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A polylipopeptide vesicle with a positively charged inner membrane and its preparation method and application

A polyester and vesicle technology, applied in the fields of polymer materials and applied chemistry, can solve the problems of poor stability and easy dissociation of nanometer systems, and achieve the effects of good stability, high encapsulation capacity and small size.

Active Publication Date: 2022-02-25
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, several polymer siRNA composite nanoparticles have entered the clinical research stage, but these nanosystems have poor stability and are prone to dissociation in vivo.

Method used

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  • A polylipopeptide vesicle with a positively charged inner membrane and its preparation method and application
  • A polylipopeptide vesicle with a positively charged inner membrane and its preparation method and application
  • A polylipopeptide vesicle with a positively charged inner membrane and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: Synthesis of polyethylene glycol-b-poly(2-aminohexadecanoic acid)-b-poly(L-lysine) (PEG-b-PAPA-b-PLL) triblock copolymer

[0042] The synthesis of PEG-b-PAPA-b-PLL mainly includes two steps. First, under nitrogen, in DMF solution, with PEG-NH 2 Sequential initiation of 2-aminohexadecanoic acid N-carboxylate anhydride monomer (APA-NCA) and ε-benzyloxycarbonyl-L-lysine N-carboxylate anhydride monomer (ZLL-NCA) for macroinitiators PEG-b-PAPA-b-PZLL triblock copolymer was prepared by ring-opening polymerization. The specific synthesis steps are as follows: under a nitrogen atmosphere, a DMF solution of 15.0 mL of APA-NCA (1.04 g, 3.52 mmol) was rapidly added to PEG-NH 2 (0.4g, 0.08mmol) in DMF (4.0mL); after reacting at 35°C for 72 hours, the second monomer ZLL-NCA (0.44g, 1.44mmol) was added, and the reaction was continued for 72 hours. After the reaction, the reaction solution was precipitated in 20 times excess glacial ether for several times, and finall...

Embodiment 2

[0045] Embodiment 2: the synthesis of CPP33-PEG-b-PAPA copolymer

[0046] The preparation of CPP33-PEG-b-PAPA polymer was divided into two steps. First, with Mal-PEG-NH 2 As a macromolecular initiator, APA-NCA ring-opening polymerization is initiated to prepare Mal-PEG-b-PAPA; from its nuclear magnetic spectrum ( image 3 ), its molecular weight can be calculated to be 6.0-11.0kg / mol; subsequently, CPP33-PEG-b-PAPA is prepared by reacting the sulfhydryl group on CPP33 with Mal. From the NMR spectrum of CPP33-PEG-b-PAPA ( Figure 4 ), the characteristic peak of CPP33 (δ6.97) can be found, and the grafting rate of CPP33 obtained by measuring 9,10-phenanthrenequinone is 87%.

[0047] Table 1. Characterization of polymers

[0048]

[0049] a Depend on 1 Calculated by H NMR.

Embodiment 3

[0050] Example 3: Preparation of polylipopeptide vesicles (siPLK1-CPP33-CLP) with asymmetric membrane structure loaded with siRNA

[0051] siRNA-loaded, CPP33-modified vesicles (CPP33-CLP) were prepared by a solvent displacement method. The brief steps are as follows: mix 0.1 mL of a mixed solution containing 20 mol.% CPP33-PEG-b-PAPA and 80 mol.% mPEG-b-PAPA-b-PLL with a concentration of 5 mg / mL and 0.1 mL of siRNA solution, Then it was added dropwise into 0.8 mL of HEPES buffer medium (5 mM, pH 6.8), and then dialyzed in HEPES medium using a dialysis bag with a molecular weight cut-off of 350k to remove unloaded siRNA. The loading capacity (SLC) and loading efficiency (SLE) of siRNA can be measured by NanoDrop UV spectrophotometer. SLC and SLE can be calculated by the following formulas:

[0052] SLC (wt.%)=(mass of siRNA loaded in vesicles / sum of polymer and siRNA mass)×100

[0053] SLE(%)=(mass of siRNA loaded in vesicles / mass of initially added siRNA)×100

[0054] Dyn...

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Abstract

The invention discloses a polylipopeptide vesicle whose inner membrane is positively charged, a preparation method and application thereof, and belongs to the technical field of polymer materials and applied chemistry. First of all, the present invention simply prepares PEG-b-PAPA-b-PLL triblock copolymers through NCA ring-opening polymerization and deprotection to prepare polylipopeptide vesicles (CLP) with an asymmetric membrane structure whose inner membrane is positively charged. ); and through the electrostatic interaction between siRNA and PLL, the siRNA can be efficiently loaded into the hydrophilic inner cavity of the vesicle, and the strong wall membrane of the vesicle can prevent the siRNA from being degraded; the lung cancer cell-selective membrane-penetrating peptide CPP33 modified with siPLK1 The vesicle (CPP33‑CLP) has good targeting ability to A549 lung cancer cells. At the same time, CPP33 can help siRNA quickly escape from endosomes and enter the cytoplasm, resulting in a significant sequence-specific gene silencing effect. Shows significantly enhanced tumor suppressive effect in vivo. This simple and stable multifunctional nanovesicle platform shows great potential in cancer gene therapy.

Description

technical field [0001] The invention relates to a polylipopeptide vesicle whose inner membrane is positively charged, a preparation method and application thereof, and belongs to the technical field of polymer materials and applied chemistry. Background technique [0002] RNA interference therapy mediated by small interfering RNA (siRNA) has shown great potential in the treatment of various diseases including cancer. However, siRNA has disadvantages such as short circulation time in vivo, easy to be degraded by enzymes, poor cell permeability and endosome escape performance. Therefore, how to safely and efficiently deliver siRNA to the lesion site is the biggest obstacle and challenge for the clinical transformation of RNA interference, a gene therapy. To address this challenge, scientists have developed various nanocarrier systems for siRNA delivery. At present, several polymer siRNA composite nanoparticles have entered the clinical research stage, but these nanosystems h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08G69/14C08G69/16C08G69/48A61K47/34A61K9/127A61K48/00A61P35/00
CPCA61K47/34A61K48/0041A61P35/00A61K9/1273C08G69/14C08G69/16C08G69/48
Inventor 邓超邱敏钟志远
Owner SUZHOU UNIV
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