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Federica Rinaldi,1,* Patrizia Nadia Hanieh,1,* Linda Maurizi,2 Catia Longhi,2 Daniela Uccelletti,3 Emily Schifano,3 Elena Del Favero,4 Laura Cantù4 ,† Caterina Ricci,4 Maria Grazia Ammendolia,5 Donatella Paolino,6 Francesca Froiio,6 Carlotta Marianecci,1 Maria Carafa1 1Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, Rome, Italy; 2Dipartimento di Sanità pubblica e Malattie infettive, Sapienza Università di Roma, Rome, Italy; 3Dipartimento di Biologia e Biotecnologie Charles Darwin, Sapienza Università di Roma, Rome, Italy; 4Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università di Milano, Milan, Italy; 5Centro Nazionale Tecnologie Innovative in Sanità Pubblica, Istituto Superiore di Sanità, Rome, Italy; 6Dipartimento di Medicina Sperimentale e Clinica, Università Magna Graecia di Catanzaro, Campus Universitario “S. Venuta”, Catanzaro, Italy*These authors contributed equally to this work†Professor Laura Cantù passed away on August 05, 2022Correspondence: Maria Carafa; Carlotta Marianecci, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, Piazzale Aldo Moro 5, Roma, 00185, Italy, Tel +390649913603 ; +390649913970, Fax +39064913133, Email maria.carafa@uniroma1.it; carlotta.marianecci@uniroma1.itPurpose: Vitamin E (VitE) may be classified in “the first line of defense” against the formation of reactive oxygen species. Its inclusion in nanoemulsions (NEs) is a promising alternative to increase its bioavailability. The aim of this study was to compare O/W NEs including VitE based on Almond or Neem oil, showing themselves antioxidant properties. The potential synergy of the antioxidant activities of oils and vitamin E, co-formulated in NEs, was explored.Patients and Methods: NEs have been prepared by sonication and deeply characterized evaluating size, ζ-potential, morphology (TEM and SAXS analyses), oil nanodroplet feature, and stability. Antioxidant activity has been evaluated in vitro, in non-tumorigenic HaCaT keratinocytes, and in vivo through fluorescence analysis of C. elegans transgenic strain. Moreover, on healthy human volunteers, skin tolerability and anti-inflammatory activity were evaluated by measuring the reduction of the skin erythema induced by the application of a skin chemical irritant (methyl-nicotinate).Results: Results confirm that Vitamin E can be formulated in highly stable NEs showing good antioxidant activity on keratinocyte and on C. elegans. Interestingly, only Neem oil NEs showed some anti-inflammatory activity on healthy volunteers.Conclusion: From the obtained results, Neem over Almond oil is a more appropriate candidate for further studies on this application.Graphical Abstract: Keywords: O/W nanodispersion, α-tocopherol, anti-inflammatory activity, HaCaT, C. elegans, healthy volunteers

Paola Di Bonito,1 Linda Petrone,1 Gabriele Casini,2 Iolanda Francolini,2 Maria Grazia Ammendolia,3 Luisa Accardi,1 Antonella Piozzi,2 Lucio D’Ilario,2 Andrea Martinelli2 1Department of Infectious, Parasitic and Immune-mediated Diseases, Italian National Institute of Health, 2Department of Chemistry, Sapienza University of Rome, Rome, Italy; 3Department of Technology and Health, Italian National Institute of Health, Rome, Italy Background: Poly(L-lactide) (PLLA) is a biodegradable polymer currently used in many biomedical applications, including the production of resorbable surgical devices, porous scaffolds for tissue engineering, nanoparticles and microparticles for the controlled release of drugs or antigens. The surfaces of lamellar PLLA single crystals (PLLAsc) were provided with amino groups by reaction with a multifunctional amine and used to adsorb an Escherichia coli-produced human papillomavirus (HPV)16-E7 protein to evaluate its possible use in antigen delivery for vaccine development.Methods: PLLA single crystals were made to react with tetraethylenepentamine to obtain amino-functionalized PLLA single crystals (APLLAsc). Pristine and amino-functionalized PLLAsc showed a two-dimensional microsized and one-dimensional nanosized lamellar morphology, with a lateral dimension of about 15–20 µm, a thickness of about 12 nm, and a surface specific area of about 130 m2/g. Both particles were characterized and loaded with HPV16-E7 before being administered to C57BL/6 mice for immunogenicity studies. The E7-specific humoral-mediated and cell-mediated immune response as well as tumor protective immunity were analyzed in mice challenged with TC-1 cancer cells.Results: Pristine and amino-functionalized PLLAsc adsorbed similar amounts of E7 protein, but in protein-release experiments E7-PLLAsc released a higher amount of protein than E7-APLLAsc. When the complexes were dried for observation by scanning electron microscopy, both samples showed a compact layer, but E7-APLLAsc showed greater roughness than E7-PLLAsc. Immunization experiments in mice showed that E7-APLLAsc induced a stronger E7-specific immune response when compared with E7-PLLAsc. Immunoglobulin G isotyping and interferon gamma analysis suggested a mixed Th1/Th2 immune response in both E7-PLLAsc-immunized and E7-APLLAsc-immunized mice. However, only the mice receiving E7-APLLAsc were fully protected from TC-1 tumor growth after three doses of vaccine.Conclusion: Our results show that APLLA single crystals improve the immunogenicity of HPV16-E7 and indicate that E7-APLLAsc could be used for development of an HPV16 therapeutic vaccine against HPV16-related tumors. Keywords: poly(L-lactide), lamellar crystals, human papillomavirus, HPV16-E7, therapeutic vaccine