Amphiphilic galactomannan nanoparticles trigger the alternative activation of murine macrophages
Graphical abstract
Introduction
Macrophages are highly plastic cells of the immune system that play a key roles in the innate and the adaptive immune response and the normal tissular and organic development and function [1]. They are a source of growth factors [2], recruit other immune cells, and are involved in different pathophysiological conditions [[3], [4], [5], [6], [7], [8]]. Macrophages display a wide range of phenotypes and change their physiological performance according to stimuli in the biological milieu [9,10]. Macrophage polarization is an in vitro model that defines two extreme phenotypes of the activation spectrum [11]. In this model, naïve macrophages (M0) can differentiate to a classically activated (M1) phenotype that interacts with T helper type 1 cells and releases pro-inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Conversely, the alternatively activated (M2) phenotype mediates T helper type 2 cell response and releases anti-inflammatory cytokines (e.g., IL-4 and IL-10) that inhibit inflammation and promote tissue regeneration [12,13]. In vivo, macrophages can display intermediate polarization states and both phenotypes can co-exist [14]. In addition, macrophages can switch their phenotype from M1 to M2 and vice versa or maintain their M0 state in the absence of external stimuli. The M1 phenotype is involved in the response to bacterial and viral infections, eliminates aging or necrotic cells [15], and improves the direct killing of cancer cells [16]. Conversely, the M2 phenotype is commonly implicated in the response to fungi, parasites, and apoptotic cells [17] and is characterized by high expression of scavenging molecules, mannose and galactose receptors, high phagocytosis capacity, production of extracellular matrix, and stimulation of tissue remodeling, repair and regeneration [18].
The ability to trigger and direct macrophage polarization by small-molecule and biological drugs has emerged as a new therapeutic approach [19,20]. However, when these therapies are not targeted to macrophages, they might be accompanied by systemic side-effects on other cell types [21].
Biomaterials with different chemical, structural and mechanical properties and in different forms (e.g., particles, films) can be used as immunomodulators because they trigger macrophage polarization, provide a more controlled temporal and spatial release of key cytokines and growth factors and mimic better complex physiological signaling patterns with minimal toxicity [[22], [23], [24], [25]]. Due to their smaller size and larger surface-to-volume ratio, nanoparticles (NPs) have been extensively investigated in drug delivery and targeting and recently, proposed to tune macrophage polarization [14]. Amphiphilic polymeric NPs (e.g., polymeric micelles) are self-assembly nanostructures formed by the spontaneous aggregation of copolymeric amphiphiles in water above the critical aggregation concentration [26,27]. They display sizes between a few tens to several hundreds of nanometers and undergo differential biodistribution in body tissues and organs [26,27]. Their surface can be tailored to selectively bind receptors overexpressed by macrophages and undergo uptake by different pathways [28,29]. Lectin-like receptors are transmembrane proteins that selectively bind glucose and mannose and they are overexpressed by M2 macrophages [20,30]. Another key β-glucan receptor in macrophages is dectin-1 [31]. For example, particulate β-glucan switches the M2 phenotype to an M1-like one [32].
Galactomannans (GMs) are polysaccharides made of a mannose backbone and side galactose residues [33]. Polymeric NPs surface-modified with hydrolyzed galactomannan (hGM) improve the intracellular delivery of encapsulated drugs to macrophages [34]. Recently, we demonstrated that amphiphilic hGM-g-poly(methyl methacrylate) (PMMA) NPs efficiently encapsulate hydrophobic drugs (e.g., imatinib) and that their accumulation in pediatric patient-derived xenograft sarcoma models in mouse correlates with the expression of glucose transporter 1 [35].
In this conceptual framework, we hypothesized that the interaction and uptake of hGM-g-PMMA NPs by macrophages triggers their polarization. Thus, in this work, we first investigated the compatibility and endocytic mechanisms by which hGM-g-PMMA NPs (PMMA content of 30% w/w) are internalized by the murine macrophage cell line RAW264.7 by using a metabolic assay, confocal laser scanning fluorescence microscopy (CLSFM) and imaging flow cytometry. Then, the ability of these NPs to induce M2-polarization in vitro was demonstrated by flow cytometry, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA). Next, their wound healing performance was assessed in an artificial wound model by a real time scratch assay. Finally, the ability of murine macrophages pre-exposed to drug-free hGM-g-PMMA NPs and polarized to a M2 phenotype to induce the migration of murine fibroblasts was studied in a macrophage/fibroblast co-culture model.
Section snippets
Materials
GM (from locust bean gum) was supplied by Glentham Life Sciences (Corsham, UK) and hydrolyzed under acid conditions to produce hGM and dialyzed before use [34,35]. Methyl methacrylate (MMA, Alfa Aesar, Heysham, UK) was distilled under vacuum at 35–40 °C to remove the free radical inhibitor. Cerium(IV) ammonium nitrate (CAN, Sigma-Aldrich, St. Louis, MO, USA), nitric acid 70% (Bio-Lab, Jerusalem, Israel) and tetramethylethylenediamine (TEMED, Alfa Aesar) were used without further purification.
Synthesis and characterization of the nanoparticles
An amphiphilic hGM-g-PMMA copolymer containing 30% w/w PMMA (hGM-PMMA30) was synthesized by the free radical graft polymerization of MMA in water [35]. The copolymer was solubilized in different physiologically relevant media (0.1% w/v) at a concentration above the critical aggregation concentration [35] to produce the NPs by self-assembly and the Dh and the PDI measured by DLS at 25 and 37 °C. In PBS, NPs display a monomodal size population (Dh of 128–130 nm, by intensity) and PDI of 0.14–0.50
Conclusions
Active drug targeting of macrophages by means of glycosylated nanoparticles that bind lectin-like receptors and other transmembrane receptors overexpressed by these immune cells has been extensively investigated. Conversely, the exploitation of the pathways to immunomodulate and polarize them has been remarkably scarce, especially with drug-free nanocarriers.
In this work, we investigated the polarization of murine macrophages by drug-free hGM-g-PMMA NPs. Imaging flow-cytometry and CLSFM
Declaration of Competing Interest
None.
Acknowledgments
A.S. thanks the financial support of Technion and the Russell Berrie Nanotechnology Institute (RBNI, Technion). We thank Nitzan Dahan and Efrat Barak at the Life Sciences and Engineering Infrastructure Center (Lorry I. Lokey Center, Technion) for technical assistance in CLSFM and imaging flow cytometry, respectively.
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