A method for manufacturing a near-infrared light responsive gold nano-coating intelligent cell culture container

Abstract

The application provides a manufacturing method of a near-infrared light responsive gold nano coating intelligent cell culture container, and solves the problems of complex manufacturing process, long cycle, high cost, low efficiency, poor biocompatibility and poor stability of the existing near-infrared light responsive cell culture container. The hydrophilic treatment of the cell culture container (substrate) can increase the number of hydroxyl groups on the surface, thereby increasing the bonding force between the gold nano structure sputtering layer and the cell culture container, making it more stable. The methoxy polyethylene glycol thiol is used to make the gold nano structure polyethylene glycol, so that the PEG is fully combined with the gold nano structure through the mercapto-gold bond to enhance the biocompatibility. Then the polyethylene glycolated gold nano structure and the polypeptide solution are mixed, the concentration of RGD is controlled to make it as much as possible to be coupled with the gold nano structure, and the targeting of the combination with the target cells is enhanced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of responsive cell culture containers, and particularly relates to a method for manufacturing a near-infrared light responsive gold nanocoating intelligent cell culture container. BACKGROUND

[0002] For adherent cells, detaching and transferring them from the wall surface of a cell culture plate, a culture bottle or a culture dish is one of the most basic and most commonly involved experimental operations in cell engineering and tissue engineering. The commonly used cell detaching method is trypsin digestion. Trypsin is a kind of protease, which plays a role of digestive enzyme in the body of vertebrates. Trypsin hydrolyzes the extracellular matrix protein (ECM) between cells and between cells and the surface of a culture container, thereby separating cells, and thus can be used to separate cells in tissues and detach adherent cells in the process of animal cell culture. However, in addition to the ability to digest the intercellular matrix protein, long-term action of trypsin can also digest cell membrane proteins, which has a damaging effect on cells. Therefore, when using trypsin to digest cells, it is necessary to strictly control the concentration of trypsin, the action time, the temperature and the number of rinsing parameters. Under the conditions of weak alkalinity and 37℃, the digestion ability of trypsin solution is the strongest, but a slight negligence in operation can easily cause damage to the membrane structure and functional integrity of cells, thereby causing a decrease in cell activity, and even leading to cell death.

[0003] In view of the above problems, in the past few decades, a variety of methods have been developed to regulate cell behavior, mainly including mechanical scratching, temperature control, pH control, ion control, magnetization control and light control. Among them, the light control method is outstanding in many separation technologies due to its strong universality, fast separation speed and good controllability. In the light control method, near-infrared light is more preferred because it causes the least damage to cells. The core of the near-infrared light control method is the design and construction of a photothermal surface, which is a surface embedded or coated with a photothermal agent such as noble metal nanomaterials, semiconductor nanomaterials, carbon-based nanomaterials and conjugated polymers. The surface can absorb light of appropriate wavelength (usually in the near-infrared region) and convert light energy to heat energy, so that the cell sheet is detached.

[0004] Noble metal nanomaterials can be used as plasmonic substrates to convert light energy into heat energy through plasmonic resonance. Plasmonic resonance refers to the excitation oscillation of electrons under external illumination when the light frequency and the oscillation frequency of electrons coincide. Gold nanomaterials are the most common plasmonic materials because they have a large electron density.

[0005] Currently, the methods for modifying gold nanostructures on the surface of cell culture plates mainly include chemical grafting and physical deposition. The chemical grafting method mainly reduces in situ on the surface of the cell culture plate by a reducing agent (Tian C, Qian W, Shao X, et al. Plasmonic Nanoparticles with Quantitatively Controlled Bioconjugation for Photoacoustic Imaging of Live Cancer Cells [J]. Advanced Science, 2016, 3(12): 1600237.); the physical deposition method mainly uses electrostatic interaction to graft gold nanoparticles on the surface of the cell culture plate (Giner-Casares J J, Henriksen-Lacey M, García I, et al. Plasmonic Surfaces for Cell Growth and Retrieval Triggered by Near-Infrared Light [J]. Angewandte Chemie International Edition, 2016, 55(3): 974-978.).

[0006] Although the above-mentioned chemical or physical methods can achieve controllable detachment of cells on the surface of the cell culture plate, there are still the following deficiencies in using the existing methods:

[0007] 1) The chemical method is complex, and needs to perform multi-step surface chemical modification on the surface of the cell culture plate, has a long production cycle, and the reagents used in the chemical method are difficult to remove after the reaction, resulting in poor biocompatibility; the price of the equipment and reagents used is high;

[0008] 2) The physical method has strict requirements for the surface performance of the cell culture plate, generally needs to synthesize polyelectrolytes with different charged groups first, and then modify the surface charge of the cell culture plate using the polyelectrolytes, so that the gold nanostructure is fixed on the cell culture plate by electrostatic interaction; the electrostatic binding force is weak, and the use stability of the modified cell culture plate is poor.

[0009] The above problems not only increase the production cost of the near-infrared light responsive cell culture plate, but also greatly reduce the production efficiency. Therefore, it is of great significance to develop and design a simple, inexpensive and efficient method to produce a near-infrared light responsive intelligent cell culture container with good use stability, for promoting the marketization development of the near-infrared light responsive cell culture container. SUMMARY

[0010] The application aims to solve the problems of the prior art, such as complex manufacturing process, long cycle, high cost, low efficiency, poor biocompatibility and poor stability of the near-infrared light responsive cell culture container, and provides a manufacturing method of a near-infrared light responsive gold nano coating intelligent cell culture container.

[0011] To achieve the above-mentioned application purposes, the technical solution provided by the application is as follows:

[0012] A manufacturing method of a near-infrared light responsive gold nano coating, which is characterized by comprising the following steps:

[0013] 1) sputtering gold nanoclusters onto a substrate with a surface hydrophilic treatment, wherein the thickness of the sputtered layer is 10-80 nm, which is an important condition for realizing the function, and the sputtered layer thickness is controlled by controlling the metal sputtering time to ensure that the plasmon resonance effect is generated when the substrate is irradiated by near-infrared light in the subsequent operation, and the light energy is converted into heat energy; at the same time, it is better to sputter gold nanoclusters immediately after the substrate is completed with the hydrophilic treatment to ensure the stability of the gold nano structure fixed on the substrate in the subsequent operation;

[0014] 2) after the substrate obtained in step 1) is cleaned, the surface of the sputtered layer is treated with a methoxy polyethylene glycol thiol (mPEG-SH) solution, and a polyethylene glycolated gold nanocluster is obtained by reaction;

[0015] 3) the polyethylene glycolated gold nanocluster obtained in step 2) is further treated with a polypeptide (RGD) solution, and after the reaction is completed, the substrate is cleaned and dried to remove the surface moisture, and a near-infrared light responsive gold nano coating is obtained on the surface of the substrate.

[0016] Further, in step 1), according to the thickness requirement of the sputtered layer, the gold nanoclusters are sputtered onto the substrate with a surface hydrophilic treatment by using a metal sputtering instrument.

[0017] Further, in step 2), the molar concentration of the methoxy polyethylene glycol thiol solution is 0.5-20 mM, preferably 1-2 mM, and the preferred value is 1 mM;

[0018] The methoxy polyethylene glycol thiol (mPEG-SH) solution is added to the surface of the gold nanoclusters in a molar ratio of (100-1000) to 1 according to the molar ratio of the mercapto polyethylene glycol (PEG-SH) to the sputtered gold nanoclusters in step 1); the preferred range is (400-600) to 1, and the preferred value is 500 to 1;

[0019] The reaction temperature is 20-40℃, and the reaction time is 1-12h, preferably 8-10h, and the preferred value is 10h.

[0020] Further, in step 3), the molar concentration of the polypeptide (RGD) solution is 0.5–20 mM, preferably 1–2 mM, with a preferred value of 1 mM; commercially available RGD lyophilized powder is purchased directly for preparation.

[0021] The RGD solution was dropped onto the surface of the polyethylene glycol-modified gold nanoclusters at a molar ratio of (100 to 10000):1; the preferred range was (1000 to 5000):1, and the preferred value was 1000:1.

[0022] The reaction temperature is 20–40℃, and the reaction time is 1–12 h, with a preferred range of 8–10 h and a preferred value of 10 h.

[0023] The drying temperature is 20–50℃.

[0024] Furthermore, in step 1), the surface hydrophilic treatment refers to treatment using a UVO cleaning machine (ultraviolet ozone cleaning machine).

[0025] In both steps 2) and 3), deionized water is used for cleaning.

[0026] Meanwhile, the present invention provides a near-infrared light-responsive gold nanocoating, characterized in that it is prepared by the above method.

[0027] The near-infrared light-responsive gold nanocoating prepared by the above method is used as a photothermal surface in the preparation of near-infrared light-responsive smart cell culture containers.

[0028] Furthermore, a method for preparing a near-infrared light-responsive smart cell culture container is described, characterized by: preparing a near-infrared light-responsive gold nano-coating on a cell culture container with a hydrophilically treated surface according to the above method, thereby obtaining the near-infrared light-responsive smart cell culture container. That is, the cell culture container is directly used as a substrate, and its bottom is modified with a near-infrared light-responsive gold nano-coating as a photothermal surface, facilitating cell detachment and transfer.

[0029] Furthermore, the cell culture container is a cell culture plate, cell culture flask, or cell culture dish, and the material of the cell culture container can be any material. For example, commercially available cell culture containers made of materials such as glass, polystyrene, polypropylene, polyvinyl chloride, and polyethylene are all suitable.

[0030] In addition, the present invention also provides a near-infrared light-responsive smart cell culture container, which is special in that it is obtained by the above-described preparation method.

[0031] And the application of the aforementioned near-infrared light-responsive smart cell culture container in cell sheet desorption and separation.

[0032] Principles of the present application:

[0033] The present application hydrophilic treatment of cell culture vessel (substrate) will increase the surface of the hydroxyl group, thereby increasing the binding force of the gold nano structure sputtering layer and the cell culture vessel, making it more stable; the methoxy polyethylene glycol thiol (mPEG-SH) is used to make the gold nano structure polyethylene glycol, and the PEG is fully combined with the gold nano structure through the mercapto-gold bond to enhance its biocompatibility; then the polyethylene glycol gold nano structure and the polypeptide (RGD) solution are mixed, and the concentration of RGD is controlled to make it as much as possible to be coupled with the gold nano structure, and the targeting of the target cell binding is enhanced; the cells are cultured on the gold nano structure layer, and the gold nano structure layer has a near infrared response function, which can heat the cell layer by absorbing near infrared light, and when a certain temperature is reached, the cell layer will be decoupled with the RGD on the gold nano structure layer, and then the complete separation is realized.

[0034] Advantages of the present application:

[0035] 1. The near infrared light responsive gold nano coating on the cell culture vessel is modified by the method of the present application, which is obtained directly by metal particle sputtering in one step, and the process steps are simple, no complex chemical synthesis method and steps are needed, the cycle is short, the efficiency is high, the raw materials are widely available, and no expensive equipment is used, the whole production cost is significantly reduced compared with the existing method, and no difficult to remove reagent is used, which not only has no obvious influence on the biocompatibility, but also makes the gold nano structure polyethylene glycol by using mPEG-SH, so that the PEG is fully combined with the gold nano structure through the mercapto-gold bond to greatly improve its biocompatibility.

[0036] 2. The present application uses oxygen plasma treatment means to hydrophilic treatment of cell culture vessel (substrate), so that the sputtered gold nano structure is combined with the substrate surface through hydrogen bond (chemical bond connection), which has good stability and is more stable.

[0037] 3. The steps of the present application are simple to operate, easy to start, and can be mass produced, which has good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 is the optical image of the original cell culture dish;

[0039] Figure 2 is the optical image of the near infrared light responsive intelligent cell culture dish prepared in example 1;

[0040] Figure 3 is the optical microscope image of the human kidney proximal tubular cells cultured by using the near infrared light responsive intelligent cell culture dish in example 1.

[0041] Figure 4is the desorption efficiency of the photothermal response of the near-infrared light responsive smart cell culture dish in Example 1 to human renal proximal tubular cells.

[0042] Figure 5 is the desorption efficiency of the photothermal response of the near-infrared light responsive smart cell culture dish in Example 1 to human renal proximal tubular cells in the second reuse.

[0043] Figure 6 is the cell activity test result of the near-infrared light responsive smart cell culture dish in Example 1 after 24h and 72h of culture of cells. DETAILED DESCRIPTION

[0044] The content of the present application is further described in detail below in combination with the drawings and specific examples:

[0045] Unless otherwise specified, the source of the raw materials used in the present application has no special requirements, and commercially available products known to those skilled in the art can be used.

[0046] The present application provides a method for manufacturing a near-infrared light responsive gold nanocoating smart cell culture container, the steps are as follows:

[0047] 1) Use a metal sputtering instrument to sputter gold nanoclusters onto a cell culture container that has been treated with a hydrophilic surface, and the thickness of the sputtered layer is 10-80nm;

[0048] 2) After washing the cell culture container sputtered in step 1) with deionized water, treat the surface of the gold nanoclusters with an mPEG-SH solution at 20-40℃ for 1-12h to obtain polyethylene glycolated gold nanoclusters on the cell culture container;

[0049] wherein the molar concentration of the methoxypolyethylene glycol thiol solution is 0.5-20mM;

[0050] According to the molar ratio of mercapto-polyethylene glycol (PEG-SH) to the sputtered gold nanoclusters in step 1) of (100-1000):1, the methoxypolyethylene glycol thiol (mPEG-SH) solution is added to the surface of the gold nanoclusters;

[0051] 3) Treat the polyethylene glycolated gold nanoclusters obtained on the cell culture container in step 2) with a polypeptide (RGD) solution at 20-40℃ for 1-12h, then wash the cell culture container treated as described above with deionized water and dry it at 20-50℃ to remove the water on the surface of the cell culture container, thereby completing the manufacturing of the near-infrared light responsive smart cell culture container;

[0052] wherein the molar concentration of the polypeptide (RGD) solution is 0.5-20mM;

[0053] The polypeptide (RGD) solution is added dropwise to the surface of the polyethylene glycolated gold nanoclusters at a ratio of (100-10000) : 1 of the molar ratio of the polypeptide (RGD) to the polyethylene glycolated gold nanoclusters.

[0054] The near-infrared light responsive intelligent cell culture container is prepared by the process of the specific example, and the process parameters are as follows:

[0055] Example 1

[0056] 1) Gold nanoclusters are sputtered onto a cell culture dish with a diameter of 35 mm and a surface treated with hydrophilic by using a metal sputtering instrument, and the thickness of the sputtered layer is 40 nm;

[0057] 2) After the cell culture dish sputtered in step 1) is washed with deionized water, the sputtered layer thereon is treated with a methoxy polyethylene glycol thiol (mPEG-SH) solution with a molar concentration of 1 mM at a ratio of 500:1 of the molar ratio of PEG-SH to the gold nanoclusters sputtered in step 1), and the reaction is carried out at room temperature for 10 hours, to obtain polyethylene glycolated gold nanoclusters on the cell culture dish;

[0058] 3) The polyethylene glycolated gold nanoclusters are treated with a polypeptide (RGD) solution with a molar concentration of 1 mM at a ratio of 1000:1 of the molar ratio of the polypeptide (RGD) to the polyethylene glycolated gold nanoclusters, and the reaction is carried out at room temperature for 10 hours; then the cell culture dish treated in the foregoing manner is washed with deionized water, and the washed cell culture dish is dried at 50°C for 5 hours to remove the water on the surface of the cell culture dish, to obtain a near-infrared light responsive intelligent cell culture container.

[0059] Example 2

[0060] 1) Gold nanoclusters are sputtered onto a cell culture dish with a diameter of 60 mm and a surface treated with hydrophilic by using a metal sputtering instrument, and the thickness of the sputtered layer is 80 nm;

[0061] 2) After the cell culture dish sputtered in step 1) is washed with deionized water, the sputtered layer thereon is treated with a methoxy polyethylene glycol thiol (mPEG-SH) solution with a molar concentration of 2 mM at a ratio of 600:1 of the molar ratio of PEG-SH to the gold nanoclusters sputtered in step 1), and the reaction is carried out at room temperature for 8 hours, to obtain polyethylene glycolated gold nanoclusters on the cell culture dish;

[0062] 3) The polyethylene glycolated gold nanoclusters are treated with a polypeptide (RGD) solution with a molar concentration of 2 mM at a ratio of 5000:1 of the molar ratio of the polypeptide (RGD) to the polyethylene glycolated gold nanoclusters, and the reaction is carried out at room temperature for 8 hours; then the cell culture dish treated in the foregoing manner is washed with deionized water, and the washed cell culture dish is dried at 37°C for 4 hours to remove the water on the surface of the cell culture dish, to obtain a near-infrared light responsive intelligent cell culture container.

[0063] Example 3

[0064] 1) Gold nanoclusters were sputtered onto a cell culture dish with a diameter of 100 mm which was surface hydrophilic treated by a metal sputtering instrument, and the thickness of the sputtered layer was 10 nm;

[0065] 2) After the cell culture dish sputtered in step 1) was cleaned with deionized water, the sputtered layer was treated with a methoxy polyethylene glycol thiol (mPEG-SH) solution with a molar concentration of 2 mM according to a molar ratio of PEG-SH to gold nanoclusters sputtered in step 1) of 400:1, and reacted at room temperature for 8 h, thereby obtaining polyethylene glycolated gold nanoclusters on the cell culture dish;

[0066] 3) The polyethylene glycolated gold nanoclusters were treated with a polypeptide (RGD) solution with a molar concentration of 2 mM according to a molar ratio of polypeptide (RGD) to polyethylene glycolated gold nanoclusters of 3000:1, and reacted at room temperature for 9 h; then the cell culture dish treated in the foregoing manner was cleaned with deionized water, and the cleaned cell culture dish was dried at 40 °C for 4 h to remove water on the surface of the cell culture dish, thereby obtaining a near-infrared light responsive intelligent cell culture container.

[0067] Example 4

[0068] 1) Gold nanoclusters were sputtered onto a cell culture dish with a diameter of 150 mm which was surface hydrophilic treated by a metal sputtering instrument, and the thickness of the sputtered layer was 50 nm;

[0069] 2) After the cell culture dish sputtered in step 1) was cleaned with deionized water, the sputtered layer was treated with a methoxy polyethylene glycol thiol (mPEG-SH) solution with a molar concentration of 2 mM according to a molar ratio of PEG-SH to gold nanoclusters sputtered in step 1) of 600:1, and reacted at room temperature for 8 h, thereby obtaining polyethylene glycolated gold nanoclusters on the cell culture dish;

[0070] 3) The polyethylene glycolated gold nanoclusters were treated with a polypeptide (RGD) solution with a molar concentration of 2 mM according to a molar ratio of polypeptide (RGD) to polyethylene glycolated gold nanoclusters of 4000:1, and reacted at room temperature for 9 h; then the cell culture dish treated in the foregoing manner was cleaned with deionized water, and the cleaned cell culture dish was dried at 20 °C for 4 h to remove water on the surface of the cell culture dish, thereby obtaining a near-infrared light responsive intelligent cell culture container.

[0071] Example 5

[0072] 1) Gold nanoclusters were sputtered onto a cell culture dish with a diameter of 35 mm which was surface hydrophilic treated by a metal sputtering instrument, and the thickness of the sputtered layer was 60 nm;

[0073] 2) After the cell culture dish sputtered in step 1) is cleaned with deionized water, the sputtered layer is treated with a 2mM methoxypolyethylene glycol thiol (mPEG-SH) solution according to a molar ratio of PEG-SH to the gold nanoclusters sputtered in step 1) of 600:1, and is reacted at room temperature for 8h to obtain polyethylene glycolated gold nanoclusters on the cell culture dish;

[0074] 3) The polyethylene glycolated gold nanoclusters are treated with a 2mM polypeptide (RGD) solution according to a molar ratio of polypeptide (RGD) to the polyethylene glycolated gold nanoclusters of 5000:1, and are reacted at room temperature for 8h; the cell culture dish treated in the foregoing manner is then cleaned with deionized water, and the cleaned cell culture dish is dried at 50°C for 5h to remove water on the surface of the cell culture dish, thereby obtaining the near-infrared light responsive intelligent cell culture container.

[0075] Figure 1 The original cell culture dish is as shown in Fig. 1. Figure 2 The near-infrared light responsive intelligent cell culture dish prepared in Example 1 (i.e. the cell culture dish after sputtering of gold nanoclusters and modification) has a film on the bottom, which is different from the original cell culture dish. The cell culture dish is then used to culture human kidney proximal tubular cells to obtain a cell sheet, as shown in Fig. 3. Figure 2 The near-infrared light responsive intelligent cell culture dish prepared in the application can successfully perform cell culture. Figure 3

[0076] To verify the near-infrared light responsive capability and stability (reusability) of the near-infrared light responsive intelligent cell culture dish prepared in the application, the cell culture dish is irradiated with near-infrared light to perform a desorption test, and the desorption efficiency is as shown in Fig. 5. Figure 4 It can be seen that the near-infrared light responsive intelligent cell culture dish prepared in the application has good near-infrared light responsive capability and can convert light energy into heat energy to desorb the cell sheet. The cell culture dish on which the cell sheet is desorbed is cleaned and then used to continue culturing the cell sheet and performing the desorption process. The desorption efficiency of the cell sheet desorbed for the second time is as shown in Fig. 6. Figure 5 It can be seen that the near-infrared light responsive intelligent cell culture dish prepared in the application still has a high desorption capability when used again, and has good stability. The biocompatibility of the near-infrared light responsive intelligent cell culture dish prepared in the application is detected, and the cell activity is detected for 24h and 72h. The detection results are as shown in Fig. 7. Figure 6 It can be seen that the cell culture dish prepared in the application has good biocompatibility.

[0077] In summary, the cell culture dish prepared in the application can intelligently respond under near-infrared light irradiation, has simple manufacturing process, low cost, high efficiency, good biocompatibility and stability. ​

[0078] The above merely provides the specific implementation of the present application, but the protection scope of the present application is not limited thereto, and any skilled person in the art can easily think of various equivalent modifications or replacements within the technical scope disclosed by the present application, and these modifications or replacements should be covered within the protection scope of the present application.

Claims

1. A method for fabricating a near-infrared light-responsive intelligent cell culture container, characterized in that: Near-infrared light-responsive gold nanocoatings were prepared on cell culture containers with hydrophilic surface treatment to obtain near-infrared light-responsive smart cell culture containers. The near-infrared light-responsive gold nanocoating was prepared according to the following method: 1) Gold nanoclusters were sputtered onto a cell culture vessel with a hydrophilic surface, wherein the thickness of the sputtered layer was 10–80 nm; 2) After cleaning the cell culture container obtained in step 1), the surface of the sputtered layer is treated with methoxy polyethylene glycol thiol (mPEG-SH) solution to react on the cell culture container to obtain polyethylene glycol-modified gold nanoclusters. 3) Treat the polyethylene glycol-modified gold nanoclusters obtained on the cell culture vessel in step 2) with a polypeptide (RGD) solution. After the reaction is complete, clean the cell culture vessel and dry it to obtain a near-infrared light-responsive gold nanocoating on the surface of the cell culture vessel.

2. The manufacturing method according to claim 1, characterized in that: In step 1), according to the sputtering layer thickness requirements, gold nanoclusters are sputtered onto the cell culture container with a hydrophilic surface using a metal sputtering instrument.

3. The manufacturing method according to claim 1 or 2, characterized in that: In step 2), the molar concentration of the methoxy polyethylene glycol thiol solution is 0.5–20 mM; A methoxy polyethylene glycol thiol (mPEG-SH) solution was dropped onto the surface of the gold nanoclusters at a molar ratio of (100-1000):1 between thiolated polyethylene glycol (PEG-SH) and sputtered gold nanoclusters in step 1). The reaction temperature is 20–40℃, and the reaction time is 1–12 h.

4. The manufacturing method according to claim 3, characterized in that: In step 3), the molar concentration of the polypeptide (RGD) solution is 0.5–20 mM; The polypeptide (RGD) solution was dropped onto the surface of the polyethylene glycol-modified gold nanoclusters at a molar ratio of (100-10000):

1. The reaction temperature is 20–40℃, and the reaction time is 1–12 h; The drying temperature is 20–50℃.

5. The manufacturing method according to claim 1, characterized in that: In step 1), the surface hydrophilic treatment refers to treatment using a UVO cleaning machine; In both steps 2) and 3), deionized water is used for cleaning.

6. A near-infrared light-responsive intelligent cell culture container, characterized in that: Obtained by any of the manufacturing methods described in claims 1-5.

7. The application of the near-infrared light-responsive intelligent cell culture container of claim 6 in cell sheet desorption and separation.

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