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Bone-targeted RNA interference compound and synthetic method thereof

A technology of RNA interference and complexes, applied in the field of biomedicine, can solve the problems of complex operation, difficulty in drug delivery to organs, and transfection of cells, and achieve easy separation and purification, avoid side effects, and expand the scope of basic research Effect

Inactive Publication Date: 2014-12-03
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007]siRNA is highly specific and can be directly injected into organs. However, this method of administration only transfects cells near the injection site, and it is difficult to achieve the transfection of the entire organ. The drug administration of the device, and the operation is complicated and has certain risks

Method used

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  • Bone-targeted RNA interference compound and synthetic method thereof
  • Bone-targeted RNA interference compound and synthetic method thereof
  • Bone-targeted RNA interference compound and synthetic method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Synthesis of siRNA-(STR-R8)-(D-Asp8) complex

[0036] Synthetic steps

[0037] a. Mix D-Asp8 containing acetylcysteine ​​residues with STR-R8 liposomes (30 μmol / ml) at a molar ratio of 3:1 and incubate at room temperature for 2 hours to obtain (STR-R8)-( D-Asp8) complex, molecular formula see figure 1 .

[0038] b. Using a Sepharose column, remove non-copolymerized D-Asp8 by size exclusion chromatography.

[0039] c. Take 0.5 ml of copolymerized (STR-R8)-(D-Asp8) liposome suspension and 0.5 ml of mannitol aqueous solution (mannitol to water molar ratio = 5:1) and freeze-dry for 48 hours.

[0040] d. Dissolve the lyophilized liposome complex (STR-R8)-(D-Asp8) with HEPES (4-hydroxyethylpiperazineethanesulfonic acid, pH 4.0, concentration 10mM) to a final concentration of 0.17 mg / mL .

[0041] e. siRNA encapsulation: Shake and mix 0.1 mg / mL siRNA solution (solvent is HEPES (pH 4.0, concentration 10mM)) and (STR-R8)-(D-Asp8) solution, incubate at room temperature for 2...

Embodiment 2

[0044] Example 2 Validation of (STR-R8)-(D-Asp8) Tissue Targeting

[0045] Experimental procedure

[0046] a. Synthesis of (STR-R8)-(D-Asp8) complex as above and modification with rhodamine

[0047] b. Tail vein injection of targeted gene transfection complex, simple rhodamine or PBS at equal doses of rhodamine, after 30min, 1h, 2h, 4h and 24h, use small animal in vivo imaging system to observe and measure the fluorescence intensity of different tissues and organs and conduct quantitative analysis .

[0048] c. 24 hours after administration as described in b, the femoral tissue was taken for fixation, embedded in hard tissue, sectioned, and the fluorescence distribution and intensity in the femur were observed with a laser confocal microscope.

[0049] d. 24 hours after administration as described in b, the femoral tissue was fixed, dehydrated, frozen and hard tissue sectioned, and TRAP stained, and the fluorescence distribution on the surface of the bone trabecula and t...

Embodiment 3

[0056] Example 3 Application of Targeted RNA Interference against Osteoclast Sema4d Gene

[0057] Experimental procedure

[0058] a. 8-week-old Kunming mice were given treatment measures, the intensity of intervention was once a week, and the intervention period was 4 weeks. Both prevention and treatment experiments were grouped as follows: ①PBS group (blank control), ②Scrambled siRNA-(STR-R8)-(D-Asp8) group (siRNA negative control), ③Sema4d siRNA-(STR-R8) group (non-targeting Negative control), ④OVX+Sema4d si RNA-(STR-R8)-(D-Asp8) group (experimental group). Calcein and alizarin red were injected subcutaneously three days after the intervention and four days before the sampling.

[0059] b. Four weeks after the intervention, the bilateral femurs were fixed with 4% PFA and microCT was performed to analyze the bone mass, bone density and bone microstructure of the distal end of the femur and vertebral cancellous bone.

[0060] c. The other half of the femur after microCT sc...

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Abstract

The invention provides a preparation method and application of a bone-targeted RNA interference compound. The RNA interference compound comprises siRNA and (D-Asp8)-(STR-R8) carriers. Specifically, the preparation method comprises the following steps: coupling bone-oriented D-Asp8 (oligopeptide of a bone tissue-targeted water-soluble polymeric drug carrier) and STR-R8 cationic polymer (a good gene transfection carrier) by adopting a chemical method, and attracting and packaging siRNAs aiming at target genes to STR-R8 through heterogeneous charge to obtain the bone-targeted drug. The bone-targeted drug directionally acts on bone tissues under the guide effect of D-Asp8, and achieves different effects by carrying corresponding siRNAs aiming at different target genes. According to the preparation method and application of the bone-targeted RNA interference compound, the application range is wide, the bone affinity is good, the synthetic steps are simple, and the conditions are easy to control.

Description

[0001] technical field [0002] The invention belongs to the field of biomedicine and relates to a bone-targeting RNA interference complex and a synthesis method thereof. Background technique [0003] Bone is a specialized connective tissue composed of massive calcified intercellular matrix and cells that provide mechanical support and participate in calcium homeostasis. It is constantly reabsorbed and rebuilt to maintain its normal function. Disturbances in this resorption / formation balance are characteristic of most bone diseases. With the increasing understanding of bone biology, many new therapeutic drugs have been used to treat bone diseases. However, bone tissue has high hardness, poor permeability, and special physiological and biochemical processes. For general drug treatment, it is difficult for drugs to reach the lesion effectively, resulting in low local drug concentration and ineffective effects. And increasing the administration concentration will increas...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K47/42A61P19/08A61P35/00A61P19/02A61P19/10A61P3/00A61P29/00
Inventor 张玉峰边专魏凌飞
Owner WUHAN UNIV
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