Macrophage Uptake of Janus Particles Depends upon Janus Balance

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Macrophage Uptake of Janus Particles Depends on Janus Balance Yuan Gao, and Yan Yu Langmuir, Just Accepted Manuscript • DOI: 10.1021/la504668c • Publication Date (Web): 12 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Macrophage Uptake of Janus Particles Depends on Janus Balance Yuan Gao and Yan Yu* Department of Chemistry, Indiana University, Bloomington, Indiana 47405, United States

ABSTRACT

Properties of synthetic particles, such size and shape, influence how immune cells uptake vaccine and drug carriers. Here we explore the role of a new property – anisotropic presentation of ligands – in particle uptake by macrophage cells. We use micron-sized Janus particles that are partially coated with ligands and investigate how ligand patch size (Janus balance) affects their uptake by macrophages. Macrophage uptake of both 1.6-µm and 3-µm Janus particles is enhanced as the size of the ligand patch increases. However, presenting ligands asymmetrically reduces particle phagocytosis – Janus particles with the same amount of ligands as uniformly coated particles are internalized less efficiently. We also show that, because of the asymmetric geometry of Janus particles, the onset of ligand-mediated phagocytosis depends on the orientation of the particles with respect to the cells. This study demonstrates Janus balance as a new parameter that we can use to manipulate the macrophage uptake of particles.

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INTRODUCTION Immune cells, such as macrophages and neutrophils, engulf bacteria and dead cells to fight against infections.1,2 This cellular uptake process, known as phagocytosis, is also responsible for removing foreign particles from the blood stream, which leads to many drug delivery failures.3,4 Therefore, there is a critical need to develop strategies to control the phagocytosis of synthetic particles, so that tumor-targeting drug carriers can evade phagocytosis, while vaccine delivery particles can be uptaken efficiently by immune cells. Using physical parameters of particles, such as size, shape, ligand density, and mechanical stiffness, to control cellular uptake of particles is a predominant strategy among many.5-26 For phagocytosis of micron-sized particles, it has been found that particles that are 1-3 µm, a size range comparable to most commonly found bacteria, are internalized most efficiently.18,27,28 For particles smaller than 2 µm, but not in other size ranges, their internalization efficiency increases with ligand density.8 Shape matters too. For non-spherical particles, initiation of phagocytosis is determined by the local shape of the particle at its contact point with the cell.9,10 Rod-like or ellipsoidal particles, for example, mostly enter cells from their tip.

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Oblate particles were

found to be internalized more efficiently than spherical or ellipsoidal ones.13 Surface chemistry of particles has also been shown to impact phagocytosis. Macrophages prefer to uptake hydrophobic or highly charged particles over the neutral hydrophilic ones.14-18 It was also reported that soft particles evade macrophage phagocytosis more easily than hard ones.19-21 In spite of the extensive studies, controlling particle phagocytosis remains a challenge. New material parameters that influence particle uptake must be identified and incorporated into the design of biomaterials.


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In this study, we explore surface anisotropy as a new parameter for manipulating particle phagocytosis. We draw our inspiration from bacteria, which are known to have polarized distributions of proteins on their surfaces,29,30 and ask whether anisotropic ligand presentation will likewise affect the phagocytosis of artificial particles. This topic is currently poorly understood, since existing studies have focused on particles with uniform surface functionality. We will use Janus particles with an anisotropic coating of ligands. In a previous study, we found that Janus particles with ligands coated on one hemisphere entered cells by a different mechanism than did uniformly coated particles.31 Based upon this observation, here we report a study of the relationship between particle phagocytosis and the Janus balance (a measure of the size of the ligand patch). Using a microcontact printing method, we fabricated Janus particles that varied over a wide range of Janus balance. We found that as ligand patch size increases, macrophage uptake of µm-sized Janus particles is enhanced, but that Janus geometry reduces particle internalization in comparison to uniformly coated particles. Given the spatial segregation of ligands on Janus particles, we found that initiation of phagocytosis depends on the orientation of the particle in relation to the cell. EXPERIMENTAL Particle fabrication. Monodisperse Janus particles (1.6 µm and 3 µm in diameter) were fabricated by using a microcontact printing method (Figure 1a). A PDMS stamp was pre-inked with bovine serum albumin (BSA)-biotin (30 µg/ml) for 20 min and then pressed against a monolayer of silica particles (purchased from Spherotech). A pressure of 1.5×104 Pa was applied for 3 min before the stamp was peeled off. Sonication was used to remove particles from the PDMS stamp, and the particles were harvested for further functionalization. To fabricate uniformly coated particles, silica particles were first functionalized with 2% (3-aminopropyl) triethoxysilane


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(APTES) dissolved in anhydrous tetrahydrofuran (THF), and then conjugated with biotin-NHS (2 mM in pH8.2 sodium bicarbonate buffer). To attach IgG onto particle surfaces, particles were sequentially incubated with bovine serum albumin (BSA) (10 µM) for 1 hour, 100 nM streptavidin for 1.5 hours and 10 nM biotinylated IgG for 1.5 hours. Detailed experimental procedures are included in SI. Cell imaging. RAW264.7 macrophage cells (purchased from ATCC) were serum starved for 3 hours prior to all experiments. To quantify particle internalization efficiency, macrophage cell membrane was labeled with VivoTrack 680 (purchased from Perkin Elmer). Particles were incubated with cells at a constant particle-to-cell ratio of 8:1 for 15 min at 37 °C before samples were fixed with 2% paraformaldehyde (PFA). Both particles and cells were scanned along the zaxis with a 0.5-µm step size by using fluorescence confocal microscopy. The internalization efficiency was obtained as the ratio of the number of internalized particles to the number of all particles that were either internalized or attached to cells. For imaging calcium influx during phagocytosis, macrophages were loaded with Fluo-4 (purchased from Life Technology) and imaged with epi-fluorescence microscopy. Integrated fluorescence intensity of individual cells was analyzed by using a custom Matlab algorithm. All live cell experiments were performed at 37 °C. Detailed experimental procedures are included in SI. RESULTS AND DISCUSSION Macrophages engulf particles via both receptor-mediated and non-specific phagocytosis; the former is significantly more efficient.8,32,33 The Janus particles studied here were partially coated with immunoglobulin G (IgG), whose Fc region binds the Fc receptors on RAW 264.7 macrophages and triggers Fc-receptor mediated phagocytosis.34,35 The remaining surface of the


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Janus particles was passivated with BSA, which can lead to non-specific phagocytosis but at low efficiency.36 Patches of IgG were generated on silica particles (1.6 µm and 3 µm in diameter) by using a microcontact printing method and subsequent biotin-streptavidin conjugation. In agreement with previous reports,37 we observed that the IgG patch size depends on the stamping pressure, with higher pressure generating larger patches. Taking advantage of this, we intentionally applied uneven pressure during each microcontact printing to generate a wide distribution of patch sizes in a single sample. By doing so, we were able to compare the uptake of particles with different patch sizes by the same batch of cells and under the same experimental conditions. Janus particles with fluorescently labeled IgG patches were imaged by using epifluorescence microscopy. Due to the 2-D projection of this imaging technique, the fluorescently labeled IgG patches appear to be of different shapes depending on the particle orientation with respect to the imaging plane (Figure 1b and Figure S1 SI). However, the base diameter of the ligand patch does not change with the particle orientation; it is equal to the longest dimension of each projected image. That enabled us to characterize the surface coverage of IgG on Janus particles – the Janus balance – by measuring the arc angle θ subtended by this longest dimension. Given the pixel size of the epi-fluorescence images (70 nm with 100⨯ magnification), we estimated the inaccuracy of the angle measurement to be ±20°. As shown in Figure 1c, the microcontact printing fabrication method resulted in a large range of ligand patch sizes. The arc angle θ of the IgG patch ranges from 60⁰ to >180⁰, while centering at the range of 100⁰ to 140⁰. Angles equal or larger than 180⁰ are indistinguishable under epi-fluorescence imaging, and were therefore grouped into one category.


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Figure 1. Fabrication of Janus particles and characterization of the Janus balance. (a) Schematic illustration of the microcontact printing method for Janus particle fabrication. A PDMS stamp pre-inked with BSA-biotin is pressed against a monolayer of particles and then peeled off. Janus particles are removed from the PDMS stamp by sonication. (b) The fluorescently labeled ligand patches appear to be of different shapes in epi-fluorescence images, depending on the particle orientation. The longest dimension of each 2-D projected image equals the base diameter of a given ligand patch. Scale bars: 1 µm. (c) Distribution of the Janus balance, defined as the arc angle θ of ligand patches. Inset is a schematic illustration of the angle θ.

We first investigated how phagocytosis efficiency of Janus particles is affected by the Janus balance. We defined the internalization efficiency (% internalization) as the ratio of the number


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of internalized particles to the number of all particles that are either internalized or attached to cells. The particle-to-cell ratio was kept constant in all experiments. We kept the surface density of IgG the same for all Janus particles but varied the ligand patch size. As shown in Figure 2a, macrophages engulf non-coated particles (θ = 0°) non-specifically at a low efficiency. As the IgG patch size increases, an increasing fraction of 1.6-µm Janus particles is internalized. Janus particles also have lower internalization efficiency than uniformly coated particles, as particles that are uniformly coated with IgG (θ = 360°) are internalized most efficiently. Our next question is: Is the reduced internalization of Janus particles due to the asymmetric presentation of ligands or a decreased amount of IgG? In order to distinguish the effect of Janus geometry from that of the amount of ligands on particles, we varied the total amount of IgG on particle surfaces and compared the phagocytosis efficiency of Janus particles to that of uniformly coated particles (referred to as “uniform allIgG”) with the same number of ligands. The total number of IgG molecules on particle surfaces was varied from 0 to ≈ 300 molecules per particle and measured by using a quantitative fluorescence method (see Experimental Section in SI). As shown in Figure 2a, internalization efficiency of uniform all-IgG particles increases with the number of ligands on the particles, in agreement with previous reports.8 However, Janus particles are internalized less efficiently than uniformly coated particles even if they have the same number of ligands on their surfaces. This demonstrates that the asymmetric presentation of IgG molecules reduces the phagocytosis efficiency of particles. We showed in our previous study that half-coated Janus particles enter Jurkat T cells at the same efficiency as uniformly coated particles if they have the same number of ligands.31 The discrepancy is likely due to the difference of cells. Phagocytes such as macrophages are capable of both receptor-mediated and non-specific phagocytosis, but non-


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phagocytic Jurkat T cells can only uptake large particles via specific receptor-mediated endocytosis.

Figure 2. The internalization efficiency (% internalization) of (a) 1.6-µm and (b) 3-µm Janus particles depends on the Janus balance. Internalization efficiency of the Janus particles (▲) is plotted against arc angle θ of the IgG patch and that of uniform all-IgG particles (○) is plotted against the total number of IgG on particle surface. Each data point is an average of >40 particles. Error bars represent standard deviations between at least two independent samples.

We confirmed that the observations can be generalized to particles of other sizes. As for 1.6 µm particles, a dependency of internalization efficiency on the ligand patch size was also observed for 3-µm Janus particles (Figure 2b). The ligand segregation of IgG on Janus particles reduces the internalization efficiency in comparison to uniformly coated particles. Overall, 3-µm Janus particles are internalized more efficiently than 1.6-µm particles, a size dependence that was also previously reported for uniformly coated particles.9,28


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Figure 3. Calcium influx during macrophage uptake of 1.6-µm Janus particles (a), uniform allIgG particles (b) and non-coated particles (c). The normalized fluorescence intensity of Fluo-4 of individual macrophage cells is color-coded and plotted against time. Cells are organized along the y-axis according to the average intensity. (d) Four parameters: the onset time of the first calcium peak, number of calcium peaks per cell, duration of calcium influx, and calcium peak intensity, were analyzed from all cells in each calcium heatmap and compared among the three types of particles as indicated. Error bars represent S.E.M. P-values were determined by using a two-tailed student’s t-test.


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Figure 4. Onset of the calcium influx during macrophage phagocytosis of 1.6-µm Janus particles depends on particle-cell orientation. In both (a) and (b), the top two panels are the schematic illustration and overlaid epi-fluorescence images showing the macrophages labelled with Fluo-4 (shown in green) and the Janus particles labeled with Alexa 568-streptavidin (shown in red). Normalized fluorescence intensity of Fluo-4, which is proportional to the intracellular [Ca2+], is plotted against time. The blue shade in both plots indicates when the particles were visibly engulfed. Time zero is defined as the time when Janus particles are added into the imaging chambers. Scale bars: 5 µm.

We noticed that Janus particles not only have lower internalization efficiency than their uniformly coated counterparts, but also take longer to enter macrophages. We hypothesize that the Janus presentation of IgG changes the initiation of particle phagocytosis. To quantify the initiation of phagocytosis, we chose to measure the onset of calcium influx into the cell, because


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studies have shown that such a transient calcium ion influx into the cell cytosol precedes macrophage phagocytosis.38,39 A calcium-sensitive dye, Fluo-4, was used to measure the intracellular concentration of calcium ions. Its fluorescence emission at 506 nm increases when it binds to calcium ions. Resting macrophages loaded with Fluo-4 exhibit occasional “blinking”, as indicated by the random fluctuation of Fluo-4 intensities (Figure S3a SI). Immediately before a macrophage engulfs an IgG-coated particle, the intracellular [Ca2+] increases abruptly and persists for tens of seconds before returning to the basal level. In contrast, phagocytosis of noncoated particles, which occur less frequently, leads to calcium peaks that are less intense and more sustained (Figure S3b SI). In over 100 cells, we found that a transient calcium elevation is always followed by a successful phagocytosis. Multiple calcium peaks were occasionally observed with a single phagocytosis event, but only the first calcium peak coincided with the onset of phagocytosis. These observations, in agreement with previous studies,38,40 confirmed that calcium influx is a sensitive temporal readout for macrophage phagocytosis. We next investigated the calcium response during the macrophage uptake of three types of 1.6-µm particles: uniformly coated all-IgG, Janus, and non-coated. The calcium responses of over 100 macrophage cells for each type of particle are shown in Figures 3a-c as heatmaps that are color-coded by Fluo-4 intensity. Each horizontal line in the calcium heatmaps represents how the concentration of intracellular calcium ions in a single cell changes as a function of time. The heterogeneous calcium response during phagocytosis regardless of the particle type reflects the intrinsic cell-to-cell variation. The comparison between heatmaps shows the obvious differences for the three types of particles. The first difference is in the onset of the first calcium peaks, which indicates the beginning of particle phagocytosis. We observed that Janus particles took an average of (276 ± 16) seconds from the time when particles were added to the cells until the


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initiation of the first phagocytic event. In contrast, the first phagocytosis occurred at (193 ± 13) seconds for the uniform all-IgG particles and at (362 ± 56) seconds for the non-coated ones (Figure 3d). The results suggest that the Janus geometry delays particle phagocytosis. We found that the delay is correlated with the orientation of the Janus particle with respect to the cell. In a representative cell that first touched the particle on the non-coated side (Figure 4a), the calcium ion concentration remained at the basal level until the particle was rotated to face the cell from its IgG-coated side. A rapid calcium transient was observed ≈70 seconds before the Janus particle was visibly engulfed by the cell. In contrast, if a cell touched the IgG-coated side first, the calcium elevation was observed sooner (Figure 4b). The combined results from ensembleaverage and single-cell analyses of calcium response indicate that the Janus presentation of IgG introduces directionality into the particle-cell recognition, which affects the time required for particle phagocytosis. In addition to the onset time of calcium peaks, we also observed that the number of calcium peaks per cell during phagocytosis varies for different types of particles (Figure 3d), even though the overall particle-to-cell ratio was consistently kept at 8:1 in all experiments. Janus particles lead to fewer calcium peaks (3.5±2.2/cell) than uniformly coated ones (5.8±3.7/cell), whereas the non-coated particles have the fewest calcium peaks (2.3±1.6/cell). The calcium results correlate well with the overall dependence we have shown (Figure 2a) between internalization efficiency and surface presentation of ligands. In addition, it was suggested previously that the duration and magnitude of the calcium peaks differ for Fc receptor-mediated and nonspecific phagocytosis.40 Indeed, we also found that phagocytosis of IgG-coated particles leads to more intense and transient calcium elevations than that of non-coated ones (Figure 3d). The characteristics of calcium elevations for Janus particle phagocytosis appear intermediate


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between those observed for particles fully coated with IgG, and those not coated. This suggests that Janus particle phagocytosis involves a mixture of both Fc-receptor mediated and nonspecific phagocytosis events. The difference between the Janus and all IgG particles is subtle, likely due to the small fraction of non-specific phagocytosis in comparison to the predominant fraction of receptor-mediated phagocytosis. CONCLUSIONS In summary, we reported here a quantitative study on macrophage uptake of Janus particles that are partially coated with ligands. The Janus particles were fabricated by using a microcontact printing method, which allowed the size of the ligand patch to be varied over a large range. The internalization efficiency of both 1.6-µm and 3-µm Janus particles was found to increase with the ligand patch size. Janus particles with the same amount of ligand as uniformly coated ones have lower internalization efficiency, which suggests that the reduced rate of particle phagocytosis is due specifically to asymmetric presentation of ligands. We also found that the initiation of Janus particle phagocytosis depends on the orientation of the particle with respect to the cell. Our results demonstrated that Janus balance can be used as a new parameter to modulate particle internalization. This study, built upon our previous observation that half-coated Janus particles use two mechanisms to enter cells, established that one can manipulate the time course of particle internalization by altering the spatial presentation of ligands, a level of control that is inaccessible with uniformly coated particles. Designing drug delivery particles whose cellular uptake fate can be controlled remains a major challenge. Past studies have focused on the properties of particles whose surfaces were uniformly coated. In contrast, this study explores the use of anisotropic presentation of ligands to control


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particle internalization. The quantitative relationship between Janus balance (a measure of the size of the ligand patch), and macrophage uptake efficiency of particles established in this study could be important for engineering multivalent particles as drug carriers – the ligands may be clustered to control cell-particle binding while leaving the remaining surface still available for other functionalization such as PEG stealth coatings that reduce macrophage uptake. This is part of the work in progress. The interplay between the Janus geometry and other physical parameters, such as size and shape, will also be investigated in our future work to further establish the role of Janus geometry in controlling particle internalization.


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ASSOCIATED CONTENT Supporting Information. Experimental section, calibration plot for quantitative fluorescence measurement, calcium data for uniformly coated and non-coated particles. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author Email: [email protected]; Tel: 812-855-0593 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank Dr. Yi Yi for helpful discussion and the use of vacuum oven, Mr. Jim Powers of the IUB Light Microscopy Imaging Center for fluorescence imaging assistance, Dr. Ardian Wibowo of the Indiana University Physical Biochemistry Instrumentation Facility for assistance with UV/Vis spectrometer, Prof. Charles Dann III for the use of NanoDrop, Prof. Richard DiMarchi for the use of lyophilizer, Dr. Stephen M. Anthony (Sandia National Laboratories) for the use of calcium-detection Matlab algorithm, and Indiana University for funding.


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Table of Contents (TOC)


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Macrophage Uptake of Janus Particles Depends upon Janus Balance

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