Three-dimensional spherical α-helical cationic polypeptide with high-efficiency gene delivery ability, its preparation method and application

Abstract

The present invention provides a three-dimensional star-shaped α-helical polypolypeptide with high-efficiency gene delivery ability and its preparation method and application. Dendrimers are used as initiators and dichloromethane is used as a reaction solvent to trigger different types of N-carboxylic acid anhydride monopolypeptides. The high-speed ring-opening polymerization of the body, and the introduction of groups with different electrical properties at the end through a click chemical reaction. The abundant amino groups on the surface of the dendrimers provide sufficient polymerization sites, allowing the polypeptide to form a three-dimensional spherical topology, and the topology provides an opportunity for the acceleration of the initial stage of the ring-opening polymerization reaction. The guanidinium / amino group modified by the side chain of the polypeptide brings a higher positive charge density, which can obtain high-efficiency gene loading capacity through the electrostatic interaction between positive and negative charges, and enhance the rigid structure of the α-helix on the secondary structure. The polypeptide has a stronger membrane-penetrating ability; these properties of this type of polypeptide make it have great development prospects in the field of biomedical materials, especially in the field of gene delivery.

Description

technical field

[0001] The invention relates to the field of gene loading and delivery, in particular to a three-dimensional spherical α-helical cationic polypeptide with high-efficiency gene delivery capability and its preparation method and application, which can be applied to combined photothermal-gene therapy of breast cancer. Background technique

[0002] Gene delivery vectors are necessary media for loading nucleic acid molecules, introducing them into target cells and successfully expressing them. They are mainly divided into two types: viral vectors and non-viral vectors. Viral vectors have been widely used due to the advantages of high transduction efficiency and expression efficiency, but their own antigenicity, potential tumorigenic risk and insufficient gene loading have seriously restricted their development. Based on this, non-viral vectors have gradually gained attention. Commonly used non-viral vectors include liposomes, nanoparticles, cationic polymers, and...

Examples

Embodiment 1

[0096] Dissolve L-glutamic acid in water (375 mL) and heat to 70 °C with stirring, then add aqueous solution (375 mL) of hydrated copper acetate (18.6 g, 103 mmol) to L-glutamic acid in solution. After that, the stirring was stopped, and after standing at room temperature for 48 h, the precipitate was stirred and washed with water, ethanol, and petroleum ether for 24 h, and the blue solid was obtained by suction filtration, that is, copper (II) L-glutamate complex, which was freeze-dried Store in a desiccator for later use;

[0097] Potassium carbonate (15.2 g, 0.11 mol) and p-hydroxybenzyl alcohol (9.3 g, 0.075 mol) were dissolved in acetone (150 mL), and propyne bromide (6.75 mL, 0.09 mol) and 18-crown Ether-6 (0.1 g). After the solution was refluxed at 75 °C for 12 h, the acetone was removed by a rotary evaporator, and water (200 mL) was added to dissolve the remaining solid. The solution was extracted with dichloromethane (30 mL×3), and the organic phases were combined....

Embodiment 2

[0102] 1, 6-Dibromohexane (1.26 mL, 8 mmol) and sodium azide (1.6 g, 24 mmol) were dissolved in DMF (19 mL), and reacted at 60 °C for 24 h. Then water (150 mL) was added to dissolve the insoluble matter, extracted with diethyl ether (20 mL×3) and the organic phase was collected. After the organic phase was dried with sodium sulfate, it was filtered and rotary evaporated to obtain a white oily substance, that is, compound 4, which was subjected to NMR with deuterated chloroform. Figure 5 its NMR spectrum.

[0103] Compound 4 (3.33 g, 20 mmol) was dissolved in a mixed solvent of diethyl ether (15 mL) and ethyl acetate (15 mL), and 5 % hydrochloric acid solution (30 mL) was added, and triphenyl Phosphorus (5.51 g, 22 mmol), ensured that the two phases were separated and stirred slowly for 1 h, then reacted at room temperature for 24 h. Subsequently, 1M hydrochloric acid solution (30 mL) was added to wash the organic phase, and the aqueous phase was collected after separation. ...

Embodiment 3

[0106] In a glove box, dissolve γ-(4-propargyloxybenzyl)-L-glutamic acid-N-carboxylic acid anhydride monomer (50 mg, 1.6 mmol) in anhydrous N,N-dimethylformaldehyde Amide (1 mL), and the third-generation dendrimer (0.68 mg, 0.001 mmol) was added, and the reaction was stirred at room temperature for 72 h. Subsequently, the reaction phase was added dropwise into ice methanol (50 mL) to precipitate, and the methanol was removed after centrifugation to obtain polymer A, which was subjected to NMR with deuterated chloroform. Figure 8 its NMR spectrum.

[0107] Dissolve γ-(4-propargyloxybenzyl)-L-glutamic acid-N-carboxylic anhydride monomer (50 mg, 1.6 mmol) in anhydrous dichloromethane (1 mL) in the glove box , and the third-generation dendrimers (0.68 mg, 0.001 mmol) were added, and the reaction was stirred at room temperature for 30 min. Subsequently, the reaction phase was added dropwise into ice methanol (50 mL) to precipitate, and the methanol was removed after centrifugati...

technical field

[0001] The invention relates to the field of gene loading and delivery, in particular to a three-dimensional spherical α-helical cationic polypeptide with high-efficiency gene delivery capability and its preparation method and application, which can be applied to combined photothermal-gene therapy of breast cancer. Background technique

[0002] Gene delivery vectors are necessary media for loading nucleic acid molecules, introducing them into target cells and successfully expressing them. They are mainly divided into two types: viral vectors and non-viral vectors. Viral vectors have been widely used due to the advantages of high transduction efficiency and expression efficiency, but their own antigenicity, potential tumorigenic risk and insufficient gene loading have seriously restricted their development. Based on this, non-viral vectors have gradually gained attention. Commonly used non-viral vectors include liposomes, nanoparticles, cationic polymers, and...

Examples

Embodiment 1

[0096] Dissolve L-glutamic acid in water (375 mL) and heat to 70 °C with stirring, then add aqueous solution (375 mL) of hydrated copper acetate (18.6 g, 103 mmol) to L-glutamic acid in solution. After that, the stirring was stopped, and after standing at room temperature for 48 h, the precipitate was stirred and washed with water, ethanol, and petroleum ether for 24 h, and the blue solid was obtained by suction filtration, that is, copper (II) L-glutamate complex, which was freeze-dried Store in a desiccator for later use;

[0097] Potassium carbonate (15.2 g, 0.11 mol) and p-hydroxybenzyl alcohol (9.3 g, 0.075 mol) were dissolved in acetone (150 mL), and propyne bromide (6.75 mL, 0.09 mol) and 18-crown Ether-6 (0.1 g). After the solution was refluxed at 75 °C for 12 h, the acetone was removed by a rotary evaporator, and water (200 mL) was added to dissolve the remaining solid. The solution was extracted with dichloromethane (30 mL×3), and the organic phases were combined....

Embodiment 2

[0102] 1, 6-Dibromohexane (1.26 mL, 8 mmol) and sodium azide (1.6 g, 24 mmol) were dissolved in DMF (19 mL), and reacted at 60 °C for 24 h. Then water (150 mL) was added to dissolve the insoluble matter, extracted with diethyl ether (20 mL×3) and the organic phase was collected. After the organic phase was dried with sodium sulfate, it was filtered and rotary evaporated to obtain a white oily substance, that is, compound 4, which was subjected to NMR with deuterated chloroform. Figure 5 its NMR spectrum.

[0103] Compound 4 (3.33 g, 20 mmol) was dissolved in a mixed solvent of diethyl ether (15 mL) and ethyl acetate (15 mL), and 5 % hydrochloric acid solution (30 mL) was added, and triphenyl Phosphorus (5.51 g, 22 mmol), ensured that the two phases were separated and stirred slowly for 1 h, then reacted at room temperature for 24 h. Subsequently, 1M hydrochloric acid solution (30 mL) was added to wash the organic phase, and the aqueous phase was collected after separation. ...

Embodiment 3

[0106] In a glove box, dissolve γ-(4-propargyloxybenzyl)-L-glutamic acid-N-carboxylic acid anhydride monomer (50 mg, 1.6 mmol) in anhydrous N,N-dimethylformaldehyde Amide (1 mL), and the third-generation dendrimer (0.68 mg, 0.001 mmol) was added, and the reaction was stirred at room temperature for 72 h. Subsequently, the reaction phase was added dropwise into ice methanol (50 mL) to precipitate, and the methanol was removed after centrifugation to obtain polymer A, which was subjected to NMR with deuterated chloroform. Figure 8 its NMR spectrum.

[0107] Dissolve γ-(4-propargyloxybenzyl)-L-glutamic acid-N-carboxylic anhydride monomer (50 mg, 1.6 mmol) in anhydrous dichloromethane (1 mL) in the glove box , and the third-generation dendrimers (0.68 mg, 0.001 mmol) were added, and the reaction was stirred at room temperature for 30 min. Subsequently, the reaction phase was added dropwise into ice methanol (50 mL) to precipitate, and the methanol was removed after centrifugati...

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