RESUMO
Lipid membrane fusion is an essential process for a number of critical biological functions. The overall process is thermodynamically favorable but faces multiple kinetic barriers along the way. Inspired by nature's engineered proteins such as SNAP receptor [soluble N-ethylmale-imide-sensitive factor-attachment protein receptor (SNARE)] complexes or viral fusogenic proteins that actively promote the development of membrane proximity, nucleation of a stalk, and triggered expansion of the fusion pore, here we introduce a synthetic fusogen that can modulate membrane fusion and equivalently prime lipid membranes for calcium-triggered fusion. Our fusogen consists of a gold nanoparticle functionalized with an amphiphilic monolayer of alkanethiol ligands that had previously been shown to fuse with lipid bilayers. While previous efforts to develop synthetic fusogens have only replicated the initial steps of the fusion cascade, we use molecular simulations and complementary experimental techniques to demonstrate that these nanoparticles can induce the formation of a lipid stalk and also drive its expansion into a fusion pore upon the addition of excess calcium. These results have important implications in general understanding of stimuli-triggered fusion and the development of synthetic fusogens for biomedical applications.
Assuntos
Cálcio/metabolismo , Membrana Celular/metabolismo , Ouro/química , Bicamadas Lipídicas/metabolismo , Nanopartículas Metálicas/química , Cálcio/química , Membrana Celular/química , Ouro/metabolismo , Humanos , Bicamadas Lipídicas/química , Fusão de Membrana , Simulação de Dinâmica Molecular , Proteínas SNARE/metabolismo , Análise Serial de TecidosRESUMO
Although immunotherapy shows great promise for the long-term control of cancer, many tumors still fail to respond to treatment. To improve the outcome, the delivery of immunostimulants to the lymph nodes draining the tumor, where the antitumor immune response is initiated, is key. Efforts to use nanoparticles as carriers for cancer immunotherapy have generally required targeting agents and chemical modification of the drug, and have unfortunately resulted in low delivery and therapeutic efficiency. Here, we report on the efficacy of gold nanoparticles with approximately 5â¯nm hydrodynamic diameter coated with a mixture of 1-octanethiol and 11-mercaptoundecanesulfonic acid for the delivery of an immunostimulatory TLR7 ligand to tumor-draining lymph nodes. The drug was loaded without modification through nonspecific adsorption into the ligand shell of the nanoparticles, taking advantage of their amphiphilic nature. After loading, nanoparticles retained their stability in solution without significant premature release of the drug, and the drug cargo was immunologically active. Upon subcutaneous injection into tumor-bearing mice, the drug-loaded particles were rapidly transported to the tumor-draining lymph nodes. There, they induced a local immune activation and fostered a cytotoxic T-cell response that was specific for the tumor. Importantly, the particle-delivered TLR7 ligand blocked the growth of large established tumors and significantly prolonged survival compared to the free form of the drug. Thus, we demonstrate for the first time that nanoparticle delivery of a TLR7 immunostimulant to the tumor-draining lymph nodes enhances antitumor immunity and improves the outcome of cancer immunotherapy.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Neoplasias do Colo/terapia , Portadores de Fármacos/química , Ouro/química , Nanopartículas Metálicas/química , Receptor 7 Toll-Like/agonistas , Adjuvantes Imunológicos/uso terapêutico , Animais , Linhagem Celular , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Imunoterapia , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Camundongos Endogâmicos C57BL , Receptor 7 Toll-Like/imunologiaRESUMO
We sought to develop a nanoparticle vehicle that could efficiently deliver small molecule drugs to target lymphocyte populations. The synthesized amphiphilic organic ligand-protected gold nanoparticles (amph-NPs) were capable of sequestering large payloads of small molecule drugs within hydrophobic pockets of their ligand shells. These particles exhibit membrane-penetrating activity in mammalian cells, and thus enhanced uptake of a small molecule TGF-ß inhibitor in T cells in cell culture. By conjugating amph-NPs with targeting antibodies or camelid-derived nanobodies, the particles' cell-penetrating properties could be temporarily suppressed, allowing targeted uptake in specific lymphocyte subpopulations. Degradation of the protein targeting moieties following particle endocytosis allowed the NPs to recover their cell-penetrating activity in situ to enter the cytoplasm of T cells. In vivo, targeted amph-NPs showed 40-fold enhanced uptake in CD8+ T cells relative to untargeted particles, and delivery of TGF-ß inhibitor-loaded particles to T cells enhanced their cytokine polyfunctionality in a cancer vaccine model. Thus, this system provides a facile approach to concentrate small molecule compounds in target lymphocyte populations of interest for immunotherapy in cancer and other diseases.
Assuntos
Sistemas de Liberação de Medicamentos , Ouro/química , Imunoconjugados/química , Nanopartículas Metálicas/química , Bibliotecas de Moléculas Pequenas/administração & dosagem , Linfócitos T/efeitos dos fármacos , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Células Cultivadas , Feminino , Ouro/farmacocinética , Imunoconjugados/farmacocinética , Camundongos Endogâmicos C57BL , Bibliotecas de Moléculas Pequenas/farmacologia , Linfócitos T/imunologia , Fator de Crescimento Transformador beta/análiseRESUMO
This corrects the article DOI: 10.1038/ncomms14069.
RESUMO
Inorganic nanoparticles (NPs) are studied as drug carriers, radiosensitizers and imaging agents, and characterizing nanoparticle biodistribution is essential for evaluating their efficacy and safety. Tracking NPs at the single-cell level with current technologies is complicated by the lack of reliable methods to stably label particles over extended durations in vivo. Here we demonstrate that mass cytometry by time-of-flight provides a label-free approach for inorganic nanoparticle quantitation in cells. Furthermore, mass cytometry can enumerate AuNPs with a lower detection limit of â¼10 AuNPs (3 nm core size) in a single cell with tandem multiparameter cellular phenotyping. Using the cellular distribution insights, we selected an amphiphilic surface ligand-coated AuNP that targeted myeloid dendritic cells in lymph nodes as a peptide antigen carrier, substantially increasing the efficacy of a model vaccine in a B16-OVA melanoma mouse model. This technology provides a powerful new level of insight into nanoparticle fate in vivo.