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Research Spotlight | How to target tumor cells


The team of Liu Yang and Shi Linqi from Nankai University and Kang Chunsheng from Tianjin Medical University recently proposed a type of double locked nanoparticles (DLNP) which can limit the activation of CRISPR / Cas13a into tumor tissues. By replacing specific cRNA targeted genes, DLNP will become a potential universal platform for safe and effective cancer immunotherapy.

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Fundamental Information:

Title: Dual-Locking Nanoparticles Disrupt the PD-1/PD-L1 Pathway for Efficient Cancer Immunotherapy

Journal: Advanced Materials

IF27.398 (2019)

Institution of the first author: Nankai University, Tianjin, China

Institution of the corresponding author: Nankai University, Tianjin, China

Tianjin Medical University, Tianjin, China

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Mouse TGF-β1


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Mouse IFN-γ


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Mouse TNF-α


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Mouse IL-12


Tissue homogenate

Background of the Research:

Genetic mutations due to cancer leads to abnormal metabolism and proliferation of cells. Researchers found that Cas13a is a CRISPR effector targeting RNA. Cas13a, holds great promise in cancer treatment due to its potential for selective destruction of tumor cells via collateral effects after target recognition. However, this "collateral effect" is not specific to tumor cells. It can be activated when the RNA of any cell is binds to the Cas13a / crRNA complex. For example systemic administration of drugs based on the Cas13a / PD-L1 system, may lead to the safety problems of unexpected cell death in tissues other than tumor. Thus, the practicable approach which could limit the activation of CRISPR / Cas13a system in tumor cells is necessary to safely apply CRISPR / Cas13a based drugs in cancer treatment.

In order to accurately control the activation of CRISPR / Cas13a system in vivo, it is very important to deliver CRISPR / Cas13a based drugs to tumor tissues.

Tumor microenvironment (TME), especially acidic microenvironment (pHe), is one of the typical characteristics of solid tumors. However, acid accumulation from acidic organelles or other external sources can also activate CRISPR / Cas13a resulting in potential safety issues and serious side effects. The excessive release of reactive oxygen species (ROS), especially hydrogen peroxide (H2O2), is another trait of cancer which initiates tumorigenesis affects DNA mutation and tumor development. The combination of pHe and H2O2 can effectively distinguish tumor from normal tissue. Therefore, it may be an ideal carrier to control the activation of CRISPR / Cas13a.

Dual locking nanoparticle DLNP has a core–shell structure, in which the CRISPR/Cas13a system (plasmid DNA, pDNA) is encapsulated inside the core with a dual-responsive polymer layer. This polymer layer endows the DLNP with enhanced stability during blood circulation or in normal tissues and facilitates cellular internalization of the CRISPR/Cas13a system and activation of gene editing upon entry into tumor tissue.

Experimental Design:

Schematic illustration of the synthesis of dual-locking nanoparticles (DLNP).

To develop a dual locking nanoparticle (DLNP) that can limit CRISPR / Cas13a activation in tumor tissue, and verify its pH and H2O2 dual response.

The crRNA targeting PD-L1 gene was selected as the model crRNA to verify the anti-tumor mechanism and effectiveness of DLNP, H2O2-NP and pH NP as CRISPR / Cas13a release platform in vitro and in vivo.

Research Findings:

1. A dual-locking nanoparticle was developed capable of restricting CRISPR/Cas13a activation in tumor tissues.

2. On injecting intravenously, with the coating of PEG-based polymer shell, a stable superior blood circulation was showed by this DLNP.

3. Microenvironment with low pHe and high concentration of H2O2 are suitable for the release of CRISPR/Cas13a system by DNLP. This trait of DNLP is beneficial for ameliorating accumulation of the CRISPR/Cas13a system, boosting its gene-editing efficiency at tumor sites, and reduction of side effects caused by undesired activations of CRISPR/Cas13a in normal tissues. CRISPR/Cas13a system can be applied in cancer immunotherapy by precisely controlling activation of CRISPR/Cas13a by DNLP.

4. Tumor suppression was enhanced in B16F10-bearing mice and their survival rate was improved with systemic administration of the DLNP/Cas13a/PD-L1.

5. This study demonstrated a safe and efficient method for precisely controlling CRISPR/Cas13a system activation to suppress tumor growth.


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