title: Size is an essential parameter in governing the UVB-protective efficacy of silver nanoparticles in human keratinocytes.
abstract: BACKGROUND
  Ultraviolet (UV) radiation from sun, particularly its UVB component (290-320 nm), is considered the major etiological cause of skin cancer that impacts over 2 million lives in the United States alone. Recently, we reported that polydisperse colloidal suspension of silver nanoparticles (AgNPs) protected the human keratinocytes (HaCaT) against UVB-induced damage, thus indicating their potential for prevention of skin carcinogenesis. Here we sought out to investigate if size controlled the chemopreventive efficacy of AgNPs against UVB-induced DNA damage and apoptosis.
  
  
  METHODS
  Percent cell viability was examined by WST-1 assay after treating the cells with various doses (1-10 μg/mL) of AgNPs of different sizes (10, 20, 40, 60 and 100 nm) for 12 and 24 h. For protection studies, cells were treated with AgNPs of different sizes at a uniform concentration of 1 μg/mL. After 3 h, cells were irradiated with UVB (40 mJ/cm(2)) and dot-blot analysis was performed to detect cyclobutane pyrimidine dimers (CPDs) as an indication of DNA damage. Apoptosis was analyzed by flow cytometry after staining the cells with 7-Amino-Actinomycin (7-AAD) and PE Annexin V. Immunoblot analysis was accomplished by processing the cells for protein extraction and Western blotting using specific antibodies against various proteins.
  
  
  RESULTS
  The data show that the pretreatment of HaCaT cells with AgNPs in the size range of 10-40 nm were effective in protecting the skin cells from UVB radiation-induced DNA damage as validated by reduced amounts of CPDs, whereas no protection was observed with AgNPs of larger sizes (60 and 100 nm). Similarly, only smaller size AgNPs (10-40 nm) were effective in protecting the skin cells from UV radiation-induced apoptosis. At the molecular level, UVB -irradiation of HaCaT cells led to marked increase in expression of pro-apoptotic protein (Bax) and decrease in anti-apoptotic proteins (Bcl-2 and Bcl-xL), while it remained largely unaffected in skin cells pretreated with smaller size AgNPs (10-40 nm).
  
  
  CONCLUSIONS
  Altogether, these findings suggest that size is a critical determinant of the UVB-protective efficacy of AgNPs in human keratinocytes.
authors.affiliation: Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Mobile, AL, 36604, USA.
authors.forename: Rohan
authors.initials: R
authors.lastname: Palanki
authors.affiliation: Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Mobile, AL, 36604, USA.
authors.forename: Sumit
authors.initials: S
authors.lastname: Arora
authors.affiliation: Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Mobile, AL, 36604, USA.
authors.forename: Nikhil
authors.initials: N
authors.lastname: Tyagi
authors.affiliation: Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Mobile, AL, 36604, USA.|Department of Chemical and Biomolecular Engineering, University of South Alabama, Mobile, AL, 36688, USA.
authors.forename: Lilia
authors.initials: L
authors.lastname: Rusu
authors.affiliation: Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Mobile, AL, 36604, USA.|Department of Biochemistry and Molecular Biology, College of Medicine, University of South Alabama, Mobile, AL, 36688, USA.
authors.forename: Ajay P
authors.initials: AP
authors.lastname: Singh
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of South Alabama, Mobile, AL, 36688, USA.
authors.forename: Srinivas
authors.initials: S
authors.lastname: Palanki
authors.affiliation: Department of Pathology, College of Medicine, University of South Alabama, Mobile, AL, 36688, USA.
authors.forename: James E
authors.initials: JE
authors.lastname: Carter
authors.affiliation: Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Mobile, AL, 36604, USA. seemasingh@health.southalabama.edu.|Department of Biochemistry and Molecular Biology, College of Medicine, University of South Alabama, Mobile, AL, 36688, USA. seemasingh@health.southalabama.edu.
authors.forename: Seema
authors.initials: S
authors.lastname: Singh
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D004249:DNA Damage / Q000187:drug effects / Q000528:radiation effects; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects* / Q000528:radiation effects*; D053768:Metal Nanoparticles / Q000008:administration & dosage / Q000737:chemistry; D010316:Particle Size; D020011:Protective Agents / Q000008:administration & dosage / Q000737:chemistry; D012834:Silver; D014466:Ultraviolet Rays
chemical_list: D020011:Protective Agents; D012834:Silver