A photoresponsive ordered porous membrane material for cell spontaneous adhesion and detachment and a preparation method thereof

Abstract

The application discloses a kind of light-responsive ordered porous membrane materials for cell spontaneous adhesion and detachment and preparation method thereof.The porous membrane material component includes polymer and light-responsive compound spiropyran, and the polymer is at least one of polylactic acid, polystyrene.The preparation method of porous membrane material is as follows: (1) polymer and light-responsive compound are dissolved in solvent; (2) the prepared solution is added dropwise to the surface of substrate, and the substrate is placed on the rotating platform of spin coater; (3) air is introduced into the spin coater, so that the air flow is blown to the solution from directly above the solution, and the air rate introduced, the temperature and relative humidity inside the chamber of spin coater are controlled, while starting the spin coater and controlling its rotating speed; (4) after the solvent is completely volatilized, stop the spin coater rotation, take out the substrate to obtain the ordered porous membrane material.The ordered porous membrane can make cell spontaneous adhesion and spontaneous detachment, so as to improve the success rate and harvest rate of cell culture.

Description

Technical Field

[0001] This invention belongs to the field of polymer porous materials and cell culture, and specifically relates to a photoresponsive ordered porous membrane material. Background Technology

[0002] The ordered pore structure of porous membranes provides an excellent substrate for cell adhesion, and cell adhesion to the surface of porous membranes is selective. Introducing stimulus-responsive functional groups into the membrane surface to construct intelligent porous membrane materials with reversible stimulus-responsive surface properties can not only avoid secondary hydrophilic treatment after membrane formation, but also potentially regulate cell adhesion and detachment behavior on the membrane surface simply by altering external stimuli. However, in cell culture, unnecessary manipulations and the ease with which cells become contaminated and introduce other variables can adversely affect cell culture. Furthermore, environmental stimuli themselves can also damage cells. Therefore, designing an ordered porous membrane that enables spontaneous cell adhesion and detachment can significantly improve the success rate and yield of cell culture. Summary of the Invention

[0003] The purpose of this invention is to address the shortcomings of existing technologies by providing a photoresponsive ordered porous membrane material and its preparation method for spontaneous cell adhesion and detachment. This results in an ordered porous membrane with photoresponsive macroscopic color and surface wettability, enabling spontaneous cell adhesion and detachment, thereby improving the success rate and yield of cell culture.

[0004] Compared to pH and temperature stimuli, light stimulation is a non-contact response, characterized by its cleanliness, ease of acquisition, strong remote controllability, and precise and rapid response regulation. Based on these advantages, introducing specific photosensitive units onto the surface of porous membranes to induce light-responsive changes and endow the membranes with specific light-controlled surface wettability holds promise for solving the aforementioned problems. Spiropyrans are a typical photochromic photosensitive unit. These compounds consist of two aromatic heterocycles connected by sp3-hybridized spirocarbon atoms. The two ring systems are orthogonal and non-conjugated, resulting in a closed-ring (SP) molecular structure. When exposed to light of a certain wavelength, the COC bond in the molecule breaks, transforming the two orthogonal ring systems into a planar structure. The entire molecule forms a conjugated system, and the molecular structure becomes an open-ring (MC) structure. This change in molecular structure leads to a significant change in its absorption spectrum, resulting in a noticeable color change on a macroscopic scale. When in darkness, spiropyrans automatically revert from an open-ring state to a closed-ring state, restoring the original color. The transition between the two states of spiropyran not only leads to a reversible change in its macroscopic color but also a reversible change in its hydrophilic / hydrophobic properties. The closed-ring state is hydrophobic, while the open-ring state is ionic and thus exhibits good hydrophilicity. It is foreseeable that if rationally designed spiropyran molecules are introduced onto the surface of ordered porous membranes, it is possible to construct photoresponsive intelligent ordered porous films. In the hydrophilic MC state, cells adhere to the porous membrane; with continued culturing, the MC state spontaneously transforms into the hydrophobic SP state, thereby causing spontaneous cell detachment.

[0005] The present invention provides a photoresponsive ordered porous membrane material for spontaneous cell adhesion and detachment, comprising a polymer and a photoresponsive compound, wherein the photoresponsive compound is a spiropyran, and the spiropyran has the following structural formula:

[0006] .

[0007] Furthermore, the polymer is at least one of polylactic acid and polystyrene, preferably polystyrene.

[0008] Furthermore, the mass ratio of spiropyran to polymer in the porous membrane material is preferably 1:4.

[0009] The method for preparing the photoresponsive ordered porous membrane material provided by this invention is as follows:

[0010] (1) Dissolve the polymer and photoresponsive compound in a solvent to prepare a homogeneous solution;

[0011] (2) Add the prepared solution to the substrate surface and place the substrate on the rotating platform of the spin coater;

[0012] (3) Introduce air into the spin coater so that the airflow blows from directly above the solution onto the solution, and control the airflow rate, the temperature and relative humidity inside the spin coater chamber, while starting the spin coater and controlling its rotation speed.

[0013] (4) After all the solvent has evaporated, stop the spin coater and remove the substrate to obtain the ordered porous membrane material.

[0014] Furthermore, the polymer is at least one of polylactic acid and polystyrene, preferably polystyrene.

[0015] Furthermore, the photoresponsive compound is spiropyran, and its synthetic route and structural formula are shown below. Figure 1 .

[0016] Furthermore, the solvent is at least one of dichloromethane, trichloromethane, and carbon disulfide, preferably trichloromethane.

[0017] Furthermore, the substrate is glass, silicon wafer, or polyester film, preferably glass.

[0018] Further, the polymer concentration in the solution obtained in step (1) is 5~50 mg / mL, and the concentration of the photoresponsive compound is 0.01~2 mg / mL; preferably, the polymer concentration in the solution is 5~30 mg / mL, and the concentration of the photoresponsive compound is 0.01~1.5 mg / mL.

[0019] Furthermore, in step (3), the air flow rate is 100~300 L / h, and the temperature inside the homogenizer chamber is 20~28℃. o C. The relative humidity inside the spin coater chamber is 50%~90%, and the spin coater speed is 0~50 rpm.

[0020] In the above technical solutions of the present invention, the solvent evaporation time varies depending on the solvent and the rate of air introduction, and generally requires at least 2 minutes.

[0021] The present invention also provides a photoresponsive ordered porous membrane material prepared by the above method.

[0022] The ordered porous membrane material described in this invention has photoresponsiveness, and the degree of response and pore size are tunable, enabling reversible changes in surface hydrophilicity and hydrophobicity under light and dark conditions.

[0023] In the above technical solutions of the present invention, the spiropyran is prepared by the following method:

[0024] (1) Synthesis of spiropyran: Under nitrogen protection, 11-bromo-1-undecanol was dissolved in acetonitrile. After heating to reflux, a mixture of 2,3,3-trimethyl-3H-indole and acetonitrile was slowly added dropwise, wherein the ratio of 11-bromo-1-undecanol to 2,3,3-trimethyl-3H-indole was 1:1.1. After the addition was complete, the reaction was continued for 36 h. The solvent was removed under reduced pressure to obtain the crude product. The crude product was completely dissolved in a small amount of dichloromethane, and then diethyl ether was added under stirring. The mixture was then refluxed for half an hour. After cooling to room temperature, the liquid was discarded to obtain the solid. The addition of diethyl ether and reflux were repeated three times to obtain the pink powdered indole salt.

[0025] (2) Under nitrogen protection, the obtained indole salt was dissolved in a mixed solvent of 5-nitrosalicylic acid triethylamine and anhydrous ethanol, wherein the molar ratio of indole salt to 5-nitrosalicylic acid triethylamine was 1:1.1. The reaction was carried out in an oil bath at 85 °C for 12 h. After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure. The obtained solid was extracted three times with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed to obtain a reddish-brown crude product. The crude product was purified by column chromatography under the following conditions: 200-300 mesh silica gel column, eluent of petroleum ether:ethyl acetate = 8:1. The column chromatography yielded an orange-yellow solid spiropyran.

[0026] Compared with the prior art, the present invention has the following beneficial effects and features:

[0027] (1) Compared with pH, ​​temperature and voltage stimulation, the light stimulation used in this invention is a non-contact response, which has the characteristics of being clean and easy to obtain, having strong remote control capability, and having precise and rapid response regulation.

[0028] (2) Spiropyran has excellent biocompatibility, and the porous membranes prepared from it can be used in the field of cell culture.

[0029] (3) The synthesized spiropyran has high photosensitivity, which greatly reduces the effect of light on cells.

[0030] (4) The surface wettability of the prepared porous membrane is photoresponsive. Through the interaction of light and dark conditions, a reversible change between hydrophilicity and hydrophobicity can be achieved. By utilizing this change in wettability to apply stress to cells, cell adhesion and detachment can be achieved. Moreover, this process is spontaneous and does not require the application of other conditions. Attached Figure Description

[0031] Figure 1 The following are the synthetic route and UV-Vis spectral characterization results of spiropyran: (a) Synthetic route diagram, (b) Initial UV characterization results, and (c) UV-Vis spectral characterization results after 1 min of 6W, 375nm UV irradiation.

[0032] Figure 2The image shows a SEM image of the porous membrane obtained in Example 1.

[0033] Figure 3 This is a SEM image of the porous membrane obtained in Example 2.

[0034] Figure 4 This is a SEM image of the porous membrane obtained in Example 3.

[0035] Figure 5 This is a SEM image of the porous membrane obtained in Example 4.

[0036] Figure 6 The cell adhesion on the porous membrane in Experiment 6 is as follows: (a) adhesion for 2 hours; (b) adhesion for 8 hours.

[0037] Figure 7 The cell adhesion on the porous membrane in Experiment 7 is as follows: (a) adhesion for 2 hours; (b) adhesion for 8 hours.

[0038] Figure 8 Photographs of cells on the porous membrane after culturing for 24 hours in Experiment 8: (a) Porous membrane from Experiment 6; (b) Porous membrane from Experiment 7. Detailed Implementation

[0039] The specific embodiments of the present invention will be further described below with reference to examples and accompanying drawings. These embodiments are only used to more clearly illustrate the technical solutions of the present invention and should not be construed as limiting the scope of protection of the present invention.

[0040] In the following examples, spiropyran molecules are arranged according to Figure 1 The synthesis is carried out using the methods described above, and all other materials can be purchased on the market.

[0041] Spiropyran is prepared by the following method:

[0042] (1) Synthesis of spiropyran: Under nitrogen protection, 11-bromo-1-undecanol (5 g, 31.4 mmol) and 10 mL of acetonitrile were mixed together, and then heated to reflux. A mixture of 2,3,3-trimethyl-3H-indole (8.67 g, 34.5 mmol) and 5 mL of acetonitrile was slowly added dropwise. After the addition was complete, the reaction was continued for 36 h. The solvent was removed under reduced pressure to obtain the crude product. The crude product was completely dissolved in a small amount of dichloromethane, and then diethyl ether was added while stirring. The mixture was refluxed for half an hour, cooled to room temperature, and the liquid was discarded to obtain the solid. This process was repeated three times to obtain the pink indole salt.

[0043] (2) Under nitrogen protection, the obtained indole salt was dissolved in a mixed solvent of 5-nitrosalicylic acid triethylamine and anhydrous ethanol, wherein the molar ratio of indole salt to 5-nitrosalicylic acid triethylamine was 1:1.1. The reaction was carried out in an oil bath at 85 °C for 12 h. After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure. The obtained solid was extracted three times with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed to obtain a reddish-brown crude product. The crude product was purified by column chromatography under the following conditions: 200-300 mesh silica gel column, eluent of petroleum ether:ethyl acetate = 8:1. The column chromatography yielded an orange-yellow solid spiropyran.

[0044] The synthesized spiropyran molecule was characterized by ultraviolet light, demonstrating that it exhibits photoresponsiveness, such as... Figure 1 As shown.

[0045] Example 1

[0046] (1) Dissolve polystyrene and spiropyran in chloroform to prepare solutions with polystyrene and spiropyran concentrations of 10 mg / mL and 0.5 mg / mL, respectively;

[0047] (2) Take 60 μL of the above solution and drop it onto the glass substrate, and place it on the rotating platform of the spin coater;

[0048] (3) Introduce air at a rate of 200 L / h and control the internal temperature of the homogenizer chamber to 25°C. o C, relative humidity 70%, simultaneously start the spin coater and control the speed at 20 rpm.

[0049] (4) After about 2 minutes, the solvent evaporates completely. Stop the spin coater and remove the substrate to obtain an ordered porous membrane.

[0050] Scanning electron microscopy observation (e.g.) Figure 2 This indicates that the surface of the porous membrane has pores, but the regularity is low and there is no regularity.

[0051] Example 2

[0052] (1) Dissolve polystyrene and spiropyran in chloroform to prepare solutions of polystyrene and spiropyran at concentrations of 10 mg / mL and 1 mg / mL, respectively;

[0053] (2) Take 60 μL of the above solution and drop it onto the glass substrate, and place it on the rotating platform of the spin coater;

[0054] (3) Introduce air at a rate of 200 L / h and control the internal temperature of the homogenizer chamber to 25°C. o C, relative humidity 70%, simultaneously start the spin coater and control the speed at 20 rpm.

[0055] (4) After about 2 minutes, the solvent evaporates completely. Stop the spin coater and remove the substrate to obtain an ordered porous membrane.

[0056] Scanning electron microscopy observation ( Figure 3 This indicates that the arrangement of the micropores on the surface of the porous membrane begins to show a certain regularity.

[0057] Example 3

[0058] (1) Dissolve polystyrene and spiropyran in chloroform to prepare solutions with polystyrene and spiropyran concentrations of 10 mg / mL and 2.5 mg / mL, respectively;

[0059] (2) Take 60 μL of the above solution and drop it onto the glass substrate, and place it on the rotating platform of the spin coater;

[0060] (3) Introduce air at a rate of 200 L / h and control the internal temperature of the homogenizer chamber to 25°C. o C, relative humidity 70%, simultaneously start the spin coater and control the speed at 20 rpm.

[0061] (4) After about 2 minutes, the solvent will evaporate completely. Stop the spin coater and remove the substrate to obtain an ordered porous membrane.

[0062] Scanning electron microscopy observation ( Figure 4 This indicates that the micropore structure on the surface of the porous membrane is arranged in an orderly manner and has a high degree of regularity.

[0063] Example 4

[0064] (1) Dissolve polystyrene and spiropyran in chloroform to prepare solutions of 10 mg / mL polystyrene and 5 mg / mL spiropyran;

[0065] (2) Take 60 μL of the above solution and drop it onto the glass substrate, and place it on the rotating platform of the spin coater;

[0066] (3) Introduce air at a rate of 200 L / h and control the internal temperature of the homogenizer chamber to 25°C. o C, relative humidity 70%, simultaneously start the spin coater and control the speed at 20 rpm.

[0067] (4) After about 2 minutes, the solvent will evaporate completely. Stop the spin coater and remove the substrate to obtain an ordered porous membrane.

[0068] Scanning electron microscopy observation ( Figure 5 This indicates that the micropore structure on the surface of the porous membrane is arranged in an orderly manner, with reduced regularity.

[0069] Examples 1-4 demonstrate that, within a certain range, the regularity of the porous membrane increases with increasing spiropyran molecule concentration in this invention; however, beyond this range, the regularity decreases. The pore size of the porous membrane also increases with increasing spiropyran molecule concentration.

[0070] Comparative Example 1

[0071] (1) Dissolve spiropyran in chloroform to prepare a solution with a spiropyran concentration of 10 mg / mL;

[0072] (2) Take 60 μL of the above solution and drop it onto the glass substrate, and place it on the rotating platform of the spin coater;

[0073] (3) Introduce air at a rate of 200 L / h and control the internal temperature of the homogenizer chamber to 25°C. o C, relative humidity 70%, simultaneously start the spin coater and control the speed at 20 rpm.

[0074] (4) After about 2 minutes, the solvent evaporates completely. Stop the spin coater and remove the substrate to obtain a pure spiropyran membrane.

[0075] The following experiments are used to illustrate the photoresponse properties of the wettability of the above-mentioned porous membrane surface.

[0076] Experiment 1

[0077] The membrane prepared in Comparative Example 1 was subjected to a water droplet contact angle test. The water droplet contact angle test showed that the initial contact angle of the porous membrane was 86°. After irradiating it with a 6 W, 375 nm UV lamp for one minute, its contact angle was measured to be 58°. After being placed in the dark at 25°C for 24 h, the contact angle recovered to 85°.

[0078] Experiment 2

[0079] The membrane prepared in Example 1 was subjected to a water droplet contact angle test. The water droplet contact angle test showed that the initial contact angle of the porous membrane was 105°. After irradiating it with a 6 W, 375 nm UV lamp for one minute, its contact angle was measured to be 103°. After being placed in the dark at 25°C for 24 h, the contact angle recovered to 105°.

[0080] Experiment 3

[0081] The membrane prepared in Example 3 was subjected to a water droplet contact angle test. The water droplet contact angle test showed that the initial contact angle of the porous membrane was 105°. After irradiating it with a 6 W, 375 nm UV lamp for one minute, its contact angle was measured to be 81°. After being placed in the dark at 25°C for 24 h, the contact angle recovered to 95°.

[0082] Experiment 4

[0083] The membrane prepared in Example 4 was subjected to a water droplet contact angle test. The water droplet contact angle test showed that the initial contact angle of the porous membrane was 95°. After irradiating it with a 6 W, 375 nm UV lamp for one minute, its contact angle was measured to be 81°. After being placed in the dark at 25°C for 24 h, the contact angle recovered to 94°.

[0084] Experiments 1-5 show that, in this invention, the higher the spiropyran content of the porous membrane, the greater its surface wettability response. The surface wettability response of the porous membrane only becomes significant when the spiropyran content reaches a certain value.

[0085] The following experiments illustrate the method for achieving controlled cell adhesion and release using the porous membrane prepared in Example 3.

[0086] Experiment 6

[0087] The inoculated cells were HeLa cells, with a cell count of approximately 1 × 10⁻⁶. 4 The spiropyran was inoculated onto the surface of the hydrophobic porous membrane in Example 3 (SP state) and cultured in a 6-well plate with 300 μL of complete culture medium for 8 hours in an incubator at 37°C with 5% CO2.

[0088] The adhesion rate of the porous membrane was characterized using the Calcein-AM method. The results are as follows: Figure 6 As shown.

[0089] Experiment 7

[0090] The inoculated cells were HeLa cells, with a cell count of approximately 1 × 10⁻⁶. 4 The sample was inoculated onto the surface of the hydrophilic porous membrane treated with a 6 W, 375 nm UV lamp for one minute (spiropyran was converted to MC state) as described in Example 3, and cultured in a 6-well plate with 300 μL of complete culture medium in an incubator at 37°C for 8 hours with 5% CO2.

[0091] The adhesion rate of the porous membrane was characterized using the Calcein-AM method. The results are as follows: Figure 7 As shown.

[0092] Examples 6 and 7 show that, in this invention, the hydrophilic porous membrane surface treated with a 6 W, 375 nm UV lamp for one minute is more suitable for the adhesion of the porous membrane, which can greatly improve the adhesion rate.

[0093] Experiment 8

[0094] Cells from Experiments 6 and 7 were cultured for another 24 hours without changing the culture conditions. Cells on the porous membrane were characterized using the Calcein-AM method. Results are as follows: Figure 8 As shown.

[0095] The cells on the porous membrane in the original experiment 6 showed almost no change because the spiropyran molecules remained unchanged (maintaining the SP state).

[0096] In the original experiment 7, most of the cells on the porous membrane detached because, under dark conditions (in the incubator), the spiropyran molecules changed from the MC state to the SP state, and the hydrophilic porous membrane became hydrophobic. This change could be sensed by the cells, thus causing them to detach.

[0097] Examples 7 and 8 show that the porous membrane prepared in this invention is suitable for automatic cell adhesion, and after cell adhesion is completed, the cells can spontaneously detach under dark conditions without the application of other conditions.

Claims

1. A photoresponsive ordered porous membrane material for spontaneous cell adhesion and detachment, characterized in that, It includes polymers and photoresponsive compounds, wherein the photoresponsive compound is a spiropyran, and the spiropyran has the following structural formula: ， The polymer is at least one of polylactic acid and polystyrene; The mass ratio of the spiropyran to the polymer is 1:4; It is prepared by the following method: (1) Dissolve the polymer and photoresponsive compound in a solvent to prepare a homogeneous solution; (2) Add the prepared solution to the substrate surface and place the substrate on the rotating platform of the spin coater; (3) Introduce air into the spin coater so that the airflow blows from directly above the solution onto the solution, and control the airflow rate, the temperature and relative humidity inside the spin coater chamber, while starting the spin coater and controlling its rotation speed. (4) After all the solvent has evaporated, stop the spin coater and remove the substrate to obtain the ordered porous membrane material.

2. The photoresponsive ordered porous membrane material according to claim 1, characterized in that, The solvent is at least one of dichloromethane, trichloromethane, and carbon disulfide.

3. The photoresponsive ordered porous membrane material according to claim 1, characterized in that, The substrate is glass, silicon wafer, or polyester film.

4. The photoresponsive ordered porous membrane material according to claim 1, characterized in that, The polymer concentration in the solution obtained in step (1) is 5–50 mg / mL, and the concentration of the photoresponsive compound is 0.01–2 mg / mL; the polymer concentration in the solution obtained is 5–30 mg / mL, and the concentration of the photoresponsive compound is 0.01–1.5 mg / mL.

5. The photoresponsive ordered porous membrane material according to claim 1, characterized in that, In step (3), the air flow rate is 100–300 L / h, and the temperature inside the homogenizer chamber is 20–28 °C. o C. The relative humidity inside the spin coater chamber is 50%–90%, and the spin coater speed is 0–50 rpm.

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