RESUMO
When compartmentally mislocalized within cells, nucleic acids can be exceptionally immunostimulatory and can even trigger the immune-mediated elimination of cancer. Specifically, the accumulation of double-stranded DNA in the cytosol can efficiently promote antitumor immunity by activating the cGAMP synthase (cGAS) / stimulator of interferon genes (STING) cellular signaling pathway. Targeting this cytosolic DNA sensing pathway with interferon stimulatory DNA (ISD) is therefore an attractive immunotherapeutic strategy for the treatment of cancer. However, the therapeutic activity of ISD is limited by several drug delivery barriers, including susceptibility to deoxyribonuclease degradation, poor cellular uptake, and inefficient cytosolic delivery. Here, we describe the development of a nucleic acid immunotherapeutic, NanoISD, which overcomes critical delivery barriers that limit the activity of ISD and thereby promotes antitumor immunity through the pharmacological activation of cGAS at the forefront of the STING pathway. NanoISD is a nanoparticle formulation that has been engineered to confer deoxyribonuclease resistance, enhance cellular uptake, and promote endosomal escape of ISD into the cytosol, resulting in potent activation of the STING pathway via cGAS. NanoISD mediates the local production of proinflammatory cytokines via STING signaling. Accordingly, the intratumoral administration of NanoISD induces the infiltration of natural killer cells and T lymphocytes into murine tumors. The therapeutic efficacy of NanoISD is demonstrated in preclinical tumor models by attenuated tumor growth, prolonged survival, and an improved response to immune checkpoint blockade therapy.
Assuntos
DNA , Sistemas de Liberação de Medicamentos , Nanopartículas , Nucleotidiltransferases , Animais , Feminino , Humanos , Camundongos , Neoplasias do Colo/terapia , Citocinas/biossíntese , Citocinas/genética , DNA/administração & dosagem , DNA/síntese química , DNA/farmacologia , DNA/uso terapêutico , Ensaios de Seleção de Medicamentos Antitumorais , Endossomos/fisiologia , Imunoterapia/métodos , Células Matadoras Naturais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Mamárias Experimentais/terapia , Melanoma Experimental/terapia , Proteínas de Membrana/fisiologia , Camundongos Endogâmicos C57BL , Nanopartículas/administração & dosagem , Nanopartículas/uso terapêutico , Neoplasias/imunologia , Nucleotidiltransferases/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Tionucleotídeos/farmacologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
INTRODUCTION: Nucleic acids have gained recognition as promising immunomodulatory therapeutics. However, their potential is limited by several drug delivery barriers, and there is a need for technologies that enhance intracellular delivery of nucleic acid drugs. Furthermore, controlled and sustained release is a significant concern, as the kinetics and localization of immunomodulators can influence resultant immune responses. Here, we describe the design and initial evaluation of poly(lactic-co-glycolic) acid (PLGA) microparticle (MP) depots for enhanced retention and sustained release of endosomolytic nanoparticles that enable the cytosolic delivery of nucleic acids. METHODS: Endosomolytic p[DMAEMA]10kD-bl-[PAA0.3-co-DMAEMA0.3-co-BMA0.4]25kD diblock copolymers were synthesized by reversible addition-fragmentation chain transfer polymerization. Polymers were electrostatically complexed with nucleic acids and resultant nanoparticles (NPs) were encapsulated in PLGA MPs. To modulate release kinetics, ammonium bicarbonate was added as a porogen. Release profiles were quantified in vitro and in vivo via quantification of fluorescently-labeled nucleic acid. Bioactivity of released NPs was assessed using small interfering RNA (siRNA) targeting luciferase as a representative nucleic acid cargo. MPs were incubated with luciferase-expressing 4T1 (4T1-LUC) breast cancer cells in vitro or administered intratumorally to 4T1-LUC breast tumors, and silencing via RNA interference was quantified via longitudinal luminescence imaging. RESULTS: Endosomolytic NPs complexed to siRNA were effectively loaded into PLGA MPs and release kinetics could be modulated in vitro and in vivo via control of MP porosity, with porous MPs exhibiting faster cargo release. In vitro, release of NPs from porous MP depots enabled sustained luciferase knockdown in 4T1 breast cancer cells over a five-day treatment period. Administered intratumorally, MPs prolonged the retention of nucleic acid within the injected tumor, resulting in enhanced and sustained silencing of luciferase relative to a single bolus administration of NPs at an equivalent dose. CONCLUSION: This work highlights the potential of PLGA MP depots as a platform for local release of endosomolytic polymer NPs that enhance the cytosolic delivery of nucleic acid therapeutics.
RESUMO
Tissue-resident memory T cells (TRM) patrol nonlymphoid organs and provide superior protection against pathogens that commonly infect mucosal and barrier tissues, such as the lungs, intestine, liver, and skin. Thus, there is a need for vaccine technologies that can induce a robust, protective TRM response in these tissues. Nanoparticle (NP) vaccines offer important advantages over conventional vaccines; however, there has been minimal investigation into the design of NP-based vaccines for eliciting TRM responses. Here, we describe a pH-responsive polymeric nanoparticle vaccine for generating antigen-specific CD8+ TRM cells in the lungs. With a single intranasal dose, the NP vaccine elicited airway- and lung-resident CD8+ TRM cells and protected against respiratory virus challenge in both sublethal (vaccinia) and lethal (influenza) infection models for up to 9 weeks after immunization. In elucidating the contribution of material properties to the resulting TRM response, we found that the pH-responsive activity of the carrier was important, as a structurally analogous non-pH-responsive control carrier elicited significantly fewer lung-resident CD8+ T cells. We also demonstrated that dual-delivery of protein antigen and nucleic acid adjuvant on the same NP substantially enhanced the magnitude, functionality, and longevity of the antigen-specific CD8+ TRM response in the lungs. Compared to administration of soluble antigen and adjuvant, the NP also mediated retention of vaccine cargo in pulmonary antigen-presenting cells (APCs), enhanced APC activation, and increased production of TRM-related cytokines. Overall, these data suggest a promising vaccine platform technology for rapid generation of protective CD8+ TRM cells in the lungs.
Assuntos
Adjuvantes Imunológicos/farmacologia , Memória Imunológica/efeitos dos fármacos , Influenza Humana/imunologia , Pulmão/imunologia , Administração Intranasal , Animais , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Humanos , Concentração de Íons de Hidrogênio , Imunização/métodos , Imunogenicidade da Vacina/efeitos dos fármacos , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Pulmão/efeitos dos fármacos , Camundongos , Nanopartículas/química , Nanopartículas/uso terapêutico , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Vacinas/imunologia , Vacinas/farmacologiaRESUMO
Cyclic dinucleotide (CDN) agonists of stimulator of interferon genes (STING) are a promising class of immunotherapeutics that activate innate immunity to increase tumour immunogenicity. However, the efficacy of CDNs is limited by drug delivery barriers, including poor cellular targeting, rapid clearance and inefficient transport to the cytosol where STING is localized. Here, we describe STING-activating nanoparticles (STING-NPs)-rationally designed polymersomes for enhanced cytosolic delivery of the endogenous CDN ligand for STING, 2'3' cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). STING-NPs increase the biological potency of cGAMP, enhance STING signalling in the tumour microenvironment and sentinel lymph node, and convert immunosuppressive tumours to immunogenic, tumoricidal microenvironments. This leads to enhanced therapeutic efficacy of cGAMP, inhibition of tumour growth, increased rates of long-term survival, improved response to immune checkpoint blockade and induction of immunological memory that protects against tumour rechallenge. We validate STING-NPs in freshly isolated human melanoma tissue, highlighting their potential to improve clinical outcomes of immunotherapy.
Assuntos
Endossomos/metabolismo , Imunoterapia , Proteínas de Membrana/agonistas , Neoplasias/imunologia , Neoplasias/terapia , Polímeros/metabolismo , Animais , Citosol/metabolismo , Feminino , Humanos , Inflamação/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/química , Nanopartículas/ultraestrutura , Metástase Neoplásica , Nucleotídeos Cíclicos/metabolismo , Linfócitos T/imunologia , Microambiente TumoralRESUMO
Cancer vaccines targeting patient-specific tumor neoantigens have recently emerged as a promising component of the rapidly expanding immunotherapeutic armamentarium. However, neoantigenic peptides typically elicit weak CD8+ T cell responses, and so there is a need for universally applicable vaccine delivery strategies to enhance the immunogenicity of these peptides. Ideally, such vaccines could also be rapidly fabricated using chemically synthesized peptide antigens customized to an individual patient. Here, we describe a strategy for simple and rapid packaging of peptide antigens into pH-responsive nanoparticles with endosomal escape activity. Electrostatically-stabilized polyplex nanoparticles (nanoplexes) can be assembled instantaneously by mixing decalysine-modified antigenic peptides and poly(propylacrylic acid) (pPAA), a polyanion with pH-dependent, membrane destabilizing activity. These nanoplexes increase and prolong antigen uptake and presentation on MHC-I (major histocompatibility complex class I) molecules expressed by dendritic cells, resulting in enhanced activation of CD8+ T cells. Using an intranasal immunization route, nanoplex vaccines inhibit formation of lung metastases in a murine melanoma model. Additionally, nanoplex vaccines strongly synergize with the adjuvant α-galactosylceramide (α-GalCer) in stimulating robust CD8+ T cell responses, significantly increasing survival time in mice with established melanoma tumors. Collectively, these findings demonstrate that peptide/pPAA nanoplexes offer a facile and versatile platform for enhancing CD8+ T cell responses to peptide antigens, with potential to complement ongoing advancements in the development of neoantigen-targeted cancer vaccines.
Assuntos
Acrilatos/uso terapêutico , Vacinas Anticâncer/uso terapêutico , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Melanoma/patologia , Nanoconjugados/uso terapêutico , Peptídeos/uso terapêutico , Polímeros/uso terapêutico , Acrilatos/administração & dosagem , Acrilatos/imunologia , Administração Intranasal , Animais , Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Linhagem Celular , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Neoplasias Pulmonares/imunologia , Masculino , Melanoma/imunologia , Melanoma/terapia , Camundongos , Camundongos Endogâmicos C57BL , Nanoconjugados/administração & dosagem , Peptídeos/administração & dosagem , Peptídeos/imunologia , Polímeros/administração & dosagemRESUMO
Vaccine design has undergone a shift towards the use of purified protein subunit vaccines, which offer increased safety and greater control over antigen specificity, but at the expense of immunogenicity. Here we report the development of a new polymer-based vaccine delivery platform engineered to enhance immunity through the co-delivery of protein antigens and the Toll-like receptor 7 (TLR7) agonist imiquimod (IMQ). Owing to the preferential solubility of IMQ in fatty acids, a series of block copolymer micelles with a fatty acid-mimetic core comprising lauryl methacrylate (LMA) and methacrylic acid (MAA), and a poly(ethylene glycol) methyl ether methacrylate (PEGMA) corona decorated with pyridyl disulfide ethyl methacrylate (PDSM) moieties for antigen conjugation were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Carriers composed of 50 mole% LMA (LMA50) demonstrated the highest IMQ loading (2.2 w/w%) and significantly enhanced the immunostimulatory capacity of IMQ to induce dendritic cell maturation and proinflammatory cytokine production. Conjugation of a model antigen, ovalbumin (OVA), to the corona of IMQ-loaded LMA50 micelles enhanced in vitro antigen uptake and cross-presentation on MHC class I (MHC-I). A single intranasal (IN) immunization of mice with carriers co-loaded with IMQ and OVA elicited significantly higher pulmonary and systemic CD8+ T cell responses and increased serum IgG titer relative to a soluble formulation of antigen and adjuvant. Collectively, these data demonstrate that rationally designed fatty acid-mimetic micelles enhance intracellular antigen and IMQ delivery and have potential as synthetic vectors for enhancing the immunogenicity of subunit vaccines.
RESUMO
Previously synthesized poly(methacrylic acid-co-cholesteryl methacrylate) P(MAA-co-CMA) copolymers were examined as potential drug delivery vehicles. P(MAA-co-CMA) copolymers were fluorescently labelled and imaged in SHEP and HepG2 cells. To understand their cell internalization pathway endocytic inhibition studies were conducted. It was concluded that P(MAA-co-CMA) are taken up by the cells via clathrin-independent endocytosis (CIE) (both caveolae mediated and cholesterol dependent endocytosis) mechanisms. The formation and characterization of P(MAA-co-CMA)-doxorubicin (DOX) nanocomplexes was investigated by fluorescence lifetime imaging microscopy (FLIM), UV-Visible spectroscopy (UV-Vis) and dynamic light scattering (DLS) studies. The toxicity screening between P(MAA-co-CMA)-DOX nanocomplexes (at varying w/w ratios) and free DOX, revealed nanocomplexes to exhibit higher cytotoxicity towards cancer cells in comparison to normal cells. FLIM and confocal microscopy were employed for investigating the time-dependent release of DOX in SHEP cells and the cellular uptake profile of P(MAA-co-CMA)-DOX nanocomplexes in cancer and normal cell lines, respectively. The endocytic pathway of P(MAA-co-CMA)-DOX nanocomplexes were examined in SHEP and HepG2 cells via flow cytometry revealing the complexes to be internalized through both clathrin-dependent (CDE) and CIE mechanisms. The drug delivery profile, reported herein, illuminates the specific endocytic route and therapeutic efficiency of P(MAA-co-CMA)-DOX nanocomplexes strongly suggesting these particles to be promising candidates for in vivo applications.
Assuntos
Ésteres do Colesterol/química , Colesterol/química , Doxorrubicina/química , Endocitose/efeitos dos fármacos , Nanopartículas/química , Polímeros/química , Ácidos Polimetacrílicos/química , Antibióticos Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Ésteres do Colesterol/farmacologia , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos , Células Hep G2 , Humanos , Espectroscopia Fotoeletrônica , Ácidos Polimetacrílicos/farmacologiaRESUMO
A library of cholesterol-derived ionic copolymers were previously synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization as 'smart' gene delivery vehicles that hold diverse surface charges. Polyplex systems formed with anionic poly(methacrylic acid-co-cholesteryl methacrylate) (P(MAA-co-CMA)) and cationic poly(dimethylamino ethyl methacrylate-co-cholesteryl methacrylate) (Q-P(DMAEMA-co-CMA)) copolymer series were evaluated for their therapeutic efficiency. Cell viability assays, conducted on SHEP, HepG2, H460, and MRC5 cell lines, revealed that alterations in the copolymer composition (CMA mol %) affected the cytotoxicity profile. Increasing the number of cholesterol moieties in Q-P(DMAEMA-co-CMA) copolymers reduced the overall toxicity (in H460 and HepG2 cells) while P(MAA-co-CMA) series displayed no significant toxicity regardless of the CMA content. Agarose gel electrophoresis was employed to investigate the formation of stable polyplexes and determine their complete conjugation ratios. P(MAA-co-CMA) copolymer series were conjugated to DNA through a cationic linker, oligolysine, while Q-P(DMAEMA-co-CMA)-siRNA complexes were readily formed via electrostatic interactions at conjugation ratios beginning from 6:1:1 (oligolysine-P(MAA-co-CMA)-DNA) and 20:1 (Q-P(DMAEMA-co-CMA)-siRNA), respectively. The hydrodynamic diameter, ζ potential and complex stability of the polyplexes were evaluated in accordance to complexation ratios and copolymer composition by dynamic light scattering (DLS). The therapeutic efficiency of the conjugates was assessed in SHEP cells via transfection and imaging assays using RT-qPCR, Western blotting, flow cytometry, and confocal microscopy. DNA transfection studies revealed P(MAA-co-CMA)-oligolysine-DNA ternary complexes to be ineffective transfection vehicles that mostly adhere to the cell surface as opposed to internalizing and partaking in endosomal disrupting activity. The transfection efficiency of Q-P(DMAEMA-co-CMA)-GFP siRNA complexes were found to be polymer composition and N/P ratio dependent, with Q-2% CMA-GFP siRNA polyplexes at N/P ratio 20:1 showing the highest gene suppression in GFP expressing SHEP cells. Cellular internalization studies suggested that Q-P(DMAEMA-co-CMA)-siRNA conjugates efficiently escaped the endolysosomal pathway and released siRNA into the cytoplasm. The gene delivery profile, reported herein, illuminates the positive and negative attributes of each therapeutic design and strongly suggests Q-P(DMAEMA-co-CMA)-siRNA particles are extremely promising candidates for in vivo applications of siRNA therapy.
Assuntos
Colesterol/química , DNA/administração & dosagem , Polímeros/química , RNA Interferente Pequeno/administração & dosagem , Transfecção/métodos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Colesterol/administração & dosagem , Colesterol/farmacologia , Colesterol/toxicidade , Ésteres do Colesterol/administração & dosagem , Ésteres do Colesterol/química , Ésteres do Colesterol/toxicidade , Relação Dose-Resposta a Droga , Terapia Genética/métodos , Células Hep G2 , Humanos , Íons/administração & dosagem , Íons/química , Íons/farmacologia , Íons/toxicidade , Metacrilatos/administração & dosagem , Metacrilatos/química , Metacrilatos/toxicidade , Modelos Moleculares , Estrutura Molecular , Tamanho da Partícula , Polímeros/administração & dosagem , Polímeros/toxicidade , Ácidos Polimetacrílicos/administração & dosagem , Ácidos Polimetacrílicos/química , Ácidos Polimetacrílicos/toxicidade , Relação Estrutura-Atividade , Propriedades de SuperfícieRESUMO
Cholesterol has been used as an effective component of therapeutic delivery systems because of its ability to cross cellular membranes. Considering this, well-defined copolymers of methacrylic acid and cholesteryl methacrylate, poly(methacrylic acid-co-cholesteryl methacrylate) P(MAA-co-CMA), were generated as potential delivery system components for pH-controlled intracellular delivery of therapeutics. Statistical copolymers with varying cholesterol contents (2, 4, and 8 mol %) were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Dynamic light scattering (DLS) analysis showed that the hydrodynamic diameters of the copolymers in aqueous solutions ranged from 5 ± 0.3 to 7 ± 0.4 nm for the copolymers having 2 and 4 mol % CMA and 8 ± 1.1 to 13 ± 1.9 nm for the copolymer having 8 mol % CMA with increasing pH (pH 4.5-7.4). Atomic force microscopy (AFM) analysis revealed that the copolymer having 8 mol % CMA formed supramolecular assemblies while the copolymers having 2 and 4 mol % CMA existed as unimers in aqueous solution. The pH-responsive behavior of the copolymers was investigated via UV-visible spectroscopy revealing phase transitions at pH 3.9 for 2 mol % CMA, pH 4.7 for 4 mol % CMA, and pH 5.4 for 8 mol % CMA. Lipid bilayers and liposomes as models for cellular membranes were generated to probe their interactions with the synthesized copolymers. The interactions were determined in a pH-dependent manner (at pH 5.0 and 7.4) using surface plasmon resonance (SPR) spectroscopy and liposome leakage assay. Both the SPR analyses and liposome leakage assays indicated that the copolymer containing 2 mol % CMA displayed the greatest polymer-lipid interactions at pH 5.0, presenting the highest binding ability to the lipid bilayer surfaces, and also demonstrating the highest membrane destabilization activity. CellTiter-Blue assay showed that the copolymers did not affect the cell viability up to 30 µM over a period of 72 h.