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HSP70 promoter-driven activation of gene expression for immunotherapy using gold nanorods and near infrared light.

Andersson HA, Kim YS, O'Neill BE, Shi ZZ, Serda RE - Vaccines (Basel) (2014)

Bottom Line: However, therapy with pro-inflammatory cytokines, such as IL-12, is limited in practice due to adverse systemic effects.Exposure to either 42 °C heat-shock or NIR light induced significant expression of the reporter gene.Overall, we demonstrate a novel opportunity for site-directed, heat-inducible expression of a gene based upon the NIR-absorbing properties of AuNRs and a HSP70 promoter-driven expression vector.

View Article: PubMed Central - PubMed

Affiliation: Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX.

ABSTRACT
Modulation of the cytokine milieu is one approach for vaccine development. However, therapy with pro-inflammatory cytokines, such as IL-12, is limited in practice due to adverse systemic effects. Spatially-restricted gene expression circumvents this problem by enabling localized amplification. Intracellular co-delivery of gold nanorods (AuNR) and a heat shock protein 70 (HSP70) promoter-driven expression vector enables gene expression in response to near infrared (NIR) light. AuNRs absorb the light, convert it into heat and thereby stimulate photothermal expression of the cytokine. As proof-of-concept, human HeLa and murine B16 cancer cells were transfected with a HSP70-Enhanced Green Fluorescent Protein (EGFP) plasmid and polyethylenimine (PEI)-conjugated AuNRs. Exposure to either 42 °C heat-shock or NIR light induced significant expression of the reporter gene. In vivo NIR driven expression of the reporter gene was confirmed at 6 and 24 h in mice bearing B16 melanoma tumors using in vivo imaging and flow-cytometric analysis. Overall, we demonstrate a novel opportunity for site-directed, heat-inducible expression of a gene based upon the NIR-absorbing properties of AuNRs and a HSP70 promoter-driven expression vector.

No MeSH data available.


Related in: MedlinePlus

Transfection of HeLa cells with the HSP70- Enhanced Green Fluorescent Protein (EGFP) expression vector and quantitative measurement of AuNR uptake in vitro. (A) Cartoon of the expression vector; (B,C) HeLa cells were transfected with the vector using lipofectamine LTX reagent, and 24 h later the cells were either left at 37 °C or heat-shocked at 42.5 °C for 30 min. The following day, cells were analyzed for EGFP expression via flow cytometric analysis or fluorescence microscopy (B); B16-luc cells were plated in 96-well plates and AuNR were added at increasing concentrations. After 24 h, the cells were washed and lysed. The number of AuNR in each well was determined using UV/VIS spectroscopy (C); A standard curve was generated by adding known numbers of AuNR to wells, and a graph showing the number of particles present in the cells at 24 h vs. the number added at 0 h is presented.
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vaccines-02-00216-f002: Transfection of HeLa cells with the HSP70- Enhanced Green Fluorescent Protein (EGFP) expression vector and quantitative measurement of AuNR uptake in vitro. (A) Cartoon of the expression vector; (B,C) HeLa cells were transfected with the vector using lipofectamine LTX reagent, and 24 h later the cells were either left at 37 °C or heat-shocked at 42.5 °C for 30 min. The following day, cells were analyzed for EGFP expression via flow cytometric analysis or fluorescence microscopy (B); B16-luc cells were plated in 96-well plates and AuNR were added at increasing concentrations. After 24 h, the cells were washed and lysed. The number of AuNR in each well was determined using UV/VIS spectroscopy (C); A standard curve was generated by adding known numbers of AuNR to wells, and a graph showing the number of particles present in the cells at 24 h vs. the number added at 0 h is presented.

Mentions: As shown in Figure 2, 60% of HSP70-EGFP transfected B16 cells showed an increase in EGFP expression following heat-shock compared to <1% of transfected cells without heat-shock. This negligible activity in the “off” state confirms that gene expression from the HSP70 promoter is tightly regulated. Figure 2C shows high uptake of the PEI-coated AuNPs during incubation, with 20%‑25% of AuNPs in solution ending up in the cells.


HSP70 promoter-driven activation of gene expression for immunotherapy using gold nanorods and near infrared light.

Andersson HA, Kim YS, O'Neill BE, Shi ZZ, Serda RE - Vaccines (Basel) (2014)

Transfection of HeLa cells with the HSP70- Enhanced Green Fluorescent Protein (EGFP) expression vector and quantitative measurement of AuNR uptake in vitro. (A) Cartoon of the expression vector; (B,C) HeLa cells were transfected with the vector using lipofectamine LTX reagent, and 24 h later the cells were either left at 37 °C or heat-shocked at 42.5 °C for 30 min. The following day, cells were analyzed for EGFP expression via flow cytometric analysis or fluorescence microscopy (B); B16-luc cells were plated in 96-well plates and AuNR were added at increasing concentrations. After 24 h, the cells were washed and lysed. The number of AuNR in each well was determined using UV/VIS spectroscopy (C); A standard curve was generated by adding known numbers of AuNR to wells, and a graph showing the number of particles present in the cells at 24 h vs. the number added at 0 h is presented.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199457&req=5

vaccines-02-00216-f002: Transfection of HeLa cells with the HSP70- Enhanced Green Fluorescent Protein (EGFP) expression vector and quantitative measurement of AuNR uptake in vitro. (A) Cartoon of the expression vector; (B,C) HeLa cells were transfected with the vector using lipofectamine LTX reagent, and 24 h later the cells were either left at 37 °C or heat-shocked at 42.5 °C for 30 min. The following day, cells were analyzed for EGFP expression via flow cytometric analysis or fluorescence microscopy (B); B16-luc cells were plated in 96-well plates and AuNR were added at increasing concentrations. After 24 h, the cells were washed and lysed. The number of AuNR in each well was determined using UV/VIS spectroscopy (C); A standard curve was generated by adding known numbers of AuNR to wells, and a graph showing the number of particles present in the cells at 24 h vs. the number added at 0 h is presented.
Mentions: As shown in Figure 2, 60% of HSP70-EGFP transfected B16 cells showed an increase in EGFP expression following heat-shock compared to <1% of transfected cells without heat-shock. This negligible activity in the “off” state confirms that gene expression from the HSP70 promoter is tightly regulated. Figure 2C shows high uptake of the PEI-coated AuNPs during incubation, with 20%‑25% of AuNPs in solution ending up in the cells.

Bottom Line: However, therapy with pro-inflammatory cytokines, such as IL-12, is limited in practice due to adverse systemic effects.Exposure to either 42 °C heat-shock or NIR light induced significant expression of the reporter gene.Overall, we demonstrate a novel opportunity for site-directed, heat-inducible expression of a gene based upon the NIR-absorbing properties of AuNRs and a HSP70 promoter-driven expression vector.

View Article: PubMed Central - PubMed

Affiliation: Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX.

ABSTRACT
Modulation of the cytokine milieu is one approach for vaccine development. However, therapy with pro-inflammatory cytokines, such as IL-12, is limited in practice due to adverse systemic effects. Spatially-restricted gene expression circumvents this problem by enabling localized amplification. Intracellular co-delivery of gold nanorods (AuNR) and a heat shock protein 70 (HSP70) promoter-driven expression vector enables gene expression in response to near infrared (NIR) light. AuNRs absorb the light, convert it into heat and thereby stimulate photothermal expression of the cytokine. As proof-of-concept, human HeLa and murine B16 cancer cells were transfected with a HSP70-Enhanced Green Fluorescent Protein (EGFP) plasmid and polyethylenimine (PEI)-conjugated AuNRs. Exposure to either 42 °C heat-shock or NIR light induced significant expression of the reporter gene. In vivo NIR driven expression of the reporter gene was confirmed at 6 and 24 h in mice bearing B16 melanoma tumors using in vivo imaging and flow-cytometric analysis. Overall, we demonstrate a novel opportunity for site-directed, heat-inducible expression of a gene based upon the NIR-absorbing properties of AuNRs and a HSP70 promoter-driven expression vector.

No MeSH data available.


Related in: MedlinePlus