CN114869879A - Small molecule hydrogel with double inhibition effects of active oxygen and inflammation and preparation method thereof - Google Patents
Small molecule hydrogel with double inhibition effects of active oxygen and inflammation and preparation method thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 230000000694 effects Effects 0.000 title claims abstract description 16
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- 230000002401 inhibitory effect Effects 0.000 claims description 2
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
- A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
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- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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Abstract
The invention discloses a small molecular hydrogel with double inhibition effects on active oxygen and inflammation and a preparation method thereof, and the preparation method comprises the following specific steps: step one, Rhein-polypeptide compound and empty carrier-polypeptide compound are prepared, and the polypeptide sequence is Rhein- D F D FG‑SS‑ERGD/Nap‑ D F D FG-SS-ERGD; step two, treating the rhein-polypeptide compound PBS solution by using reduced glutathione; and step three, blending the treated solution and the EGCG solution of epigallocatechin gallate, and co-assembling under the action of reduced glutathione to form the small molecule drug hydrogel. The micromolecule drug hydrogel prepared by the invention can continuously improve the microenvironment of active oxygen and inflammation caused by ischemia reperfusion, thereby realizing the treatment of ischemia in perfusion injury.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to a small molecular hydrogel with double inhibition effects on active oxygen and inflammation and a preparation method thereof.
Background
Excessive production of reactive oxygen species and outbreaks of inflammation are the major causes of ischemia reperfusion injury, and Toll-like receptor 4(TLR4), one of the TLR receptors, has received little success as a single therapeutic strategy to scavenge reactive oxygen species or inhibit inflammation, is an important link through reactive oxygen species and inflammation. Previous researches find that Rhein (Rhein) has a strong inhibiting effect on a TLR 4/NF-kB pathway, but Rhein has poor water solubility, so that the application of Rhein is limited. The polypeptide self-assembly based on the bisphenylalanine (FF) is widely applied in the field of life sciences due to the advantages of good biocompatibility, low price, easy design and preparation and the like. The introduction of the system provides possibility for solving the limit of the rhein. Meanwhile, in order to enhance the antioxidant capacity of the polypeptide, epigallocatechin gallate (EGCG) is introduced, and finally the target compound EGCG @ Rh-gel is obtained. The double inhibition of ROS and TLR4 is expected to block active oxygen-inflammatory circulation and realize the treatment of myocardial ischemia-reperfusion injury.
Disclosure of Invention
Based on the prior art, the invention aims to provide the small-molecule hydrogel with the double inhibition effects of active oxygen and inflammation and the preparation method thereof.
In order to achieve the above purpose, the invention adopts the technical scheme that:
a small molecular hydrogel with double inhibition effects on active oxygen and inflammation and a preparation method thereof comprise the following steps:
step one, Rhein-polypeptide compound (Rh-pep) and empty carrier-polypeptide compound are prepared, and the polypeptide sequence is Rhein- D F D FG-SS-ERGD/Nap- D F D FG-SS-ERGD；
Step two, preparing a PBS solution from the rhein-polypeptide compound;
and step three, blending the prepared solution and epigallocatechin gallate (EGCG) solution, and co-assembling under the action of reduced glutathione to form the small molecule drug hydrogel.
In order to better implement the present invention, further, the steps of preparing rhein-polypeptide compound and empty carrier-polypeptide compound in the step one are as follows:
1) synthesizing Fmoc-SS by using sodium bicarbonate, cystamine dihydrochloride, 1, 4-butanedioic anhydride, dioxane and 9-fluorenylmethyl-N-succinimide carbonate (Fmoc-Osu) as raw materials;
2) synthesis of Rhein- D F D FG-SS-ERGD and Nap- D F D FG-SS-ERGD。
In order to better implement the present invention, further, in the first step, the rhein-polypeptide compound has the structural formula:
the empty carrier-polypeptide compound has the structural formula:
in order to better implement the invention, the synthesis steps of Fmoc-SS are as follows:
(1) synthesis of intermediate reactant 1: weighing cystamine dihydrochloride and sodium bicarbonate according to a molar ratio of 1: 2-3, adding the cystamine dihydrochloride to a 250ml flask, adding water with the mass being 20 times that of the two substances, stirring at room temperature until the mixture is clear, adding dioxane with the volume being 1/5, uniformly stirring, weighing 1, 4-succinic anhydride with the same mole as that of cystamine dihydrochloride, adding the 1, 4-succinic anhydride into the reaction bottle, and reacting at room temperature for 8-12 hours to obtain an intermediate product 1.
(2) Fmoc-SS synthesis: after the intermediate product 1 is completely reacted, weighing 9-fluorenylmethyl-N-succinimide carbonate (Fmoc-Osu) and sodium bicarbonate with the same mole of cystamine dihydrochloride in the first step; sodium bicarbonate was added directly to the flask of intermediate 1, Fmoc-Osu was first mixed uniformly in dioxane of the same volume as in the first step, and the mixture was dropped into the flask using a constant pressure dropping funnel and allowed to react overnight at room temperature. After the reaction was completed, the reaction solution was filtered under reduced pressure, and the filtrate was retained. And (3) concentrating the filtrate by using a rotary evaporator, adjusting the pH of the concentrated filtrate to 1-2 by using 1M hydrochloric acid, standing at room temperature, performing suction filtration under reduced pressure to retain a solid after the white solid is completely separated out, and freeze-drying to obtain the Fmoc-SS.
In order to better implement the invention, the Fmoc solid phase synthesis method comprises the following steps
1) The dichloro resin was weighed (load: 1.4mmol/g) into a dry solid phase tube, adding a proper amount of Dichloromethane (DCM), placing on a shaker for 30min, introducing nitrogen for 5min to fully expand the dichloro resin, and extruding dichloromethane solvent;
2) according to the volume of the dichloro resin in the solid phase tube, weighing aspartic acid Asp (D) (Fmoc-Asp (tBu) -OH), adding equimolar DIPEA, dissolving with dichloromethane, mixing, adding into a solid phase reactor, adding equivalent DIPEA into the reaction solution, and reacting for 1-2 h;
3) after the reaction is finished, pressing and drying the reaction solution, washing for 5 times by using dichloromethane, washing for 1min each time, then adding a sealing solution to react for 15-30 min so as to seal the active groups on the resin, and pressing and drying the reaction solution; the confining liquid consists of dichloromethane, methanol and DIPEA, and the volume ratio of DCM to MEOH to DIEA is 17-20: 2: 1.
4) Extruding the sealing solution, washing with dichloromethane for 5 times (1 min each time), and washing with N, N-Dimethylformamide (DMF) for 5 times (1 min each time);
5) cutting off the Fmoc protecting group: cutting with 20% piperidine (solvent is DMF) for 20-40 min, removing the protecting group Fmoc on aspartic acid to expose amino group, drying the reaction solution, washing with DMF for 5 times and 1min each time;
6) weighing the next amino acid glycine Gly (G) (Fmoc-Gly-OH) and equimolar HBTU in a dried vial, adding equimolar DIEA, dissolving with DMF, performing ultrasonic assisted dissolution, adding a solid phase tube for reaction for 2h, extruding the solution, and washing with DMF for 5 times, 1 min/time; cutting with 20% piperidine (solvent is DMF) for 20-40 min, removing the protecting group Fmoc to expose amino group, drying the reaction solution, washing with DMF for 5 times and each time for 1 min;
7) repeating the above step 6), followed by sequentially adding Fmoc-Arg (pbf) -OH, Fmoc-Glu (tBu) -OH, Fmoc-SS, Fmoc-Gly-OH, Fmoc- D Phe (tBu) -OH (D-configuration), Fmoc- D Phe (tBu) -OH (D-configuration), and finally rhein. The reaction difficulty is gradually increased along with the extension of the peptide chain, so that the dosage of the amino acid after the reaction is gradually increased to 3-6 times of the dosage of the first amino acid so as to improve the yield of the target polypeptide;
8) after the reaction is finished, washing the reaction product with DMF for 5 times, then washing the reaction product with DCM for 5 times, and each time for 1min, preparing a proper amount of cutting fluid, wherein the cutting fluid is 95% of trichloroacetic acid TFA, and preparing a mixed solution of triisopropylsilane TIS and water, and the volume fraction of the mixed solution is 95% of TFA: 2.5% TIS: 2.5% H 2 And O, cutting for 1h, and collecting cutting fluid. It was washed 3 times with 2 min/time more with DCM and the liquid was collected.
9) Spin-drying the collected liquid in a rotary evaporator, adding a proper amount of anhydrous ether, standing for 3-5min to separate out a white precipitate, centrifuging by using a high-speed centrifuge, removing a supernatant, freeze-drying the white precipitate to obtain a crude product, and purifying the crude product by using a high performance liquid chromatograph.
In order to better implement the invention, in the second step, the PBS solution is a mixed phosphate solution of sodium dihydrogen phosphate and disodium hydrogen phosphate, which is prepared into 38.0g of sodium dihydrogen phosphate, and 5.04g of disodium hydrogen phosphate, and water is added to the mixture to reach a constant volume of 1000ml, and the pH value of the solution is 7.4.
In order to better realize the invention, further, the concentration of the rhein-polypeptide compound solution prepared in the step two is 10.0-15.0 g of Rh-pep dissolved in every 1000ml of PBS solution.
In order to better realize the invention, the mass ratio of the dosages of the EGCG and the Rh-pep in the blending in the step three is further 1: 20-25.
In order to better implement the present invention, further, the hydrogel preparation steps in the third step are as follows:
1) pouring Rh-pep into a gel-forming small bottle, and dissolving with a PBS solution;
2) EGCG which is adjusted to neutral pH by 1mol/l sodium carbonate and added;
3) adding 4eq of GSH solution at pH 7.4;
4) after being mixed evenly, the mixture is inverted to form stable hydrogel.
Advantageous effects
The invention has the following beneficial effects:
(1) the micromolecule drug hydrogel prepared by the invention can continuously improve the microenvironment of active oxygen and inflammation caused by ischemia reperfusion, thereby realizing the treatment of ischemia in perfusion injury.
(2) The micromolecule drug hydrogel prepared by the invention is a hydrogel molecule formed by co-assembling EGCG and Rh-pep, has a slow release effect when in use, and is greatly improved compared with the aspect of singly using Rh-pep to remove active oxygen and improve an inflammation microenvironment, so that the use effect of organism injury is improved.
Drawings
FIG. 1: EXAMPLE 4 pictures of rhein-polypeptide and EGCG and rhein-polypeptide before and after gelling
FIG. 2: transmission electron micrograph of EGCG and Rhein-Polypeptides from example 4
FIG. 3: example 4 rhein polypeptide hydrogel and EGCG and rhein-polypeptide Co-assembled hydrogel to remove DPPH free radical
FIG. 4: rhein polypeptide hydrogel obtained in example 4 and co-assembled hydrogel of EGCG and rhein-polypeptide show fluorescence diagram of intracellular reactive oxygen species level after hypoxia reoxygenation of H9C2 cells
FIG. 5: EXAMPLE 4 qPCR detection of intracellular inflammatory factor expression after hypoxia reoxygenation of H9C2 cells by Rhein polypeptide hydrogel and EGCG and Rhein-polypeptide co-assembled hydrogel obtained in EXAMPLE 4
Detailed Description
The present invention will be described in further detail with reference to specific examples, which are provided by way of illustration only and are not intended to be limiting.
Example 1
Synthesis of Fmoc-SS:
(1) synthesis of intermediate reactant 1: weighing cystamine dihydrochloride and sodium bicarbonate according to a molar ratio of 1: 2-3, adding the cystamine dihydrochloride to a 250ml flask, adding water with the mass being 20 times that of the two substances, stirring at room temperature until the mixture is clear, adding dioxane with the volume being 1/5, uniformly stirring, weighing 1, 4-succinic anhydride with the same mole as that of cystamine dihydrochloride, adding the 1, 4-succinic anhydride into the reaction bottle, and reacting at room temperature for 8-12 hours to obtain an intermediate product 1.
(2) Fmoc-SS synthesis: after the intermediate product 1 is completely reacted, weighing 9-fluorenylmethyl-N-succinimide carbonate (Fmoc-Osu) and sodium bicarbonate with the same mole of cystamine dihydrochloride in the first step; sodium bicarbonate was added directly to the flask of intermediate 1, Fmoc-Osu was first mixed uniformly in dioxane of the same volume as in the first step, and the mixture was dropped into the flask using a constant pressure dropping funnel and allowed to react overnight at room temperature. After the reaction was completed, the reaction solution was filtered under reduced pressure, and the filtrate was retained. And (3) concentrating the filtrate by using a rotary evaporator, adjusting the pH of the concentrated filtrate to 1-2 by using 1M hydrochloric acid, standing at room temperature, performing suction filtration under reduced pressure to retain a solid after the white solid is completely separated out, and freeze-drying to obtain Fmoc-SS, wherein the weight of the Fmoc-SS is 4.7g (10mmol), and the final yield is 70%.
Example 2
Polypeptide compound Rhein- D F D FG-SS-ERGD/Nap- D F D Solid phase Synthesis of FG-SS-ERGD:
1) 1g of dichloro resin (load: 1.4mmol/g) into a dry solid phase tube, adding a proper amount of dichloromethane DCM, placing on a shaking table for 30min, introducing nitrogen for 5min to fully expand the dichloro resin, and extruding the DCM solvent;
2) aspartic acid Asp (D) (Fmoc-Asp (tBu) -OH)0.411g (1mmol) is weighed, 165 ul (1mmol) DIPEA is added, DCM is used for dissolution, the mixture is mixed and added into a solid phase reactor, 165 ul (1mmol) DIPEA is added into the reaction solution for reaction for 2 h;
3) after the reaction is finished, pressing and drying the reaction solution, washing for 5 times by using dichloromethane, washing for 1min each time, then adding a sealing solution to react for 15min so as to seal the active groups on the resin, and pressing and drying the reaction solution; wherein the confining liquid consists of dichloromethane, methanol and DIPEA, the volume ratio of DCM to MEOH to DIEA is 17:2:1, and 10ml of confining liquid is prepared;
4) extruding the sealing solution, washing with dichloromethane for 5 times (1 min each time), and washing with N, N-Dimethylformamide (DMF) for 5 times (1 min each time);
5) cleavage of the Fmoc protecting group: cutting with 20% piperidine (solvent is DMF) for 30min, removing protecting group Fmoc on aspartic acid to expose amino group, drying reaction solution, washing with DMF for 5 times each for 1 min;
6) weighing 0.298g (1mmol) of the next amino acid glycine Gly (G) (Fmoc-Gly-OH) and 0.38g (1mmol) of HBTU in a dry vial, adding 330 ul (1mmol) of DIEA, dissolving with DMF, performing ultrasonic assisted dissolution, adding a solid phase tube for reaction for 2h, extruding the solution, and washing with DMF for 5 times and 1 min/time; cutting with 20% piperidine (solvent is DMF) for 30min, removing protecting group Fmoc to expose amino group, drying reaction solution, washing with DMF for 5 times each for 1 min;
7) repeating the above step 6), followed by sequentially adding Fmoc-Arg (pbf) -OH, Fmoc-Glu (tBu) -OH, Fmoc-SS, Fmoc-Gly-OH, Fmoc-Phe (tBu) -OH (D-configuration), and finally rhein. Along with the extension of the peptide chain, the reaction difficulty is gradually increased, so that the dosage of the amino acid after the reaction is gradually increased, and the dosage is increased by 1.2 times each time;
8) after the reaction was completed, the mixture was washed 5 times with DMF and 5 times with DCM for 1min each, and 10ml of a cleavage solution containing 95% TFA (trichloroacetic acid) was prepared as a mixed solution of triisopropylsilane TIS and water, the volume fraction of which was 95% TFA: 2.5% TIS: 2.5% H 2 And O, cutting for 1h, and collecting cutting fluid. It was washed 3 times with DCM for 2 min/time and the liquid was collected.
9) And (3) spin-drying the collected liquid on a rotary evaporator, adding 20ml of anhydrous ether, standing for 5min to separate out a white precipitate, centrifuging by using a high-speed centrifuge, removing a supernatant, freeze-drying the white precipitate to obtain a crude product, and purifying the crude product by using a high performance liquid chromatograph.
The crude product obtained was 3.16g, purity 61.7%, and purified by liquid chromatography to give 1.62g of pure product, yield 83.08%.
Example 3
Polypeptide compound Rhein- D F D FG-SS-ERGD/Nap- D F D Solid phase Synthesis of FG-SS-ERGD:
1) 2g of dichloro resin (load: 1.4mmol/g) into a dry solid phase tube, adding a proper amount of dichloromethane DCM, placing on a shaking table for 30min, introducing nitrogen for 5min to fully expand the dichloro resin, and extruding the DCM solvent;
2) aspartic acid Asp (D) (Fmoc-Asp (tBu) -OH)0.411g (1mmol) is weighed, 165 ul (1mmol) DIPEA is added, DCM is used for dissolution, the mixture is mixed and added into a solid phase reactor, 165 ul (1mmol) DIPEA is added into the reaction solution for reaction for 1 h;
3) after the reaction is finished, pressing and drying the reaction solution, washing for 5 times by using dichloromethane, washing for 1min each time, then adding a sealing solution to react for 15min so as to seal the active groups on the resin, and pressing and drying the reaction solution; wherein the confining liquid consists of dichloromethane, methanol and DIPEA, the volume ratio of DCM to MEOH to DIEA is 20:2:1 is prepared, and 10ml of confining liquid is prepared;
4) extruding the sealing solution, washing with dichloromethane for 5 times (1 min each time), and washing with N, N-Dimethylformamide (DMF) for 5 times (1 min each time);
5) cutting off the Fmoc protecting group: cutting with 20% piperidine (solvent is DMF) for 40min, removing protecting group Fmoc on aspartic acid to expose amino group, drying reaction solution, washing with DMF for 5 times each for 1 min;
6) weighing 0.298g (1mmol) of the next amino acid glycine Gly (G) (Fmoc-Gly-OH) and 0.38g (1mmol) of HBTU in a dry vial, adding 330 ul (1mmol) of DIEA, dissolving with DMF, performing ultrasonic assisted dissolution, adding a solid phase tube for reaction for 2h, extruding the solution, and washing with DMF for 5 times and 1 min/time; cutting with 20% piperidine (solvent is DMF) for 40min, removing protecting group Fmoc to expose amino group, drying reaction solution, washing with DMF for 5 times each for 1 min;
7) repeating the above step 6), followed by sequentially adding Fmoc-Arg (pbf) -OH, Fmoc-Glu (tBu) -OH, Fmoc-SS, Fmoc-Gly-OH, Fmoc- D Phe (tBu) -OH (D-configuration), Fmoc- D Phe (tBu) -OH (D-configuration),and finally rhein. Along with the extension of the peptide chain, the reaction difficulty is gradually increased, so that the dosage of the amino acid after the reaction is gradually increased, and the dosage is increased by 1.1 times each time;
8) after the reaction was completed, the mixture was washed 5 times with DMF and 5 times with DCM for 1min each, and 10ml of a cleavage solution containing 95% TFA of trichloroacetic acid was prepared as a mixed solution of TIS of triisopropylsilane and water, the volume fraction of which was 95% TFA: 2.5% TIS: 2.5% H 2 And O, cutting for 1h, and collecting cutting fluid. It was washed 3 times with DCM for 2 min/time and the liquid was collected.
9) And (3) spin-drying the collected liquid on a rotary evaporator, adding 20ml of anhydrous ether, standing for 5min to separate out a white precipitate, centrifuging by using a high-speed centrifuge, removing a supernatant, freeze-drying the white precipitate to obtain a crude product, and purifying the crude product by using a high performance liquid chromatograph.
The crude product obtained was 3.48g, purity 56.1%, and purified by liquid chromatography to give 1.54g of pure product, yield 78.97%.
Example 4
Preparing 1 wt% of EGCG and rhein polypeptide co-assembled hydrogel, which comprises the following steps:
1) weighing 5mg of Rh-pep, pouring the Rh-pep into a gel forming bottle, and dissolving the Rh-pep by PBS;
2) adjusting pH to neutral with 1mol/l sodium carbonate, and adding 0.2mg EGCG;
3) 4eq of GSH solution (pH 7.4) was added and finally made up to 400. mu.l with PBS;
4) whether a stable hydrogel was formed was then observed by inverting the vial, and the results are shown in fig. 1.
The hydrogel obtained in example 4 was subjected to a free radical and inflammation scavenging test, the contents of which were as follows:
DPPH scavenging experiment
A DPPH solution with a concentration of 0.01mmol/L was prepared using 80% methanol. Mu.l of the hydrogel was then blended with 380. mu.l of DPPH solution and incubated for 60 minutes in the dark. The control group was 400. mu.l of DPPH solution. And detecting the absorbance of the solution at the wavelength of 400-600 nm by using a microplate reader.
The scavenging effect of the hydrogel on DPPH free radicals is calculated according to the following formula:
the scavenging effect (%) - (A515nm control-A515 nm hydrogel)/A515nm control × 100%
The result is shown in fig. 3, compared with the simple rhein polypeptide hydrogel, the scavenging efficiency of DPHH of the co-assembled hydrogel of EGCG and rhein polypeptide is obviously high, reaching 45%, while the rhein polypeptide hydrogel is only 7.6%, which indicates that the active oxygen scavenging capacity of the hydrogel is effectively improved by adding EGCG.
(II) measurement of active oxygen scavenging ability of cells
1) H9C2 cells were plated in 24-well plates at a cell density of 2X 105/well and cultured for 72 hours;
2) after the incubation was completed, the cells were reaerated and carefully purged 3 times with PBS. Then adding 10 mu M DHE and DCFH-DA staining working solution respectively, 1 ml/hole, keeping out of the sun, and culturing for 30 minutes in an incubator at 37 ℃;
3) then sucking out the working solution, washing for 3 times by PBS, then staining nuclei by Hoechst staining solution with 500 mul/hole, and culturing for half an hour in an incubator in the dark;
4) the Hoechst staining solution was aspirated and washed 3 times with PBS, and then observed under a fluorescence microscope, DHE showed red fluorescence, which overlapped with and represented that the stronger the red fluorescence, the higher the level of superoxide anion in the cells, and the higher the level of reactive oxygen species. DCFH-DA showed green fluorescence, the stronger its fluorescence intensity, indicating a higher total active oxygen level.
The result is shown in fig. 4, the co-assembled hydrogel of EGCG and rhein polypeptide has strong superoxide anion and active oxygen scavenging effect compared with the co-assembled hydrogel of rhein polypeptide, which indicates that the addition of EGCG is an essential ring.
(III) qPCR detection of changes in intracellular inflammatory factor expression levels
H9C2 cells are paved on a 6-hole plate, after the cell density reaches 80%, anoxic reoxygenation treatment is carried out, then 80 mu M rhein polypeptide co-assembled hydrogel is added, after empty carrier hydrogel and EGCG and rhein polypeptide co-assembled hydrogel are treated for 48 hours, the expression condition of inflammatory factors is detected through qPCR, the result is shown in figure 5, the expression of inflammatory factors TNF-alpha, IL-1 beta and IL-6 of the EGCG and rhein polypeptide co-assembled hydrogel is lower than that of other groups, and the fact that the co-assembled hydrogel has excellent inflammation inhibition effect is shown.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A small molecular hydrogel with double inhibition effects on active oxygen and inflammation and a preparation method thereof are characterized by comprising the following steps:
step one, Rhein-polypeptide compound (Rh-pep) and empty carrier-polypeptide compound are prepared, and the polypeptide sequence is Rhein- D F D FG-SS-ERGD/Nap- D F D FG-SS-ERGD；
Step two, preparing a PBS solution from the rhein-polypeptide compound;
and step three, blending the prepared solution and epigallocatechin gallate (EGCG) solution, and co-assembling under the action of reduced glutathione to form the small molecule drug hydrogel.
2. The small molecule hydrogel with double inhibition effects on active oxygen and inflammation and the preparation method thereof according to claim 1, wherein the steps of preparing the rhein-polypeptide compound and the empty carrier-polypeptide compound in the step one are as follows:
1) synthesizing Fmoc-SS by using sodium bicarbonate, cystamine dihydrochloride, 1, 4-butanedioic anhydride, dioxane and 9-fluorenylmethyl-N-succinimide carbonate (Fmoc-Osu) as raw materials;
2) synthesis of Rhein- D F D FG-SS-ERGD and Nap- D F D FG-SS-ERGD。
3. The small molecule hydrogel with double inhibition effects on active oxygen and inflammation and the preparation method thereof according to claim 1, wherein the rhein-polypeptide compound in the first step has a structural formula as follows:
the empty carrier-polypeptide compound has the structural formula:
4. the small molecule hydrogel with double inhibition of active oxygen and inflammation and the preparation method thereof according to claim 1 or 2, wherein the synthesis of Fmoc-SS in the first step comprises:
(1) synthesis of intermediate reactant 1: weighing cystamine dihydrochloride and sodium bicarbonate according to a molar ratio of 1: 2-3, adding the cystamine dihydrochloride to a 250ml flask, adding water with the mass being 20 times that of the two substances, stirring at room temperature until the mixture is clear, adding dioxane with the volume being 1/5, uniformly stirring, weighing 1, 4-succinic anhydride with the same mole as that of cystamine dihydrochloride, adding the 1, 4-succinic anhydride into the reaction bottle, and reacting at room temperature for 8-12 hours to obtain an intermediate product 1.
(2) Fmoc-SS synthesis: after the intermediate product 1 is completely reacted, weighing 9-fluorenylmethyl-N-succinimide carbonate (Fmoc-Osu) and sodium bicarbonate with the same mole of cystamine dihydrochloride in the first step; sodium bicarbonate was added directly to the flask of intermediate 1, Fmoc-Osu was first mixed uniformly in dioxane of the same volume as in the first step, and the mixture was dropped into the flask using a constant pressure dropping funnel and allowed to react overnight at room temperature. After the reaction was completed, the reaction solution was filtered under reduced pressure, and the filtrate was retained. And (3) concentrating the filtrate by using a rotary evaporator, adjusting the pH of the concentrated filtrate to 1-2 by using 1M hydrochloric acid, standing at room temperature, performing suction filtration under reduced pressure to retain a solid after the white solid is completely separated out, and freeze-drying to obtain the Fmoc-SS.
5. The small molecule hydrogel with double inhibition of active oxygen and inflammation as claimed in claim 1 or 2, wherein the Fmoc solid phase synthesis step in the first step is
1) The dichloro resin was weighed (load: 1.4mmol/g) into a dry solid phase tube, adding a proper amount of Dichloromethane (DCM), placing on a shaker for 30min, introducing nitrogen for 5min to fully expand the dichloro resin, and extruding dichloromethane solvent;
2) according to the volume of the dichloro resin in the solid phase tube, weighing aspartic acid Asp (D) (Fmoc-Asp (tBu) -OH), adding equimolar DIPEA, dissolving with dichloromethane, mixing, adding into a solid phase reactor, adding equivalent DIPEA into the reaction solution, and reacting for 1-2 h;
3) after the reaction is finished, pressing and drying the reaction solution, washing for 5 times by using dichloromethane, washing for 1min each time, then adding a sealing solution to react for 15-30 min so as to seal the active groups on the resin, and pressing and drying the reaction solution; the confining liquid consists of dichloromethane, methanol and DIPEA, and the volume ratio of DCM to MEOH to DIEA is 17-20: 2: 1.
4) Extruding the sealing solution, washing with dichloromethane for 5 times (1 min each time), and washing with N, N-Dimethylformamide (DMF) for 5 times (1 min each time);
5) cutting off the Fmoc protecting group: cutting with 20% piperidine (solvent is DMF) for 20-40 min, removing the protecting group Fmoc on aspartic acid to expose amino group, drying the reaction solution, washing with DMF for 5 times and 1min each time;
6) weighing the next amino acid glycine Gly (G) (Fmoc-Gly-OH) and equimolar HBTU in a dried vial, adding equimolar DIEA, dissolving with DMF, performing ultrasonic assisted dissolution, adding a solid phase tube for reaction for 2h, extruding the solution, and washing with DMF for 5 times, 1 min/time; cutting with 20% piperidine (solvent is DMF) for 20-40 min, removing the protecting group Fmoc to expose amino group, drying the reaction solution, washing with DMF for 5 times and each time for 1 min;
7) repeating step 6) above, followed by the sequential addition of Fmoc-Arg (pbf) -OH, Fmoc-Glu(tBu)-OH，Fmoc-SS，Fmoc-Gly-OH，Fmoc- D Phe (tBu) -OH (D-configuration), Fmoc- D Phe (tBu) -OH (D-configuration), and finally rhein. The reaction difficulty is gradually increased along with the extension of the peptide chain, so that the dosage of the amino acid after the reaction is gradually increased to 3-6 times of the dosage of the first amino acid so as to improve the yield of the target polypeptide;
8) after the reaction is finished, washing the reaction product with DMF for 5 times, then washing the reaction product with DCM for 5 times, and each time for 1min, preparing a proper amount of cutting fluid, wherein the cutting fluid is 95% of trichloroacetic acid TFA, and preparing a mixed solution of triisopropylsilane TIS and water, and the volume fraction of the mixed solution is 95% of TFA: 2.5% TIS: 2.5% H 2 And O, cutting for 1h, and collecting cutting fluid. It was washed 3 times with DCM for 2 min/time and the liquid was collected.
9) Spin-drying the collected liquid on a rotary evaporator, adding a proper amount of anhydrous ether, standing for 3-5min to separate out a white precipitate, centrifuging by using a high-speed centrifuge, removing a supernatant, freeze-drying the white precipitate to obtain a crude product, and purifying the crude product by using a high performance liquid chromatograph.
6. The small molecule hydrogel having double inhibitory effects on active oxygen and inflammation according to claim 1, wherein the PBS solution in step two is a mixed phosphate solution of sodium dihydrogen phosphate and disodium hydrogen phosphate, and is prepared by mixing 38.0g of sodium dihydrogen phosphate with 5.04g of disodium hydrogen phosphate, adding water to a volume of 1000ml, and adjusting the pH of the solution to 7.4.
7. The small molecule hydrogel with double inhibition effects on active oxygen and inflammation and the preparation method thereof according to claim 1, wherein the concentration of the rhein-polypeptide compound solution prepared in the second step is 10.0-15.0 g of dissolved Rh-pep per 1000ml of PBS solution.
8. The small-molecule hydrogel with the dual inhibition effects on active oxygen and inflammation and the preparation method thereof as claimed in claim 1, wherein the mass ratio of the dosages of EGCG and Rh-pep in the blending in the third step is 1: 20-25.
9. The small molecule hydrogel with double inhibition effects on active oxygen and inflammation and the preparation method thereof according to claim 1, wherein the hydrogel preparation steps in the third step are as follows:
1) pouring Rh-pep into a gel-forming small bottle, and dissolving with a PBS solution;
2) EGCG which is adjusted to neutral pH by 1mol/l sodium carbonate and added;
3) adding 4eq of GSH solution at pH 7.4;
4) after being mixed evenly, the mixture is inverted to form stable hydrogel.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115531291A (en) * | 2022-09-02 | 2022-12-30 | 南京医科大学 | Medicine-carrying lysine rhein self-assembled hydrogel and preparation method and application thereof |
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