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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

Cell viability and GFP expression after in vitro NIR treatment. (A) B16-Luc cells (1 × 105 per well in 24-well glass bottom plates) were incubated with HSP70-GFP and 1010, 1011, or 1012 AuNR per well. The following day, the cells were treated with NIR laser with 10 pulses at 0, 25, 50, or 75 J/cm2 (B) B16-Luc cells were incubated overnight with or without 1011 AuNR per well and treated with NIR laser with 10 or 20 pulses. Cells were analyzed for GFP expression and viability by flow cytometery the following day; (C) Mean GFP fluorescence intensity (MFI) and percent viable cells (measured by uptake of propidium iodide) as a function of laser fluence (25, 50 and 75 J/cm2; left), time after NIR treatment (6 h or 24 h; left) and pulse duration (30, 100, or 400 ms; right).
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vaccines-02-00216-f003: Cell viability and GFP expression after in vitro NIR treatment. (A) B16-Luc cells (1 × 105 per well in 24-well glass bottom plates) were incubated with HSP70-GFP and 1010, 1011, or 1012 AuNR per well. The following day, the cells were treated with NIR laser with 10 pulses at 0, 25, 50, or 75 J/cm2 (B) B16-Luc cells were incubated overnight with or without 1011 AuNR per well and treated with NIR laser with 10 or 20 pulses. Cells were analyzed for GFP expression and viability by flow cytometery the following day; (C) Mean GFP fluorescence intensity (MFI) and percent viable cells (measured by uptake of propidium iodide) as a function of laser fluence (25, 50 and 75 J/cm2; left), time after NIR treatment (6 h or 24 h; left) and pulse duration (30, 100, or 400 ms; right).

Mentions: The results of the in vitro NIR optimization are shown in Figure 3. We found that the optimal dose to reach the highest level of EGFP expression with the lowest cell death/toxicity is achieved by using approximately 1011 AuNR and 10 pulses at 50 J/cm2, with a 30 ms pulse length. In general, cells are driven to increase production of EGFP with increased heating up to some threshold, at which point cell damage rapidly reduces expression and eventually viability. Heating efficiency increases as expected with increased loading of AuNPs (Figure 3A) and pulse numbers (Figure 3B). At a laser fluence of 25 J/cm2 relatively little gene expression occurs, with increases in fluence to 50 J/cm2 inducing increased expression and relatively high viability (approximately 80%). While gene expression was highest at 75 J/cm2, cell viability dropped to 44% and 37% at 6 h and 24 h, respectively. Increasing the length of the pulses from 30 ms to 400 ms actually decreases the GFP expression (Figure 3C; right), demonstrating that peak power is more important than total power. In Figure 3C, the peak power is decreasing (the same energy, 50 J, is spread over a longer pulse), even though the total power is the same. In general, some loss of viability appears as a necessary price for high expression.


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)

Cell viability and GFP expression after in vitro NIR treatment. (A) B16-Luc cells (1 × 105 per well in 24-well glass bottom plates) were incubated with HSP70-GFP and 1010, 1011, or 1012 AuNR per well. The following day, the cells were treated with NIR laser with 10 pulses at 0, 25, 50, or 75 J/cm2 (B) B16-Luc cells were incubated overnight with or without 1011 AuNR per well and treated with NIR laser with 10 or 20 pulses. Cells were analyzed for GFP expression and viability by flow cytometery the following day; (C) Mean GFP fluorescence intensity (MFI) and percent viable cells (measured by uptake of propidium iodide) as a function of laser fluence (25, 50 and 75 J/cm2; left), time after NIR treatment (6 h or 24 h; left) and pulse duration (30, 100, or 400 ms; right).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
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vaccines-02-00216-f003: Cell viability and GFP expression after in vitro NIR treatment. (A) B16-Luc cells (1 × 105 per well in 24-well glass bottom plates) were incubated with HSP70-GFP and 1010, 1011, or 1012 AuNR per well. The following day, the cells were treated with NIR laser with 10 pulses at 0, 25, 50, or 75 J/cm2 (B) B16-Luc cells were incubated overnight with or without 1011 AuNR per well and treated with NIR laser with 10 or 20 pulses. Cells were analyzed for GFP expression and viability by flow cytometery the following day; (C) Mean GFP fluorescence intensity (MFI) and percent viable cells (measured by uptake of propidium iodide) as a function of laser fluence (25, 50 and 75 J/cm2; left), time after NIR treatment (6 h or 24 h; left) and pulse duration (30, 100, or 400 ms; right).
Mentions: The results of the in vitro NIR optimization are shown in Figure 3. We found that the optimal dose to reach the highest level of EGFP expression with the lowest cell death/toxicity is achieved by using approximately 1011 AuNR and 10 pulses at 50 J/cm2, with a 30 ms pulse length. In general, cells are driven to increase production of EGFP with increased heating up to some threshold, at which point cell damage rapidly reduces expression and eventually viability. Heating efficiency increases as expected with increased loading of AuNPs (Figure 3A) and pulse numbers (Figure 3B). At a laser fluence of 25 J/cm2 relatively little gene expression occurs, with increases in fluence to 50 J/cm2 inducing increased expression and relatively high viability (approximately 80%). While gene expression was highest at 75 J/cm2, cell viability dropped to 44% and 37% at 6 h and 24 h, respectively. Increasing the length of the pulses from 30 ms to 400 ms actually decreases the GFP expression (Figure 3C; right), demonstrating that peak power is more important than total power. In Figure 3C, the peak power is decreasing (the same energy, 50 J, is spread over a longer pulse), even though the total power is the same. In general, some loss of viability appears as a necessary price for high expression.

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