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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

In vivo imaging of luciferase and GFP expression after NIR laser treatment. (A) B16-luciferase cells were transfected with the HSP70-GFP vector 24 h before NIR treatment and AuNR (106/cell) were added 12 h after transfection. Cells were washed before being harvested and 2 × 107 cells were injected in each flank of two nude mice. Immediately following injection, the right flank of each mouse was treated with NIR laser for 10 pulses at 30 ms at either 40 or 50 J/cm2. The left flank was left non-treated. Mice were imaged at 6 h via IVIS and Maestro in vivo imaging systems. At 24 h after treatment, one mouse was imaged as just described, and the other mouse was sacrificed and the tumor analyzed for GFP expression via flow cytometry analysis (B).
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vaccines-02-00216-f005: In vivo imaging of luciferase and GFP expression after NIR laser treatment. (A) B16-luciferase cells were transfected with the HSP70-GFP vector 24 h before NIR treatment and AuNR (106/cell) were added 12 h after transfection. Cells were washed before being harvested and 2 × 107 cells were injected in each flank of two nude mice. Immediately following injection, the right flank of each mouse was treated with NIR laser for 10 pulses at 30 ms at either 40 or 50 J/cm2. The left flank was left non-treated. Mice were imaged at 6 h via IVIS and Maestro in vivo imaging systems. At 24 h after treatment, one mouse was imaged as just described, and the other mouse was sacrificed and the tumor analyzed for GFP expression via flow cytometry analysis (B).

Mentions: As shown in Figure 5, strong EGFP expression could be detected 6 hours after NIR treatment at both 40 J/cm2 and 50 J/cm2 laser at 30 ms pulse length. This was confirmed with FACS on the 40 J/cm2 and was confirmed to last at least 24 h in the 50 J/cm2 animal. The laser treatment at the higher power seemed to result in more physical damage to the skin of the animal. Bioluminescent imaging (BLI) was also used to monitor luciferase expression in the cells with IVIS200. Interestingly, the luciferase signal was intact on the non-treated left flanks in both mice, but suppressed on the NIR treated right flanks. This finding is in agreement with previous studies reporting that luciferase is highly sensitive to temperature, and thus can function as an indicator of a successful thermal treatment [28]. Further experiments indicated that detectable expression may be achieved with half the gold loading, and using 5 instead of 10 pulses, still at 40 J/cm2. Reducing the laser fluence to 30 J/cm2 or below resulted in no observable expression. Treatment with laser of cells without AuNPs did not result in any increase in EGFP expression or loss of bioluminescence.


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)

In vivo imaging of luciferase and GFP expression after NIR laser treatment. (A) B16-luciferase cells were transfected with the HSP70-GFP vector 24 h before NIR treatment and AuNR (106/cell) were added 12 h after transfection. Cells were washed before being harvested and 2 × 107 cells were injected in each flank of two nude mice. Immediately following injection, the right flank of each mouse was treated with NIR laser for 10 pulses at 30 ms at either 40 or 50 J/cm2. The left flank was left non-treated. Mice were imaged at 6 h via IVIS and Maestro in vivo imaging systems. At 24 h after treatment, one mouse was imaged as just described, and the other mouse was sacrificed and the tumor analyzed for GFP expression via flow cytometry analysis (B).
© Copyright Policy
Related In: Results  -  Collection

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

vaccines-02-00216-f005: In vivo imaging of luciferase and GFP expression after NIR laser treatment. (A) B16-luciferase cells were transfected with the HSP70-GFP vector 24 h before NIR treatment and AuNR (106/cell) were added 12 h after transfection. Cells were washed before being harvested and 2 × 107 cells were injected in each flank of two nude mice. Immediately following injection, the right flank of each mouse was treated with NIR laser for 10 pulses at 30 ms at either 40 or 50 J/cm2. The left flank was left non-treated. Mice were imaged at 6 h via IVIS and Maestro in vivo imaging systems. At 24 h after treatment, one mouse was imaged as just described, and the other mouse was sacrificed and the tumor analyzed for GFP expression via flow cytometry analysis (B).
Mentions: As shown in Figure 5, strong EGFP expression could be detected 6 hours after NIR treatment at both 40 J/cm2 and 50 J/cm2 laser at 30 ms pulse length. This was confirmed with FACS on the 40 J/cm2 and was confirmed to last at least 24 h in the 50 J/cm2 animal. The laser treatment at the higher power seemed to result in more physical damage to the skin of the animal. Bioluminescent imaging (BLI) was also used to monitor luciferase expression in the cells with IVIS200. Interestingly, the luciferase signal was intact on the non-treated left flanks in both mice, but suppressed on the NIR treated right flanks. This finding is in agreement with previous studies reporting that luciferase is highly sensitive to temperature, and thus can function as an indicator of a successful thermal treatment [28]. Further experiments indicated that detectable expression may be achieved with half the gold loading, and using 5 instead of 10 pulses, still at 40 J/cm2. Reducing the laser fluence to 30 J/cm2 or below resulted in no observable expression. Treatment with laser of cells without AuNPs did not result in any increase in EGFP expression or loss of bioluminescence.

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