title: High-throughput manufacturing of size-tuned liposomes by a new microfluidics method using enhanced statistical tools for characterization.
abstract: Microfluidics has recently emerged as a new method of manufacturing liposomes, which allows for reproducible mixing in miliseconds on the nanoliter scale. Here we investigate microfluidics-based manufacturing of liposomes. The aim of these studies was to assess the parameters in a microfluidic process by varying the total flow rate (TFR) and the flow rate ratio (FRR) of the solvent and aqueous phases. Design of experiment and multivariate data analysis were used for increased process understanding and development of predictive and correlative models. High FRR lead to the bottom-up synthesis of liposomes, with a strong correlation with vesicle size, demonstrating the ability to in-process control liposomes size; the resulting liposome size correlated with the FRR in the microfluidics process, with liposomes of 50 nm being reproducibly manufactured. Furthermore, we demonstrate the potential of a high throughput manufacturing of liposomes using microfluidics with a four-fold increase in the volumetric flow rate, maintaining liposome characteristics. The efficacy of these liposomes was demonstrated in transfection studies and was modelled using predictive modeling. Mathematical modelling identified FRR as the key variable in the microfluidic process, with the highest impact on liposome size, polydispersity and transfection efficiency. This study demonstrates microfluidics as a robust and high-throughput method for the scalable and highly reproducible manufacture of size-controlled liposomes. Furthermore, the application of statistically based process control increases understanding and allows for the generation of a design-space for controlled particle characteristics.
authors.affiliation: Medicines Research Unit, School of Life and Health Sciences, Aston University, Birmingham B4 7ET, UK.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Kastner
authors.affiliation: Medicines Research Unit, School of Life and Health Sciences, Aston University, Birmingham B4 7ET, UK.
authors.forename: Randip
authors.initials: R
authors.lastname: Kaur
authors.affiliation: Medicines Research Unit, School of Life and Health Sciences, Aston University, Birmingham B4 7ET, UK.
authors.forename: Deborah
authors.initials: D
authors.lastname: Lowry
authors.affiliation: Medicines Research Unit, School of Life and Health Sciences, Aston University, Birmingham B4 7ET, UK.
authors.forename: Behfar
authors.initials: B
authors.lastname: Moghaddam
authors.affiliation: Medicines Research Unit, School of Life and Health Sciences, Aston University, Birmingham B4 7ET, UK.
authors.forename: Alexander
authors.initials: A
authors.lastname: Wilkinson
authors.affiliation: Medicines Research Unit, School of Life and Health Sciences, Aston University, Birmingham B4 7ET, UK. Electronic address: y.perrie@aston.ac.uk.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Perrie
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004247:DNA / Q000008:administration & dosage / Q000235:genetics; D004337:Drug Carriers / Q000737:chemistry; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry; D057166:High-Throughput Screening Assays; D008081:Liposomes; D044085:Microfluidics / Q000379:methods* / Q000706:statistics & numerical data*; D015999:Multivariate Analysis; D010316:Particle Size; D010714:Phosphatidylethanolamines / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D013678:Technology, Pharmaceutical / Q000379:methods* / Q000706:statistics & numerical data*; D014162:Transfection
chemical_list: C410262:1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine; D004337:Drug Carriers; D005229:Fatty Acids, Monounsaturated; D008081:Liposomes; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; D004247:DNA; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane
keywords: DNA delivery; Design of experiment; High-throughput manufacturing; Liposomes; Microfluidics