RESUMO
Current strategies for treating autoimmunity involve the administration of broad-acting immunosuppressive agents that impair healthy immunity. Intravenous (i.v.) administration of poly(lactide- co-glycolide) nanoparticles (NPs) containing disease-relevant antigens (Ag-NPs) have demonstrated antigen (Ag)-specific immune tolerance in models of autoimmunity. However, subcutaneous (s.c.) delivery of Ag-NPs has not been effective. This investigation tested the hypothesis that codelivery of the immunomodulatory cytokine, transforming growth factor beta 1 (TGF-ß), on Ag-NPs would modulate the immune response to Ag-NPs and improve the efficiency of tolerance induction. TGF-ß was coupled to the surface of Ag-NPs such that the loadings of Ag and TGF-ß were independently tunable. The particles demonstrated bioactive delivery of Ag and TGF-ß in vitro by reducing the inflammatory phenotype of bone marrow-derived dendritic cells and inducing regulatory T cells in a coculture system. Using an in vivo mouse model for multiple sclerosis, experimental autoimmune encephalomyelitis, TGF-ß codelivery on Ag-NPs resulted in improved efficacy at lower doses by i.v. administration and significantly reduced disease severity by s.c. administration. This study demonstrates that the codelivery of immunomodulatory cytokines on Ag-NPs may enhance the efficacy of Ag-specific tolerance therapies by programming Ag presenting cells for more efficient tolerance induction.
Assuntos
Antígenos/administração & dosagem , Encefalomielite Autoimune Experimental/tratamento farmacológico , Fatores Imunológicos/administração & dosagem , Esclerose Múltipla/tratamento farmacológico , Nanoconjugados/administração & dosagem , Poliglactina 910/administração & dosagem , Fator de Crescimento Transformador beta/administração & dosagem , Animais , Antígenos/química , Antígenos/uso terapêutico , Células Cultivadas , Encefalomielite Autoimune Experimental/imunologia , Feminino , Tolerância Imunológica/efeitos dos fármacos , Fatores Imunológicos/química , Fatores Imunológicos/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/imunologia , Nanoconjugados/química , Nanoconjugados/uso terapêutico , Poliglactina 910/química , Poliglactina 910/uso terapêutico , Fator de Crescimento Transformador beta/química , Fator de Crescimento Transformador beta/uso terapêuticoRESUMO
Polymeric nanoparticles (NPs) have demonstrated their potential to induce antigen (Ag)-specific immunological tolerance in multiple immune models and are at various stages of commercial development. Association of Ag with NPs is typically achieved through surface coupling or encapsulation methods. However, these methods have limitations that include high polydispersity, uncontrollable Ag loading and release, and possible immunogenicity. Here, using antigenic peptides conjugated to poly(lactide-co-glycolide), we developed Ag-polymer conjugate NPs (acNPs) with modular loading of single or multiple Ags, negligible burst release, and minimally exposed surface Ag. Tolerogenic responses of acNPs were studied in vitro to decouple the role of NP size, concentration, and Ag loading on regulatory T cell (Treg) induction. CD4+CD25+Foxp3+ Treg induction was dependent on NP size, but CD25 expression of CD4+ T cells was not. NP concentration and Ag loading could be modulated to achieve maximal levels of Treg induction. In relapsing-remitting experimental autoimmune encephalomyelitis (R-EAE), a murine model of multiple sclerosis, acNPs were effective in inhibiting disease induced by a single peptide or multiple peptides. The acNPs provide a simple, modular, and well-defined platform, and the NP physicochemical properties offer potential to design and answer complex mechanistic questions surrounding NP-induced tolerance.
Assuntos
Antígenos/farmacologia , Preparações de Ação Retardada/química , Encefalomielite Autoimune Experimental/terapia , Imunoconjugados/farmacologia , Proteína Proteolipídica de Mielina/farmacologia , Nanopartículas/química , Ovalbumina/farmacologia , Animais , Antígenos/química , Antígenos/imunologia , Biomarcadores/metabolismo , Antígenos CD4/genética , Antígenos CD4/imunologia , Preparações de Ação Retardada/administração & dosagem , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Expressão Gênica , Tolerância Imunológica/efeitos dos fármacos , Imunoconjugados/química , Imunoconjugados/metabolismo , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína Proteolipídica de Mielina/química , Proteína Proteolipídica de Mielina/imunologia , Nanopartículas/administração & dosagem , Ovalbumina/química , Ovalbumina/imunologia , Tamanho da Partícula , Poliglactina 910/química , Poliglactina 910/metabolismo , Cultura Primária de Células , Baço/efeitos dos fármacos , Baço/imunologia , Baço/patologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologiaRESUMO
Flash NanoPrecipitation (FNP) is a scalable, single-step process that uses rapid mixing to prepare nanoparticles with a hydrophobic core and amphiphilic stabilizing shell. Because the two steps of particle self-assembly - (1) core nucleation and growth and (2) adsorption of a stabilizing polymer onto the growing core surface - occur simultaneously during FNP, nanoparticles formulated at core loadings above approximately 70% typically exhibit poor stability or do not form at all. Additionally, a fundamental limit on the concentration of total solids that can be introduced into the FNP process has been reported previously. These limits are believed to share a common mechanism: entrainment of the stabilizing polymer into the growing particle core, leading to destabilization and aggregation. Here, we demonstrate a variation of FNP which separates the nucleation and stabilization steps of particle formation into separate sequential mixers. This scheme allows the hydrophobic core to nucleate and grow in the first mixing chamber unimpeded by adsorption of the stabilizing polymer, which is later introduced to the growing nuclei in the second mixer. Using this Sequential Flash NanoPrecipitation (SNaP) technique, we formulate stable nanoparticles with up to 90% core loading by mass and at 6-fold higher total input solids concentrations than typically reported.
Assuntos
Nanopartículas , Polímeros , Tamanho da Partícula , Polímeros/química , Nanopartículas/química , Interações Hidrofóbicas e HidrofílicasRESUMO
To mitigate antimicrobial resistance, we developed polymeric nanocarrier delivery of the chemorepellent signaling agent, nickel, to interfere with Escherichia coli transport to a surface, an incipient biofilm formation stage. The dynamics of nickel nanocarrier (Ni NC) chemorepellent release and induced chemorepellent response required to effectively modulate bacterial transport for biofilm prevention were characterized in this work. Ni NCs were fabricated with the established Flash NanoPrecipitation method. NC size was characterized with dynamic light scattering. Measured with a zincon monosodium salt colorimetric assay, NC nickel release was pH-dependent, with 62.5% of total encapsulated nickel released at pH 7 within 0-15 min, competitive with rapid E. coli transport to the surface. Confocal laser scanning microscopy of E. coli (GFP-expressing) biofilm growth dynamics on fluorescently labeled Ni NC coated glass coupled with a theoretical dynamical criterion probed the biofilm prevention outcomes of NC design. The Ni NC coating significantly reduced E. coli attachment compared to a soluble nickel coating and reduced E. coli biomass area by 61% compared to uncoated glass. A chemical-in-plug assay revealed Ni NCs induced a chemorepellent response in E. coli. A characteristic E. coli chemorepellent response was observed away from the Ni NC coated glass over 10 µm length scales effective to prevent incipient biofilm surface attachment. The dynamical criterion provided semiquantitative analysis of NC mechanisms to control biofilm and informed optimal chemorepellent release profiles to improve NC biofilm inhibition. This work is fundamental for dynamical informed design of biofilm-inhibiting chemorepellent-loaded NCs promising to mitigate the development of resistance and interfere with the transport of specific pathogens.