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Published in final edited form as: Nano Lett. 2020 Jul 31;20(8):6170–6175. doi: 10.1021/acs.nanolett.0c02536

Endosomal Organization of CpG-constructs Correlates with Enhanced Immune Activation

Kwahun Lee 1,3, Ziyin N Huang 2,3, Chad A Mirkin 1,2,3, Teri W Odom 1,2,3,*
PMCID: PMC7609249  NIHMSID: NIHMS1641088  PMID: 32787186

Abstract

This paper describes how the endosomal organization of immuno-stimulatory nanoconstructs can tune the in vitro activation of macrophages. Nanoconstructs composed of gold nanoparticles conjugated with unmethylated cytosine-phosphate-guanine (CpG) oligonucleotides have distinct endosomal distributions depending on surface curvature. Mixed-curvature constructs produce a relatively high percentage of hollow endosomes, where constructs accumulated primarily along the interior edges. These constructs achieved a higher level of toll-like receptor (TLR) 9 activation along with enhanced secretion of proinflammatory cytokines and chemokines compared to constant-curvature constructs that aggregated mostly in the center of the endosomes. Our results underscore the importance of intra-endosomal interactions in regulating immune responses and targeted delivery.

Keywords: immunostimulation, CpG, intracellular targeting, nanoparticle, surface curvature

Graphical Abstract

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INTRODUCTION

The delivery of biomolecules into cells plays a key role in the manipulation of cell function1 for numerous cancer therapies.2–4 Nanoconstructs consisting of inorganic nanoparticles (NPs) with a shell of biomolecular ligands show improved delivery efficiency because of increased cellular uptake and multi-valent interactions with surface receptors.5–15 The physicochemical properties of nanoconstructs can influence intracellular trafficking16 and the distribution within different cellular compartments such as the cytoplasm,17 lysosomes,12 and nucleus.18, 19 During subcellular transport, constructs are concentrated in vesicular compartments, which often results in aggregation because of the high concentration of enzymes, acids, and salts.20–24 As a result, a significant fraction of the ligands are precluded from binding targeted receptors23, 25 and also NP aggregates are not readily cleared from cells.16, 26, 27

To correlate how the intracellular arrangement of nanoconstructs affects downstream cell signaling, we focused on targeting toll-like receptor (TLR) 9 in the endosome of macrophages as a model system. TLR9 recognizes unmethylated cytosine-phosphate-guanine (CpG) moieties of oligodeoxynucleotide (ODN) sequences that induce an immunostimulatory (IS) response from macrophages that clear microbial pathogens28, 29 or induce anti-tumor immunity.30 TLR9 signaling requires the dimerization of two CpG-bound TLR9 receptors.31, 32 Therefore, we hypothesized that the spatial organization of the CpG-constructs is critical to facilitate TLR9 clustering and hence to stimulate IS activity. Previous studies have only correlated the amount of endocytosed CpG ODNs with IS response.33–35 Elucidating effects of endosomal organization of nanoconstructs on TLR9-mediated IS responses is important to enhance IS activity and receptor-targeted delivery.

Here we show how the spatial organization of CpG-nanoconstructs in the endosome increases access and binding to TLR9, resulting in enhanced IS responses of macrophages. Two types of nanoconstructs with different surface curvatures were investigated: spiky NPs (mixed curvature) and spherical NPs (constant curvature). Mixed-curvature constructs resulted in hollow endosomes (ca. 60%) across the macrophage population, where constructs accumulated primarily along the interior edges of the endosomes. In contrast, constant-curvature constructs resulted in a lower percentage of hollow endosomes (ca. 10%). The mixed-curvature constructs that produced a higher percentage of hollow endosomes also achieved stronger IS activity based on alkaline phosphatase secretion, which correlates with higher levels of proinflammatory cytokines. These results indicate that regulating the endosomal organization of constructs via differences in surface curvature can impact intracellular delivery and cell responses.

RESULTS AND DISCUSSION

We synthesized CpG-constructs by loading thiolated CpG ODNs on gold NPs to target endosomal TLR9 in macrophages based on published protocols (Scheme 1a).34, 36 We focused on mixed-curvature and constant-curvature constructs of similar sizes and physicochemical properties (Figure S1 and Table S1) to compare how surface curvature affects binding to TLR9 (Scheme 1b), endosomal organization (Scheme 1c), and hence TLR9 activation. We tuned the loading of CpG on the NPs by modifying the pH of the citrate buffer or adjusting the ratio of NPs to CpG ODNs. After ODN functionalization, the constructs were back-filled with mercaptoundecyl hexa(ethylene glycol) (MUHEG) to prevent aggregation in solution by steric repulsion.34, 36 The protein adsorption profiles of the two differently shaped constructs were also similar; only twenty additional proteins were observed on mixed-curvature constructs and at extremely low concentrations (Table S2 and S3).

Scheme 1. Comparison of Surface Curvature Effects on In Vitro Macrophage Activation of CpG-constructs.

Scheme 1.

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(a) Surface composition, (b) CpG ODN presentation, and (c) endosomal organization of CpG constructs can affect TLR9 signaling. After endocytosis, CpG-constructs that bind TLR9 activate macrophages through the transcription factor NF-κB. Different arrangements of CpG-constructs with different surface curvatures affect IS responses of macrophages.

We visualized the spatial organization of CpG-constructs in the endosomes using transmission electron microscopy (TEM) after treating macrophages with 200 nM CpG (250 CpG / NP) for 24 h (Supporting Information, METHODS). Mixed-curvature constructs resulted in hollow endosomes, where constructs accumulated along the interior surface of the endosome, which we refer to as the endosomal edge (Figure 1). Constant-curvature constructs yielded dense endosomes, where constructs aggregated in the center. When the two types of constructs were combined in a single treatment, the mixed-curvature constructs were again located along the endosomal edge while the constant-curvature constructs were in the center (Figure S2). This notable, segregated distribution of the two types of constructs was not affected by their order of incubation with macrophages. Incubation with constant-curvature constructs followed by mixed-curvature ones or the reverse sequence resulted in more mixed-curvature constructs along the endosomal edge (Figure S2d–f). One explanation for these results is that the surface curvature of the CpG-constructs affects the local chemical environment,37 which influences the separation of the two types of CpG-constructs in the endosomes.

Figure 1. Mixed-curvature constructs accumulate along the interior edge of endosomes.

Figure 1.

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TEM images of macrophages after treatment with (a) mixed-curvature and (b) constant-curvature CpG-constructs. Images in boxes indicate zoomed-in views. The two types of CpG-constructs were incubated at the same concentration of CpG (200 nM). The representative TEM images were collected from three independent treatments.

To understand the impact of the different endosomal organizations of the CpG-constructs, we classified the endosomes by construct distribution; we quantified percentages of constructs along the endosomal edge and the sizes of endosomes after different incubation times (4, 8, 16, and 24 h). A higher percentage of hollow endosomes (ca. 60%) was observed with mixed-curvature constructs compared to constant-curvature ones (ca. 10%) after 4 h (Figure 2a). Prolonged incubation (8, 16, and 24 h) caused small increases in the percentage of hollow and dense endosomes (ca. 6-10%) while decreasing the percentage of endosomes with a small number of NPs (ca. 9%; scattered). More mixed-curvature constructs were located along the endosomal edge compared to constant-curvature constructs at all incubation times (Figure 2b; p < 0.001). After 4 h, the percentage of mixed-curvature constructs at the endosomal edge (55%) was higher than that of constant-curvature constructs (36%) (p < 0.0001). After 24 h, the percentage of mixed-curvature constructs along the endosomal edge decreased (34%) but was still higher than that of the constant-curvature ones (26%) (p < 0.0001). Hence, mixed-curvature constructs can produce hollow endosomes that provide better access and binding to TLR9 on the endosomal membrane compared to constant-curvature constructs.

Figure 2. Hollow endosomes have a higher percentage of CpG-constructs along the endosomal edge and are larger than dense endosomes.

Figure 2.

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(a) Endosomes are classified by the CpG-construct distributions within them (hollow, dense, and scattered) after different incubation times (4, 8, 16, and 24 h; [CpG] = 200 nM). Scale bars = 200 nm. (b) Percentage of constructs along the endosomal edge and (c) diameter of endosomes were quantified. These characteristics of endosomes with different types of CpG-constructs show statistically significant differences (p < 0.0001, student’s t test) – otherwise marked with “n.s.” (not significant). Error bars in (b) and (c) indicate one standard deviation. All experiments were repeated at least twice.

We also quantified the diameter of the endosomes because large endosome sizes correlate with endosome maturation,38 and maturation is essential for TLR9-triggered macrophage activation.39, 40 The average diameters of the endosomes at 4 h were similar for both types of CpG-constructs (Figure 2c; p = 0.37). From 4 to 24 h, the average diameters of the endosomes with mixed-curvature constructs increased faster and ended up 30% larger (ca. 780 nm) than those treated with constant-curvature constructs (ca. 600 nm) after 24 h (Figure 2c). The larger endosomes containing mixed-curvature constructs suggest that these endosomes are more mature, which is beneficial for macrophage activation.

To identify the factors that control the formation of hollow endosomes, we tested the influence of: (1) multi-valent binding by CpG-constructs with varying surface-densities; (2) non-specific binding by GpC (a non-immune active) constructs; and (3) inhibiting TLR9 binding by CpG-constructs with added TLR9 antagonists. At high CpG-densities (250 CpG or 500 CpG / NP), mixed-curvature constructs produced a higher percentage of hollow endosomes (ca. 60%); at a lower CpG-density (150 CpG / NP), they produced a lower percentage (ca. 25%) of hollow endosomes (Figure 3a). Even when mixed-curvature constructs with high ODN surface-density (250 ODNs / NP) were applied, the non-specific and inhibited TLR9 bindings of these constructs yielded a low percentage of hollow endosomes (17% and 28%, respectively). For constant-curvature constructs, changes in CpG-density (400, 250, and 160 CpG / NP) did not drastically affect the percentage of hollow endosomes that were formed; less than 13% of hollow endosomes were formed in all cases (Figure S3). The smaller effect of CpG-surface density on the endosomal distribution of constant-curvature constructs is because positive curvature facilitates dense packing of ODNs that results in similar ODN presentations at various surface densities.41, 42

Figure 3. Formation of hollow endosomes requires specific and strong multi-valent binding between mixed-curvature constructs and TLR9 proteins.

Figure 3.

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(a) Endosomal distributions of CpG-constructs were quantified after 24 h incubation under various conditions. The number in each row represents the number of ODNs per nanoparticle. (b) The percentage of constructs at the endosomal edge was plotted against the diameter of endosomes ([CpG] = 200 nM, 24 h incubation). Error bars represent standard error of the mean. All experiments were repeated twice.

Figure 3b indicates how both the endosomal distribution of CpG-constructs and endosome sizes are affected by binding between CpG-constructs and TLR9 proteins. Endosomes containing mixed-curvature constructs with higher CpG-densities (500 CpG / NP and 250 CpG / NP; open symbols) displayed a higher percentage of constructs along the endosomal edge (ca. 33%) as well as a larger average diameter (ca. 750 nm). In contrast, endosomes containing mixed-curvature constructs with lower CpG-density (150 CpG / NP), non-specific GpC sequence, or added TLR9 antagonists displayed a lower percentage of constructs along the endosomal edge (< 30%) and a smaller average diameter (< 650 nm) (Figure 3b; solid symbols). These results imply that the formation of hollow endosomes requires specific and strong multi-valent binding between the CpG-constructs and TLR9.

Because binding of CpG to TLR9 promotes an IS response,43 we hypothesized that macrophage populations with a higher proportion of hollow endosomes would show enhanced IS activity. To test our hypothesis, we characterized alkaline phosphatase (AP) and cytokine secretions from macrophages treated with constructs having different surface curvatures and CpG-densities. AP and cytokine secretion levels represent the way macrophages communicate to clear pathogens28, 29 or to induce anti-cancer immunity30 and provide a quantitative readout of IS activity. We observed that mixed-curvature constructs resulted in significantly higher AP secretion levels (ca. 1.0) at higher CpG-densities (500 CpG and 250 CpG / NP) but a lower level (ca. 0.4) at a lower CpG-density (150 CpG / NP) (Figure 4a). Constant-curvature constructs resulted in intermediate AP secretion levels (ca. 0.7) with only small changes observed at different CpG-densities (400, 250, and 160 CpG / NP). These data are consistent with previous work where IS activities of constant-curvature constructs saturated at lower CpG densities (5% CpG in a mixture of CpG and GpC).36

Figure 4. CpG-constructs that form a high percentage of hollow endosomes also result in enhanced IS activity.

Figure 4.

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(a) Alkaline phosphatase (AP) and (b) cytokine secretion of macrophages after a 24 h treatment with CpG-constructs at varied CpG densities and surface curvatures. The results indicate that CpG-density changes the IS activity of constructs with mixed curvatures, but not with constant curvature. Error bars were from six independent experiments. ([CpG] = 200 nM, 24 h incubation)

We also measured cytokine secretion with different constructs and estimated the relative enhancement compared to mixed-curvature constructs at 150 CpG / NP, where the AP secretion level was the lowest. Figure 4b shows that only mixed-curvature constructs at 500 CpG / NP resulted in a significant relative enhancement of proinflammatory cytokines (2 times for G-CSF, 1.3 times for IL-6, and 1.3 times for IL-1α), anti-inflammatory cytokines (1.9 times for LIF), chemokines (1.3 times for RANTES), and ENA-78, but no enhancements of TNF-α and MIP-2. Mixed-curvature constructs at 250 CpG / NP resulted in small enhancements of G-CSF and ENA-78. Enhanced levels of proinflammatory cytokines and chemokines suggest stronger IS responses. We note enhanced secretion of an anti-inflammatory cytokine (LIF) at 500 CpG / NP, but LIF has only a minor effect on the IS response because the amount (ca. 50 pg / mL) was much smaller than other proinflammatory cytokines (> 1000 pg / mL) (Figure S4).44 Constant-curvature constructs showed small changes in cytokine levels at different CpG densities. Our results indicate that cytokine secretion levels are enhanced only with mixed-curvature constructs at higher CpG densities, which is consistent with AP secretion levels (Figure 4a). A higher CpG-density threshold was observed for enhanced cytokine secretions (500 CpG / NP) compared to AP secretion (250 CpG / NP) because of their different pathways.39 Taken together, mixed-curvature constructs resulted in the formation of hollow endosomes, and the location of the constructs facilitated more efficient access to TLR9, which increased IS activity.

In summary, we identified NP surface curvature as a key structural feature that determines the endosomal organization of CpG-constructs as well as the in vitro IS responses of macrophages. Mixed-curvature constructs resulted in a higher percentage of hollow endosomes and a higher IS response at higher CpG-densities (> 250 CpG / NP). Constant-curvature constructs resulted in a lower percentage of hollow endosomes and a lower IS response at all tested CpG-densities. One disadvantage of the mixed-curvature structures is their preparation, which requires an additional synthetic step. Nevertheless, our results highlight that such structures offer a means to control their local organization inside endosomes, which can improve immune cell responses and intracellular delivery. Both the local chemical environment and ODN presentation contribute to the endosomal distribution of CpG-constructs. In future work, we aim to understand how these effects influence the accumulation process and final nanoconstruct organization.

Supplementary Material

SI

Acknowledgments.

This material is based on research sponsored by the National Cancer Institute of the National Institutes of Health under Award Number U54CA199091 (K.L., T.W.O., Z.N.H., C.A.M) and R01CA208783 (Z.N.H., C.A.M). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The project was also supported by the Prostate Cancer Foundation and the Movember Foundation under award 17CHAL08 (Z.N.H., C.A.M). We thank Charlene Wilke for processing TEM samples. Z.N.H. acknowledges support by the Northwestern University Graduate School Cluster in Biotechnology, Systems, and Synthetic Biology, which is affiliated with the Biotechnology Training Program funded by NIGMS grant T32 GM008449. Fluorescence and absorbance measurements were carried out in the High Throughput Analysis Laboratory. TEM imaging was performed at the Biological Imaging Facility. Gold analysis was conducted at the Northwestern University Quantitative Bio-elemental Imaging Center generously supported by NASA Ames Research Center NNA06CB93G. Proteomics services were performed by the Northwestern Proteomics Core Facility, generously supported by NCI CCSG P30 CA060553 awarded to the Robert H. Lurie Comprehensive Cancer Center and the National Resource for Translational and Developmental Proteomics supported by P41 GM108569.

Footnotes

Supporting Information Available: Experimental methods; characterization and protein corona profiles of CpG-constructs; TEM images of CpG-constructs in the endosomes when mixed-curvature and constant-curvature constructs were combined in a single treatment; characteristics of endosomes under various treatment conditions; cytokine produced by mixed-curvature constructs (150 CpG / NP). This materials is available free of charge via the Internet at http://pubs.acs.org.

The authors declare no competing financial interest.

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