RESUMO
Nanomedicines are well recognised for their ability to improve therapeutic outcomes. Yet, due to their complexity, nanomedicines are challenging and costly to produce using traditional manufacturing methods. For nanomedicines to be widely exploited, new manufacturing technologies must be adopted to reduce development costs and provide a consistent product. Within this study, we investigate microfluidic manufacture of nanomedicines. Using protein-loaded liposomes as a case study, we manufacture liposomes with tightly defined physico-chemical attributes (size, PDI, protein loading and release) from small-scale (1 mL) through to GMP volume production (200 mL/min). To achieve this, we investigate two different laminar flow microfluidic cartridge designs (based on a staggered herringbone design and a novel toroidal mixer design); for the first time we demonstrate the use of a new microfluidic cartridge design which delivers seamless scale-up production from bench-scale (12 mL/min) through GMP production requirements of over 20 L/h using the same standardised normal operating parameters. We also outline the application of tangential flow filtration for down-stream processing and high product yield. This work confirms that defined liposome products can be manufactured rapidly and reproducibly using a scale-independent production process, thereby de-risking the journey from bench to approved product.
Assuntos
Doxorrubicina/química , Lipídeos/química , Microfluídica , Nanomedicina , Nanopartículas , Ovalbumina/química , Doxorrubicina/administração & dosagem , Doxorrubicina/normas , Composição de Medicamentos , Liberação Controlada de Fármacos , Lipídeos/normas , Lipossomos , Microfluídica/instrumentação , Microfluídica/normas , Nanomedicina/instrumentação , Nanomedicina/normas , Ovalbumina/administração & dosagem , Ovalbumina/normas , Tamanho da Partícula , Controle de Qualidade , SolubilidadeRESUMO
We present a programmable droplet-based microfluidic device that combines the reconfigurable flow-routing capabilities of integrated microvalve technology with the sample compartmentalization and dispersion-free transport that is inherent to droplets. The device allows for the execution of user-defined multistep reaction protocols in 95 individually addressable nanoliter-volume storage chambers by consecutively merging programmable sequences of picoliter-volume droplets containing reagents or cells. This functionality is enabled by "flow-controlled wetting," a droplet docking and merging mechanism that exploits the physics of droplet flow through a channel to control the precise location of droplet wetting. The device also allows for automated cross-contamination-free recovery of reaction products from individual chambers into standard microfuge tubes for downstream analysis. The combined features of programmability, addressability, and selective recovery provide a general hardware platform that can be reprogrammed for multiple applications. We demonstrate this versatility by implementing multiple single-cell experiment types with this device: bacterial cell sorting and cultivation, taxonomic gene identification, and high-throughput single-cell whole genome amplification and sequencing using common laboratory strains. Finally, we apply the device to genome analysis of single cells and microbial consortia from diverse environmental samples including a marine enrichment culture, deep-sea sediments, and the human oral cavity. The resulting datasets capture genotypic properties of individual cells and illuminate known and potentially unique partnerships between microbial community members.
Assuntos
Hidrodinâmica , Metagenoma/genética , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Sequência de Bases , Primers do DNA/genética , Genótipo , Sedimentos Geológicos/microbiologia , Humanos , Processamento de Imagem Assistida por Computador , Metagenômica/métodos , Microscopia de Fluorescência , Dados de Sequência Molecular , Boca/microbiologia , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Tensoativos , MolhabilidadeRESUMO
Lipid nanoparticles (LNP) are the leading systems for in vivo delivery of small interfering RNA (siRNA) for therapeutic applications. Formulation of LNP siRNA systems requires rapid mixing of solutions containing cationic lipid with solutions containing siRNA. Current formulation procedures employ macroscopic mixing processes to produce systems 70-nm diameter or larger that have variable siRNA encapsulation efficiency, homogeneity, and reproducibility. Here, we show that microfluidic mixing techniques, which permit millisecond mixing at the nanoliter scale, can reproducibly generate limit size LNP siRNA systems 20 nm and larger with essentially complete encapsulation of siRNA over a wide range of conditions with polydispersity indexes as low as 0.02. Optimized LNP siRNA systems produced by microfluidic mixing achieved 50% target gene silencing in hepatocytes at a dose level of 10 µg/kg siRNA in mice. We anticipate that microfluidic mixing, a precisely controlled and readily scalable technique, will become the preferred method for formulation of LNP siRNA delivery systems.