Influence of stabilizer, size and physical state of lipid nanoparticles on uptake by THP-1 macrophages

Abstract

Macrophages, as cells of the innate immune response, are involved in the pathology of various medical conditions (tumors, intracellular infections, inflammatory diseases etc.). Due to their phagocytic nature, a drug targeting approach to macrophages based on lipid nanoparticles appears promising. Thus, the uptake of different kinds of fluorescently labeled lipid nanoparticles by THP-1 macrophages was assessed with flow cytometry and confocal laser scanning microscopy. 100 nm trilaurin nanoemulsions were produced with different stabilizers to compare the influence of nanoparticle coating on the uptake by THP-1 macrophages. After 2 and 24 h, stabilization with poloxamer 407 led to a significantly reduced uptake compared to a mixture of phosphatidylcholine with sodium glycocholate (PC/SGC) or with the ‘eat me’ signal phosphatidylserine (PC/PS). The benefit of PC/PS-stabilization over PC/SGC was less pronounced after 24 h, but still significant. A droplet size increase from 100 to 200 nm did not lead to a distinctly higher uptake of the PC/PS-stabilized trilaurin nanodroplets. Changing the matrix lipid of the emulsion from trilaurin to trimyristin (C12 vs. C14 fatty acid chains) resulted in enhanced uptake after 24 h, but not after 2 h. A change in the physical state of the matrix lipid by crystallization of supercooled trimyristin nanoparticles inhibited the cellular uptake of PC/SGC- and P407-stabilized 100 nm particles. A change in particle shape upon crystallization may contribute to this effect. Thus, triglyceride nanoemulsions stabilized with PC/PS and a size of 100 or 200 nm seem most promising regarding the uptake by macrophages.

Introduction

Macrophages are a heterogeneous group of phagocytes of the innate immune system. They reside in various tissues (liver, lung, brain etc.) and have numerous functions including phagocytosis, immunomodulation, antigen presentation and tissue homeostasis (Chen et al., 2023). Due to their defensive nature and involvement in inflammatory processes, they affect the pathology of several diseases. These include inflammatory diseases, like atherosclerosis and heart attack (Chen et al., 2022) as well as infectious diseases (Kumar and Herbein, 2014, Weiss and Schaible, 2015, Pham and Monack, 2023). Attention has also been drawn to tumor-associated macrophages, which are part of the tumor microenvironment and interesting targets for therapy (Mantovani et al., 2022).

The phagocytic uptake of colloidal drug carriers by macrophages of the Mononuclear Phagocyte System (MPS) has often been considered an obstacle as it results in reduced blood circulation time (Lainé et al., 2014, Lacoeuille et al., 2007, Gabizon et al., 1994). It can, however, also be regarded as an advantage for targeting approaches – depending on the incorporated drug and medical condition to be treated. Hence, beneficial effects of drugs formulated in nanoparticles regarding the efficacy and safety have been observed. This involved anti-inflammatory drugs for the treatment of atherosclerosis (Rafique et al., 2019, Wang et al., 2016, Alaarg et al., 2017) or anti-tumor therapy in mice (Wang et al., 2019). Further, intracellular infections with, e.g., Mycobacteria in mice could be controlled with liposomal formulations of anti-tuberculosis drugs in contrast to free drugs (Briones et al., 2008).

What is common to the treatment of the different conditions exploiting macrophages is the need of the uptake of the nanoparticles to deliver the incorporated drug to the inside of the cells. Agents used to stabilize the nanoparticles are known to have a major influence on their uptake by macrophages as they mediate contact between the particles and their environment. PEGylated nanocarriers possess stealth properties preventing their phagocytosis due to a hydrophilic particle surface resulting in a lack of opsonization (Lainé et al., 2014, Klibanov et al., 1990, Stolnik et al., 2001). On the other hand, phosphatidylserine, which is a physiological promotor for the phagocytosis of apoptotic cells (Fadok et al., 1992), is known as ‘eat me’ signal (Bagalkot et al., 2016, Bender et al., 2024). Beyond that, charge is reported to be an important characteristic in promoting particle uptake by macrophages (Bender et al., 2024, He et al., 2010). Regarding the influence of particle size, different observations are described in the literature. For example, the uptake of liposomes tended to be higher with smaller particle sizes according to Allen et al. (Allen et al., 1991), whereas other studies observed a correlation of uptake with the size of colloidal particles (He et al., 2010, Chono et al., 2007, Soni et al., 2024). In general, the different uptake mechanisms of macrophages (clathrin- or calveolin-mediated endocytosis or phagocytosis) seem to dictate the size-dependent uptake efficiency of particles (Baranov et al., 2020).

Triglyceride nanoemulsions have been used for parenteral nutrition and also as carriers for poorly water soluble drugs for many years (Bunjes, 2010). To promote the understanding of the macrophage uptake of such nanoparticles in the context of passive drug targeting, different trilaurin nanoemulsions were produced for the current study. After melt-homogenization, trilaurin, which is solid at room temperature in the bulk state (melting point: 47 °C), displays a high supercooling tendency. The resulting nanoparticles remain in the supercooled liquid state, even upon cooling to refrigerator temperature (Westesen and Bunjes, 1995). Trilaurin nanoemulsions varying in either stabilizer or size were prepared to independently compare the influence of these properties on uptake by a THP-1 macrophage model.

A great specialty of these supercooled systems is that the liquid nanoparticles can be easily converted into solid ones by cooling the dispersion below the crystallization temperature of the matrix lipid (Westesen and Bunjes, 1995). This made it possible to compare the influence of the physical state of similarly sized and stabilized nanoparticles regarding the uptake by macrophages. The use of a second triglyceride (trimyristin) further allowed a comparison of the uptake in dependence on slight changes in the chemical nature of the liquid nanoparticles.

For detection of nanoparticle uptake by THP-1 macrophages with confocal laser scanning microscopy and flow cytometry, the fluorescent probe DiI was incorporated into the nanoparticles. Due to its high lipophilicity (ClogP 22.5, ChemDraw), the transfer of this dye into other lipophilic compartments was very low (Petersen et al., 2010). The retention of the majority of the fluorescent probe within the lipid nanoparticles makes it suitable for the tracing of their cellular uptake.

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Materials

Trimyristin (TM, Dynasan 114®) was a kind gift by IOI Oleo (Witten, Germany). Trilaurin (TL, Dynasan 112®) was obtained from Hüls AG (Witten, Germany). Poloxamer 407 (P407, Kolliphor® P407) was donated by BASF (Ludwigshafen, Germany). Phosphatidylcholine from soybean (PC, Lipoid S100) and phosphatidylserine (PS, Lipoid PS 18:1/18:1, 1,2-dioleyl-sn-glycero-3-phopho-L-serine, sodium salt) were kindly donated by Lipoid (Ludwigshafen, Germany). Sodium glycocholate, DiIC18(3) (DiI, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyaninperchlorate), fetal bovine serum, antibiotic antimycotic solution, tetrahydrofuran (HPLC grade), Hoechst 33,342 (2́-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5́-bi-1H-benzimidazol-trihydrochloridtrihydrate), phosphate buffered saline (PBS), paraformaldehyde, chloroform (HPLC grade), bovine serum albumin (BSA) and Accutase® solution were obtained from Sigma Aldrich (Taufkirchen, Germany). Anhydrous glycerol, sucrose and TRIS Cellpure were purchased from Carl Roth (Karlsruhe, Germany). L-Glutamine was bought from PAN Biotech (Aidenbach, Germany), and phorbol 12-myristate 13-acetetae (PMA) was obtained from Hello Bio (Bristol, UK). RPMI 1640 medium and dimethyl sulfoxide were purchased from Thermo Fisher Scientific (Karlsruhe, Germany). Normal goat serum was bought from Biozol (Eching, Germany). Polysorbate 20 (Tween® 20) was obtained from Caesar & Loretz (Hilden, Germany). The primary antibody (Anti-CD11b antibody [EPR19387], ab184308) and the Alexa Fluor® 594-coupled secondary antibody (Goat Anti-Rabbit IgG H&L, ab150080) for immunofluorescent staining were purchased from Abcam (Cambridge, UK). Ultra-pure water was produced in-house with the EASYpure TM LF (Barnstead, Dubuque, IA, USA).

Nina Baumann, Stephan Reichl, Heike Bunjes, Influence of stabilizer, size and physical state of lipid nanoparticles on uptake by THP-1 macrophages, International Journal of Pharmaceutics, 2026, 126919, ISSN 0378-5173, https://doi.org/10.1016/j.ijpharm.2026.126919.

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