating the tension of cell membranes is to change the elastic modulus of the substrate on which the cells are seeded.

2. The method according to claim 1, characterized in that The cells are at least one of primary cells, stem cells, immortal cells, and passage cells.

3. The method according to claim 2, characterized in that The cells are mBMSCs cells and / or L929 cells.

4. The method according to claim 3, characterized in that The elastic modulus of the substrate is 17-1080 kPa; the membrane tension of the cell membrane is 0.20-0.87 mN m -1 .

5. The method according to claim 4, characterized in that The exogenous substance is a bioactive substance delivery carrier, including at least one of nanoparticles, liposomes, polymer microspheres, inorganic nanomaterials and composite carriers thereof.

6. The method according to claim 5, characterized in that The bioactive substance delivery carrier is at least one of polystyrene microspheres, lipid nanoparticles, and gold nanoparticles.

7. The method according to claim 6, characterized in that The material of the substrate is at least one of polyvinyl alcohol, polydimethylsiloxane, polycaprolactone, gelatin, cellulose, and silicon dioxide.

8. The method according to claim 4, characterized in that When the cells are mBMSCs cells, the substrate material is polyvinyl alcohol, and the elastic modulus is 24kPa to 33kPa; the cell membrane tension is 0.31 to 0.42 mN m -1 The substrate is prepared by dissolving polyvinyl alcohol powder in deionized water to form a 7% to 9% solution, freezing it at (-25 to -15)°C for 20 to 30 hours, and thawing it at room temperature for 10 to 15 hours to obtain a polyvinyl alcohol substrate with an elastic modulus of 24kPa to 33kPa.

9. The method according to claim 4, characterized in that When the cells are L929 cells, the substrate material is polydimethylsiloxane; the cell membrane tension is 0.66-0.76 mN m -1 ; The substrate is prepared by mixing a main agent and a curing agent in a mass ratio of (18-22):1, vacuuming the resulting mixed colloid to remove bubbles, pouring the prepared colloid into a mold and heating and curing it at 100° C. for 2 hours to complete the curing, thereby obtaining a polydimethylsiloxane substrate with an elastic modulus of 730 kPa to 860 kPa; The main agent is 184 Silicone Elastomer Base, and the curing agent is 184 Silicone Elastomer Curing Agent.

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