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Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy
Published on: May 3, 2013
Intelligent Bio-Responsive Fluorescent Au-shRNA Complexes for Regulated Autophagy and Effective Cancer Bioimaging and
Weijuan Cai1, Liang Yin2, Hui Jiang1
1State Key Laboratory of Bioelectronics (Chien-Shiung Wu Lab), School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.
Abstract:
The long non-coding RNA (lncRNA) MALAT1 acts as an oncogene. RNA interference (RNAi) is an effective method to control the expression of specific genes and can be used for the treatment of tumors, but an effective and safe carrier system is a significant obstacle to gene therapy. Herein, we explored the possibility of constructing an in situ bio-responsive self-assembled fluorescent gold-short hairpin RNA nanocomplex (Au-shRNA NCs) delivery system by co-incubating gold and MALAT1-shRNA for precise hepatocellular carcinoma (HCC) imaging and treatment. Due to the characteristics of the cancer microenvironment, Au-shRNA NCs self-assembled in HCC cells (HepG2) but did not occur in control cells (L02) under the same conditions. The in situ bio-responsive self-assembled Au-shRNA NCs delivery system can realize cancer cell bioimaging and promote cell uptake and endosomal escape mechanism, thereby realizing effective transfection. They effectively silenced target gene MALAT1, and with the downregulation of MALAT1, we found that several molecules involved in autophagic flux were also regulated. In vitro and tumor-bearing mouse model experiments demonstrated that the as-prepared fluorescent Au-shRNA NCs can readily realize tumor bioimaging and effectively silence the target gene MALAT1, and those autophagy-related pathway molecules were significantly downregulated, thereby exerting a tumor suppressor efficiency. This raises the possibility of realizing accurate multi-scale bio-imaging from the molecular-level with targeted gene-recognition to cancer cell imaging as well as in vivo tumor tissue imaging for the simultaneous precise cancer therapy.
Insights
This study developed a novel gold-short hairpin RNA nanocomplex for hepatocellular carcinoma therapy. The system precisely targets cancer cells, enabling imaging and effective silencing of the oncogene MALAT1, leading to tumor suppression.
Area of Science:
- Biomedical Engineering
- Nanotechnology
- Cancer Research
Background:
- Long non-coding RNA MALAT1 functions as an oncogene in cancer development.
- RNA interference (RNAi) offers therapeutic potential for tumors, but effective gene delivery remains a challenge.
- Developing safe and efficient carriers for gene therapy is crucial for cancer treatment.
Purpose of the Study:
- To explore the construction of an in situ bio-responsive self-assembled fluorescent gold-short hairpin RNA nanocomplex (Au-shRNA NCs) for hepatocellular carcinoma (HCC) imaging and treatment.
- To investigate the self-assembly, bioimaging, and gene silencing capabilities of Au-shRNA NCs within HCC cells.
- To evaluate the therapeutic efficacy of Au-shRNA NCs in vitro and in vivo models.
Main Methods:
- Co-incubation of gold nanoparticles and MALAT1-shRNA to form Au-shRNA NCs.
- Utilizing the cancer microenvironment for in situ self-assembly of Au-shRNA NCs in HCC cells (HepG2).
- Assessing cell uptake, endosomal escape, gene silencing of MALAT1, and regulation of autophagy-related molecules.
- Conducting in vitro and tumor-bearing mouse model experiments for bioimaging and therapeutic evaluation.
Main Results:
- Au-shRNA NCs demonstrated selective self-assembly within HCC cells, not control cells, triggered by the cancer microenvironment.
- The nanocomplex facilitated cancer cell bioimaging, enhanced cellular uptake, and promoted endosomal escape for effective transfection.
- Effective silencing of MALAT1 was achieved, leading to the downregulation of key molecules in the autophagic flux.
- Fluorescent Au-shRNA NCs enabled accurate tumor bioimaging and demonstrated significant tumor suppressor efficiency by downregulating MALAT1 and autophagy-related pathways.
Conclusions:
- The developed fluorescent Au-shRNA NCs serve as an effective in situ bio-responsive delivery system for precise HCC imaging and therapy.
- This system enables multi-scale bio-imaging from molecular to in vivo tumor levels.
- Targeted silencing of MALAT1 and regulation of autophagy pathways contribute to the observed tumor suppressor effects, offering a promising strategy for simultaneous precise cancer therapy.
