Plasmid Transfection in Mammalian Cells Spatiotemporally Tracked by a Gold Nanoparticle

Recent advances in cell transfection have suggested that delivery of a gene on a gold nanoparticle (AuNP) can enhance transfection efficiency. The mechanism of transfection is poorly understood, particularly when the gene is appended to a AuNP, as expression of the desired exogenous protein is dependent not only on the efficiency of the gene being taken into the cell but also on efficient endosomal escape and cellular processing of the nucleic acid. Design of a multicolor surface energy transfer (McSET) molecular beacon by independently dye labeling a linearized plasmid and short duplex DNA (sdDNA) appended to a AuNP allows spatiotemporal profiling of the transfection events, providing insight into package uptake, disassembly, and final plasmid expression. Delivery of the AuNP construct encapsulated in Lipofectamine2000 is monitored in Chinese hamster ovary cells using live-cell confocal microscopy. The McSET beacon signals the location and timing of the AuNP release and endosomal escape events for the plasmid and the sdDNA discretely, which are correlated with plasmid transcription by fluorescent protein expression within the cell. It is observed that delivery of the construct leads to endosomal release of the plasmid and sdDNA from the AuNP surface at different rates, prior to endosomal escape. Slow cytosolic diffusion of the nucleic acids is believed to be the limiting step for transfection, impacting the time-dependent expression of protein. The overall protein expression yield is enhanced when delivered on a AuNP, possibly due to better endosomal escape or lower degradation prior to endosomal escape

Plasmid Transfection in Mammalian Cells Spatiotemporally Tracked by a Gold Nanoparticle

Recent advances in cell transfection have suggested that delivery of a gene on a gold nanoparticle (AuNP) can enhance transfection efficiency. The mechanism of transfection is poorly understood, particularly when the gene is appended to a AuNP, as expression of the desired exogenous protein is dependent not only on the efficiency of the gene being taken into the cell but also on efficient endosomal escape and cellular processing of the nucleic acid. Design of a multicolor surface energy transfer (McSET) molecular beacon by independently dye labeling a linearized plasmid and short duplex DNA (sdDNA) appended to a AuNP allows spatiotemporal profiling of the transfection events, providing insight into package uptake, disassembly, and final plasmid expression. Delivery of the AuNP construct encapsulated in Lipofectamine2000 is monitored in Chinese hamster ovary cells using live-cell confocal microscopy. The McSET beacon signals the location and timing of the AuNP release and endosomal escape events for the plasmid and the sdDNA discretely, which are correlated with plasmid transcription by fluorescent protein expression within the cell. It is observed that delivery of the construct leads to endosomal release of the plasmid and sdDNA from the AuNP surface at different rates, prior to endosomal escape. Slow cytosolic diffusion of the nucleic acids is believed to be the limiting step for transfection, impacting the time-dependent expression of protein. The overall protein expression yield is enhanced when delivered on a AuNP, possibly due to better endosomal escape or lower degradation prior to endosomal escape

Determining the Cytosolic Stability of Small DNA Nanostructures <i>In Cellula</i>

DNA nanostructures have proven potential in biomedicine. However, their intracellular interactionsespecially cytosolic stabilityremain mostly unknown and attempts to discern this are confounded by the complexities of endocytic uptake and entrapment. Here, we bypass the endocytic uptake and evaluate the DNA structural stability directly in live cells. Commonly used DNA structurescrosshairs and a tetrahedronwere labeled with a multistep Förster resonance energy transfer dye cascade and microinjected into the cytosol of transformed and primary cells. Energy transfer loss, as monitored by fluorescence microscopy, reported the structure’s direct time-resolved breakdown in cellula. The results showed rapid degradation of the DNA crosshair within 20 min, while the tetrahedron remained consistently intact for at least 1 h postinjection. Nuclease assays in conjunction with a current understanding of the tetrahedron’s torsional rigidity confirmed its higher stability. Such studies can inform design parameters for future DNA nanostructures where programmable degradation rates may be required

Cell-Penetrating Peptide-Modified Gold Nanoparticles for the Delivery of Doxorubicin to Brain Metastatic Breast Cancer

As therapies continue to increase the lifespan of patients with breast cancer, the incidence of brain metastases has steadily increased, affecting a significant number of patients with metastatic disease. However, a major barrier toward treating these lesions is the inability of therapeutics to penetrate into the central nervous system and accumulate within intracranial tumor sites. In this study, we designed a cell-penetrating gold nanoparticle platform to increase drug delivery to brain metastatic breast cancer cells. TAT peptide-modified gold nanoparticles carrying doxorubicin led to improved cytotoxicity toward two brain metastatic breast cancer cell lines with a decrease in the IC50 of at least 80% compared to free drug. Intravenous administration of these particles led to extensive accumulation of particles throughout diffuse intracranial metastatic microsatellites with cleaved caspase-3 activity corresponding to tumor foci. Furthermore, intratumoral administration of these particles improved survival in an intracranial MDA-MB-231-Br xenograft mouse model. Our results demonstrate the promising application of gold nanoparticles for improving drug delivery in the context of brain metastatic breast cancer

A: MTT assay of HB1.F3.CD cells. 10, 20, and 50 SD particles per cell were added into HB1.F3.CD cells for 24 hours. MF treatment consists of applying a 1T field rotating at 20 Hz for 30 min. B: Representative optical microscopy images of HB1.F3.CD cells before and after MF treatment.

<p>The top panel contains SD and the bottom panels are control without SD.</p

7-AAD staining of neural stem cells.

<p>The cells are incubated with 50 SD/cell for 24h and then receive or not the MF treatment for 30sec. The SD alone do not cause damage to the cells, only when exposed to a MF.</p

SD do not impair NSC migratory function.

<p>Cells are plated in a migration chamber for 24h before removal of the barrier. The control NSC and the NSC loaded with 50SD/cell demonstrate no difference in migration ability.</p

A: Confocal microscopy images of U87 glioma cells uptake of released SD particles (50 SD/NSC). The U87 cells (green) are able to effectively internalize particles that have been released by the NSCs. NSCs (grey) are visibly damaged. B: U87 survival rate determined by luciferase. C: Timeline of the experiment.

<p>A: Confocal microscopy images of U87 glioma cells uptake of released SD particles (50 SD/NSC). The U87 cells (green) are able to effectively internalize particles that have been released by the NSCs. NSCs (grey) are visibly damaged. B: U87 survival rate determined by luciferase. C: Timeline of the experiment.</p

SD uptake and MF effect on glioma cells, U87.

<p>The cells are treated with 50SD/cell and allowed to incubate for 24h. Afterwards, the cells are exposed to MF treatment for 30 seconds and then stained with 7-AAD. The cells that receive SD + no MF do not have compromised membranes. The cells with SD and MF stain red.</p