PH-responsive siRNA delivery system

A delivery system and responsive technology, applied in the direction of organic active ingredients, medical formulations with non-active ingredients, and medical formulations containing active ingredients, etc., can solve the problem of prolonged siRNA circulation time, low tumor site concentration, and poor biodistribution and other problems, to achieve the effect of enhancing target cell uptake, process stability, and inhibiting non-specific adsorption

Pending Publication Date: 2022-07-15
ZHUJIANG HOSPITAL SOUTHERN MEDICAL UNIV
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Problems solved by technology

[0005] Aiming at the problems of low efficiency, poor biodistribution, and low concentration at the tumor site, the inventors developed a brand new pH-responsive siRNA delivery system through intensive research and development, which can prolong the siRNA delivery system. In vivo circulation time, while inhibiting the metabolic accumulation of siRNA in the kidney, improving the efficiency of tumor-targeted delivery, so as to better realize the silencing effect of siRNA

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0049] The preparation of embodiment 1mPEG-hdy-Mal

[0050] The synthetic reaction path of mPEG-hdy-Mal is as follows figure 1 As shown, the specific: the first step: the methoxy polyethylene glycol propionaldehyde (mPEG-CHO) and 4-(benzylamino) benzoic hydrazide according to the molar ratio of 1:2 in anhydrous ethanol system Stir, add an equivalent of acetic acid as a catalyst; reflux the reaction mixture at 80°C for 6-8 hours under nitrogen protection, cool the reaction mixture to room temperature, collect the precipitate by filtration, and remove residual residues by simple washing with dichloromethane Starting materials and impurities yielded a PEG derivative (mPEG-hdy) containing a hydrazone bond (hdy). In the second step, mPEG-hdy and 3-maleimidopropionic acid obtained above were added to the dichloromethane solution at a molar ratio of 1:5, and the mixture was stirred for 4 hours. The mixture was washed with saturated ammonium chloride solution and then twice with sat...

Embodiment 2

[0051] Example 2 Preparation of c(RGDfk)-Mal

[0052] c(RGDfk) polypeptide (12.1 mg) and 8-amino-3,6-dioxaoctanoic acid (3.02 mg) were stirred in polyethylene glycol solution under nitrogen at room temperature for 4-8 h until all starting peptides were After consumption, 3-maleimidopropionic acid (6.76 mg) was added to continue stirring the reaction for 4-8 h. The resulting crude product was purified by RP-MPLC. (Chromatographic conditions: injection volume 50 μL; column temperature 40 °C; flow rate 2.5 mL / min; mobile phase: phase A 0.1% TFA acetonitrile, phase B 0.1% water; elution program: 0~30 min, 5%~50% A ; 30~32min, 50%~5%A).

[0053] The obtained c(RGDfk)-Mal was characterized by mass spectrometry (UHPLC-Q Exactive Orbitrap quadrupole combined electrostatic orbitrap LC / MS was used for detection, electrospray ion source (H-ESI) was used, and the sheath gas flow rate was 30L / min, curtain gas flow rate 10L / min, nozzle voltage +4000V, capillary temperature 320ºC, curtai...

Embodiment 3c

[0054] Example 3c Synthesis of (RGDfk)-PEG-siRNA

[0055] The synthetic route of c(RGDfk)-PEG-siRNA is as follows Image 6 shown. Specifically: The intermediates and mPEG-hdy-Mal and c(RGDfk)-Mal were successfully synthesized by the above-mentioned Example 1 and Example 2, and the sulfhydryl (-SH) modified nucleic acid single chain was constructed by conventional nucleic acid chemical synthesis method. Monomer modification at the 5' end of the nucleic acid chain: After phosphorylation of the hydroxyl group at the 5' position of the pentose sugar in the nucleotide unit at the 5' end of the siRNA sense strand, a sulfhydryl (-SH) group is attached. Monomer modification at the 3' end of the nucleic acid strand: After phosphorylation of the hydroxyl group at the 5-position of the pentose sugar in the nucleotide unit at the 3' end of the siRNA antisense strand, a sulfhydryl (-SH) group is attached. Synthesize single-stranded sense strand ssRNA and antisense strand asRNA according ...

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Abstract

The invention relates to the technical field of biology, in particular to a pH responsive siRNA delivery system for a tumor growth factor receptor. The delivery system provided by the invention comprises a molecule formed by a positive-sense strand ssRNA modified by a polypeptide derivative and an antisense strand asRNA modified by a PEG derivative, polypeptide in the delivery system is c (RGDfK), PEG derivative modified antisense strand asRNA is formed by connecting and reacting methoxy polyethylene glycol propionaldehyde (mPEG-CHO) molecules, coupling molecules and asRNA, the PEG molecular weight in the mPEG-CHO is 500-20000, hydrazone bonds are formed between the mPEG-CHO and the coupling molecules, the coupling molecules comprise 8-amino-3, 5-dimethoxy-3, 5-dimethoxy-3, 5-dimethoxy-3, 5-dimethoxy-3, 5-dimethoxy-3, 5-dimethoxy-3, 5-dimethoxy-3, 5-dimethoxy-3, 5-dimethoxy-3, 5-dimethoxy-3, 5-dimethoxy-3, 5-dimethoxy-3, 5-dimethoxy-3, 5-dimethoxy-3, 5-dimethoxy-3, 5-dimethoxy-3, the organic solvent is one or more of 2, 6-dioxaoctanoic acid, 4-(benzyl amino) benzoyl hydrazine, N-maleyl-beta-alanine and 3-maleimidopropionic acid. The delivery system can prolong the in-vivo circulation time of the siRNA, inhibit metabolic accumulation of the siRNA in the kidney and improve the tumor targeted delivery efficiency, thereby better achieving the silence effect of the siRNA.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a pH-responsive siRNA delivery system for tumors. Background technique [0002] Small interfering nucleic acid molecules (siRNAs), sometimes called short interfering RNAs or silencing RNAs, are a class of double-stranded RNA molecules, usually double-stranded RNAs composed of 19 to 23 base pairs (bp). siRNA is mainly involved in RNA interference (RNAi). ) phenomenon that regulates gene expression in a specific manner with sequence homology-driven gene knockout capabilities. It is based on this ability that it has broad prospects for clinical application. However, due to the large molecular weight and negative charge of siRNA, siRNA molecules have poor stability in vivo (easy to be degraded by nucleases in serum), Off-target effects, immunogenicity, easy accumulation in liver and kidney metabolism, and difficulty in cellular uptake. These problems seriously hinder the clinic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/64A61K47/60A61K31/713A61K48/00A61P35/00
CPCA61K47/64A61K47/60A61K31/713A61P35/00
Inventor 贺帅苏晴陈俊晓范仪圻
Owner ZHUJIANG HOSPITAL SOUTHERN MEDICAL UNIV
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