A liposome delivery system for treating cartilage diseases and its preparation method

A liposome and cartilage technology, applied in the field of medicine, can solve problems such as the inability of liposomes to enter cartilage tissue

Active Publication Date: 2018-10-09
魏垒 +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The bone-targeted delivery system prepared by this patent is intramuscular or intravenous injection of liposomes, and then liposomes bring liposomes into bone tissue through targeting molecules, thereby acting on bone tissue, but the bone-targeted delivery system Inability to target liposomes into cartilage tissue

Method used

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  • A liposome delivery system for treating cartilage diseases and its preparation method
  • A liposome delivery system for treating cartilage diseases and its preparation method
  • A liposome delivery system for treating cartilage diseases and its preparation method

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Embodiment 1: A liposome delivery system for the treatment of cartilage diseases, including liposomes, liposome modifications and small nucleic acid drugs, the liposomes are phosphatidylcholine DPPC, cholesterol, Dlin-KC2 -DMA; the liposome modification is polyethylene glycol lipid conjugate (C16 PEG2000 Ceramide) C-PEG, and the small nucleic acid drug is Ihh siRNA; its target sequence is SEQ ID NO: 2, namely Ihh The 1708-1734 base sequence of siRNA; the concentration of phosphatidylcholine DPPC is 35mg / ml, accounting for 11.72% of the mass percentage of liposomes and liposome modifications; the concentration of cholesterol is 9.5mg / ml, accounting for The mass percent of liposomes and liposome modifications is 23.85%; the concentration of polyethylene glycol lipid conjugate C-PEG is 50 mg / ml, accounting for 16.74% of the mass percent of liposomes and liposome modifications ; The concentration of Dlin-KC2-DMA is 28.5mg / ml, accounting for 47.69% of the mass percent of lip...

experiment example 1

[0034] Experimental example 1: Verification of liposome delivery system into chondrocytes

[0035] 1. Liposome delivery of siRNA into chicken hypertrophic chondrocytes

[0036] Experimental materials: 1 / 3 chondrocytes of 17-day chicken embryo sternum, lipid nanoparticles (LNP), negative control siRNA (Negative Control siRNA). 0.3% EDTA-free trypsin, 0.9% collagenase, 0.3% hyaluronidase [Type I, Sigma, Cat. No. H3506-1G].

[0037] Experimental method: The LNP-siRNA complex was prepared as described in Example 1. Obtain chondrocytes from 1 / 3 of the sternum of 15 17-day-old chicken embryos, cut them into pieces, put them in a 50ml centrifuge tube, and then add 1ml of each of the three digestive solutions, 3ml in total (digestive solution preparation: 0.3% EDTA-free pancreatic Protease, 0.9% collagenase, 0.3% hyaluronidase (prepared by volume 1:1:1), placed in a 37°C water bath for 30 minutes, carefully sucked up the upper layer liquid, and then added 1ml each of the three diges...

experiment example 2

[0046] Experimental Example 2: Verification of the expression of Ihh mRNA in rats after the prepared LNP-siRNA complex system treated rats:

[0047] Experimental materials: 10 Wistar rats. Lipid nanoparticle (LNP), Ihh.siRNA solution: Take 10ul of Ihh.siRNA with a concentration of 20uM, dilute to 40ul and set aside.

[0048] Experimental method: The LNP-Ihh.siRNA complex was prepared as described in Example 1. 40ul of the LNP-Ihh.siRNA complex was injected into the right knee joint of the rat, and 40ul of the Ihh.siRNA solution was injected into the left knee joint. After 48 hours, the rats were sacrificed, the whole knee cartilage was scraped, and the total RNA was extracted, reverse-transcribed into cDNA, and detected by Real-time PCR.

[0049] Ihh mRNA primers: sense strand: 5'-CAGGAAGGACCCATTCCGTC-3'; antisense strand: 5'-AAGTCACAAACCCAGGTCCC-3'.

[0050] The results showed that the expression of Ihh mRNA decreased by 80% in the rat right knee joint compared with the le...

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Abstract

The invention belongs to the technical field of medicine. In order to solve the lack of an effective and safe cartilage siRNA delivery system and limit the application of RNAi in the treatment of cartilage diseases, the invention provides a cartilage siRNA liposome delivery system and a preparation method thereof. Including liposomes, liposome modifications and small nucleic acid drugs, liposomes are phosphatidylcholine, cholesterol, Dlin-KC2-DMA; liposome modifications are polyethylene glycol lipid conjugates, small nucleic acid The drug is Ihh siRNA. The invention modifies the liposome nano-based cartilage RNA interference delivery system, and the formed complex has positive charges, is safe, small in size and can be distributed into the full-thickness cartilage tissue. Carrier experiments have confirmed that siRNA can be delivered into chondrocytes, and the corresponding genes in chondrocytes can be successfully knocked out, breaking through the bottleneck of gene knockout in cartilage tissue; it can be used as a targeted gene to treat PTOA.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a liposome delivery system for treating cartilage diseases and a preparation method thereof. Background technique [0002] Indian Hedgehog (Ihh) is associated with post-traumatic osteoarthritis (PTOA), and the expression of Ihh increases in the early stage of knee joint anterior cruciate ligament (ACL) injury, and eventually after ACL injury Will develop into PTOA. In animal experiments, the use of conditional gene knockout mice to knock out the Ihh gene in the articular cartilage of adult mice can slow down ACL injury and reduce the occurrence of PTOA. Therefore, knocking out the Ihh gene can be used as a targeted gene to treat PTOA. However, the current Ihh gene knockout technology cannot be used to treat PTOA in animals other than transgenic mice and humans, and chemical Ihh inhibitors can cause serious side effects, including: holoprosencephaly, cleft lip and p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K9/127A61K48/00A61K31/713A61P19/04
Inventor 魏垒卫小春王少伟张戈孙晓娟
Owner 魏垒
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