CN110066315A - A kind of novel hydrogels and preparation method thereof - Google Patents

Abstract

The invention discloses a kind of novel hydrogels and preparation method thereof, it is related to gel technique field, the novel hydrogels are prepared by a kind of synthetic peptide, the sequence of the synthetic peptide are as follows: glutamine-valine-glutamic acid-valine-lysine-valine-glutamic acid-valine-lysine, it, which has, is easily-synthesized, belong to all aminoacid sequence, its end group does not contain Nap- (naphthalene), Fmoc- (fluorenes methoxy carbonyl acyl group), Cbz- (benzyloxy carbonyl acyl group), the groups such as aliphatic chain, the specific immunity easily caused can effectively be avoided or reduced, no cytotoxicity, and the synthetic peptide has high gelation, hydrogel can be quickly formed by pH induction.

Description

A new type of hydrogel and its preparation method

technical field

The present invention relates to the technical field of gels, in particular, to a novel hydrogel and a preparation method thereof.

Background technique

Polypeptide hydrogels have significant potential in life science fields such as tissue engineering, drug delivery, and biosensing. As a new type of material, synthetic peptides are easy to program. Some current research work controls the self-assembly process of polypeptides by regulating the amino acid sequence of synthetic polypeptides to form fibers with a defined thickness to form hydrogels with tunable mechanical properties. Synthetic polypeptides can be designed to respond to external stimuli, such as salt ions, light, temperature, or enzymes, and the resulting hydrogels can be used for coagulation, drug molecule release, and more. Furthermore, by controlling the degradation characteristics of peptides with specific sequences in the human body, synthetic peptide-based hydrogels are ideal candidates for use as topical drug delivery vehicles.

In the current research, the peptide sequences used to synthesize peptide hydrogels are long, more than 10 amino acids, and the synthesis is complex and difficult; many synthetic short peptide molecules can easily cause specific immunity of the body, and even have cytotoxicity.

SUMMARY OF THE INVENTION

The first object of the present invention is to provide a synthetic peptide, which is easy to synthesize, belongs to a complete amino acid sequence, and does not contain Nap-(naphthalene), Fmoc-(fluorenylmethoxycarbonyl), Cbz-(benzyloxycarbonyl) at the end group ), aliphatic chain and other groups, can effectively avoid or reduce easily induced specific immunity, no cytotoxicity, and the synthetic peptide has high gelation, and can quickly form a hydrogel through pH induction.

The second object of the present invention is to provide a material for synthesizing a hydrogel, which can quickly and efficiently synthesize a hydrogel with good cytocompatibility, and the synthesized hydrogel has no obvious cytotoxicity.

The third object of the present invention is to provide a preparation method of a synthetic hydrogel, which can effectively synthesize a non-cytotoxic hydrogel.

The fourth object of the present invention is to provide a novel hydrogel which is non-cytotoxic and has good cytocompatibility.

The fifth object of the present invention is to provide an application of the above-mentioned synthetic peptide, material or hydrogel in the field of food processing.

The present invention is realized in this way:

A synthetic peptide whose sequence is: glutamine-valine-glutamic acid-valine-lysine-valine-glutamic acid-valine-lysine. Specifically, the chemical structure of the synthetic peptide at pH 7 is shown in FIG. 1 .

In the embodiment of the present invention, the synthetic peptide belongs to the complete amino acid sequence, that is, the terminal groups (N-terminal and C-terminal) of the synthetic peptide do not contain Nap-(naphthalene), Fmoc-(fluorenylmethoxycarbonyl), Cbz- (benzyloxycarbonyl), aliphatic chain and other groups, synthetic peptide molecules with the above-mentioned end groups are easy to cause specific immunity of the body, and some peptides have been proved to be cytotoxic.

However, the synthetic peptide provided in the embodiment of the present invention has no cytotoxicity or obvious cytotoxicity, and the synthetic peptide has a short sequence, is easy to synthesize, has good cytocompatibility requirements, and can be used under acidic or neutral conditions. form a gel.

The embodiment of the present invention also provides a material for synthesizing hydrogel, which includes the above-mentioned synthetic peptide. Preferably, the material for synthesizing the hydrogel may also include water, a pH-adjusting solution, such as NaOH solution, and the like.

Embodiments of the present invention also provide a method for preparing a novel hydrogel, which includes a hydrogel preparation step and a hydrogel formation step. details as follows:

Hydrogel preparation steps:

The above-mentioned synthetic peptides or the above-mentioned materials are used to prepare a gel aqueous solution. Specifically, the sequence of the synthetic peptide is: glutamine-valine-glutamic acid-valine-lysine-valine-glutamic acid-valine-lysine, and the specific effects are the same as above.

Stir the synthetic peptide with water to fully dissolve the peptide, and prepare the synthetic peptide at a concentration of 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, and 10 mg/mL. , 11 mg/mL, 12 mg/mL, 13 mg/mL, 14 mg/mL, 15 mg/mL, 16 mg/mL, 17 mg/mL, 18 mg/mL, 19 mg/mL, or 20 mg/mL in water. Specifically, the prepared concentration of the aqueous solution can be selected in the range of 3-20 mg/mL, and preferably, the prepared solution of the aqueous solution is in the range of 5-20 mg/mL.

Further, the pH value of the aqueous solution is adjusted to 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8 or 5.0, and the pH value scheduling range of the aqueous solution is between 3.6 and 5. Preferably, the pH value scheduling range of the aqueous solution is Between 3.8 and 4.5. Within this pH range, the aqueous solution of synthetic peptides can better form hydrogels.

Hydrogel formation steps:

The aqueous solution obtained in the hydrogel preparation step is allowed to stand at room temperature to form a gel. Specifically, the room temperature refers to 15 to 25 degrees Celsius.

The hydrogel prepared by the above method has good cytocompatibility and no cytotoxicity or obvious cytotoxicity.

The embodiment of the present invention also provides a novel hydrogel, which is prepared by the above-mentioned preparation method, and the novel hydrogel has the characteristics of no cytotoxicity.

The embodiments of the present invention also provide applications of the above synthetic peptides, the above materials or the above hydrogels in the field of food processing, such as directional release in the digestive tract as nutrients or embedded carriers.

The present invention has the following beneficial effects:

The embodiment of the present invention provides a synthetic peptide, which is easy to synthesize, belongs to a complete amino acid sequence, and does not contain Nap-(naphthalene), Fmoc-(fluorenylmethoxycarbonyl), Cbz-(benzyloxycarbonyl), Groups such as aliphatic chains can effectively avoid or reduce specific immunity that is easily caused, and have no cytotoxicity, and the synthetic peptide has high gelation, and can quickly form a hydrogel through pH induction.

In addition, the embodiments of the present invention provide a new type of hydrogel, its synthetic material, its preparation method and its application. The material can quickly and effectively synthesize a hydrogel with good cell compatibility, and the synthesized hydrogel has no Significant cytotoxicity.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the following briefly introduces the accompanying drawings used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore do not It should be regarded as a limitation of the scope, and for those of ordinary skill in the art, other related drawings can also be obtained according to these drawings without any creative effort.

Fig. 1 is the chemical structure schematic diagram of synthetic peptide in pH7.0 environment in the embodiment of the present invention;

Fig. 2 is the infrared spectrogram of synthetic peptide in embodiment 7 in 1550-1800cm ^-1 waveband;

Figure 3 is a graph showing the results of the survival rate of Caco-2 cells provided by Comparative Example 1 of the present invention after culturing in a synthetic peptide aqueous solution and a whey protein environment for 24 hours;

Fig. 4 is the different states of synthetic peptide aqueous solution under different pH environment in Comparative Example 2 of the present invention;

Fig. 5 is the physical image (Fig. 5A) and the rheological diagram (Fig. 5B) of the gel formation in the synthetic peptide aqueous solution with the concentration of 10 mg/mL in the comparative example 3 of the present invention under the condition of pH 4.0;

Figure 6 is the rheological diagram of gel formation in Comparative Example 3 of the present invention, under the condition of pH 4.0, the concentration of synthetic peptide aqueous solution is (A) 20 mg/mL (B) 10 mg/mL (C) 5 mg/mL, (D) ) is the comparison chart of the storage modulus G' of short peptide gels with different concentrations when the stress is 1% and 0.1 rad/s.

Detailed ways

In order to make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

The features and performances of the present invention will be further described in detail below in conjunction with the embodiments.

Example 1

A preparation method of a novel hydrogel, which comprises a hydrogel preparation step and a hydrogel formation step, as follows:

Hydrogel preparation steps:

A gel solution was prepared using synthetic peptides. Specifically, the sequence of the synthetic peptide is: glutamine-valine-glutamic acid-valine-lysine-valine-glutamic acid-valine-lysine, the sequence abbreviation of synthetic peptide For: QVEVKVEVK. The preparation of synthetic peptides adopts general preparation technology, and the peptide synthesizer is used for synthesis.

Mix the synthetic peptide with pure water to fully dissolve the synthetic peptide. Specifically, the synthetic peptide was formulated as an aqueous solution with a concentration of 10 mg/mL. Further, the pH value of the above aqueous solution was adjusted to 4.

Hydrogel formation steps:

The aqueous solution obtained in the hydrogel preparation step is allowed to stand at room temperature to form a gel.

Example 2

A preparation method of a novel hydrogel, which includes a hydrogel preparation step and a hydrogel formation step, which is roughly the same as the preparation method provided in Example 1, except that the parameters are different, and the differences are as follows:

Hydrogel preparation steps:

The sequence of the synthetic peptide was: glutamine-valine-glutamic acid-valine-lysine-valine-glutamic acid-valine-lysine. Mix the synthetic peptide with pure water to fully dissolve the synthetic peptide. Specifically, the synthetic peptide was formulated as an aqueous solution with a concentration of 5 mg/mL. Further, the pH value of the above aqueous solution was adjusted to 4.

Hydrogel formation steps:

The aqueous solution obtained in the hydrogel preparation step is allowed to stand at room temperature to form a gel.

Example 3

A preparation method of a novel hydrogel, which includes a hydrogel preparation step and a hydrogel formation step, which is roughly the same as the preparation method provided in Examples 1 and 2, except that the parameters are different, and the differences are as follows:

Hydrogel preparation steps:

The sequence of the synthetic peptide was: glutamine-valine-glutamic acid-valine-lysine-valine-glutamic acid-valine-lysine. Mix the synthetic peptide with pure water to fully dissolve the synthetic peptide. Specifically, the synthetic peptide was formulated as an aqueous solution with a concentration of 20 mg/mL. Further, the pH value of the above aqueous solution was adjusted to 4.

Hydrogel formation steps:

The aqueous solution obtained in the hydrogel preparation step is allowed to stand at room temperature to form a gel.

Example 4

A preparation method of a new type of hydrogel, which includes a hydrogel preparation step and a hydrogel formation step, which is roughly the same as the preparation method provided in Examples 1 to 3, except that the parameters are different, and the differences are as follows:

Hydrogel preparation steps:

The sequence of the synthetic peptide was: glutamine-valine-glutamic acid-valine-lysine-valine-glutamic acid-valine-lysine. Mix the synthetic peptide with pure water to fully dissolve the synthetic peptide. Specifically, the synthetic peptide was formulated as an aqueous solution with a concentration of 10 mg/mL. Further, the pH value of the above aqueous solution was adjusted to 4.5.

Hydrogel formation steps:

The aqueous solution obtained in the hydrogel preparation step is allowed to stand at room temperature to form a gel.

Example 5

A preparation method of a new type of hydrogel, which includes a hydrogel preparation step and a hydrogel formation step, which is roughly the same as the preparation method provided in Examples 1 to 4, except that the parameters are different, and the differences are as follows:

Hydrogel preparation steps:

The sequence of the synthetic peptide was: glutamine-valine-glutamic acid-valine-lysine-valine-glutamic acid-valine-lysine. Mix the synthetic peptide with pure water to fully dissolve the synthetic peptide. Specifically, the synthetic peptide was formulated as an aqueous solution with a concentration of 10 mg/mL. Further, the pH value of the above aqueous solution was adjusted to 3.9.

Hydrogel formation steps:

The aqueous solution obtained in the hydrogel preparation step is allowed to stand at room temperature to form a gel.

Example 6

A preparation method of a new type of hydrogel, which includes a hydrogel preparation step and a hydrogel formation step, which is roughly the same as the preparation method provided in Examples 1 to 5, except that the parameters are different, and the differences are as follows:

Hydrogel preparation steps:

The sequence of the synthetic peptide was: glutamine-valine-glutamic acid-valine-lysine-valine-glutamic acid-valine-lysine. Mix the synthetic peptide with pure water to fully dissolve the synthetic peptide. Specifically, the synthetic peptide was formulated as an aqueous solution with a concentration of 10 mg/mL. Further, the pH value of the above aqueous solution was adjusted to 5.

Hydrogel formation steps:

The aqueous solution obtained in the hydrogel preparation step is allowed to stand at room temperature to form a gel.

Example 7

Secondary structure characterization of the synthetic peptide molecules provided in Example 1 was performed using FTIR.

The hydrogel prepared by the preparation method provided in Example 1 was freeze-dried, and then mixed with dry KBR powder at a mass ratio of 1:100, ground and pressed, and then Fourier infrared scanning was performed, 128 scans, with a resolution of 4 cm ^-1 , the scanning range is 4000 to 650 cm ^-1 . The characterization results are shown in Figure 2.

It can be seen from the figure that there is an obvious absorption peak at 1630cm ^-1 , indicating that the synthetic peptide molecule can form a β-sheet secondary structure under the given conditions. In addition, since the synthetic peptide was cleaved using TFA during the synthesis, there was an absorption of TFA at 1675 cm ⁻¹ that could not be removed in the system.

Comparative Example 1

The biocompatibility of the synthetic peptides provided in Example 1 was verified.

The Caco-2 cell line with the morphological and enzymatic characteristics of typical normal human intestinal cells was used to conduct MTT cytotoxicity experiments to determine the effect of the synthetic peptide on the growth and proliferation of Caco-2 cells. The synthetic peptide was mixed with pure water to a concentration of 10 mg/mL and 5 mg/mL aqueous solutions, the experiment took whey protein isolate (WPI) under the same treatment conditions as the control group, and cultured Caco-2 cells. After 24 hours, the cell viability was detected.

Please refer to Figure 3 for the experimental results. It can be seen that when the concentration of the aqueous solution prepared by the synthetic peptide is 10 mg/mL (Example 1) and 5 mg/mL (Example 2), the survival rate of Caco-2 cells is about 134.96% and 168.543%, respectively. %, while the cell viability was about 65.939% and 73.799% when the concentration of WPI was 10 mg/mL and 5 mg/mL. This shows that the synthetic peptides provided in Examples 1-2 of the present invention all have low cytotoxicity and good biocompatibility.

Comparative Example 2

Verify the state of the aqueous solution of the synthetic peptide provided in Example 1 under different pH environments.

Take 10 mg of the synthetic peptide provided in Example 1, add 1 mL of pure water and stir to make the synthetic peptide fully solution, add a certain amount of NaOH solution dropwise, and gradually adjust the pH value of the aqueous solution from 2 to 10.

Please refer to Figure 4, it can be seen that under different pH environments, the aqueous solution presents different states. When the pH is 2.5 to 3.0, the aqueous solution is a clear and flowing liquid. When the pH is 3.5 to 4.91, the turbidity and viscosity of the aqueous solution When the pH is 5.00, the aqueous solution is still turbid and viscous; when the pH is 5.45-9.82, the aqueous solution is a clear flowing liquid, which cannot or is difficult to form a hydrogel.

Comparative Example 3

The properties of the hydrogels provided in Examples 1-3 were verified.

The stress of 1% was selected from the linear viscoelastic region of the hydrogels prepared by the preparation methods provided in Examples 1 to 3, and then frequency scanning was performed in the range of 0.1Hz-100rad/s. All solutions were placed in a 37°C water bath for 1 Measured after hours.

The experimental results are shown in Fig. 5 (Fig. 5A and Fig. 5B) and Fig. 6. Under the condition of pH 4.0, the storage modulus G' and the energy dissipation modulus G" of 10 mg/mL synthetic peptide aqueous gel are in the whole range. There is a good linear viscoelasticity relationship in the range of frequency scanning, and the storage modulus G' is larger than the energy dissipation modulus G" by an order of magnitude, which proves that it has a tendency to gel.

And under the same treatment conditions, different peptide concentrations (20, 10, 5 mg/mL) formed the rheological difference of the gel. The results showed that the storage modulus G' and the energy dissipation modulus G" of the 20 mg/mL short peptide gel had a good linear viscoelastic relationship, and the storage modulus G' reached 500 Pa.

In summary, the embodiments of the present invention provide a synthetic peptide, which is easy to synthesize, belongs to a complete amino acid sequence, and does not contain Nap-(naphthalene), Fmoc-(fluorenylmethoxycarbonyl), Cbz-(benzyloxycarbonyl) at the end group acyl), aliphatic chain and other groups, can effectively avoid or reduce easily induced specific immunity, no cytotoxicity, and the synthetic peptide has high gelation, and can synthesize hydrogel with good cytocompatibility.

In addition, the embodiments of the present invention also provide a new type of hydrogel, its synthetic material, its preparation method and its application. The material can quickly and effectively synthesize a hydrogel with good cell compatibility, and the synthesized hydrogel No obvious cytotoxicity.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (10)

1. a kind of synthetic peptide, which is characterized in that the sequence of the synthetic peptide are as follows: glutamine-valine-glutamic acid-valine- Lysine-valine-glutamic acid-valine-lysine.

2. a kind of material of synthetic hydrogel, which is characterized in that it includes synthetic peptide described in claim 1.

3. a kind of preparation method of novel hydrogels, which is characterized in that it includes hydrogel preparation steps；The hydrogel is matched Step processed includes being configured to using the material of synthetic peptide described in claim 1 or synthetic hydrogel as claimed in claim 2 Gel solution.

4. preparation method according to claim 3, which is characterized in that the hydrogel preparation steps include: by the conjunction The aqueous solution of 3~20mg/mL is configured at peptide.

5. the preparation method according to claim 4, which is characterized in that the hydrogel preparation steps include will be described water-soluble The pH value of liquid is adjusted to 3.6~5.

6. preparation method according to claim 5, which is characterized in that the hydrogel preparation steps include will be described water-soluble The pH value of liquid is adjusted to 3.8~4.5.

7. preparation method according to claim 5, which is characterized in that after the hydrogel preparation steps, the preparation Method further includes hydrogel forming step；

The hydrogel forming step includes: to be stored at room temperature aqueous solution made from the hydrogel preparation steps, solidifying to be formed Glue.

8. a kind of novel hydrogels, which is characterized in that it is made by the described in any item preparation methods of claim 3~7.

9. novel hydrogels according to claim 8, which is characterized in that the novel hydrogels no cytotoxicity.

10. synthetic peptide as described in claim 1, the material of synthetic hydrogel as claimed in claim 2 or claim 8 or Application of the novel hydrogels described in 9 in food processing field.