Modelling protein therapeutic co-formulation and co-delivery with PLGA nanoparticles continuously manufactured by microfluidics

摘要

Formulating protein therapeutics into nanoparticles (NPs) of poly(lactic- -glycolic acid) (PLGA) provides key features such as protection against clearance, sustained release and less side effects by possible at- tachment of targeting ligands. These NPs also offer the potential for protein combination therapy, which is expected to exploit synergetic bioresponses, avoid multiple dosage regimens and consequent mis-dosing. Since the conventional manufacture of protein-loaded PLGA NPs is still associated with low-throughput, new continuous manufacturing methods such as microfluidics have been established. Herein, PLGA NPs continuously manufactured through microfluidics are proposed for co-formulation and -delivery of two model proteins consisting of bovine serum albumin (BSA) conjugated to either fluorescein (FITC) or tetra- methylrhodamine (TRITC) isothiocyanates. Protein co-formulated NPs of 100 nm, monodispersed and with 70% of association efficiency were obtained. The microfluidic setup allowed a production rate of around 7 g of particles per day and demonstrated scale-up capacity. Model proteins were released in a controlled manner and without significant changes in their secondary structure. Studies in macrophage-like cells proved that protein co-formulated PLGA NPs did not impair metabolic activity (>70%). The cellular associa- tion of the proteins was around 2-times higher when co-formulated into PLGA NPs, compared to the free protein controls. Moreover, the cellular association of the co-formulated proteins was 4-times higher than the physical mixture of NPs individually loaded with each protein type. This work has demonstrated the ef- fectiveness of continuously manufactured PLGA NPs for co-formulating and enhancing the cellular associ- ation of co-delivered model proteins, providing a proof-of-concept foundation for future protein combi- nation nanotherapies.