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Review
. 2023 Sep 18;4(3):031304.
doi: 10.1063/5.0150345. eCollection 2023 Sep.

Microfluidic approaches for producing lipid-based nanoparticles for drug delivery applications

Caterina Piunti, Elisa Cimetta
Review

Microfluidic approaches for producing lipid-based nanoparticles for drug delivery applications

Caterina Piunti et al. Biophys Rev (Melville). .

Abstract

The importance of drug delivery for disease treatment is supported by a vast literature and increasing ongoing clinical studies. Several categories of nano-based drug delivery systems have been considered in recent years, among which lipid-based nanomedicines, both artificial and cell-derived, remain the most approved. The best artificial systems in terms of biocompatibility and low toxicity are liposomes, as they are composed of phospholipids and cholesterol, the main components of cell membranes. Extracellular vesicles-biological nanoparticles released from cells-while resembling liposomes in size, shape, and structure, have a more complex composition with up to hundreds of different types of lipids, proteins, and carbohydrates in their membranes, as well as an internal cargo. Although nanoparticle technologies have revolutionized drug delivery by enabling passive and active targeting, increased stability, improved solubilization capacity, and reduced dose and adverse effects, the clinical translation remains challenging due to manufacturing limitations such as laborious and time-consuming procedures and high batch-to-batch variability. A sea change occurred when microfluidic strategies were employed, offering advantages in terms of precise particle handling, simplified workflows, higher sensitivity and specificity, and good reproducibility and stability over bulk methods. This review examines scientific advances in the microfluidics-mediated production of lipid-based nanoparticles for therapeutic applications. We will discuss the preparation of liposomes using both hydrodynamic focusing of microfluidic flow and mixing by herringbone and staggered baffle micromixers. Then, an overview on microfluidic approaches for producing extracellular vesicles and extracellular vesicles-mimetics for therapeutic applications will describe microfluidic extrusion, surface engineering, sonication, electroporation, nanoporation, and mixing. Finally, we will outline the challenges, opportunities, and future directions of microfluidic investigation of lipid-based nanoparticles in the clinic.

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Conflict of interest statement

The authors have no conflicts to disclose.

Figures

FIG. 1.
FIG. 1.
Microfluidic-based liposome production. (A-a) Schematic of a fully integrated microfluidic device made of PDMS and cellulose for liposome synthesis using HFF, buffer exchange via microdialysis and drug loading and (A-b) photograph of the fabricated device. Reproduced with permission from Hood et al., Lab Chip 14(17), 3359–3367 (2014). Copyright 2014 Royal Society of Chemistry. (B) Liposome formation through nanoprecipitation. (B-a) Schematic of a device. Liposomes form through self-assembly when the lipid solution is met with the aqueous buffer from the adjacent channels. (B-b) Fluorescently labeled liposomes with functional groups (PEG-Lip, FA-Lip, TAT-Lip, and FA-TAT-Lip) produced using an HFF-based device and tested on SKOV3 tumor spheroids; scale bars: 200 μm. (B-c) Flow cytometry results demonstrated an increased uptake by the FA-TAT-Lip of 37% and 98% compared to the single ligand TAT-Lip and FA-Lip, respectively. Reproduced with permission from Ran et al., Eur. J. Pharm. Biopharm. 130, 1–10 (2018). Copyright 2018 Elsevier. (C-a) Liposomal curcumins (Lipo-Cur) were prepared by a microfluidic platform equipped with a staggered herringbone micromixer. Reproduced with permission from Stroock et al., Science 295(5555), 647–651 (2002). Copyright 2002 The American Association for the Advancement of Science. (C-b) Lipo-Cur antitumor activity was evaluated in tumor models in mice with EMT6 murine breast tumor cells inoculated into BALB/c mice. 7 days post tumor inoculation, the mice received an injection of either saline, free cisplatin (CDDP), Lipo-Cur, or combination of CDDP and Lipo-Cur. The combination treatment displayed enhanced effect as demonstrated by tumor growth kinetics. Lipo-Cur had also a protective effect against CDDP-induced kidney toxicity. (C-c) The kidney isolated from BALB/C mice was treated with either saline, Lipo-Cur, CDDP, or combination of Lipo-Cur and CDDP. CDDP induced significant acute tubular necrosis as indicated by the arrows, while the kidney histology was normal in other groups. Reproduced with permission from Hamano et al., Mol. Pharm. 16(9), 3957–3967 (2019). Copyright 2019 American Chemical Society. (D-a) Schematic of the iLiNP device featuring a two-dimensional baffle mixer enabling liposomes production and precise size tuning within a range of 20–100 nm, with intervals as small as 10 nm. (D-b) Computational fluid dynamic simulation of ethanol dilution in the iLiNP device at different flow rates demonstrated that the dilution performance was dramatically accelerated at 500 μl/min, and ethanol was completely diluted within 3 ms. Reproduced with permission from Kimura et al., ACS Omega, 3(5), 5044–5051 (2018). Copyright 2018 American Chemical Society.
FIG. 2.
FIG. 2.
Microfluidic-based production of engineered EVs. (A-a) and (A-b) Schematic of the microfluidic system for the generation of self-assembled nanovesicles by slicing cells with 500 nm-thick silicon nitride blades. During self-assembly, the plasma membrane fragments envelop exogenous materials (here, fluorescent polystyrene beads) that is then successfully delivered across the plasma membrane of recipient cells (MEF-GFP, (A-c); scale bars: 20 μm [Yoon et al., Biomaterials, 59, 12–20 (2015). Copyright 2015 Authors, licensed under a Creative Commons Attribution (CC BY) license]. (b-a) Schematic of the 3D molded microfluidic device enabling real-time harvesting, antigenic modification (e.g., gp-100), and subsequent photo-release of engineered antigenic EVs. A fluorescent solution was used to demonstrate the flow of immunomagnetic beads mixing with cell culture media (B-b), while bright-field microscopy images showed the serpentine microchannel (B-c) and the on-chip cultured leukocytes (B-d); and (B-e) SEM image of the engineered EVs, visible in (f) labeled with green membrane dye PKH67 in confocal images showing that gp100 enhanced cellular uptake from dendritic monocytes by ∼2 fold compared to native EVs; scale bars: 5 μm. Reproduced with permission from Zhao et al., Lab Chip 19(10), 1877–1886 (2019). Copyright 2019 Royal Society of Chemistry. (c-a) Nanoporation device for the generation of EVs for targeted nucleic acid delivery. EVs containing PTEN mRNA (Exo-T) were used to treat PTEN-deficient glioma models: mice treated with Exo-Ts showed inhibited tumor growth (C-b) and prolonged survival (C-c) compared with non-targeted EVs (exosomes), empty EVs (E-Exo-T), TurboFect nanoparticles (Turbo), or PBS. Reproduced with permission from Armstrong et al., Nat. Biomed. Eng. 4(1), 69–83 (2020). Copyright 2019 Springer Nature Limited. (D-a) Schematic of a device to prepare DOX-carrying EVs via generation of membrane nanopores by mechanical compression; (D-b) the effect of DOX-EVs was analyzed using a tumor spheroid model after 48 h incubation: DOX-EVs treated spheroids measured a 17% decrease in cross section, indicating an EVs-mediated inhibitory effect on tumor growth; scale bars: 100 μm. Reproduced with permission from Hao et al., Small 17, 2102150 (2021). Copyright 2021 Wiley-VCH GmbH.
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