Preparation method and application of double-lock nanoparticles with limited activation of CRISPR/Cas13a

A nanoparticle and double-lock technology, applied in the field of biomedicine, can solve problems such as toxic and side effects

Active Publication Date: 2020-01-31
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve the problem of serious side effects associated with the clinical application of the CRISPR / Cas13a system, and to provide a preparation method and application of double-locked nanoparticles (DLNP) that can limit the activation of the CRISPR / Cas13a system

Method used

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  • Preparation method and application of double-lock nanoparticles with limited activation of CRISPR/Cas13a
  • Preparation method and application of double-lock nanoparticles with limited activation of CRISPR/Cas13a
  • Preparation method and application of double-lock nanoparticles with limited activation of CRISPR/Cas13a

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Experimental program
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Embodiment 1

[0035] attached figure 1 A schematic diagram of the function of the double-lock nanoparticles of the present invention is shown, and the particle diameter of the double-lock nanoparticles prepared by the method of the present invention is 128.9±13.93nm. DLNP can maintain excellent blood circulation stability in blood. DLNP can only be produced in the presence of low pH and high H 2 o 2 The release of CRISPR / Cas13a system in the tumor microenvironment of high concentration can significantly reduce the toxic and side effects of CRISPR / Cas13a system.

[0036] See attached figure 1 , the present invention restricts the preparation method of the double-lock nanoparticle that activates CRISPR / Cas13a system, comprises the following steps:

[0037] 1) PEI 1.8k - synthesis of HPBA / pDNA,

[0038] Pre-prepared PEI 1.8k -HPBA 2.0 Dissolve in 10mM phosphate buffered saline (PBS) to configure a 1mg / ml solution, and mix the plasmid DNA (pDNA) encoding the CRISPR / Cas13a system with PE...

Embodiment 2

[0053] Embodiment 2: Observation of double-lock nanoparticles at pHe / H 2 o 2 Application of dual responsiveness.

[0054] In order to better evaluate the pHe / H of DLNP 2 o 2 Dual-responsiveness, two types of comparative nanoparticles, namely non-pH but H 2 o 2 Response to nanoparticles (denoted as H 2 o 2 -NP) and non-H 2 o 2 But pH-responsive nanoparticles (denoted as pH-NP).

[0055] For zeta potential analysis, first pDNA, PEI 1.8k - HPBA / pDNA, DLNP, H 2 o 2 -NP and pH-NP (50μg / mL pDNA, 2mL) at 37°C under different conditions (pH 7.4, pH 7.4 / H 2 o 2 , pH 6.8, pH 6.8 / H 2 o 2 ) for 8 hours. The medium was then exchanged with PBS (10 mM, 7.4) by centrifugal ultrafiltration, followed by zeta potential testing.

[0056] For non-specific protein adsorption analysis, first pDNA, PEI 1.8k - HPBA / pDNA, DLNP, H 2 o 2 -NP and pH-NP (50μg / mL pDNA, 2mL) in different conditions (pH 7.4, pH 7.4 / H 2 o 2 , pH 6.8, pH 6.8 / H 2 o 2 ) for 8 hours. The culture medium was t...

Embodiment 3

[0060] Example 3: Observing the application of double-lock nanoparticles in cell uptake and gene transfection

[0061] For CLSM observations, U87 cells were incubated at 1 × 10 per well prior to co-culture with nanoparticles 5 Cells were seeded at a density of 35 mm confocal dishes (Ф = 15 mm) and incubated for one day. Then the same amount of YOYO-1 labeled DLNP, H 2 o 2 -NP and pH-NP (1 μg pDNA) were added to the cells and incubated in complete medium under different conditions (pH 7.4, pH 7.4 / H 2 o 2 , pH 6.8, pH 6.8 / H 2 o 2 ) for 2 hours. Subsequently, cells were rinsed with ice-cold PBS and fixed with fresh 4% paraformaldehyde for 15 min at room temperature. Nuclei were further stained with DAPI and cellular actin was stained with rhodamine phalloidin according to the manufacturer's protocol. CLSM images were captured on an Olympus CLSM with a ×60 objective.

[0062] For FACS-based assays, U87 cells were incubated at 1 × 10 per well prior to co-cultivation with n...

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Abstract

The invention discloses a preparation method and application of double-lock nanoparticles with limited activation of CRISPR / Cas13a. Activation of CRISPR / Cas13a can be limited by the nanoparticles (DLNP) only in tumor tissue so that effective cancer immunotherapy is carried out. The DLNP is designed into a core-shell structure, wherein CRISPR / Cas13a is encapsulated in the core, and is covered witha dual-response polymer network layer. Under the protection action of a polyethylene glycol shell, the DLNP exhibits blood circulation stability, and CRISPR / Cas13a is released selectively by the DLNPin a tumor microenvironment of low pH values and high content of H2O2; and an effective inhibition effect on tumor growth of mice is observed without significant side effects through the double-lock nanoparticles. Because the clinical application of CRISPR / Cas13a is mainly restricted by toxic and side effects which are caused by uncontrolled activation of CRISPR / Cas13a, and a feasible method is provided for precise control on activation of CRISPR / Cas13a and application of CRISPR / Cas13a in effective cancer treatment through the double-lock nanoparticles.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to a preparation method and application of a double-lock nanoparticle capable of restricting and activating CRISPR / Cas13a. Background technique [0002] Cancer is characterized by abnormal metabolism and proliferation resulting from multiple genetic alterations or mutations. Genetic mutations in tumor cells during drug metabolism or repair of drug targets can cause cell resistance to traditional therapies. Recently, a novel clustered regularly interspaced short palindromic repeat (CRISPR) / CRISPR-associated enzyme (Cas), Cas13a (formerly known as C2c2), was identified as an RNA-guided, RNA-targeting CRISPR effector. The Cas13a / CRISPR RNA (crRNA) complex also activates general RNase activity after targeting RNA, leading to nonspecific cleavage of cellular RNA and ultimately programmed cell death or dormancy (i.e., “collateral effects”). This unique "side effect" automatically bypasses the c...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61P35/00A61P37/04B82Y5/00B82Y30/00C12N15/113
CPCA61K48/0041B82Y5/00B82Y30/00A61P35/00A61P37/04C12N15/113C12N2310/20
Inventor 陈红云康春生张展展刘阳
Owner NANKAI UNIV
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