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Biodegradable nanosyringes for intracellular amplification-based dual-diagnosis and gene therapy in single living cells.

ABSTRACT: The efficient delivery of biomolecules into living cells as well as their easy biodegradation have been challenges for the application of intracellular amplification for sensitive multiple-diagnosis and gene therapy for cancer. Herein, new strategies of amplification-based dual-detection of cancer biomarkers (Let-7a miRNA and VEGF) and gene therapy for cancers are put forward. These are achieved through biodegradable nanosyringes (NSs), rigid and sharp in vitro but degradable in vivo, which are applied for the efficient loading, delivery and release of biomolecules (enzymes, nucleic acids, and even silencing RNA) into living cells. After penetrating cell membranes and escaping from endosomes through their rigid and sharp tips, NSs release biomolecules for fast and easy "one-step" rolling circle amplification (ring formation and amplification) in single living cells. Therefore, based on signals from two probes, FAM-Probe and Cy5-Probe, that selectively bind to amplification products, 100 aM of Let-7a and 100 fM of VEGF could be detected, which are much lower than reported values. Furthermore, siRNAs can also be delivered by NSs for gene therapy, and their therapeutic effect was evaluated by their in vivo antitumor efficacy in CCRF-CEM subcutaneous xenograft nude mice. Rigid in vitro and degradable in vivo, NSs show potential for achieving fast, sensitive and safe cancer diagnosis and efficient therapy.

SUBMITTER: Shen X

PROVIDER: S-EPMC6585592 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Biodegradable nanosyringes for intracellular amplification-based dual-diagnosis and gene therapy in single living cells.

Shen Xiaotong X Zhang Ying Y Sun Jianghui J Lu Hua H Ouyang Jin J Na Na N

Chemical science 20190506 24

The efficient delivery of biomolecules into living cells as well as their easy biodegradation have been challenges for the application of intracellular amplification for sensitive multiple-diagnosis and gene therapy for cancer. Herein, new strategies of amplification-based dual-detection of cancer biomarkers (Let-7a miRNA and VEGF) and gene therapy for cancers are put forward. These are achieved through biodegradable nanosyringes (NSs), rigid and sharp <i>in vitro</i> but degradable <i>in vivo</ ...[more]

PMID: 31360417

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Project description:Non-destructive analysis of cells at the molecular level is of critical importance for cell research. At present, immunoassay-based and aptamer-based methods can achieve non-structural destructive cell analysis, but still lead to changes in cells at the molecular level. Here, we have proposed a dual-terminal amplification (DTA) strategy, which enables nondestructive analysis of membrane protein MUC1 without the effect on protein expression and cell viability in living cells. Methods: A fluorophore (Cy5)-labeled DNA ternary complex consisting of three oligonucleotides is designed. It can recognize MUC1 through its aptamer region, and thus make the MUC1 of cells visible under a fluorescence microscope. When DNA polymerase is added, dual-terminal amplification is performed. One direction dissociates aptamer from MUC1, and the other direction, also known as rolling circle amplification (RCA), produces long linear DNA strands, which can be further adopted for quantitative analysis of MUC1. In this way, all reagents are removed from the surface of the cells after the analysis, which allows nondestructive analysis. We named this strategy dual-terminal amplification (DTA) analysis. Results: By using the DTA analysis, both in situ fluorescence imaging analysis and ex situ fluorescence quantitative analysis of MUC1 were achieved. In addition, the aptamer-containing DNA ternary complex stays on cell surface only during the analysis and leaves the cell after the analysis is complete. The cells can be maintained in a non-interfering state for the rest of the time. So after the analysis, it is found that there are no effect on the physiological activity of cells and the expression of target protein even after two rounds of repeatable imaging and quantitative analysis. Conclusion: In summary, we have successfully constructed a strategy for nondestructive analysis of membrane protein in living cells. We believe that this method provides a promising way for the analysis of the key membrane proteins of cells and the versatile utilization of precious cell samples.

Dataset Information

Biodegradable nanosyringes for intracellular amplification-based dual-diagnosis and gene therapy in single living cells.

Publications

Biodegradable nanosyringes for intracellular amplification-based dual-diagnosis and gene therapy in single living cells.

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