Comparing continuous micromixing and extrusion downsizing for PEGylated nanoliposomes remotely loaded with doxorubicin or the steroid pro-drug methylprednisolone hemisuccinate

Abstract

Since the FDA approval of the first nanodrug Doxil® in 1995, twenty subsequent liposome-and lipid nanoparticle (LNP) based drugs (of which 10 are nanodrugs), were approved by the FDA. The application of such drug-products was considerably boosted by the mRNA-LNP based vaccines used to stop the COVID-19 pandemic. Research on lipid-based vesicles and nanoparticles for drug delivery dates to the 1970s and has culminated in both continuous flow and extrusion-based fabrication processes for current state-of-the-art GMP industrial production of nanoliposomes and lipid nanoparticles.

In this study, we compare these two approaches for the preparation of two PEGylated nanoliposome-based drug-products, keeping all other production steps leading to the final drug-product identical. One of these products, generic Doxil®, is remotely and actively loaded with the anthracycline doxorubicin (an amphipathic weak base) driven by a transmembrane ammonium gradient, while the other is methylprednisolone hemisuccinate (an amphipathic weak acid) remotely and actively loaded via a transmembrane acetate gradient. We demonstrate that a microfluidics-based micromixer approach yields equivalent or even better drug-products, especially since the downsizing by microfluidic is not performed above the temperature range of lipid phase transition.

The main difference in the physico-chemical features is that size distribution of the microfluidics prepared pegylated nano liposomes was significantly narrower and morphological analysis by cryo-TEM confirmed higher homogeneity. An additional advantage of the microfluidic approach is that it is a continuous production. Therefore, it enables the direct production of large volumes of high-quality nano-liposomal based drug- products.

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Materials

Unless otherwise indicated, all chemicals and lipids were used as supplied by the vendor without further purification. Sucrose (EMPROVE® ESSENTIAL, Ph. Eur., BP, ChP, JP, NF), ammonium sulfate (EMSURE® ACS, ISO, Reag. Ph Eur.), calcium acetate hydrate (extra pure), Dowex™ 50 WX-4 cation exchange resin (Na+ form, 200–400 mesh), Dowex 2×-800 anion exchanger and absolute ethanol (EMPROVE® EXPERT Ph Eur., BP, ChP, JP, USP) were obtained from Merck KGaA (Darmstadt, Germany). Doxorubicin hydrochloride (DXR) was purchased from Teva Pharmaceuticals (Israel). Methylprednisolone 21 hemisuccinate (MPS; Solu-Medrol®) was obtained from Pfizer. The lipid mixture of hydrogenated soybean phosphatidylcholine (HSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)2000] (DSPE-mPEG2000) and cholesterol was provided partly commercially, but mostly free of charge by Lipoid GmbH (Ludwigshafen am Rhein, Germany). Triton® X-100 for liposome lysis was obtained from Carl Roth GmbH & Co. KG (Karlsruhe, Germany). The highly pure water used for the drug stock and salt solutions was dispensed from a Milli-Q Plus purification system (Merck Millipore) with a QPAK® 2 purification cartridge (Merck KGaA, Darmstadt, Germany). All solutions were filtered through sterile filters (Stericup Filter Unit Millipore Durapore 0.22 μm) from Merck KGaA before use in the micromixer to prevent contamination-induced clogging.

Keren Turjeman, Alexander-Nicholas Egler-Kemmerer, Dima Shamrakov, Christine Paulus, Raphael Thiermann, Yechezkel Barenholz, Regina Bleul, Comparing continuous micromixing and extrusion downsizing for PEGylated nanoliposomes remotely loaded with doxorubicin or the steroid pro-drug methylprednisolone hemisuccinate,
Journal of Controlled Release, 2025, 113707, ISSN 0168-3659, https://doi.org/10.1016/j.jconrel.2025.113707.

Read also our introduction article on Lipid Nanoparticles here: