Cell penetrating peptide-insulin conjugate as well as preparation method and application thereof
An insulin and membrane-penetrating peptide technology, which is applied in the field of cell-penetrating peptide insulin conjugates and their preparation, can solve the problems of unsatisfactory oral bioavailability of insulin, achieve favorable activity retention, improve transmembrane absorption and The effect of simple transport and synthesis methods
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[0036] According to another aspect of the present invention, a method for preparing a cell-penetrating peptide insulin conjugate is provided, comprising the following steps:
[0037] (1) Reaction of CPPs with dimethylmaleic anhydride (DMMA) to protect the amino groups on CPPs and expose sulfhydryl groups;
[0038] (2) React the amino-protected CPPs with the cross-linking agent NHS-PEG-MAL to generate NHS-PEG-CPPs;
[0039] (3) Reaction of NHS-PEG-CPPs with insulin and deamination protection to obtain cell penetrating peptide insulin conjugates.
[0040] Preferably, the preparation method of the cell-penetrating peptide insulin conjugate specifically includes the following steps:
[0041](1) Dissolve an appropriate amount of CPPs in a PBS solution with a pH of 8.5, add DMMA, and react at room temperature for 2 to 4 hours; then use dialysis to remove unreacted DMMA to obtain solution A; the molar ratio of DMMA to CPPs 20~100: 1;
[0042] (2) Add an appropriate amount of cross...
Embodiment 1
[0064] Prepare the cell-penetrating peptide insulin conjugate as follows:
[0065] (1) Dissolve 10 mg of the membrane-penetrating peptide Tat (sequence GRKKRRQRRRPPQC) in 1 mL of PBS solution at pH 8.5, add 35 mg of DMMA, and react at room temperature for 3 h; then in 2% (m / v) chlorine at room temperature Sodium chloride solution (containing 20 mM EDTA) was dialyzed for 48 h to remove unreacted DMMA to obtain solution A;
[0066] (2) Add 65 mg of cross-linking agent NHS-PEG-MAL (the molecular weight of PEG is 2000) to solution A, and incubate at room temperature for 2 hours, so that the sulfhydryl groups on the CPPs molecules react with MAL to obtain NHS-PEG-CPPs;
[0067] (3) Dissolve 8 mg of insulin in 1 mL of PBS solution with pH 7.4, add it dropwise into NHS-PEG-CPPs, and incubate at room temperature for 1 hour to allow the amino group of insulin to react with the NHS group, and then place the reactant at 37°C , DMMA was removed under the condition of pH 5.5, dialyzed in ...
Embodiment 2
[0069] Prepare the cell-penetrating peptide insulin conjugate as follows:
[0070] (1) Dissolve 10 mg of the membrane-penetrating peptide Tat (sequence GRKKRRQRRRPPQC) in 1 mL of PBS solution at pH 8.5, add 40 mg of DMMA, and react at room temperature for 3 h; then in 2% (m / v) chlorine at room temperature Sodium chloride solution (containing 20 mM EDTA) was dialyzed for 48 h to remove unreacted DMMA to obtain solution A;
[0071] (2) Add 140 mg of cross-linking agent NHS-PEG-MAL (the molecular weight of PEG is 5000) to solution A, and incubate at room temperature for 2 hours, so that the sulfhydryl groups on the CPPs molecules react with MAL to obtain NHS-PEG-CPPs;
[0072] (3) Dissolve 10 mg of insulin in 1 mL of PBS solution with pH 7.4, add it dropwise into NHS-PEG-CPPs, and incubate at room temperature for 1 h to allow the amino group of insulin to react with the NHS group, and then react the reactant at 37 DMMA was removed under the conditions of pH 5.5, dialyzed in 2% (...
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