Utilize este identificador para referenciar este registo: http://hdl.handle.net/10451/26686
Título: Anti-tumor immunity induced by a novel nanoparticulate vaccine : a mechanistic approach
Autor: Zupančič, Eva, 1984-
Orientador: Florindo, Helena Isabel Fialho, 1979-
Moreira, João Nuno Sereno de Almeida
Videira, Mafalda de Castro Ascensão Marques, 1963-
Palavras-chave: Teses de doutoramento - 2016
Data de Defesa: 2016
Resumo: Biodegradable polymeric nanoparticles (NP) are promising tools for tumor eradication. Different nanovaccines based on the aliphatic-polyester (poly(lactic-co-glycolic acid) (PLGA) have been developed and optimized to investigate how different methods for antigen association to nanoparticulate carriers affect antigen uptake by antigen presenting cells (APC) and the generation and nature of antigen-specific immune responses. The different experimental parameters have been tested in order to predict the effect of the nature of protein association (adsorbed vs. entrapped) and polymers/surfactant concentrations, on NP average mean diameter, polydispersity index, surface charge, encapsulation efficiency, protein integrity and surfactant residual amount. This development procedure allowed for the rational identification of particle composition and experimental conditions that led to the antigenassociated nanocarrier presenting the ideal product specifications previously identified for optimal immune cell modulation, having in consideration literature evidences and our previous data. We used PLGA and pegylated (PEG)-PLGA as a matrix polymer. When preparing NP with double-emulsion solvent-evaporation technique, we used α-lactalbumin (LALBA) as an antigen, glycol chitosan (CS) to increase the viscosity of the internal aqueous phase (IP) and polyvinyl alcohol (PVA) or Pluronic F127 (PF127) to stabilization the interphase of double emulsion. Various concentrations of surfactants has been used (2, 4, 5, 8, 10, 12 % v/v) to best meet the criteria of the most optimal nanoparticulate delivery system. We aimed for NP below 200 nm, polydispersity index (PDI) under 0.2 and ζ potential close to neutrality. The formulations with 10 % v/v PVA solution in IP and 0.3 % v/v PVA or PF127 in external aqueous phase (EP) presented stable and repetitive NP formulations with desired physiochemical properties. The highest encapsulation efficacy (EE) values were obtained for the formulation prepared with PVA (EntrapLALBA(PVA)), reaching 80 % of the integrated protein, while NP with PF127 (EntrapLALBA(PF127)) presented around 70 % EE. As regards the protein adsorbed, NP formulations prepared with PVA (AdsLALBA(PVA)) or PF127 (AdsLALBA(PF127), reached the values of 40 % and 50 %, respectively. Dynamic light scattering (DLS) and laser doppler velocimetry (LDV) were used to determine average particle size, polydispersity (PDI) and ζ potential, respectively. EntrapLALBA(PVA) presented the average size of 171 nm with PDI around 0.15 and ζ potential 0.51 mV. EntrapLALBA(PF127) had 185 nm with PDI 0.17 and ζ potential 0.37 mV. On the other side AdsLALBA(PVA) and AdsLALBA(PF127) presented slightly biggersize 188 nm and 195 nm, respectively, with more negative ζ potential, -1,89 and -1,42 mV, respectively. NP size, PDI and geometry were also confirmed by atomic force microscopy (AFM). Protein integrity was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fourier transform infrared spectroscopy (FTIR) was further used to study the interaction of protein adsorption onto surfaces of polymeric NP. The presence of bands at the wavelengths 1662 cm-1 and 1562 cm-1, that are specific for primary amides and amines, were detected in formulations AdsLALBA(PVA) and AdsLALBA(PF127), where LALBA was adsorbed onto the surface of the NP. While in NP EntrapLALBA(PVA) and EntrapLALBA(PF127) this bands were absent indicating the protein incorporation into the polymeric matrix. Differential scanning calorimetry (DSC) showed that the formulation process did not alter the structure of the polymers used in the preparation of NP. The ability of the optimal formulations for systemic delivery and activation of DC was further evaluated in vitro and in vivo on bone marrow derived dendritic cells (BMDC). Similar activation and maturation profile of APC were obtained after the internalization of NP with antigen entrapped (EntrapNP) or adsorbed onto NP surface (AdsNP) evaluated at 3, 6, 16 and 24 h (ImageStreamTM and flow cytometry). The DC-NP interaction increased with the incubation time, presenting internalization values between 50-60% and 30-40%, in vitro and in vivo, respectively. Interestingly, MHCII was upregulated after the immunization with AdsNP and MHCI after the vaccination with EntrapNP, suggesting more efficient crosspresentation. To evaluate the activation of antigen-specific immune response, we replaced LALBA for ovalbumin (OVA). It is a highly immunogenic model antigen for which, in immunology, we have well-established animal models that can help to study the effect of the antigen on immune system without any unspecific immune response. Phenotype, frequency and efficacy of immune cell stimulation induced by NP loaded with OVA (EntrapNP or AdsNP) and with/without TLR ligands (CpG and Monophosphoryl Lipid A (MPLA)) were characterized and their specificity clarified by the engrafted OT-I (CD8+) and OT-II (CD4+) T cells. EntrapNP with OVA and adjuvants presented the strongest antigen-specific cytotoxic immune response, following NP with AdsOVA-Ajds. Moreover, long-lasting memory of cytotoxic T lymphocytes (CTL), was evaluated 8 weeks after a single immunization in response to the different ways of antigen delivery, PBS, OVA in solution, OVA & Adjs in solution and NP AdsOVA, AdsOVA-Adjs, EntrapOVA, or EntrapOVA-Adjs. Immunizations where we used antigen alone did not induce efficient CTL reactivation. Overall, Entrap OVA-Adj NP presented the most robust immune response in inducing the memory, followed by AdsOVAAdjs NP and OVA & Adjs in solution. To explore whether the immunization with formulated NP results in efficient crosspriming, an in vivo killing assay in steady-state conditions was performed. 24 h prior to immunization, animals were engrafted with CD8+ (OT-I) cells. 5 days p.i., mice were injected with an equal mix of targeted and untargeted CD45.1 splenocytes. Targeted cells were pulsed with OVA peptide, SIINFEKL, and labeled with higher dose of CFSE (CFSEhi) while untargeted control cells were labeled with a lower dose of CFSE (CSFElow). The in vivo clearance of both type of grafted cells was evaluated 16 h later in LN and spleens by flow cytometry. All NPs, with or without adjuvants, generated the most efficient in vivo killing activity towards the SIINFEKL-pulsed cells, as shown by the disappearance of the CFSEhi peak of cells. Indicating the successful induction of cross-priming and activation of antigenspecific CTL that could potentially lead to a broad and effective immune response pivotal in case of tumor rejections. To test the efficacy of the therapeutic vaccine and to have a good read-out of the antigen-specific T cells, we used B16.MO5 melanoma-bearing mice using different vaccination schedules, single and three time immunization with OVA and adjuvants- loaded NP. The groups immunized with PBS and Empty NP served as a control and presented similar tumor growth. All treated groups showed a significant reduction in tumor growth. EntrapOVA-Adjs NP (3x immunization) presented the slowest tumor growth (more than 12x smaller final tumor volume), followed by the group immunized with AdsOVA-Adjs NP (3x immunization) (more than 9x smaller final tumor volume) and single vaccination with EntrapOVA-Adjs NP (more than 8x smaller final tumor volume). The highest amount of tumor infiltrating lymphocytes (TILs) at the tumor site was detected for EntrapOVA-Adjs NP (3x immunization), suggesting the best remission and survival prognosis of the treated group. Two inflammatory cytokines (TNF-α and IFN-γ) produced by TILs were quantified in tumor microenvironment. 4-time high levels of TNF-α upon 3-time immunizations were detected in tumor microenvironment which kept the tumor growth under control. On the other side in the spleens, repeated vaccination prevented upregulation of both inflammatory markers, TNF-α and IFN-γ, indicating absence of systemic inflammation. Overall, the present work shows that the design of safe but efficacious nanoparticulate cancer vaccines requires a deep understanding of NP biological effect on immune cells, both under steady-state and cancerous environment. A detailed characterization of the imune cell-related pathways modulated by the optimal antigen-loaded NP and their correlation with the nature and anti-tumor efficacy of the induced immune response was performed in vivo in healthy and pathological conditions, showing that the nature of antigen delivery is crucial for a specific T cell activation and targeted cytotoxic effect.
Descrição: Tese de doutoramento, Farmácia (Tecnologia Farmacêutica), Universidade de Lisboa, Faculdade de Farmácia, 2016
URI: http://hdl.handle.net/10451/26686
Designação: Doutoramento em Farmácia
Aparece nas colecções:FF - Teses de Doutoramento

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