Dipeptide hydrogel and application thereof as cell culture medium

Abstract

The invention discloses dipeptide hydrogel and application thereof as a cell culture medium, and belongs to the field of material science. According to the invention, the dipeptide hydrogel is designed and assembled, and the dipeptide hydrogel is prepared by the following steps: carrying out self-assembly on phenylalanine dipeptide modified by 9-fluorenyl methoxy carbonyl protecting group (Fmoc), and then adding tetracarboxyl phenyl porphyrin (TCPP) and distearoyl phosphatidyl ethanolamine (DSPE) for co-assembly. When the dipeptide hydrogel prepared by the invention is applied as a cell culture medium, the proliferation activity of cultured cells can be improved, so that the growth speed and density of the cells are improved, and the dipeptide hydrogel has good biological safety. The possibility is provided for promoting the high-level development of the cell culture field.

Description

Technical Field

[0001] The invention belongs to the field of materials science, and specifically relates to a dipeptide hydrogel and an application thereof as a cell culture matrix. Background Art

[0002] Peptides are a class of compounds composed of amino acids connected by peptide bonds, and are also the hydrolysis products of proteins. Hydrogels are a class of materials that are hydrophilic but cannot dissolve in water and can only swell. They are a type of material form that is ubiquitous in nature. Peptide self-assembly is a spontaneous thermodynamic and kinetic process, and is also regulated by environmental factors such as temperature, pH, ultrasound, ionic strength and ultraviolet light. A large number of reports have shown that peptides or peptide derivatives can self-assemble to form various nanostructures such as hydrogels through non-covalent interactions including electrostatic interactions, hydrophobic interactions, hydrogen bonding interactions, stacking and π-π van der Waals forces.

[0003] Culture matrix usually refers to an amorphous colloid composed of biomacromolecules, which is colorless and transparent, has a certain viscosity, and contains tissue fluid in the pores. It is mainly used to fix and support cells and tissues, and plays a certain role in physical support and protection. The hydrogel matrix provides cells with all-round growth conditions, which is more conducive to cell growth than traditional culture matrix. It has become a new favorite in cell culture research because of its excellent properties of simulating extracellular matrix.

[0004] Dipeptide is a compound composed of two amino acid residues connected by an amide bond and is the simplest polypeptide molecule. Compared with polypeptide (non-dipeptide) hydrogel, dipeptide hydrogel has similar performance and is easier to synthesize and has lower cost. However, it also has limitations such as insufficient gel strength, poor solubility and single functional properties, which limit the potential application value of dipeptide hydrogel. By regulating the assembly mode of polypeptides, dipeptide hydrogels with good biocompatibility and cell culture effects can be prepared, which is of great significance for promoting the development of the field of cell culture to a higher level. Summary of the invention

[0005] In view of the above prior art, the object of the present invention is to provide a dipeptide hydrogel and its application as a cell culture matrix.

[0006] To achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a dipeptide hydrogel, wherein the dipeptide hydrogel is self-assembled by a phenylalanine dipeptide modified with a 9-fluorenylmethoxycarbonyl protecting group, and then co-assembled by adding tetracarboxyl porphyrin and distearoylphosphatidylethanolamine.

[0007] The preparation method of the dipeptide hydrogel comprises the following steps: (1) Pre-dissolving the phenylalanine dipeptide modified with a 9-fluorenylmethoxycarbonyl protecting group in a DMSO organic solvent, adding water to mix, then ultrasonically dissolving, and standing to obtain an Fmoc-FF hydrogel; (2) Tetracarboxyl porphyrin and distearoyl phosphatidylethanolamine are added to the Fmoc-FF hydrogel obtained in step (1) for co-assembly to obtain a dipeptide hydrogel.

[0008] In step (1), the mass volume ratio of the phenylalanine dipeptide modified with the 9-fluorenylmethoxycarbonyl protecting group to the DMSO organic solvent is (100-500):1.

[0009] In step (1), the static treatment is to place the mixture at 35°C for 10-15 hours.

[0010] In step (2), the mass ratio of the added amount of tetracarboxyl porphyrin to that of Fmoc-FF hydrogel is 1:(2-2.5).

[0011] In step (2), the mass ratio of the added amount of distearoyl phosphatidylethanolamine to the added amount of Fmoc-FF hydrogel is 1:(5-10).

[0012] In step (2), the co-assembly conditions are: temperature 30-37°C, humidity 40%-80%, and co-assembly time 5-12 h.

[0013] The second aspect of the present invention provides the use of the dipeptide hydrogel in preparing a cell culture matrix for enhancing cell proliferation activity.

[0014] The cultured cells are any one of 293T, MRC-5, Vero and human fibroblasts. Beneficial effects of the present invention: (1) The present invention designs and assembles a dipeptide hydrogel, which is self-assembled by phenylalanine dipeptide modified with 9-fluorenylmethoxycarbonyl protecting group (Fmoc), and then co-assembled by adding tetracarboxyphenylporphyrin (TCPP) and distearoylphosphatidylethanolamine (DSPE). When the dipeptide hydrogel prepared by the present invention is used as a cell culture matrix, it can improve the proliferation activity of cultured cells and has good biosafety, which provides the possibility of promoting the development of the cell culture field to a higher level.

[0015] (2) When designing and assembling the dipeptide hydrogel, the tetracarboxylphenylporphyrin (TCPP) added in the present invention is an aromatic porphyrin with four carboxyl groups, and its structure contains an aromatic ring and four carboxyl groups. The addition of TCPP can enhance the hydrophilicity of the dipeptide hydrogel, and TCPP also has the ability to image bacterial infection in real time. By utilizing the fluorescence characteristics of TCPP, real-time monitoring and imaging can be performed when bacterial infection occurs, which can help to more accurately judge the infection situation during cell culture and take corresponding treatment measures. The addition of distearoylphosphatidylethanolamine (DSPE) can improve the biocompatibility of the dipeptide hydrogel with cultured cells, and synergize with TCPP to help enhance the proliferation activity of cells, thereby increasing cell growth rate and density. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the SEM image of the dipeptide hydrogel prepared in Example 1. DETAILED DESCRIPTION

[0017] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0018] The following detailed description is for illustrative purposes only and is intended to provide further explanation of the present application, rather than to limit the scope of the present invention.

[0019] The Fmoc-FF used in the examples of the present invention was purchased from Shanghai Coraman Reagent Co., Ltd. with a purity of 98%. The TCPP used was purchased from Xi'an Qiyue Biotechnology Co., Ltd. with a purity of 95%. The DSPE used was purchased from Shanghai Langxu Biotechnology Co., Ltd. with a purity of 98%.

[0020] Example 1: Preparation of dipeptide hydrogel (1) Dissolve 5 mg of 9-fluorenylmethoxycarbonyl protective group-modified phenylalanine dipeptide (Fmoc-FF) in 10 uL DMSO, add 500 uL water and mix, then place under ultrasound at 20 kHz for 1 minute, take out the solution and place it at 35 °C for 10 hours to obtain Fmoc-FF hydrogel; (2) Take 5 mg of the Fmoc-FF hydrogel prepared in step (1), add 2.5 mg of tetracarboxyphenylporphyrin (TCPP) and 0.5 mg of distearoylphosphatidylethanolamine (DSPE), stir evenly, and assemble for 5 h at 37 ° C and 40% humidity to obtain a dipeptide hydrogel. The SEM image of the dipeptide hydrogel is shown in Figure 1 .

[0021] Example 2: Preparation of dipeptide hydrogel (1) Dissolve 10 mg of 9-fluorenylmethoxycarbonyl protective group-modified phenylalanine dipeptide (Fmoc-FF) in 100 uL DMSO, add 500 uL water and mix, then place under ultrasound at 20 kHz for 1 minute, take out the solution and place it at 35 °C for 15 hours to obtain Fmoc-FF hydrogel; (2) Take 10 mg of the Fmoc-FF hydrogel prepared in step (1), add 4 mg of tetracarboxyphenylporphyrin and 2 mg of distearoylphosphatidylethanolamine, stir evenly, and assemble at 37°C and 40% humidity for 8 h to obtain a dipeptide hydrogel.

[0022] Experimental Example 3: Determination of water absorption rate based on dipeptide hydrogel The mass of the dipeptide hydrogel prepared in Example 1 and Example 2 in a dry environment was recorded as S1. The dipeptide hydrogel was immersed in a PBS solution at 37°C for 24 hours, and the remaining liquid on the surface of the dipeptide hydrogel was removed with filter paper. The dipeptide hydrogel after immersion was weighed again and recorded as S2. The water absorption rate was calculated according to the formula = (S2-S1) / S1×100%. The test results are shown in Table 1: Table 1: Water absorption test The test results show that the dipeptide hydrogel prepared by the present invention has excellent water absorption performance and has application potential as a cell culture matrix.

[0023] Comparative Example 1: Preparation of dipeptide hydrogel The difference between Comparative Example 1 and Example 1 is that only tetracarboxyphenylporphyrin is added to the Fmoc-FF hydrogel, specifically as follows: (1) Dissolve 5 mg of 9-fluorenylmethoxycarbonyl protective group-modified phenylalanine dipeptide (Fmoc-FF) in 10 uL DMSO, add 500 uL water and mix, then place under ultrasound at 20 kHz for 1 minute, take out the solution and place it at 35 °C for 10 hours to obtain Fmoc-FF hydrogel; (2) Take 5 mg of the Fmoc-FF hydrogel prepared in step (1), add 2.5 mg of tetracarboxyphenylporphyrin and stir evenly. Under the conditions of 37°C and 40% humidity, assemble for a total of 5 hours to obtain a dipeptide hydrogel.

[0024] Comparative Example 2: Preparation of dipeptide hydrogel The difference between Comparative Example 2 and Example 1 is that only distearoyl phosphatidylethanolamine is added to the Fmoc-FF hydrogel, specifically as follows: (1) Dissolve 5 mg of 9-fluorenylmethoxycarbonyl protective group-modified phenylalanine dipeptide (Fmoc-FF) in 10 uL DMSO, add 500 uL water and mix, then place under ultrasound at 20 kHz for 1 minute, take out the solution and place it at 35 °C for 10 hours to obtain Fmoc-FF hydrogel; (2) Take 5 mg of the Fmoc-FF hydrogel prepared in step (1), add 0.5 mg of distearoyl phosphatidylethanolamine and stir evenly. At 37°C and 40% humidity, assemble for a total of 5 hours to obtain a dipeptide hydrogel.

[0025] Test Example 1: Proliferation Activity Detection According to the methods described in Example 1, Comparative Example 1 and Comparative Example 2, dipeptide hydrogels were prepared in 96-well plates, respectively, and recorded as Example 1 group, Comparative Example 1 group and Comparative Example 2 group. DMEM liquid culture medium was added to the dipeptide hydrogels prepared in each group and soaked for 3 hours. 1×10 5 293T cells were inoculated on the soaked material at a cell density of 1×10 cells / well. The culture plate was then placed on an oscillator and shaken for 10 min to allow the cells to be evenly inoculated on the dipeptide hydrogel cell culture matrix material. 200 μL of DMEM liquid culture medium was added to the edge to immerse the material, and then placed in an incubator for culture. 5 A group of 293T cells were inoculated into regular DMEM liquid culture medium at a cell density of 1:1 as the control group.

[0026] The cell counting kit (CCK-8) was used to measure the proliferation activity of each group of cells after 24 hours of culture. The measurement steps were as follows: 10 μL of CCK-8 reagent and 90 μL of DMEM mixture were dripped into each well and incubated in the dark for 90 minutes. After shaking for 15 minutes, the dipeptide hydrogel cell culture matrix material in the 96-well plate was removed, and the remaining liquid was transferred to a new 96-well plate. The absorbance was tested at 450 nm to reflect the proliferation activity of each group of cells.

[0027] Table 2: Proliferation activity assay The test results show that the dipeptide hydrogel prepared by the present invention can improve the proliferation activity of cultured cells as a cell culture matrix material, has good biosafety, and provides the possibility for promoting the development of the cell culture field to a higher level.

[0028] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification made within the spirit and principle of the present application shall be deemed to be appropriate. Equivalent replacements, improvements, etc. should all be included in the protection scope of this application.

Claims

1. A dipeptide hydrogel, characterized in that: The dipeptide hydrogel is prepared by self-assembly of phenylalanine dipeptide modified with a 9-fluorenylmethoxycarbonyl protective group, and then adding tetracarboxyl porphyrin and distearoyl phosphatidylethanolamine for co-assembly.

2. The dipeptide hydrogel according to claim 1, characterized in that The preparation method of the dipeptide hydrogel comprises the following steps: (1) Pre-dissolving the phenylalanine dipeptide modified with a 9-fluorenylmethoxycarbonyl protecting group in a DMSO organic solvent, adding water to mix, then ultrasonically dissolving, and standing to obtain an Fmoc-FF hydrogel; (2) Tetracarboxyl porphyrin and distearoyl phosphatidylethanolamine are added to the Fmoc-FF hydrogel obtained in step (1) for co-assembly to obtain a dipeptide hydrogel.

3. The dipeptide hydrogel according to claim 2, characterized in that: In step (1), the mass volume ratio of the phenylalanine dipeptide modified with the 9-fluorenylmethoxycarbonyl protecting group to the DMSO organic solvent is (100-500):

1.

4. The dipeptide hydrogel according to claim 2, characterized in that In step (1), the static treatment is to place the mixture at 35°C for 10-15 hours.

5. The dipeptide hydrogel according to claim 2, characterized in that: In step (2), the mass ratio of the added amount of tetracarboxyl porphyrin to that of Fmoc-FF hydrogel is 1:(2-2.5).

6. The dipeptide hydrogel according to claim 2, characterized in that: In step (2), the mass ratio of the added amount of distearoyl phosphatidylethanolamine to the added amount of Fmoc-FF hydrogel is 1:(5-10).

7. The dipeptide hydrogel according to claim 2, characterized in that: In step (2), the co-assembly conditions are: temperature 30-37°C, humidity 40%-80%, and co-assembly time 5-12 h.

8. Use of the dipeptide hydrogel according to claim 1 in preparing a cell culture matrix for enhancing cell proliferation activity.

9. The use according to claim 8, characterized in that: The cultured cells are any one of 293T, MRC-5, Vero and human fibroblasts.

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