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Preparation method used for enhancing multifunctional lipidosome used for enhancing siRNA (Small interfering Ribonucleic Acid) delivery

A liposome and multi-functional technology, applied in the direction of liposome delivery, medical preparations with non-active ingredients, medical preparations containing active ingredients, etc., can solve the problems of unsatisfactory membrane penetration and achieve targeting Strong, low cytotoxicity, high transfection efficiency

Pending Publication Date: 2020-07-28
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the strong polarity of R8, its own film penetration effect is not ideal

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] 1. Cationic liposome preparation

[0017] Add DODMA, OA-R8, Chol and DSPE-PEG to the phospholipid solution dissolved in ethanol 2000 , so that the molar ratio is 20 / 25 / 35 / 2, wherein the molar ratio of lecithin is 18. After mixing evenly, use HEPES buffer solution with a pH of 7.2 to 7.4 to remove ethanol. After dialyzing at room temperature for 2 to 3 hours, take out the lipid plastid. The measured average particle size is 98.8 nm, the average polydispersity coefficient is 0.107, and the Zeta potential is 38.5 mV.

[0018] 2. Preparation of multifunctional liposome-loaded siRNA delivery system

[0019] Protamine and siRNA were respectively dissolved in citrate buffer (20 mM, pH 4), and the lipid mixture in step 1 was mixed with the protamine solution, vortexed for 30-50 seconds; then the siRNA solution was added dropwise To the above mixture, vortex for 20-30 seconds. Ethanol and free siRNA were removed by dialysis, and liposomes were taken out after dialysis at roo...

Embodiment 2

[0021] 1. Cationic liposome preparation

[0022] Add DODMA, LA-R8, Chol and DSPE-PEG to the phospholipid solution dissolved in ethanol 2000 , so that the molar ratio is 20 / 25 / 35 / 2, wherein the molar ratio of lecithin is 18. After mixing evenly, use HEPES buffer solution with a pH of 7.2 to 7.4 to remove ethanol. After dialyzing at room temperature for 2 to 3 hours, take out the lipid plastid. The measured average particle size is 105.3 nm, the average polydispersity coefficient is 0.159, and the Zeta potential is 33.4 mV.

[0023] 2. Preparation of multifunctional liposome-loaded siRNA delivery system

[0024] Protamine and siRNA were dissolved in citrate buffer (20 mM, pH 4), and the lipid mixture in step 1 was mixed with the protamine solution, vortexed for 30-50s; then the siRNA solution was added dropwise to In the above mixture, vortex for 20-30 seconds. Ethanol and free siRNA were removed by dialysis, and liposomes were taken out after dialysis at room temperature fo...

Embodiment 3

[0026] 1. Preparation of multifunctional liposomes and control liposomes

[0027] Four groups of liposomes were prepared by ethanol dilution method, respectively (1) Multifunctional liposomes (TOLP) double-modified with modified penetrating peptide and transferrin: according to DODMA / OA-R8 / Egg-PC / Chol / DSPE-PEG 2000 = 20 / 25 / 18 / 35 / 2 molar ratio to prepare ethanol solution, after mixing with HEPES buffer, dialyze to remove ethanol. After 2-3 hours of dialysis at room temperature, transferrin-cholesterol and phospholipids were added to the prepared liposomes at a molar ratio of 1:100, and incubated at 37 degrees Celsius for 1 hour to obtain TOLP; (2) Unmodified lipids Plastid (LP): Prepare ethanol solution according to Egg-PC / Chol=1 / 3, inject HEPES buffer solution and vortex for 30-50 seconds to obtain LP; (3) only modified membrane-penetrating peptide OA-R8 Liposome (OLP): Prepare ethanol solution according to OA-R8 / ePC / Chol = 45 / 18 / 35, inject HEPES buffer solution and vortex ...

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Abstract

The invention provides a preparation method used for enhancing multifunctional lipidosome used for enhancing siRNA (Small interfering Ribonucleic Acid) delivery. A solid-phase peptide synthesis methodis adopted, and an amido bond is used for carrying out covalent linkage on R8 and fatty acids of different hydrophobic properties to prepare three types of amphipathic cell-penetrating peptides; polycation protamine is adopted for compressing the siRNA, the siRNA prevents from being degraded by nuclease, and the encapsulation efficiency of the siRNA is improved; then, through an ethyl alcohol dilution method, an inner core is coated in a lipidosome interlayer, the modified cell-penetrating peptide of the interlayer can assist the lipidosome to penetrate through a cell membrane, and conditionally ionized cation phospholipid can assist endosome to escape; and finally, the lipidosome is modified by transferrin and polyethylene glycol, and while the lipidosome can carry out targeting on tumors, circulation time in the blood can be prolonged. The novel multifunctional lipidosome integrates the advantages of various vector systems, and can carry out the in vivo delivery of the siRNA to a tumor position to perform an efficient RNA interference effect.

Description

technical field [0001] The invention discloses a preparation method of a multifunctional liposome for enhancing siRNA delivery, which is a multifunctional liposome and belongs to the technical field of pharmaceutical production. Background technique [0002] With the rise of siRNA research, therapeutic strategies targeting specific genes have become a major research and development hotspot in the international pharmaceutical field. In the process of practical application, the biggest obstacle in gene therapy is the lack of safe and efficient vector system. Currently used vectors are mainly divided into two categories, namely viral vectors and non-viral vectors. Viral vectors are widely used due to their high transduction efficiency and expression efficiency, but they have unavoidable safety problems, such as short expression time of foreign genes, strong immunogenicity, and easy to trigger strong inflammatory reactions and immune responses. reaction, toxicity, etc. In con...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/69A61K47/64A61K31/7088A61K47/42A61P35/00
CPCA61K31/7088A61K47/42A61K47/644A61K47/6911A61P35/00
Inventor 谢晶赵雅蓉张欢毕野
Owner JILIN UNIV
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