Alpha spiral cation polypeptide molecule and preparation method and application thereof
A cationic polypeptide, helical technology, applied in the field of alpha helical cationic polypeptide molecule and its preparation method and application, to achieve the effect of simple preparation method, high charge density and mild synthesis conditions
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Embodiment 1
[0056] The general molecular formula of the polypeptide in claim 1 is taken as n1=3, n2=1, x=1, y=1, z=0, B1 is Ala, and B2 is Lys. The structure of the polypeptide molecule is:
[0057]
[0058] Abbreviated as RC16. This example illustrates the synthesis of RC16 and its use in gene delivery. RC16 was synthesized by a peptide synthesizer and purified by HPLC.
[0059] Dissolve 10mg of RC16 in 20mM PBS buffer (pH 5.0), mix the RC16 solution with DNA according to a certain nitrogen-phosphorus ratio (N / P), and the N / P value is 0, 1, 5, 10, 20 , 30, 40 and 50. The mixture was left at room temperature for 30 minutes to fully combine RC16 with DNA to form a stable RC16 / DNA complex. The complex had an average particle diameter of 540 nm and a zeta potential of 22 mV.
[0060] HepG2 cells were seeded in 24-well plates (8×10 4 cells / well), put into an incubator, and when the confluence of the cells reaches 70%, remove the medium, rinse the cells twice with PBS buffer, and then...
Embodiment 2
[0062] The molecular general formula of the polypeptide in claim 1 is set as n1=5, n2=4, x=7, y=12, z=5, B1 is His, and B2 is Arg. n2=4 means that the molecule is a tetra-crosslinked polypeptide, referred to as tetra-RC49, and the corresponding monomer molecule is referred to as RC49.
[0063] Its structural formula is:
[0064]
[0065] This example illustrates the synthesis of tetra-RC49 and its use in gene delivery. The structural formula of tetra-RC49 is:
[0066]
[0067] Firstly, RC49 was synthesized by a peptide synthesizer, and purified by preparative liquid chromatography to obtain a monomeric polypeptide molecule.
[0068] Dissolve 10mg of purified RC49 in deionized water at a concentration of 10mg / ml; dissolve 400mg of azodicarbonamide (N,N,N',N'-tetramethylazodicarboxamide) in DMSO to a concentration of 0.1mg / ml. Then the two solutions were mixed so that the molar ratio of RC49 and azodicarbonamide was about 1:100. After stirring at room temperature for ...
Embodiment 3
[0072] In claim 1, the molecular general formula of the polypeptide takes n1=4, n2=2, x=2, y=4, z=3, B1 is His, and B2 is Lys. n2=2 means that the molecule is a di-crosslinked polypeptide, referred to as di-RC30, and the corresponding monomer molecule is referred to as RC30, and its structural formula is:
[0073]
[0074] This example illustrates the synthesis of di-RC30 and its use in gene delivery. The structural formula of di-RC30 is:
[0075]
[0076] Firstly, RC30 was synthesized by a peptide synthesizer and purified by HPLC.
[0077] Dissolve 10 mg of purified RC30 in deionized water at a concentration of 50 mg / ml; dissolve 24 mg of azodicarboxamide (N, N, N', N'-tetramethylazodicarboxamide) in DMSO at a concentration of 2 mg / ml. Then the two solutions were mixed so that the molar ratio of RC30 and azodicarbonamide was about 1:52. After stirring at room temperature for 3 days, they were dialyzed with deionized water (MWCO=500Da) to remove by-products. After 1 da...
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