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Liver targeting small nucleic acid drug sustained-release delivery system and application thereof

A small nucleic acid drug and liver-targeted technology, which is applied in drug delivery, drug combination, digestive system, etc., can solve the problems of poor stability and sustained release, and achieve good long-term sustained release effect, low cytotoxicity, Easy to prepare

Pending Publication Date: 2022-05-06
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved by the present invention is that the existing N-acetylated galactosamine-modified siRNA small molecule drug has poor stability and slow-release performance in vivo

Method used

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  • Liver targeting small nucleic acid drug sustained-release delivery system and application thereof
  • Liver targeting small nucleic acid drug sustained-release delivery system and application thereof
  • Liver targeting small nucleic acid drug sustained-release delivery system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Physical properties of embodiment 1 DP7-C / siRNA

[0084] 1. Experimental methods Materials and methods

[0085] 1.1 Synthesis of DP7-C

[0086] DP7-C was synthesized on CSBio 136XT automatic peptide synthesizer by standard peptide solid-phase synthesis method, and purified by high-performance liquid chromatography to make the purity of DP7-C more than 95%. After synthesis, it can be directly dissolved in MilliQ water to form a 10mg / ml mother solution, and stored in a refrigerator at 4°C for a short period of time; for long-term storage, the powder should be stored in a refrigerator at -20°C, ready for immediate use.

[0087] 1.2 Determination of particle size and Zeta potential.

[0088] The particle size and Zeta potential value were measured with a Malvern particle sizer (equalize at 25°C for 2 minutes, and tested 3 times, taking the middle value).

[0089] 1.3 Gel retardation experiment

[0090] (1) Configure 1% agarose gel for use;

[0091] (2) DP7-C and siRNA ...

Embodiment 2

[0104] Embodiment 2, DP7-C in vitro transfection siRNA experiment

[0105] 1. In vitro transfection efficiency test method

[0106] 1.1 Inoculation

[0107] Hep3B cells in good growth state were inoculated into 6-well plates, 2×10 per well 5 cells, adhered overnight.

[0108] 1.2 Processing

[0109] The medium in the wells was replaced with double-free medium 30 minutes in advance. Then DP7-C, liposome lipofectamine2000 were incubated with fluorescent Cy3-GalNAc-siRNA for 15 min and added to the wells, and a blank control group was set at the same time. After 4 h, 1 ml of new medium was added.

[0110] 1.3 shoot fluorescence

[0111] After 24 hours, fluorescent photographs were taken under a fluorescent microscope.

[0112] 1.4 Flow cytometry detection

[0113] Collect the cells that have been photographed into a flow tube, wash twice with PBS solution, centrifuge at 1500 rpm for 5 min, discard the waste liquid after completion, and add 300 μl PBS to each tube for flow...

Embodiment 3

[0151] Example 3 In vivo sustained release and liver targeted therapy experiments

[0152] 1. In vivo sustained release and liver targeting effect verification experimental method

[0153] 1.1 In vivo imaging was performed for 21 days to monitor the sustained release of fluorescent scramble siRNA at the injection site.

[0154] According to Table 2, the drug was subcutaneously injected into the back of the mice, and the day of injection was recorded as Day 0. At different time points, the luminescence of the injection site and the liver was observed using a small animal in vivo imaging system. In the group with hydrogel, the prepared liquid mixture was injected into the animals, and the gel formed immediately in the animals.

[0155] The preparation method of each group is

[0156] Cy3-GalNAc-siRNA group: 30 μg of Cy3-labeled GalNAc-scramble siRNA was dissolved in 200 μl of water.

[0157] DP7-C / Cy3-GalNAc-siRNA group: Dissolve 30 μg Cy3-GalNAc-siRNA and 120 μg DP7-C in 200...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a liver-targeted small nucleic acid drug sustained-release delivery system and application thereof. The invention aims to solve the technical problems of poor in-vivo uptake rate and stability of a GalNAc-small nucleic acid drug. The technical means for solving the technical problem is to provide the liver targeting small nucleic acid drug slow-release delivery system. The polypeptide DP7-C modified by cholesterol can be used for efficiently delivering a GalNAc-small nucleic acid drug to liver cells. According to the invention, the DP7-C / GalNAc-small nucleic acid drug compound is dispersed in the hydrogel to form a new delivery system, so that the stability of the GalNAc-small nucleic acid drug can be more effectively maintained, a long-acting slow-release effect is achieved, and the hydrogel has a very good practical application effect in vivo. According to the technical scheme, preparation is convenient, low toxicity and safety are achieved, the preparation period is short, and compared with an existing delivery system, the transfection efficiency is higher, the cytotoxicity is lower, the long-acting slow-release effect is better, and the good application prospect is achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a liver-targeted small nucleic acid drug sustained-release delivery system and its application. Background technique [0002] The liver is the largest metabolic organ in the human body and is involved in the synthesis and metabolism of various chemical substances in the body. Abnormal liver function can lead to a series of liver-related diseases, including cancer and metabolic diseases. Liver targeting has always been a research hotspot in the treatment of liver-related diseases. Since its discovery in 1998, siRNA interference (RNAi) therapy has emerged as a promising therapeutic candidate for a variety of diseases. However, since siRNAs are relatively polyanionic molecules, it is difficult for these molecules to enter cells through passive diffusion mechanisms. Furthermore, in vivo targeted delivery of siRNA to tissues and cells remains the greatest challenge for its c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K9/51A61K31/7088A61K47/42A61K47/54A61K9/06A61P1/16A61P3/00A61P7/00A61P35/00B82Y5/00B82Y30/00B82Y40/00A61K47/61
CPCA61K31/7088A61K47/549A61K9/5169A61K9/06A61K9/0019B82Y5/00B82Y30/00B82Y40/00A61P35/00A61P7/00A61P1/16A61P3/00A61K47/61
Inventor 杨莉周佰玲
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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