title: Green synthesis and evaluation of silver nanoparticles as adjuvant in rabies veterinary vaccine.
abstract: BACKGROUND
  Green synthesis of nanoparticles by plant extracts plays a significant role in different applications. Recently, several studies were conducted on the use of nanoparticles as adjuvant. The main aim of this study was to evaluate green synthesized silver nanoparticles (AgNPs) as adjuvant in rabies veterinary vaccine and compare the results with the existing commercially available alum adjuvant.
  
  
  METHODS
  In the current study, AgNPs were prepared by the reduction of aqueous silver nitrate by leaf extract of Eucalyptus procera. The formation of AgNPs was confirmed by ultraviolet (UV)-visible spectrophotometer, scanning electron microscopy, dynamic light scattering, and X-ray diffraction analysis. Then, different amounts of AgNPs (200 µg, 400 µg, 600 µg, and 800 µg) were added to 1 mL of inactivated rabies virus. The loaded vaccines (0.5 mL) were injected intraperitoneally into six Naval Medical Research Institute mice in each group on days 1 and 7. On the 15th day, the mice were intracerebrally challenged with 0.03 mL of challenge rabies virus (challenge virus strain-11, 20 lethal dose [20 LD50]), and after the latency period of rabies disease in mice (5 days), the mice were monitored for 21 days. Neutralizing antibodies against rabies virus were also investigated using the rapid fluorescent focus inhibition test method. The National Institutes of Health test was performed to determine the potency of optimum concentration of AgNPs as adjuvant. In vitro toxicity of AgNPs was assessed in L929 cell line using MTT assay. In addition, in vivo toxicity of AgNPs and AgNPs-loaded vaccine was investigated according to the European Pharmacopeia 8.0.
  
  
  RESULTS
  AgNPs were successfully synthesized, and the identity was confirmed by UV-visible spectrophotometry and X-ray diffraction analysis. The prepared AgNPs were spherical in shape, with an average size of 60 nm and a negative zeta potential of -14 mV as determined by dynamic light scattering technique. The highest percentage of viability was observed at 15 mg/kg and 20 mg/kg of AgNPs-loaded vaccine concentrations after injecting into the mice. The calculated potencies for alum-containing vaccine and AgNPs-loaded vaccine (dose 15 mg/kg) were 1.897 and 1.303, respectively. MTT assay demonstrated that alum at the concentration of 10 mg/mL was toxic, but AgNPs were not toxic. The in vivo toxicity also elucidated the safety of AgNPs and AgNPs-loaded vaccine in mice and dogs, respectively.
  
  
  CONCLUSIONS
  In the current study, for the first time, the adjuvanticity effect of green synthesized AgNPs on veterinary rabies vaccine potency with no in vivo toxicity was elucidated according to the European Pharmacopeia 8.0.
authors.affiliation: Virology Research Group, Department of Rabies, Pasteur Institute of Iran; Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran.
authors.forename: Vahid
authors.initials: V
authors.lastname: Asgary
authors.affiliation: Virology Research Group, Department of Rabies, Pasteur Institute of Iran.
authors.forename: Alireza
authors.initials: A
authors.lastname: Shoari
authors.affiliation: Department of Genetics and Biotechnology, School of Biological Science, Varamin-Pishva Branch, Islamic Azad University, Varamin.
authors.forename: Fahimeh
authors.initials: F
authors.lastname: Baghbani-Arani
authors.affiliation: Young Researchers and Elite Club, East Tehran Branch, Islamic Azad University.
authors.forename: Seyed Ataollah
authors.initials: SA
authors.lastname: Sadat Shandiz
authors.affiliation: Department of Laboratory of Animal Sciences, Pasteur Institute of Iran.
authors.forename: Mohammad Sadeq
authors.initials: MS
authors.lastname: Khosravy
authors.affiliation: Virology Research Group, Department of Rabies, Pasteur Institute of Iran.
authors.forename: Alireza
authors.initials: A
authors.lastname: Janani
authors.affiliation: Department of Genetic, Science and Research Branch, Islamic Azad University.
authors.forename: Razieh
authors.initials: R
authors.lastname: Bigdeli
authors.affiliation: Virology Research Group, Department of Rabies, Pasteur Institute of Iran.
authors.forename: Rouzbeh
authors.initials: R
authors.lastname: Bashar
authors.affiliation: Virology Research Group, Department of Rabies, Pasteur Institute of Iran; New Technologies Research Group, Department of Pilot Nanobiotechnology, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Reza Ahangari
authors.initials: RA
authors.lastname: Cohan
mesh_terms: D000276:Adjuvants, Immunologic / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D000818:Animals; D004285:Dogs; D005052:Eucalyptus / Q000737:chemistry*; D005260:Female; D055772:Green Chemistry Technology; D053768:Metal Nanoparticles / Q000008:administration & dosage / Q000737:chemistry; D051379:Mice; D008855:Microscopy, Electron, Scanning; D010936:Plant Extracts / Q000737:chemistry; D018515:Plant Leaves / Q000737:chemistry; D011818:Rabies / Q000517:prevention & control / Q000662:veterinary; D011819:Rabies Vaccines / Q000276:immunology / Q000494:pharmacology; D012834:Silver / Q000737:chemistry / Q000276:immunology* / Q000494:pharmacology; D013056:Spectrophotometry, Ultraviolet; D014961:X-Ray Diffraction
chemical_list: D000276:Adjuvants, Immunologic; D010936:Plant Extracts; D011819:Rabies Vaccines; D012834:Silver
keywords: adjuvant; green synthesis; nanoparticles; rabies virus