CN107529533B - PH-sensitive polypeptide capable of self-assembling into hydrogel and application thereof as drug loading material - Google Patents
PH-sensitive polypeptide capable of self-assembling into hydrogel and application thereof as drug loading material Download PDFInfo
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Abstract
The invention relates to a pH sensitive polypeptide capable of self-assembling into hydrogel, a preparation method thereof and application thereof as a drug loading material. The synthesis of the target polypeptide is rapidly realized by a solid-phase synthesis method. The pH sensitive polypeptide loaded glucose oxidase, catalase and insulin provided by the invention form a glucose response insulin release system, self-assembly is carried out under a neutral condition to form hydrogel, self-assembly is rapidly carried out in a local slightly acidic environment formed after glucose oxidase catalyzes glucose oxidation to generate gluconic acid, wrapped insulin is released, and glucose response insulin release is realized.
Description
Technical Field
The invention relates to the fields of pharmacology and polypeptide synthesis related to diabetes, in particular to pH sensitive polypeptides capable of self-assembling into hydrogel and application of the pH sensitive polypeptides as drug loading materials.
Background
Diabetes is a metabolic disease characterized by persistent hyperglycemia. Currently, there are about 3 billion diabetic patients worldwide. Clinically, type 1 diabetes patients and type 2 middle and late stage diabetes patients must be treated by subcutaneous insulin injection, but long-term multiple subcutaneous injections of insulin cause great pain to patients and cannot effectively stabilize the blood sugar level of patients. At present, the solution methods comprise oral administration, nasal administration, transdermal administration and the like, but in the administration systems, the blood sugar concentration and the insulin release are not directly related, and the blood sugar level of a patient is difficult to be fundamentally stabilized. Therefore, in order to avoid complications in diabetic patients due to unstable blood glucose levels, the construction of a glucose-responsive insulin delivery system is an urgent need in the field of diabetes treatment.
At present, most of materials applied to a glucose responsive insulin release system are high molecular polymer materials, are difficult to degrade and have poor biocompatibility; there are also few applications of natural polymers such as chitosan, but their glucose responsiveness is poor.
The pH sensitive self-assembly polypeptide can form a highly ordered nano structure by utilizing the non-covalent interaction between polypeptide molecules under the neutral condition, and can form macroscopic hydrogel through further crosslinking, while the polypeptide hydrogel is decomposed under the acidic condition, the forming and decomposing processes of the polypeptide hydrogel are reversible, and the pH sensitive self-assembly polypeptide is stable in structure, easy to degrade and good in biocompatibility, so that the pH sensitive self-assembly polypeptide is a more ideal insulin release actuator material.
In a glucose response insulin release system based on glucose oxidase, pH sensitive self-assembly polypeptide serving as an insulin release actuator material can be self-assembled to form hydrogel under a neutral condition, and is rapidly self-assembled in a local slightly acidic environment formed after the glucose oxidase oxidizes glucose to generate gluconic acid, so that insulin is released, and thus glucose response insulin release is realized.
In the patent, a novel pH sensitive polypeptide capable of self-assembling into hydrogel is designed and synthesized. The pH-sensitive self-assembly polypeptide is used as an insulin release material, and the glucose oxidase is used as a glucose receptor to construct a glucose-responsive insulin release system.
In this patent, be applied to glucose response insulin release system with novel sensitive self-assembling polypeptide of pH, insulin loading capacity is big, and the system is quick, accurate to the response of high sugar environment, and effective duration is long, has successfully realized the glucose response nature insulin release inside and outside the body. Therefore, the glucose-responsive insulin release system constructed by the novel pH-sensitive self-assembled polypeptide is applied to the treatment of diabetes, can effectively control the blood sugar level within a normal range, reduces the pain of a patient caused by multiple injections, improves the compliance of the patient, and has wide development and application prospects.
Disclosure of Invention
The invention relates to a pH sensitive polypeptide capable of self-assembling into hydrogel, which is characterized in that the amino acid sequence of the polypeptide is as follows:
Ac-Arg-Ala-Thr-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Thr-Ala-Arg-Ala-Asp-Ala-NH2
(SEQ. ID NO. 1); or
Ac-Arg-Ala-Ser-Ala-Arg-Ala-Glu-Ala-Arg-Ala-Ser-Ala-Arg-Ala-Glu-Ala-NH2
(SEQ. ID NO. 2); or
Ac-Arg-Val-Thr-Val-Arg-Val-Glu-Val-Arg-Val-Thr-Val-Arg-Val-Glu-Val-NH2
(SEQ. ID NO. 3); or
Ac-Arg-Val-Thr-Val-Arg-Val-Asp-Val-Arg-Val-Thr-Val-Arg-Val-Asp-Val-NH2
(SEQ. ID NO. 4); or
Ac-Arg-Val-Ser-Val-Arg-Val-Asp-Val-Arg-Val-Ser-Val-Arg-Val-Asp-Val-NH2
(SEQ. ID NO. 5); or
Ac-Xaa-Ala-Thr-Ala-Xaa-Ala-Glu-Ala-Xaa-Ala-Thr-Ala-Xaa-Ala-Glu-Ala-NH2
(SEQ. ID NO. 6); or
Ac-Xaa-Ala-Thr-Ala-Xaa-Ala-Asp-Ala-Xaa-Ala-Thr-Ala-Xaa-Ala-Asp-Ala-NH2
(SEQ. ID NO. 7); or
Ac-Xaa-Val-Thr-Val-Xaa-Val-Glu-Val-Xaa-Val-Thr-Val-Xaa-Val-Glu-Val-NH2
(SEQ. ID NO. 8); or
Ac-Xaa-Val-Thr-Val-Xaa-Val-Asp-Val-Xaa-Val-Thr-Val-Xaa-Val-Asp-Val-NH2
(SEQ. ID NO. 9); or
Ac-Xaa-Ile-Thr-Ile-Xaa-Ile-Glu-Ile-Xaa-Ile-Thr-Ile-Xaa-Ile-Glu-Ile-NH2
(SEQ. ID NO. 10); or
Ac-Xaa-Ile-Thr-Ile-Xaa-Ile-Asp-Ile-Xaa-Ile-Thr-Ile-Xaa-Ile-Asp-Ile-NH2
(SEQ.ID NO.11)；
Wherein
Xaa is Orn, and the structure is as follows:
in a preferred embodiment of the present invention, the present invention is characterized in that,
Ac-Arg-Val-Thr-Val-Arg-Val-Glu-Val-Arg-Val-Thr-Val-Arg-Val-Glu-Val-NH2
(SEQ. ID NO. 3); or
Ac-Arg-Val-Thr-Val-Arg-Val-Asp-Val-Arg-Val-Thr-Val-Arg-Val-Asp-Val-NH2
(SEQ. ID NO. 4); or
Ac-Arg-Val-Ser-Val-Arg-Val-Asp-Val-Arg-Val-Ser-Val-Arg-Val-Asp-Val-NH2
(SEQ. ID NO. 5); or
Ac-Xaa-Val-Thr-val-Xaa-Val-Glu-val-Xaa-Val-Thr-Val-Xaa-Val-Glu-Val-NH2
(SEQ. ID NO. 8); or
Ac-Xaa-Val-Thr-Val-Xaa-Val-Asp-Val-Xaa-val-Thr-Val-Xaa-Val-Asp-Val-NH2
(SEQ.ID NO.9)；
Wherein
Xaa is Orn, and the structure is as follows:
in a preferred embodiment of the present invention, the present invention is characterized in that,
Ac-Xaa-Val-Thr-Val-Xaa-Val-Asp-Val-Xaa-Val-Thr-Val-Xaa-Val-Asp-Val-NH2
(SEQ.ID NO.9)；
wherein
Xaa is Orn, and the structure is as follows:
in one embodiment, the present invention relates to a pH sensitive polypeptide that can self-assemble into a hydrogel having the sequence:
Ac-Arg-Ala-Thr-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Thr-Ala-Arg-Ala-Asp-Ala-NH2
(SEQ. ID NO. 1); or
Ac-Arg-Ala-Ser-Ala-Arg-Ala-Glu-Ala-Arg-Ala-Ser-Ala-Arg-Ala-Glu-Ala-NH2
(SEQ. ID NO. 2); or
Ac-Arg-Val-Thr-Val-Arg-Val-Glu-Val-Arg-Val-Thr-Val-Arg-Val-Glu-Val-NH2
(SEQ. ID NO. 3); or
Ac-Arg-Val-Thr-Val-Arg-Val-Asp-Val-Arg-Val-Thr-Val-Arg-Val-Asp-Val-NH2
(SEQ. ID NO. 4); or
Ac-Arg-Val-Ser-Val-Arg-Val-Asp-Val-Arg-Val-Ser-Val-Arg-Val-Asp-Val-NH2
(SEQ. ID NO. 5); or
Ac-Orn-Ala-Thr-Ala-Orn-Ala-Glu-Ala-Orn-Ala-Thr-Ala-Orn-Ala-Glu-Ala-NH2
(SEQ. ID NO. 6); or
Ac-Orn-Ala-Thr-Ala-Orn-Ala-Asp-Ala-Orn-Ala-Thr-Ala-Orn-Ala-Asp-Ala-NH2
(SEQ. ID NO. 7); or
Ac-Orn-Val-Thr-Val-Orn-Val-Glu-Val-Orn-Val-Thr-Val-Orn-Val-Glu-Val-NH2
(SEQ. ID NO. 8); or
Ac-Orn-Val-Thr-Val-Orn-Val-Asp-Val-Orn-Val-Thr-Val-Orn-Val-Asp-Val-NH2
(SEQ. ID NO. 9); or
Ac-Orn-Ile-Thr-Ile-Orn-Ile-Glu-Ile-Orn-Ile-Thr-Ile-Orn-Ile-Glu-Ile-NH2
(SEQ. ID NO. 10); or
Ac-Orn-Ile-Thr-Ile-Orn-Ile-Asp-Ile-Orn-Ile-Thr-Ile-Orn-Ile-Asp-Ile-NH2
(SEQ.ID NO.11)。
The invention discloses a pH sensitive polypeptide capable of self-assembling into hydrogel, which is used as a polypeptide material and is applied to loading of insulin serving as a medicament for treating diabetes.
The invention also constructs a glucose response insulin release system, which takes glucose oxidase as a glucose receptor and pH sensitive self-assembly polypeptide as an insulin release material.
The glucose-responsive insulin release system is constructed by applying the pH-sensitive self-assembly polypeptide, the pH-sensitive polypeptide is loaded with glucose oxidase, catalase and insulin, self-assembly is carried out under a neutral condition to form hydrogel, self-assembly is rapidly carried out in a local slightly acidic environment formed after the glucose oxidase catalyzes glucose oxidation to generate gluconic acid, and wrapped insulin is released, so that glucose-responsive insulin release is realized.
The invention uses the pH sensitive self-assembly polypeptide to construct a glucose response insulin release system, wherein the mass ratio of the polypeptide, the glucose oxidase, the catalase and the insulin is 100: 0-30: 0-10: 0-20, and the optimal ratio is 100: 10-20: 3-5: 5-15.
The invention provides a preparation method of the compound, and the target polypeptide is efficiently and quickly synthesized by adopting a solid-phase synthesis strategy.
The following description of the biological test example illustrates the present invention.
The experimental procedures for the specific conditions in the test examples of the present invention are generally carried out under conventional conditions or under conditions recommended by commercial manufacturers. Reagents with no specific source are indicated, and are commonly purchased in the market.
Test example 1 pH Range of hydrogel-Forming Polypeptides
11 polypeptides were each prepared as a 20mg/mL solution with ultrapure water, the pH of the solution was adjusted with 0.1M NaOH/HCl solution, and the pH range at which clear, transparent hydrogels were formed was observed and recorded, and the results are shown in Table 1.
TABLE 1 pH range for hydrogel formation of polypeptides
And (4) conclusion: in the invention, all the polypeptides can self-assemble at a pH value of about 7.4 to form clear and transparent hydrogel.
In order to facilitate the detection of the insulin release test, the insulin is labeled with Fluorescein Isothiocyanate (FITC) to obtain the fluorescent FITC-insulin. The in vitro insulin release of the hydrogel system loaded with glucose oxidase, catalase and FITC-insulin was studied, and the experiment included test example 2 and test example 3.
Test example 2 insulin release test in high sugar solution
Preparing FITC-insulin solutions with different concentrations by using 400mg/dL glucose solution, and detecting the OD value of each solution. Drawing a standard curve, and obtaining the result shown in the table 2, wherein y is 670337x, R20.9909 > 0.98, can be used for quantification. Glucose oxidase, catalase and FITC-insulin are loaded by pH sensitive polypeptide, the mass ratio is 100: 15: 3.75: 10, drug-loaded hydrogel is prepared, the drug-loaded hydrogel is placed in 400mg/dL glucose solution, the OD value is detected by a multifunctional enzyme-labeling instrument at 0, 3, 6, 9 and 12 hours, and the result is shown in table 3.
TABLE 2 FTTC-insulin-OD Standard Curve
TABLE 3 Release results of drug-loaded hydrogels in 400mg/dL glucose solution
Results are expressed as mean±SD.
The result shows that each drug-loaded hydrogel can respond to high-concentration glucose and release insulin, and the drug-loaded hydrogel of SEQ.ID NO.9 has the largest insulin release amount and strong glucose sensitivity.
Test example 3 insulin release test under high-sugar and low-sugar alternate stimulation
Preferably, the carrier hydrogel is prepared by loading the carrier hydrogel of SEQ ID NO.9 with glucose oxidase, catalase and FITC-insulin at a mass ratio of 100: 15: 3.75: 10, placing the carrier hydrogel of SEQ ID NO.9 in PBS solution with alternating glucose concentrations (glucose concentration is 400mg/dL → 100mg/dL → 400mg/dL, once every 3 h), controlling the temperature at 37 +/-0.5 ℃, and detecting the release amount of FITC-insulin, wherein the results are shown in Table 4.
TABLE 4 insulin release from drug-loaded hydrogels stimulated by pulse changes in glucose concentration
Results are expressed as mean±SD.
The results show that the drug-loaded hydrogel of SEQ.ID NO.9 shows a tendency of pulse release of insulin under the alternate stimulation of high sugar and low sugar. The amount of insulin released under high glucose conditions is greater than the amount of insulin released under low glucose conditions.
Test example 4 evaluation of in vivo Long-term hypoglycemic Effect in diabetic rats
Preferably, the carrier SEQ.ID NO.9 is used for loading glucose oxidase, catalase and insulin in a mass ratio of 100: 15: 3.75: 10 to prepare the drug-loaded hydrogel. STZ-induced diabetes model rats were randomly divided into three groups, the hydrogel loaded with enzyme and insulin by subcutaneous injection was set to SEQ.ID NO.9(enzyme + insulin), the hydrogel loaded with insulin by subcutaneous injection was set to SEQ.ID NO.9(insulin), and the physiological saline by subcutaneous injection was set to negative control (control). Meanwhile, normal rats injected with saline subcutaneously were used as a blank control (normal) group. The loading amount of the hydrogel with insulin is 2mg/mL, and the administration amount of the rat insulin is 5 mg/kg. The hydrogel was injected subcutaneously for 0h, and the blood glucose level was measured using a glucometer at 0, 0.5, 1, 2, 4, 6, 8, 10 and 12h, respectively, with the results shown in table 5, and the serum was centrifuged after blood was taken from orbital venous plexus, and the content of insulin in the serum was tested by ELISA kit with the results shown in table 6. The blood glucose value of the rat is measured the day before injection and is recorded as 0d, the blood glucose value of the rat is measured at the 2 nd hour after injection and is recorded as 1d, then the blood glucose value of the rat is measured at intervals of 24h, the result is shown in table 7, the content of insulin in blood serum is detected at each blood glucose measuring time point, and the result is shown in table 8.
TABLE 5 Long-term hypoglycemic experiments on drug-loaded hydrogels-results of blood glucose test in-12 h
Results are expressed as mean±SD，*P＜0.05 vs control.
TABLE 6 Long-term hypoglycemic test of drug-loaded hydrogel-results of insulin detection within-12 h
Resultsare expressed as mean±SD，*P＜0.05 vs control.
TABLE 7 Long-term hypoglycemic test of drug-loaded hydrogel-14 d blood sugar test results
Results are expressed as mean±SD，*P＜0.05 vs control.
TABLE 8 Long-term hypoglycemic test of drug-loaded hydrogel-14 d insulin test results
Results are expressed as mean±SD，*P＜0.05 vs control.
The result shows that the hydrogel SEQ.ID NO.9(enzyme + insulin) loaded with glucose oxidase, catalase and insulin can control the blood sugar of a diabetes model rat within a normal stable range for up to 8 days, and successfully realizes the in vivo glucose-responsive insulin release.
The invention has the advantages that:
1. the pH-sensitive self-assembly polypeptide is used as a carrier and applied to delivery of insulin, and skillfully utilizes the property that the pH-sensitive polypeptide can be self-assembled into hydrogel under a neutral condition, and the property that the polypeptide hydrogel is decomposed under an acidic condition establishes direct connection between insulin release and blood glucose concentration.
2. The response mode with the glucose oxidase as the glucose sensor is provided, and in a high-sugar environment, the glucose oxidase oxidizes glucose to generate gluconic acid, so that a local slightly acidic environment is formed, and the response is quick and accurate.
3. The glucose-responsive insulin release system is composed of glucose oxidase, catalase and insulin loaded with pH-sensitive polypeptide, and can successfully realize glucose-responsive insulin release and realize accurate treatment of diabetic patients.
In conclusion, the glucose-responsive insulin release system constructed by the pH-sensitive self-assembled polypeptide has the advantages of large insulin loading capacity, quick and accurate response of the system to a high-glucose environment, long blood glucose reduction action duration, no hypoglycemia side effect, successful realization of in-vivo and in-vitro glucose-responsive insulin release, capability of reducing pain of patients caused by multiple injections of insulin, capability of developing a new dosage form of a diabetes treatment medicament and new breakthrough in the field of diabetes treatment.
Detailed Description
The present invention is illustrated by the following examples, which are not to be construed as limiting the invention in any way.
The following abbreviations are used throughout the specification:
ala: alanine; arg: arginine; asp: aspartic acid; DCM: dichloromethane; DIEA: n, N' -diisopropylethylamine; DMF: dimethylformamide; ESI-MS: electrospray mass spectrometry; fmoc: n-9-fluorenylmethyloxycarbonyl; glu: glutamic acid; gly: glycine; HBTU: benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate; HOBt: 1-hydroxy-benzotriazole; HPLC: high performance liquid chromatography; ile: isoleucine; NaCl: sodium chloride; NaOH: sodium hydroxide; NMP: n-methyl pyrrolidone; orn: ornithine; ser: serine; TFA: trifluoroacetic acid; thr: threonine; val: valine.
Example 1
Ac-Arg-Ala-Thr-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Thr-Ala-Arg-Ala-Asp-Ala-NH2
(SEQ.ID NO.1)
Solid phase synthesis and preparation of drug-loaded hydrogel.
1. Synthesis of polypeptide peptide chain
1.1 swelling of the resin
Weighing 50mg of Fmoc-Rink amide-MBHA Resin (the substitution degree is 0.4mmol/g), swelling with 7mL of DCM for 30min, filtering off DCM by suction, swelling with 10mL of NMP for 30min, and finally washing with 7mL of NMP, DCM and NMP respectively.
1.2 removal of Fmoc protecting group
Putting the swelled resin into a reactor, adding 7mL of a 25% piperidine/NMP (V/V) solution containing 0.1M HOBt, reacting for 1min, and filtering the solution after the reaction is finished; then 7mL of a 25% piperidine/NMP (V/V) solution containing 0.1M HOBt was added, the reaction was carried out for 4min, and after completion, the solution was filtered off and washed with NMP. The resin was obtained with the Fmoc protecting group initially attached removed.
1.3 Synthesis of Fmoc-Ala-Rink amide-MBHA Resin
Fmoc-Ala-OH (12.5mg, 0.04mmol), HBTU (15.1mg, 0.04mmol), HOBt (5.4mg, 0.04mmol) and DIPEA (13.9. mu.L, 0.08mmol) were dissolved in NMP 10mL, and this solution was added to the resin from step 1.1, reacted for 7min, after which the reaction was filtered off and the resin was washed 3 times with 7mL each of DCM and NMP.
1.4 detection of coupling efficiency
Washing a small amount of resin particles with DMF, putting into a transparent vial, adding 3 drops of 1% bromophenol blue solution, shaking at normal temperature for 3 minutes, and determining that the resin is positive if it is blue and transparent if it is negative. If negative, the next coupling cycle can be entered.
1.5 elongation of peptide chain
And repeating the steps of deprotection and coupling according to the sequence of the peptide chain, sequentially connecting corresponding amino acids, and sequentially connecting corresponding amino acids until the peptide chain is synthesized, thereby obtaining the resin connected with the polypeptide chain.
1.6 acetylation of the N-terminus of the polypeptide chain
Adding 20% acetic anhydride solution of DCM into the resin connected with polypeptide chain obtained in step 1.5, reacting for 30min, filtering out the reaction solution after the reaction is finished, washing the resin 3 times with 7mL each of DCM and NMP, and completing N-terminal acetylation.
1.7 cleavage of the Polypeptides on the resin
The resin with polypeptide chain obtained above was placed in a reaction flask, 10mL of cleavage agent Reagent K (TFA/thioanisole/water/phenol/EDT, 82.5: 5: 2.5, V/V) was added, shaken at 0 ℃ for 30min, and reacted at room temperature for 3 h. After the reaction was completed, the reaction mixture was filtered with suction, washed three times with a small amount of TFA and DCM, and the filtrates were combined. Adding the filtrate into a large amount of glacial ethyl ether to separate out white flocculent precipitate, freezing and centrifuging to obtain a crude product of the target polypeptide. 30.1mg of crude polypeptide is finally obtained, and the yield is 91.8%.
2. Purification of polypeptides
Weighing a proper amount of the crude target polypeptide, preparing the crude target polypeptide into a solution of about 10mg/mL by using purified water, filtering the solution by using a 0.45-micron microporous membrane, and purifying the solution by using a preparative reverse phase HPLC system. The chromatographic conditions are as follows: reverse phase C18 preparation of a column (340 mm. times.28 mm, 5 μm), UV detection wavelength 214nm, mobile phase A: 0.1% TFA/water (V/V), mobile phase B: 0.1% TFA/acetonitrile (V/V); the flow rate is 5 mL/min; the elution time was 45 min. Eluting with linear gradient, collecting target peak, evaporating under reduced pressure to remove organic solvent, mixing water phases, and vacuum freeze drying to obtain pure product. The theoretical relative molecular mass is 1683.9. ESI-MS m/z: calcd [ M +2H ]]2+843.0，[M+3H]3+562.3；Found[M+2H]2+843.7，[M+3H]3+562.8。
3. Preparation of drug-loaded hydrogel
Preparing a mixed solution of glucose oxidase (final concentration of 3mg/mL) and catalase (final concentration of 0.75mg/mL) with 150mM NaCl solution, putting 1mL of the double-enzyme mixed solution into a 1.5mL of EP tube, adding 2mg of insulin, adding 20mg of polypeptide, and fully and uniformly mixing. Adjusting the pH value to about 7.4 by NaOH, centrifuging at 1000 Xg for 2min to remove air bubbles, and standing for 30min to obtain clear and transparent drug-loaded hydrogel.
Example 2
Ac-Arg-Ala-Ser-Ala-Arg-Ala-Glu-Ala-Arg-Ala-Ser-Ala-Arg-Ala-Glu-Ala-NH2
(SEQ.ID NO.2)
The synthesis method and the preparation method of the drug-loaded hydrogel are the same as those of example 1, and the theoretical relative molecular mass is 1683.9. ESI-MSm/z: calcd [ M +2H ]]2+843.0，[M+3H]3+562.3；Found[M+2H]2+843.6，[M+3H]3+562.8。
Example 3
Ac-Arg-Val-Thr-Val-Arg-Val-Glu-Val-Arg-Val-Thr-Val-Arg-Val-Glu-Val-NH2
(SEQ.ID NO.3)
The synthesis method and the preparation method of the drug-loaded hydrogel are the same as those of example 1, and the theoretical relative molecular mass is 1936.2. ESI-MSm/z: calcd [ M +2H ]]2+969.1，[M+3H]3+646.4；Found[M+2H]2+969.6，[M+3H]3+646.8。
Example 4
Ac-Arg-Val-Thr-Val-Arg-Val-Asp-Val-Arg-Val-Thr-Val-Arg-Val-Asp-Val-NH2
(SEQ.ID NO.4)
The synthesis method and the preparation method of the drug-loaded hydrogel are the same as those of example 1, and the theoretical relative molecular mass is 1908.1. ESI-MSm/z: calcd [ M +2H ]]2+955.1，[M+3H]3+637.1；Found[M+2H]2+955.7，[M+3H]3+637.6。
Example 5
Ac-Arg-Val-Ser-Val-Arg-Val-Asp-Val-Arg-Val-Ser-Val-Arg-Val-Asp-Val-NH2
(SEQ.ID NO.5)
The synthesis method and the preparation method of the drug-loaded hydrogel are the same as those of example 1, and the theoretical relative molecular mass is 1880.1. ESI-MSm/z: calcd [ M +2H ]]2+941.1，[M+3H]3+627.7；Found[M+2H]2+942.0，[M+3H]3+628.3。
Example 6
Ac-Orn-Ala-Thr-Ala-Orn-Ala-Glu-Ala-Orn-Ala-Thr-Ala-Orn-Ala-Glu-Ala-NH2
(SEQ.ID NO.6)
The synthesis method and the preparation method of the drug-loaded hydrogel are the same as those of example 1, and the theoretical relative molecular mass is 1543.8. ESI-MSm/z: calcd [ M +2H ]]2+772.9，[M+3H]3+515.6；Found[M+2H]2+773.4，[M+3H]3+516.4。
Example 7
Ac-Orn-Ala-Thr-Ala-Orn-Ala-Asp-Ala-Orn-Ala-Thr-Ala-Orn-Ala-Asp-Ala-NH2
(SEQ.ID NO.7)
The synthesis method and the preparation method of the drug-loaded hydrogel are the same as those of example 1, and the theoretical relative molecular mass is 1515.8. ESI-MSm/z: calcd [ M +2H ]]2+758.9，[M+3H]3+506.3；Found[M+2H]2+759.5，[M+3H]3+506.8。
Example 8
Ac-Orn-Val-Thr-Val-Orn-Val-Glu-Val-Orn-Val-Thr-Val-Orn-Val-Glu-Val-NH2
(SEQ.ID NO.8)
The synthesis method and the preparation method of the drug-loaded hydrogel are the same as those of example 1, and the theoretical relative molecular mass is 1768.1. ESI-MSm/z: calcd [ M +2H ]]2+885.1，[M+3H]3+590.4；Found[M+2H]2+885.7，[M+3H]3+590.7。
Example 9
Ac-Orn-Val-Thr-Val-Orn-Val-Asp-Val-Orn-Val-Thr-Val-Orn-Val-Asp-Val-NH2
(SEQ.ID NO.9)
The synthesis method and the preparation method of the drug-loaded hydrogel are the same as those of example 1, and the theoretical relative molecular mass is 1740.1. ESI-MSm/z: calcd [ M +2H ]]2+871.0，[M+3H]3+581.0；Found[M+2H]2+871.7，[M+3H]3+581.8。
Example 10
Ac-Orn-Ile-Thr-Ile-Orn-Ile-Glu-Ile-Orn-Ile-Thr-Ile-Orn-Ile-Glu-Ile-NH2
(SEQ.ID NO.10)
The synthesis method and the preparation method of the drug-loaded hydrogel are the same as those of example 1, and the theoretical relative molecular mass is 1880.2. ESI-MSm/z: calcd [ M +2H ]]2+941.1，[M+3H]3+627.7；Found[M+2H]2+942.1，[M+3H]3+628.6。
Example 11
Ac-Orn-Ile-Thr-Ile-Orn-Ile-Asp-Ile-Orn-Ile-Thr-Ile-Orn-Ile-Asp-Ile-NH2
(SEQ.ID NO.11)
The synthesis method and the preparation method of the drug-loaded hydrogel are the same as those of example 1, and the theoretical relative molecular mass is 1852.2. ESI-MSm/z: calcd [ M +2H ]]2+927.1，[M+3H]3+618.4；Found[M+2H]2+927.8，[M+3H]3+619.1。
Claims (6)
1. A pH-sensitive polypeptide that can self-assemble into a hydrogel, wherein the amino acid sequence of the polypeptide is:
Ac-Xaa-Val-Thr-Val-Xaa-Val-Asp-Val-Xaa-Val-Thr-Val-Xaa-Val-Asp-Val-NH2
(SEQ ID NO.9)；
wherein
Xaa is Orn, and the structure is as follows:
2. use of the pH sensitive polypeptide self-assemblable into a hydrogel of claim 1 for loading insulin as a medicament for the treatment of diabetes.
3. Use of the pH sensitive polypeptide self-assemblable into a hydrogel of claim 1 for the construction of a glucose responsive insulin release system.
4. The use of claim 3, wherein the glucose responsive insulin release system is constructed by loading a pH sensitive polypeptide that self-assembles into a hydrogel with glucose oxidase, catalase, and insulin.
5. The use of claim 4, wherein the weight ratio of the pH sensitive polypeptide, glucose oxidase, catalase and insulin that can self-assemble into the hydrogel is 100: (10-20): (3-5): (5-15).
6. A method for preparing the polypeptide of claim 1, comprising a liquid phase synthesis or solid phase synthesis preparation method.
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| CN201710821024.7A CN107529533B (en) | 2017-09-07 | 2017-09-07 | PH-sensitive polypeptide capable of self-assembling into hydrogel and application thereof as drug loading material |
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| CN201710821024.7A CN107529533B (en) | 2017-09-07 | 2017-09-07 | PH-sensitive polypeptide capable of self-assembling into hydrogel and application thereof as drug loading material |
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