A dipeptide hydrogel and its application as a cell culture matrix

Abstract

The present invention discloses a dipeptide hydrogel and its application as a cell culture matrix, belonging to the field of materials science. The present invention designs and assembles a dipeptide hydrogel, which is co-assembled by self-assembly of phenylalanine dipeptide modified with 9-fluorenylmethoxycarbonyl protecting group (Fmoc), and then adding tetracarboxyphenyl porphyrin (TCPP) and distearoyl phosphatidylethanolamine (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, thereby increasing the cell growth rate and density, and has good biosafety. It provides the possibility for promoting the development of the cell culture field to a higher level.

Description

Technical Field

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

[0002] Polypeptides are a class of compounds formed by 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, and are a common form of substances in nature. The self-assembly of polypeptides 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 demonstrated that polypeptides or derivatives of polypeptides can self-assemble into diverse nanostructures such as hydrogels through non-covalent interactions including electrostatic interactions, hydrophobic interactions, hydrogen bond interactions, stacking, and π-π van der Waals forces.

[0003] A culture matrix generally refers to an amorphous gel formed by biological macromolecules, which is colorless and transparent, has a certain viscosity, and has tissue fluid in the pores. It is mainly used to fix and support cells and tissues, playing a certain physical support and protection role. The hydrogel matrix provides all-round growth conditions for cells and is more conducive to cell growth than traditional culture matrices, and has become a new favorite in cell culture research due to its excellent characteristics of mimicking the extracellular matrix.

[0004] Dipeptides are compounds formed by two amino acid residues connected by an amide bond and are the simplest polypeptide molecules. Dipeptide hydrogels have similar properties to polypeptide (non-dipeptide) hydrogels, and are synthesized more simply and at lower cost. However, they also have limitations such as insufficient gel strength, poor solubility, and single functional properties, which limit the potential application value of dipeptide hydrogels. Preparing dipeptide hydrogels with good biocompatibility and cell culture effects by regulating the assembly mode of polypeptides is of great significance for promoting the development of the cell culture field to a higher level. Summary of the Invention

[0005] Aiming at the above-mentioned prior art, the purpose of the present invention is to provide a dipeptide hydrogel and its application as a cell culture matrix.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, a dipeptide hydrogel is provided, which is formed by self-assembling a phenylalanine dipeptide modified with a 9-fluorenylmethoxycarbonyl protecting group, and then co-assembling with tetracarboxyl porphyrin and distearoyl phosphatidylethanolamine.

[0008] The preparation method of the dipeptide hydrogel includes the following steps:

[0009] (1) Pre-dissolve the phenylalanine dipeptide modified with 9-fluorenylmethoxycarbonyl protecting group in DMSO organic solvent, add water and mix, then perform ultrasonic dissolution, and let it stand to obtain Fmoc-FF hydrogel.

[0010] (2) Add tetracarboxyl porphyrin and distearoyl phosphatidylethanolamine to the Fmoc-FF hydrogel obtained in step (1) for co-assembly to obtain a dipeptide hydrogel.

[0011] In step (1), the mass-volume ratio of the phenylalanine dipeptide modified with 9-fluorenylmethoxycarbonyl protecting group to the DMSO organic solvent is (100 - 500):1.

[0012] In step (1), the standing treatment is to stand at 35 °C for 10 - 15 h.

[0013] In step (2), the mass ratio of the tetracarboxyl porphyrin to the added amount of Fmoc-FF hydrogel is 1:(2 - 2.5).

[0014] In step (2), the mass ratio of the distearoyl phosphatidylethanolamine to the added amount of Fmoc-FF hydrogel is 1:(5 - 10).

[0015] In step (2), the conditions for the co-assembly are: temperature 30 - 37 °C, humidity 40% - 80%, and co-assembly time 5 - 12 h.

[0016] In the second aspect of the present invention, there is provided the application of the above-mentioned dipeptide hydrogel in the preparation of a cell culture matrix for enhancing cell proliferation vitality.

[0017] The cultured cells are any one of 293T, MRC-5, Vero, and human fibroblasts.

[0018] The beneficial effects of the present invention:

[0019] (1) The present invention designs and assembles a dipeptide hydrogel, which is formed by self-assembly of a phenylalanine dipeptide modified with 9-fluorenylmethoxycarbonyl (Fmoc) protecting group, and then co-assembled with tetracarboxyl phenyl porphyrin (TCPP) and distearoyl phosphatidylethanolamine (DSPE). When the dipeptide hydrogel prepared by the present invention is used as a cell culture matrix, it can improve the proliferation vitality of cultured cells, has good biosafety, and provides the possibility for promoting the development of the cell culture field to a higher level.

[0020] (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

[0021] Figure 1 This is the SEM image of the dipeptide hydrogel prepared in Example 1. DETAILED DESCRIPTION

[0022] 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.

[0023] 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.

[0024] 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%.

[0025] Example 1: Preparation of dipeptide hydrogel

[0026] (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;

[0027] (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 .

[0028] Example 2: Preparation of Dipeptide Hydrogel

[0029] (1) Dissolve 10 mg of phenylalanine dipeptide modified with 9-fluorenylmethoxycarbonyl protecting group (Fmoc-FF) in 100 μL of DMSO, add 500 μL of water and mix, then sonicate at 20 kHz for 1 minute. Take out the solution and let it stand at 35 °C for 15 h to obtain Fmoc-FF hydrogel;

[0030] (2) Take 10 mg of the Fmoc-FF hydrogel prepared in step (1), add 4 mg of tetracarboxyphenyl porphyrin and 2 mg of distearoyl phosphatidylethanolamine, stir evenly, and co-assemble at 37 °C and 40% humidity for 8 h to obtain the dipeptide hydrogel.

[0031] Experimental Example 3: Measurement of Water Absorption Rate of Dipeptide Hydrogel

[0032] Statistically record the mass of the dipeptide hydrogel prepared in Example 1 and Example 2 in a dry environment as S1. Immerse the dipeptide hydrogel in PBS solution at 37 °C for 24 h, take it out and absorb the remaining liquid on the surface of the dipeptide hydrogel with filter paper, and weigh the soaked dipeptide hydrogel again and record it as S2. Calculate the water absorption rate according to the formula = (S2 - S1) / S1 × 100%. The test results are shown in Table 1:

[0033] Table 1: Detection of Water Absorption Rate

[0034]

[0035] The test results show that the dipeptide hydrogel prepared by the present invention has excellent water absorption performance and has the application potential as a cell culture matrix.

[0036] Comparative Example 1: Preparation of Dipeptide Hydrogel

[0037] The difference between Comparative Example 1 and Example 1 is only that only tetracarboxyphenyl porphyrin is added to the Fmoc-FF hydrogel, specifically as follows:

[0038] (1) Dissolve 5 mg of phenylalanine dipeptide modified with 9-fluorenylmethoxycarbonyl protecting group (Fmoc-FF) in 10 μL of DMSO, add 500 μL of water and mix, then sonicate at 20 kHz for 1 minute. Take out the solution and let it stand at 35 °C for 10 h to obtain Fmoc-FF hydrogel;

[0039] (2) Take 5 mg of the Fmoc-FF hydrogel prepared in step (1), add 2.5 mg of tetracarboxyphenyl porphyrin, stir evenly, and co-assemble at 37 °C and 40% humidity for 5 h to obtain the dipeptide hydrogel.

[0040] Comparative Example 2: Preparation of Dipeptide Hydrogel

[0041] The difference between Comparative Example 2 and Example 1 is only that the Fmoc-FF hydrogel only adds distearoyl phosphatidylethanolamine, specifically as follows:

[0042] (1) Dissolve 5 mg of phenylalanine dipeptide modified with 9-fluorenylmethoxycarbonyl protecting group (Fmoc-FF) in 10 μL of DMSO, add 500 μL of water and mix, then place it under ultrasound at 20 kHz for 1 minute. Take out the solution and let it stand at 35 °C for 10 h to obtain the Fmoc-FF hydrogel;

[0043] (2) Take 5 mg of the Fmoc-FF hydrogel prepared in step (1), add 0.5 mg of distearoyl phosphatidylethanolamine and stir evenly. Under the conditions of 37 °C and 40% humidity, co-assemble for 5 h to obtain the dipeptide hydrogel.

[0044] Test Example 1: Detection of Proliferation Activity

[0045] According to the methods described in Example 1, Comparative Example 1 and Comparative Example 2, prepare dipeptide hydrogels in 96-well plates respectively, and label them as the Example 1 group, the Comparative Example 1 group and the Comparative Example 2 group. Add DMEM liquid medium to the dipeptide hydrogels prepared in each group and soak for 3 hours. Inoculate 293T cells onto the soaked materials at a cell density of 1×10 5 . Then place the above culture plates on an oscillator and oscillate for 10 min to make the cells evenly inoculated on the dipeptide hydrogel cell culture matrix material. Add 200 μL of DMEM liquid medium to the edge to immerse the material, and then place it in an incubator for culture. A group without dipeptide hydrogel, inoculating 293T cells onto the conventional DMEM liquid medium at a cell density of 1×10 5 is used as the control group.

[0046] Use a cell counting kit (CCK-8) to measure the proliferation activity of the cells in each group after culturing for 24 h. The measurement steps are as follows: Drop a mixture of 10 μL of CCK-8 reagent and 90 μL of DMEM into each well, and incubate in the dark for 90 min. After shaking for 15 min, remove the dipeptide hydrogel cell culture matrix material in the 96-well plate, transfer the remaining liquid to a new 96-well plate, and measure the absorbance at 450 nm to reflect the proliferation activity of the cells in each group.

[0047] Table 2: Detection of Proliferation Activity

[0048]

[0049] The detection results show that the dipeptide hydrogel prepared by the present invention, as a cell culture matrix material, can improve the proliferation vitality of cultured cells, has good biosafety, and provides the possibility for promoting the development of the cell culture field to a higher level.

[0050] The foregoing are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various modifications and changes can be made to the present application. Any modification

[0051] equivalent replacement, improvement, etc. shall be included within the protection scope of the present application.

Claims

1. A dipeptide hydrogel, characterized in that: The dipeptide hydrogel is self-assembled by phenylalanine dipeptide modified with 9-fluorenylmethoxycarbonyl protecting group, and then co-assembled by adding tetracarboxyl porphyrin and distearoyl phosphatidylethanolamine; 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) adding tetracarboxyl porphyrin and distearoyl phosphatidylethanolamine to the Fmoc-FF hydrogel obtained in step (1) for co-assembly to obtain a dipeptide hydrogel; In step (2), the co-assembly conditions are: temperature 30-37°C, humidity 40%-80%, and co-assembly time 5-12 h.

2. The dipeptide hydrogel according to claim 1, 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.

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

4. The dipeptide hydrogel according to claim 1, 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).

5. The dipeptide hydrogel according to claim 1, 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).

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

7. The use according to claim 6, characterized in that: The cultured cells are any one of 293T, MRC-5, Vero and human fibroblasts.

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