CN103992486B - Click chemical-synthesis preparation method of light-operated tetrazole-alkene for polypeptide hydrogel - Google Patents

Abstract

The invention discloses a method for preparing polypeptide hydrogels synthesized by light-controlled tetrazole-ene click chemistry. 6,8,10) mixing, tetrazole is connected on the polypeptide molecule by esterification; 2) The double-stranded DNA of the obtained polypeptide tetrazole derivative and the end group modified methacrylic group is according to molar ratio 1:(2 , 3, 4) mixed under conditions of pH 7.4 and 37°C, and irradiated with an ultraviolet lamp with a wavelength of 300-400nm for 100-200s to trigger a tetrazole-ene reaction and rapidly cross-link to form a gel; the beneficial effects of the present invention are: water The preparation process of the gel has space-time controllability, without catalysts such as photoinitiators and copper salts, a fluorescent polypeptide hydrogel is obtained, which has strong traceability, and nucleic acid aptamers in double-stranded DNA molecules It has a targeting function and has potential application value in the fields of biomedicine and targeted therapy.

Description

Preparation method of peptide hydrogel synthesized by light-controlled tetrazole-ene click chemistry

technical field

The invention relates to a method for preparing polypeptide hydrogel, in particular to a method for preparing polypeptide hydrogel synthesized by light-controlled tetrazole-ene click chemistry.

Background technique

Hydrogel is a kind of gel with water as the dispersion medium. It has a three-dimensional network structure. It has sensitivity to environmental stimuli, high swelling, permeability and similarity to human tissue. It has a high load capacity and can realize Efficient entrapment of drugs; these characteristics make hydrogels widely used in tissue engineering, drug delivery and targeted therapy. At present, the methods used to prepare hydrogels are mainly physical cross-linking methods and chemical cross-linking methods, among which. Physical crosslinking includes hydrophobic interaction, stereocomplexation of polylactic acid, electrostatic interaction, etc. Typical reactions of chemical crosslinking methods to form hydrogels include: enzyme crosslinking reaction, free radical polymerization reaction, Schiff base reaction, disulfide bond reaction, Michael addition reaction, etc.

Peptides are natural biological macromolecules. The products of complete degradation of peptide-based hydrogels are only amino acids, so they will not have adverse effects in the human body, nor will they cause adverse reactions in the body and some tissue inflammation, making peptides play an increasingly important role in the development of innovative medical technologies. Important roles, such as controlled release technology of drugs, research on new scaffolds for cell therapy, tissue engineering, etc. Moreover, polypeptide hydrogels have good biocompatibility, which can provide scaffolds for the distribution of cells and the accumulation of extracellular matrix, and these biocompatible and biodegradable scaffolds have great potential in tissue repair and tissue engineering. Very wide range of applications. At present, the preparation methods of polypeptide hydrogel mainly include self-assembly method and chemical cross-linking method. Among them, the self-assembly method is mostly used for injectable polypeptide hydrogel. The Chinese invention patent with the authorized notification number CN102408575B discloses Solubilization, the ion-compensating polypeptide is dissolved in a NaCl aqueous solution with a concentration of 3-20mM, and then the resulting solution is self-assembled to obtain an injectable polypeptide hydrogel; chemical cross-linking methods include enzyme-catalyzed cross-linking and click chemistry. Among them, the Chinese invention patent with the authorized announcement number CN102688525B discloses a polypeptide hydrogel prepared by an enzyme-catalyzed cross-linking method, and Macromolecules, 2012, 45, 9579-9584 describes a polypeptide hydrogel prepared by a click chemistry method. There are certain deficiencies in the above-mentioned method for preparing polypeptide hydrogels: (1) the self-assembled hydrogels of polypeptides are all combined by weaker intermolecular non-covalent interactions, which makes the mechanical properties of hydrogels relatively weak. (2) Enzyme-catalyzed cross-linking may cause toxicity and immune reactions; (3) Copper catalysts are added to the traditional click chemical synthesis of polypeptide hydrogels, and their presence may induce viruses or oligosaccharides. Nucleotide degradation and cytotoxicity; while polypeptide hydrogels prepared by copper-free click chemistry are usually limited by longer gel time and lower storage modulus.

Contents of the invention

Aiming at the above-mentioned prior art, the present invention provides a method for preparing polypeptide hydrogels synthesized by light-controlled tetrazole-ene click chemistry. The "click chemistry" has the characteristics of rapidity, high efficiency, specificity and mild reaction conditions, and does not require catalysts such as photoinitiators and copper salts, and the obtained polypeptide hydrogel is fluorescent and easy to observe. It is suitable for drug carriers, biomedical and drug release.

The present invention is achieved through the following technical solutions:

A method for preparing polypeptide hydrogels synthesized by light-controlled tetrazole-ene click chemistry, comprising the following steps: Synthesis of polypeptide tetrazole derivatives:

(1) Under the protection of nitrogen, add 0.2 to 0.4 g of tetrazole to a 50 mL two-necked bottle, and stir 15 to 20 ml of dichloromethane until dissolved to obtain a dichloromethane solution of tetrazole;

(2) Add 150-200 mg of DCC (N,N-dicyclohexylcarbodiimide) to the dichloromethane solution of tetrazole and continue stirring for 15-20 minutes to obtain a tetrazole solution;

(3) Dissolve the polypeptide in 5-10mL of dichloromethane to obtain a polypeptide solution, wherein the amino acid sequence of the polypeptide is ThrSerThrSerThrSerThrSerThrSerThrSer, and each amino acid contains a side chain hydroxyl group, and the above polypeptide solution is added to step (2) Stir in the obtained tetrazole solution for 15-20 minutes; add 10-15 mg DMAP (4-dimethylaminopyridine) to the mixture of polypeptide and tetrazole, react for 10-12 hours, filter, and precipitate the filtrate with ice ether, dry Obtaining tetrazole derivatives of polypeptide molecules;

Light-controlled click synthesis of peptide hydrogels:

(4) Dissolve the polypeptide tetrazole derivative obtained in step (3) and the allyl-modified double-stranded DNA in Tris-HCl buffer until completely dissolved, wherein the molar ratio of polypeptide to DNA is 1:2 ~1:4;

(5) After mixing the mixed solution obtained in step (4) at 37°C, place it under a UV lamp with a wavelength of 300-400nm under vacuum conditions, and illuminate it for 100-200s to trigger the tetrazole-ene click chemical reaction, and quickly Cross-linking forms a polypeptide hydrogel with fluorescent properties.

Preferably, the molar ratio of the selected polypeptide to tetrazole in step (3) is 1:6.

One of the double-stranded DNAs is a nucleic acid aptamer strand.

The nucleic acid aptamer chain is AS1411 aptamer or sgc8 aptamer.

Preferably, the molar ratio of polypeptide to DNA in step (4) is 1:3.

The pH7.4 of Tris-HCl damping solution described in step (4), contains 10mM NaCl.

Preferably, the wavelength of the ultraviolet light in step (5) is 375nm.

Preferably, the illumination time in step (5) is 180s.

The present invention also provides a light-controlled tetrazole-ene click chemically synthesized polypeptide hydrogel prepared by the above method.

The present invention connects functional small molecules of tetrazole to polypeptide molecules through a simple esterification reaction to obtain tetrazole derivatives; then, the mixed solution of polypeptide and DNA is irradiated by ultraviolet light to trigger tetrazole-ene reaction to rapidly cross-link to form water gel, which has the following advantages:

1. During the synthesis process of polypeptide hydrogel, time and space are controllable;

2. In the synthesis process of polypeptide hydrogel, light-controlled click chemical reaction is used, the reaction conditions are mild, and copper salt catalyst is not required;

3. In the process of light-controlled synthesis of polypeptide hydrogel, no photoinitiator is needed, and the reaction is directly initiated by ultraviolet irradiation;

4. The prepared polypeptide hydrogel has strong fluorescence properties and strong traceability;

5. The nucleic acid aptamer chain in the obtained polypeptide hydrogel can realize the targeting function to cancer cells.

Description of drawings

Figure 1 is a schematic diagram of the photo-controlled tetrazole-ene click synthesis reaction;

Fig. 2 is the synthesizing schematic diagram of the tetrazole derivative of polypeptide;

Fig. 3 is a schematic diagram of light-controlled tetrazole-ene click synthesis polypeptide hydrogel;

Among them, 1. Tetrazole derivatives of polypeptides, 2. DNA with allyl groups modified at the end groups, one of which is a nucleic acid aptamer chain, and the other chain is its complementary chain, 3. Hydrogel.

Figure 4 is a schematic diagram of the synthesis of patterned hydrogels;

Figure 5 is a schematic diagram of drug-loaded hydrogel targeted drug release to cancer cells;

Among them, 1. Anticancer drugs, 2. Nucleic acid aptamers, 3. Receptors, 4. Cell membranes, 5. Cell nuclei, 6. Amino acid small molecules.

Detailed ways

The present invention will be further described below in conjunction with embodiment.

Example 1:

A preparation method for light-controlled tetrazole-ene click chemical synthesis of polypeptide hydrogel, comprising the following steps:

The synthesis of polypeptide tetrazole derivatives is shown in Figure 2:

(1) Under the protection of nitrogen, add 0.4g tetrazole to a 50mL two-necked bottle, and stir 20ml of dichloromethane until dissolved;

(2) Add 150 mg of DCC (N,N-dicyclohexylcarbodiimide) to the tetrazole solution and continue stirring for 15 minutes;

(3) The polypeptide molecule ThrSerThrSerThrSerThrSerThrSerThrSer with a specific amino acid sequence, as shown in SEQ ID NO.1, is dissolved in 5mL of dichloromethane to obtain a polypeptide solution, and the above polypeptide solution is added to the tetrazole solution, wherein the molar amount of the polypeptide and tetrazole The ratio is 1:6, stirring for 15 minutes; adding 12 mg DMAP (4-dimethylaminopyridine) to the mixture of polypeptide and tetrazole, reacting for 12 hours, filtering, and the filtrate is precipitated with glacial ether, and dried to obtain the tetrazole derivative of the polypeptide molecule. thing;

Light-controlled click synthesis of polypeptide hydrogels, as shown in Figures 1, 3 and 4:

(4) Dissolve the double-stranded DNA of the polypeptide tetrazole derivative and the end-group modified allyl group obtained in step (3) in 300 μL Tris-HCl buffer solution (pH 7.4, 10 mM NaCl) until completely dissolved, wherein the polypeptide and The molar ratio of DNA is 1:3;

(5) Mix the mixture obtained in step (4) uniformly at 37°C, cover it with a photomask with a pattern under vacuum, place it under a UV lamp with a wavelength of 375nm, and illuminate it for 180s to induce tetrazole- The ene click chemical reaction quickly cross-linked to form a patterned hydrogel, and the patterned fluorescent gel can be observed under an inverted fluorescence microscope, indicating that the synthesis of polypeptide hydrogels is controllable in time and space.

One of the double-stranded DNAs is a nucleic acid aptamer strand.

The nucleic acid aptamer chain is AS1411 aptamer.

Example 2:

A preparation method for light-controlled tetrazole-ene click chemical synthesis of polypeptide hydrogel, comprising the following steps:

Synthesis of Polypeptide Tetrazole Derivatives:

(1) Under the protection of nitrogen, add 0.2g tetrazole to a 50mL two-necked bottle, and stir 15ml of dichloromethane until dissolved;

(2) Add 200 mg of DCC (N,N-dicyclohexylcarbodiimide) to the tetrazole solution and continue stirring for 20 minutes;

(3) The polypeptide molecule ThrSerThrSerThrSerThrSerThrSerThrSer with a specific amino acid sequence is dissolved in 10 mL of dichloromethane to obtain a polypeptide solution, and the above polypeptide solution is added to the tetrazole solution, wherein the molar ratio of the polypeptide to tetrazole is 1:8, and stirred for 20 minutes ; Add 15mg DMAP (4-dimethylaminopyridine) to the mixed solution of polypeptide and tetrazole, react for 12h, filter, and the filtrate is precipitated with glacial ether, and the tetrazole derivative of polypeptide molecule is obtained after drying;

Light-controlled click synthesis of peptide hydrogels:

(4) Dissolve the double-stranded DNA of the polypeptide tetrazole derivative and the end-group modified allyl group obtained in step (3) in 300 μL Tris-HCl buffer solution (pH 7.4, 10 mM NaCl) until completely dissolved, wherein the polypeptide and The molar ratio of DNA is 1:4;

(5) Mix the mixture obtained in step (4) evenly at 37°C, place it under a UV lamp with a wavelength of 350nm under vacuum conditions, and illuminate it for 200s to trigger a tetrazole-ene click chemical reaction and rapidly cross-link to form water gel.

One of the double-stranded DNAs is a nucleic acid aptamer strand.

The nucleic acid aptamer chain is sgc8 aptamer.

Example 3:

A preparation method for light-controlled tetrazole-ene click chemical synthesis of polypeptide hydrogel, comprising the following steps:

Synthesis of Polypeptide Tetrazole Derivatives:

(1) Under the protection of nitrogen, add 0.3g of tetrazole to a 50mL two-necked bottle, and stir 18ml of dichloromethane until dissolved;

(2) Add 175 mg of DCC (N,N-dicyclohexylcarbodiimide) to the tetrazole solution and continue stirring for 18 minutes;

(3) The polypeptide molecule ThrSerThrSerThrSerThrSerThrSerThrSer with a specific amino acid sequence was dissolved in 7mL of dichloromethane to obtain a polypeptide solution, and the above polypeptide solution was added to the tetrazole solution, wherein the molar ratio of polypeptide to tetrazole was 1:4, and stirred for 17min Add 10mg DMAP (4-dimethylaminopyridine) to the mixed solution of polypeptide and tetrazole, react for 10h, filter, and the filtrate is precipitated with glacial ether, and the tetrazole derivative of polypeptide molecule is obtained after drying;

Light-controlled click synthesis of peptide hydrogels:

(4) Polypeptide tetrazole derivatives, double-stranded DNA modified with allyl groups at the end groups, and doxorubicin were dissolved in 300 μL of Tris-HCl buffer solution containing 10 mM NaCl at pH 7.4 until completely dissolved to obtain a mixture, in which the peptide The molar ratio of tetrazole derivatives, DNA and doxorubicin is 1:3:3;

(5) After mixing the mixture obtained in step (4) evenly at 37°C, place it under a UV lamp with a wavelength of 375nm under vacuum conditions, and illuminate it for 180s to trigger a tetrazole-ene click chemical reaction, which rapidly cross-links and forms Hydrogels loaded with doxorubicin.

One of the double-stranded DNAs is a nucleic acid aptamer strand.

The nucleic acid aptamer chain is AS1411 aptamer.

As shown in Figure 5, collect the logarithmic phase cells and cancer cells, adjust the concentration of the cell suspension, add 100ul of the cell suspension to each well, and plate to adjust the density of the cells to be tested to 1000-10000 wells (the edge wells are filled with sterile Tris) ; incubate at 5% CO ₂ at 37°C until the cell monolayer covers the bottom of the well (96-well flat-bottomed plate). Add the gel into corresponding wells, set up 7 gradients, 100ul per well, and set up 5 duplicate wells; then incubate at 5% CO ₂ at 37°C for 24-72 hours; add 20uL 5mg/mL3-(4,5- Dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT) solution, continue to cultivate for 4 hours, terminate the culture, carefully suck off the culture medium in the well; add 150uL dimethyl sulfoxide to each well, Shake on a shaker at a low speed for 10 minutes to fully dissolve the crystals, and measure the absorbance of each well at OD570nm in an enzyme-linked immunosorbent detector.

The results show that the survival rate of normal cells reaches about 100%, and the cells can proliferate normally with the prolongation of culture time, indicating that the hydrogel has no cytotoxicity; the survival rate of cancer cells is significantly reduced, only 10%, indicating that the hydrogel has no cytotoxicity. The glue can well realize targeted drug release, and can effectively inhibit the proliferation of cancer cells and kill cancer cells.

Although the specific implementation of the present invention has been described above in conjunction with the accompanying drawings, it does not limit the protection scope of the present invention. Those skilled in the art should understand that on the basis of the technical solution of the present invention, those skilled in the art do not need to pay creative work Various modifications or variations that can be made are still within the protection scope of the present invention.

Claims (9)

1. the preparation method of light-operated tetrazolium-alkene clicking chemistry improvement on synthesis hydrogel, is characterized in that, comprise the following steps:

The synthesis of polypeptide terazole derivatives:

(1) under the protection of nitrogen, in 50mL two neck bottle, add 0.2 ~ 0.4g tetrazolium, methylene dichloride 15 ~ 20ml is stirred to dissolving, obtains the dichloromethane solution of tetrazolium;

(2) in the dichloromethane solution of tetrazolium, add N, N-dicyclohexylcarbodiimide 150 ~ 200mg continues stirring 15 ~ 20min, obtains tetrazolium solution;

(3) polypeptide is dissolved in 5 ~ 10mL methylene dichloride, obtain polypeptide solution, wherein, the aminoacid sequence of polypeptide is ThrSerThrSerThrSerThrSerThrSerThrSer, aforementioned polypeptides solution is joined in the tetrazolium solution of step (2) gained, stir 15 ~ 20min; In the mixed solution of polypeptide and tetrazolium, add 10 ~ 15mg DMAP, reaction 10 ~ 12h, filter, filtrate uses ice ether sedimentation, obtains the terazole derivatives of peptide molecule after drying;

The light-operated click synthesis of polypeptide hydrogel:

(4) respectively step (3) gained polypeptide terazole derivatives and end group being modified allylic double-stranded DNA is dissolved in Tris-HCl damping fluid, and to dissolving completely, wherein the mol ratio of polypeptide and DNA is 1:2 ~ 1:4;

(5) after step (4) gained mixed solution being mixed at 37 DEG C, under vacuum condition, under being placed in the ultraviolet lamp of wavelength 300 ~ 400nm, illumination 100 ~ 200s, cause tetrazolium-alkene clicking chemistry reaction, be cross-linked to form rapidly the polypeptide hydrogel with fluorescence property.

2. the preparation method of light-operated tetrazolium as claimed in claim 1-alkene clicking chemistry improvement on synthesis hydrogel, is characterized in that, the mol ratio of step (3) described polypeptide and tetrazolium is 1:6.

3. the preparation method of light-operated tetrazolium as claimed in claim 1-alkene clicking chemistry improvement on synthesis hydrogel, is characterized in that, one in described double-stranded DNA is aptamer chain.

4. the preparation method of light-operated tetrazolium as claimed in claim 1-alkene clicking chemistry improvement on synthesis hydrogel, is characterized in that, described aptamer chain is that AS1411 is fit or sgc8 is fit.

5. the preparation method of light-operated tetrazolium as claimed in claim 1-alkene clicking chemistry improvement on synthesis hydrogel, is characterized in that, the mol ratio of step (4) described polypeptide and DNA is 1:3.

6. the preparation method of light-operated tetrazolium as claimed in claim 1-alkene clicking chemistry improvement on synthesis hydrogel, is characterized in that, the pH7.4 of the described Tris-HCl damping fluid of step (4), containing 10mM NaCl.

7. the preparation method of light-operated tetrazolium as claimed in claim 1-alkene clicking chemistry improvement on synthesis hydrogel, is characterized in that, step (5) described ultraviolet wavelength is 375nm.

8. the preparation method of light-operated tetrazolium as claimed in claim 1-alkene clicking chemistry improvement on synthesis hydrogel, is characterized in that, step (5) described light application time is 180s.

9. the light-operated tetrazolium of one-alkene clicking chemistry improvement on synthesis hydrogel prepared by the method described in any one of claim 1-8.