Preparation method and application of a double-locked nanoparticle that can restrict 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, and achieve the effects of reducing toxic and side effects, easy to popularize and apply, and excellent in blood circulation stability.

Active Publication Date: 2022-07-26
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 a double-locked nanoparticle that can restrict activation of CRISPR/Cas13a
  • Preparation method and application of a double-locked nanoparticle that can restrict activation of CRISPR/Cas13a
  • Preparation method and application of a double-locked nanoparticle that can restrict activation of CRISPR/Cas13a

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Embodiment 1

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

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

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

[0038] pre-prepared PEI 1.8k -HPBA 2.0 Dissolved in 10 mM phosphate buffered saline (PBS) to prepare a 1 mg / ml solution, the plasmid DNA (pDNA) encoding the CRISPR / Cas13a system was mi...

Embodiment 2

[0053] Example 2: Observation of double-locked nanoparticles in pHe / H 2 O 2 Dual responsiveness applications.

[0054] To better evaluate the pHe / H of DLNP 2 O 2 Dual-responsive, two types of contrasting nanoparticles were prepared following a similar approach to DLNP, i.e. non-pH but H 2 O 2 Responsive nanoparticles (denoted as H 2 O 2 -NP) and non-H 2 O 2 But pH-responsive nanoparticles (denoted as pH-NPs).

[0055] For zeta potential analysis, first pDNA, PEI 1.8k -HPBA / pDNA, DLNP, H 2 O 2 -NP and pH-NP (50 μg / mL pDNA, 2 mL) 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 culture broth was then exchanged with PBS (10 mM, 7.4) by centrifugal ultrafiltration, followed by zeta potential testing.

[0056] For nonspecific protein adsorption analysis, first pDNA, PEI 1.8k -HPBA / pDNA, DLNP, H 2 O 2 -NP and pH-NP (50 μg / mL pDNA, 2 mL) at 37°C under different conditions (pH 7.4, pH 7.4 / H 2 O 2 , pH 6.8, pH 6....

Embodiment 3

[0060] Example 3: Observation of the application of double-locked nanoparticles in cellular uptake and gene transfection

[0061] For CLSM observations, U87 cells were grown at 1 × 10 per well prior to co-culture with nanoparticles 5 A density of individual cells was seeded in 35 mm confocal dishes (Φ=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 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 rhodamine phalloidin for cellular actin according to the manufacturer's protocol. CLSM images were taken on an Olympus CLSM with a ×60 objective.

[0062] For FACS-based assays, U87 cells were grown at 1 × 10 per well prior to co-culture with nanoparticles 5 1 c...

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Abstract

A preparation method and application of a double-locked nanoparticle that can restrict the activation of CRISPR / Cas13a, this nanoparticle (DLNP) can only restrict the activation of CRISPR / Cas13a in tumor tissue for effective cancer immunotherapy. DLNPs are designed as core-shell structures, in which CRISPR / Cas13a is encapsulated in the core and covered by a layer of dual-responsive polymer network. Under the protection of polyethylene glycol shell, DLNP exhibited blood circulation stability. at low pH and high H 2 O 2 In the tumor microenvironment, DLNP selectively releases CRISPR / Cas13a. Using this double-locked nanoparticles, potent tumor growth inhibition was observed in mice with no apparent side effects. Since the clinical application of CRISPR / Cas13a is mainly limited by the toxic side effects caused by uncontrollable activation, this double-locked nanoparticle provides a feasible method for precisely controlling the activation of CRISPR / Cas13a and its application in effective cancer therapy.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to a preparation method and application of double-locked nanoparticles capable of restricting activation of CRISPR / Cas13a. Background technique [0002] Cancer is characterized by abnormal metabolism and proliferation caused by multiple genetic alterations or mutations. Gene mutations in tumor cells during drug metabolism or repair of drug targets can lead to cell resistance to conventional therapies. Recently, a novel clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated enzyme (Cas), Cas13a (formerly C2c2), was identified as an RNA-guided RNA-targeting CRISPR effector. The Cas13a / CRISPR RNA (crRNA) complex also activates general RNase activity after targeting the RNA of interest, resulting in nonspecific cleavage of cellular RNAs and ultimately programmed cell death or dormancy (i.e., "collateral effects"). This unique "collateral effect" automatically byp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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