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Intelligent targeting and environmental dual-responsibility siRNA [short interfering RNA (ribonucleic acid)] delivery system for tumor, preparation method and application

A systematic and amphiphilic technology, applied in the fields of polymer chemistry and biomedical engineering, can solve the problems of low release, high siRNA concentration, and waste, and achieve the goal of improving blood circulation time, tumor enrichment, and siRNA interference effect Effect

Active Publication Date: 2016-12-07
SUN YAT SEN UNIV
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Problems solved by technology

However, these obvious disadvantages restrict the further application of this system
First of all, the surface potential reversal characteristics of the complex system have very strict requirements on the ratio of the three components, and the reversal of the surface potential of the complex can only be achieved within a very limited ratio range, which is difficult to control; secondly, PEI, as an siRNA carrier, relies on protons to slow The gene drug is released by action, the process is slow and the final release amount is too low, resulting in a high concentration of siRNA used and serious waste; moreover, the use of 25kDa molecular weight branched PEI, its toxicity problem is not suitable for long-term treatment

Method used

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  • Intelligent targeting and environmental dual-responsibility siRNA [short interfering RNA (ribonucleic acid)] delivery system for tumor, preparation method and application
  • Intelligent targeting and environmental dual-responsibility siRNA [short interfering RNA (ribonucleic acid)] delivery system for tumor, preparation method and application
  • Intelligent targeting and environmental dual-responsibility siRNA [short interfering RNA (ribonucleic acid)] delivery system for tumor, preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1 3

[0039] The synthesis of embodiment 1 triblock polymer

[0040] (1) Synthesis of benzyloxycarbonyl aspartic anhydride (BLA-NCA), the reaction mechanism and process are as follows:

[0041]

[0042] Synthesize β-aspartic acid benzyl ester first. Take a 500mL one-mouth eggplant-shaped bottle, add 100mL anhydrous ether, 12mL H 2 SO 4 (98%) and 120 mL of benzyl alcohol (1.2 mol, 1.04 g / mL, 108.06 g / mol) were stirred and distilled off ether. Under stirring, a total of 16 g of L-aspartic acid (0.12 mol, 133.04 g / mol) was added three times. After stirring and reacting at room temperature for 12 h, 240 mL of 95% ethanol was added to dilute, and then 60 mL of pyridine was added dropwise, and a white precipitate was precipitated. After freezing in the refrigerator overnight, the solid was extracted and washed with pure water. The obtained crude product was recrystallized twice with pure water at 80°C to obtain pure benzyl aspartate.

[0043] Then BLA-NCA was synthesized from ben...

Embodiment 2

[0053] Preparation and characterization of embodiment 2 nanocomposites

[0054] Dissolve the triblock polymer prepared in Example 1 in PBS at pH 5.0, then prepare nanoparticles according to the nanocomposite preparation method, select N / P composites of 18, 24 and 30, and measure their hydraulic diameters and surface potential, the results are as follows image 3 As shown, due to the confinement of subsurface disulfide bonds, the particle size of triblock polymers has no obvious pH dependence, all of which are less than 100 nm. Potential results showed that when the pH value was 5.0, the tertiary amine of the acid-sensitive segment of the polymer was completely protonated, which could well complex the siRNA, and the complex was positively charged; The PDPA segment is hydrophobic and will form a micelle-like structure to wrap siRNA in the micelle core. At this time, the surface of the nanoparticle will be negatively charged due to the presence of siRNA, that is, a stable siRNA ...

Embodiment 3 3

[0055] siRNA Release Research in Example 3 Triblock Polymer

[0056] In order to confirm the sensitive release of siRNA, an in vitro mock release experiment was performed. According to the nanocomposite preparation method, the polymer and FITC-siRNA were prepared at an N / P ratio of 18 to prepare complexes, and incubated under different conditions: (1) the same amount of pure FITC-siRNA; (2) pH 5.0; (3) pH 5.0+10mM GSH; (4) pH 7.4; (5) pH 7.4+5μM GSH; (6) pH7.4+10mM GSH, after 1.5h, the mixture was subjected to gel retardation electrophoresis. The result is as Figure 4, at pH 5.0, even in the presence of a high concentration of reducing agent, there was no release of siRNA (no free bands), indicating that at lysosome pH, siRNA was still protected by polymers to avoid degradation by enzymes; only when the pH The value rose to 7.4, and when the concentration of GHS was higher (10mM), a bright siRNA free band ran out. This experiment well demonstrates that high reducing agent ...

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Abstract

The invention particularly discloses an intelligent targeting and environmental dual-responsibility siRNA [short interfering RNA (ribonucleic acid)] delivery system for tumor, a preparation method and application. The siRNA delivery system is characterized in that siRNA is concentrated and compounded in nanometer particle nucleuses by the aid of acid-sensitive amphiphilic three-block polymers, and intermolecular disulfide bonds are formed by PAsp(MEA) on sub-surfaces, so that the siRNA can be protected, and the intelligent targeting and environmental dual-responsibility siRNA delivery system can respond to release of the siRNA in reductive cytoplasm. The acid-sensitive amphiphilic three-block polymers comprise polyethylene glycol block-intermediate block-acid-sensitive block three-block copolymers, intermediate blocks comprise polyaspartate acyl mercaptoethylamine, and acid-sensitive blocks comprise poly (diisopropyl amine) ethyl methacrylate. The intelligent targeting and environmental dual-responsibility siRNA delivery system, the preparation method and the application have the advantages that the siRNA delivery system can be applied to preparing intelligent targeting siRNA nanometer medicines for the tumor and is low in N / P ratio dependence degree, and the siRNA can be quickly and completely released at targets; a novel idea can be provided for gene delivery systems, and the intelligent targeting and environmental dual-responsibility siRNA delivery system, the preparation method and the application have important significance on preparing clinical diagnosis and treatment medicines for the tumor.

Description

technical field [0001] The invention relates to the fields of polymer chemistry and biomedical engineering, and more specifically, to a tumor intelligent targeting and environment double responsive siRNA delivery system, preparation method and application. Background technique [0002] Malignant tumor, also known as cancer, has become a major disease that threatens human health and life. The prevention, diagnosis and treatment of malignant tumors have always been a hotly concerned issue. In recent years, with the advancement of science and technology and the attention of governments in various countries, although significant progress has been made in this area, various treatment methods have encountered different problems. Difficult to achieve high efficiency, low side effects and high specificity at the same time. [0003] In recent years, RNA interference has provided an important reference method for tumor therapy because of its specific targeting, and has shown efficien...

Claims

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Application Information

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IPC IPC(8): C08G81/02C08G69/16C08F220/34A61K48/00A61K31/713A61K47/34A61P35/00
CPCA61K47/34A61K31/713C08F220/34C08G69/16C08G81/028
Inventor 帅心涛王勇吴腾苏振伟李博程度
Owner SUN YAT SEN UNIV
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