title: Functional characterization of biodegradable nanoparticles as antigen delivery system.
abstract: BACKGROUND
  Peptide based vaccines may suffer from limited stability and inefficient delivery to professional antigen-presenting cells (APCs), such as dendritic cells (DCs). In order to overcome such limitations, several types of biodegradable nanoparticles (NPs) have been developed as carrier system for antigens. The present study describes for the first time the extensive biological characterization of cationic NPs made of poly (D,L-lactide-co-glycolide) (PLGA) and polyethylenimine (PLGA/PEI) as delivery system for protein/peptide antigens, with potential in therapeutic cancer vaccine development.
  
  
  RESULTS
  Flow cytometry as well as confocal laser scanning microscopy (CLSM) showed that PLGA/PEI NPs are more readily taken up than PLGA NPs by both human CD14(+) monocytes and mouse Hepa 1-6 hepatoma cell line. No signs of toxicity were observed in either cellular setting. Sequential image acquisition by TEM showed an intracellular apical localization for PLGA NPs and a perinuclear localization for PLGA/PEI NPs. Both NPs showed a clathrin-dependent as well as a caveolin-dependent internalization pathway and, once in the cells, they formed multivesicular endosomes (MVE). Finally, an ex vivo priming experiment showed that PLGA/PEI NPs are comparable to PLGA NPs in delivering a non-self antigen (i.e., ovalbumin - OVA) to immature dendritic cells (imDCs), which matured and induced autologous naïve CD4(+) T cells to differentiate to memory (i.e., central memory and effector memory) cells. Such a differentiation was associated with a Th1 phenotype suggesting a downstream activation and amplification of a CD8(+) T cell cytotoxic response. The same OVA antigen in a soluble form was unable to induce maturation of DCs, indicating that both NP formulations provided an intrinsic adjuvanting effect combined to efficient antigen delivery.
  
  
  CONCLUSIONS
  Our study represents the first report on side-by-side comparison of PLGA and PLGA/PEI NPs as strategy for protein antigen delivery. PLGA/PEI NPs are superior for cellular uptake and antigen delivery as compared to PLGA NPs. Such an evidence suggests their great potential value for vaccine development, including therapeutic cancer vaccines.
authors.affiliation: Laboratoy Molecular Biology and Viral Oncology, Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori "Fondazione Pascale" - IRCCS, Via Mariano Semmola 142, 80131, Naples, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Petrizzo
authors.affiliation: Department of Pharmacy, University of Napoli Federico II, Via Domenico Montesano 49, 80131, Naples, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Conte
authors.affiliation: Laboratoy Molecular Biology and Viral Oncology, Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori "Fondazione Pascale" - IRCCS, Via Mariano Semmola 142, 80131, Naples, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Tagliamonte
authors.affiliation: Laboratory of Clinical Immunology, Istituto Nazionale per lo Studio e la Cura dei Tumori "Fondazione Pascale" - IRCCS, Via Mariano Semmola 142, 80131, Naples, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Napolitano
authors.affiliation: Laboratory of Tumor Progression, Istituto Nazionale per lo Studio e la Cura dei Tumori "Fondazione Pascale" - IRCCS, Via Mariano Semmola 142, 80131, Naples, Italy.
authors.forename: K
authors.initials: K
authors.lastname: Bifulco
authors.affiliation: Laboratory of Tumor Progression, Istituto Nazionale per lo Studio e la Cura dei Tumori "Fondazione Pascale" - IRCCS, Via Mariano Semmola 142, 80131, Naples, Italy.
authors.forename: V
authors.initials: V
authors.lastname: Carriero
authors.affiliation: National Research Council Institutional Sustainable Plant Protection, Bari, Italy.
authors.forename: A
authors.initials: A
authors.lastname: De Stradis
authors.affiliation: Laboratoy Molecular Biology and Viral Oncology, Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori "Fondazione Pascale" - IRCCS, Via Mariano Semmola 142, 80131, Naples, Italy.
authors.forename: M L
authors.initials: ML
authors.lastname: Tornesello
authors.affiliation: Laboratoy Molecular Biology and Viral Oncology, Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori "Fondazione Pascale" - IRCCS, Via Mariano Semmola 142, 80131, Naples, Italy.
authors.forename: F M
authors.initials: FM
authors.lastname: Buonaguro
authors.affiliation: Department of Pharmacy, University of Napoli Federico II, Via Domenico Montesano 49, 80131, Naples, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Quaglia
authors.affiliation: Laboratoy Molecular Biology and Viral Oncology, Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori "Fondazione Pascale" - IRCCS, Via Mariano Semmola 142, 80131, Naples, Italy. l.buonaguro@istitutotumori.na.it.
authors.forename: L
authors.initials: L
authors.lastname: Buonaguro
mesh_terms: D000818:Animals; D017951:Antigen Presentation / Q000276:immunology*; D000941:Antigens / Q000008:administration & dosage / Q000276:immunology*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D019496:Cancer Vaccines / Q000276:immunology*; D051242:Caveolin 1 / Q000378:metabolism; D045744:Cell Line, Tumor; D002966:Clathrin / Q000378:metabolism; D003713:Dendritic Cells / Q000276:immunology*; D006801:Humans; D007156:Immunologic Memory / Q000276:immunology; D019344:Lactic Acid / Q000494:pharmacology*; D051379:Mice; D018613:Microscopy, Confocal; D057146:Multivesicular Bodies / Q000378:metabolism; D053758:Nanoparticles; D010047:Ovalbumin / Q000276:immunology; D011094:Polyethyleneimine / Q000494:pharmacology*; D011100:Polyglycolic Acid / Q000494:pharmacology*; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D022223:Vaccines, Subunit / Q000276:immunology
chemical_list: D000941:Antigens; D019496:Cancer Vaccines; D051242:Caveolin 1; D002966:Clathrin; D022223:Vaccines, Subunit; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D019344:Lactic Acid; D011094:Polyethyleneimine; D010047:Ovalbumin