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Cationic liposome containing DHA and HMGB1-silent siRNA, preparation method and application of cationic liposome

A cationic liposome, silent technology, applied in the biological field, can solve the problems of low bioavailability, poor solubility, and single target

Active Publication Date: 2019-08-16
ZHEJIANG PHARMA COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that dihydroartemisinin (DHA) can improve the symptoms of MRL / lpr mice by inhibiting the TLR4 signaling pathway. my country has made a research breakthrough in the treatment of lupus erythematosus with artemisinin and its derivatives, but the traditional artemisinin Poor solubility, low bioavailability, and single target of action of steroid antimalarial drugs limit the application and promotion of these drugs in the field of autoimmune disease treatment

Method used

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  • Cationic liposome containing DHA and HMGB1-silent siRNA, preparation method and application of cationic liposome
  • Cationic liposome containing DHA and HMGB1-silent siRNA, preparation method and application of cationic liposome
  • Cationic liposome containing DHA and HMGB1-silent siRNA, preparation method and application of cationic liposome

Examples

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Embodiment 1

[0066] Embodiment 1, the preparation of the cationic liposome (CLs-DHA / siRNA) of co-carrying DHA and HMGB1 silencing siRNA

[0067] Will DOTAP, Chol, PEG 2000 - DSPE and DHA were dissolved in chloroform according to the molar ratio (2:1:0.15), and the lipid film was formed by rotary evaporation, and a certain amount of PBS was added to hydrate for 30 minutes, so that the final concentration of DHA was 1mg / ml, and then 200nm and 100nm The polycarbonate membrane extruded to obtain the cationic liposome containing DHA with uniform particle size.

[0068] The cationic liposomes prepared above and HMHGB1 silencing siRNA were mixed according to different mass ratios of DOTAP and siRNA (5:1 / 10:1 / 15:1 / 20:1 / 30:1) and incubated at room temperature for 30 minutes to combine them through electrostatic binding. Cationic liposomes containing DHA and HMGB1-silencing siRNA were prepared by binding HMGB1-silencing siRNA to liposomes. The ratio of DOTAP to HMGB1 silencing siRNA was examined b...

Embodiment 2

[0070] Example 2, preparation of TAT peptide (AYGRKKRRQRRR) modified cationic liposomes (TAT-CLs-DHA / siRNA) loaded with DHA and HMGB1 silencing siRNA

[0071] 1 TAT-mPEG 2000 -DSPE synthesis

[0072] One-step synthesis of TAT-mPEG 2000 -DSPE. We added a cysteine ​​(cys) to the N-terminus of the TAT peptide to introduce a sulfhydryl group for compatibility with DSPE-PEG 2000 The maleimide at the -Mal terminal undergoes direct addition reaction. Specifically, TAT and DSPE-PEG 2000 -Mal was dissolved in PBS at a molar ratio of 1:1, under nitrogen protection, at 4°C, and reacted overnight to prepare TAT-mPEG 2000 -DSPE.

[0073] 2 Preparation of TAT peptide-modified cationic liposomes co-loaded with DHA and HMGB1 silencing siRNA

[0074] DOTAP, Chol, the above TAT-mPEG 2000 -DSPE and DHA were dissolved in chloroform according to the molar ratio of 2:1:0.15:0.33, and the lipid film was formed by rotary evaporation, and a certain amount of PBS was added to hydrate for 30 min...

Embodiment 3

[0077] Embodiment 3, the cationic liposome (CLs-DHA / siRNA) of co-loading DHA and HMGB1 silencing siRNA and the TAT peptide modified cationic liposome (TAT-CLs-DHA / siRNA) of co-loading DHA and HMGB1 silencing siRNA ) Stability study

[0078] Prepare the complex of the optimal prescription (DOTAP / siRNA mass ratio is 15:1), mix it with 20% fetal bovine serum at 1:1 (volume ratio), so that the final serum concentration is 10%, and place the sample in a 37°C water bath Incubation. After incubation for a certain period of time, an appropriate amount of Triton-100 solution was added to the sample to extract siRNA, and immediately mixed with 6x Loading Buffer, analyzed by agarose gel electrophoresis and photographed by a gel imaging system.

[0079] For cationic liposomes co-loaded with DHA and siRNA, by Figure 5 A shows that the siRNA encapsulated in the liposome remains stable within 36h in serum and is gradually degraded after 36h; while naked siRNA is generally degraded in 6h i...

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Abstract

The invention provides a cationic liposome containing dihydroartemisinin (DHA) and HMGB1-silent siRNA, and a preparation method and an application of the cationic liposome, thereby constructing a novel drug delivery carrier. The cationic liposome not only can realize high drug loading and co-encapsulation of multiple drugs, but also can avoid liver first pass effect and improve pharmacokinetic characteristics; and the HMGB1-silent siRNA and the DHA are constructed in the same drug delivery carrier, and multiple targets act on the TLR4 signal axis, and thus a synergistic effect on LN treatmentis played, and a creative research method for treating autoimmune diseases is provided.

Description

[0001] 【Technical field】 [0002] The invention relates to the field of biotechnology, in particular to a cationic liposome containing DHA and HMGB1 silencing siRNA, a preparation method and application thereof. [0003] 【Background technique】 [0004] Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by excessive activation of B lymphocytes and the production of a large number of autoantibodies. Most patients with lupus have multi-system and multi-organ involvement. Lupus nephritis (LN) is the main complication and cause of death in SLE. The etiology and pathogenesis of LN are still unclear, but it is currently recognized that LN is a complex immune complex-mediated nephritis. At present, there is no effective radical treatment plan clinically, and glucocorticoids and immunosuppressants are mainly used for treatment, but the side effects are large, it is difficult to adhere to long-term administration, and the disease is still active or relapses. [...

Claims

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Application Information

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IPC IPC(8): A61K31/357A61K48/00A61K31/713A61K9/127A61K47/42A61K47/18A61P37/02A61P13/12
CPCA61K31/357A61K31/713A61K9/127A61K47/42A61K47/18A61P37/02A61P13/12A61K2300/00
Inventor 胡英刁璐
Owner ZHEJIANG PHARMA COLLEGE
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