title: Development and in vivo safety assessment of tenofovir-loaded nanoparticles-in-film as a novel vaginal microbicide delivery system.
abstract: Topical pre-exposure prophylaxis (PrEP) with antiretroviral drugs holds promise in preventing vaginal transmission of HIV. However, significant biomedical and social issues found in multiple past clinical trials still need to be addressed in order to optimize protection and users' adherence. One approach may be the development of improved microbicide products. A novel delivery platform comprising drug-loaded nanoparticles (NPs) incorporated into a thin polymeric film base (NPs-in-film) was developed in order to allow the vaginal administration of the microbicide drug candidate tenofovir. The system was optimized for relevant physicochemical features and characterized for biological properties, namely cytotoxicity and safety in a mouse model. Tenofovir-loaded poly(lactic-co-glycolic acid) (PLGA)/stearylamine (SA) composite NPs with mean diameter of 127nm were obtained with drug association efficiency above 50%, and further incorporated into an approximately 115μm thick, hydroxypropyl methylcellulose/poly(vinyl alcohol)-based film. The system was shown to possess suitable mechanical properties for vaginal administration and to quickly disintegrate in approximately 9min upon contact with a simulated vaginal fluid (SVF). The original osmolarity and pH of SVF was not affected by the film. Tenofovir was also released in a biphasic fashion (around 30% of the drug in 15min, followed by sustained release up to 24h). The incorporation of NPs further improved the adhesive potential of the film to ex vivo pig vaginal mucosa. Cytotoxicity of NPs and film was significantly increased by the incorporation of SA, but remained at levels considered tolerable for vaginal delivery of tenofovir. Moreover, histological analysis of genital tissues and cytokine/chemokine levels in vaginal lavages upon 14days of daily vaginal administration to mice confirmed that tenofovir-loaded NPs-in-film was safe and did not induce any apparent histological changes or pro-inflammatory response. Overall, obtained data support that the proposed delivery system combining the use of polymeric NPs and a film base may constitute an exciting alternative for the vaginal administration of microbicide drugs in the context of topical PrEP.
  
  
  
  The development of nanotechnology-based microbicides is a recent but promising research field seeking for new strategies to circumvent HIV sexual transmission. Different reports detail on the multiple potential advantages of using drug nanocarriers for such purpose. However, one important issue being frequently neglected regards the development of vehicles for the administration of microbicide nanosystems. In this study, we propose and detail on the development of a nanoparticle-in-film system for the vaginal delivery of the microbicide drug candidate tenofovir. This is an innovative approach that, to our best knowledge, had never been tested for tenofovir. Results, including those from in vivo testing, sustain that the proposed system is safe and holds potential for further development as a vaginal microbicide product.
authors.affiliation: i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Porto, Portugal.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Machado
authors.affiliation: CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde & Instituto Universitário de Ciências da Saúde, Gandra, Portugal.
authors.forename: Cassilda
authors.initials: C
authors.lastname: Cunha-Reis
authors.affiliation: i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Porto, Portugal; ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal.
authors.forename: Francisca
authors.initials: F
authors.lastname: Araújo
authors.affiliation: i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Porto, Portugal; ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal.
authors.forename: Rute
authors.initials: R
authors.lastname: Nunes
authors.affiliation: CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde & Instituto Universitário de Ciências da Saúde, Gandra, Portugal.
authors.forename: Vítor
authors.initials: V
authors.lastname: Seabra
authors.affiliation: Laboratório de Tecnologia Farmacêutica, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal.
authors.forename: Domingos
authors.initials: D
authors.lastname: Ferreira
authors.affiliation: i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Porto, Portugal; CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde & Instituto Universitário de Ciências da Saúde, Gandra, Portugal. Electronic address: j.dasneves@ineb.up.pt.
authors.forename: José
authors.initials: J
authors.lastname: das Neves
authors.affiliation: i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Porto, Portugal; CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde & Instituto Universitário de Ciências da Saúde, Gandra, Portugal. Electronic address: bruno.sarmento@ineb.up.pt.
authors.forename: Bruno
authors.initials: B
authors.lastname: Sarmento
mesh_terms: D000282:Administration, Intravaginal; D000818:Animals; D000890:Anti-Infective Agents / Q000494:pharmacology*; D002460:Cell Line; D018925:Chemokines / Q000378:metabolism; D016503:Drug Delivery Systems; D065546:Drug Liberation; D005260:Female; D006801:Humans; D019344:Lactic Acid / Q000737:chemistry; D051379:Mice; D053758:Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D011100:Polyglycolic Acid / Q000737:chemistry; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D013052:Spectrometry, X-Ray Emission; D034421:Sus scrofa; D000068698:Tenofovir / Q000494:pharmacology*; D013997:Time Factors; D044364:Vaginal Douching
chemical_list: D000890:Anti-Infective Agents; D018925:Chemokines; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D019344:Lactic Acid; D000068698:Tenofovir
keywords: Antiretroviral; HIV/AIDS; Nanotechnology; Pre-exposure prophylaxis; Safety; Vaginal drug administration