Arginine-Based Poly(I:C)-Loaded Nanocomplexes for the Polarization of Macrophages Toward M1-Antitumoral Effectors

Abstract

Background: Tumor-associated macrophages (TAMs), with M2-like immunosuppressive profiles, are key players in the development and dissemination of tumors. Hence, the induction of M1 pro-inflammatory and anti-tumoral states is critical to fight against cancer cells. The activation of the endosomal toll-like receptor 3 by its agonist poly(I:C) has shown to efficiently drive this polarization process. Unfortunately, poly(I:C) presents significant systemic toxicity, and its clinical use is restricted to a local administration. Therefore, the objective of this work has been to facilitate the delivery of poly(I:C) to macrophages through the use of nanotechnology, that will ultimately drive their phenotype toward pro-inflammatory states. Methods: Poly(I:C) was complexed to arginine-rich polypeptides, and then further enveloped with an anionic polymeric layer either by film hydration or incubation. Physicochemical characterization of the nanocomplexes was conducted by dynamic light scattering and transmission electron microscopy, and poly(I:C) association efficiency by gel electrophoresis. Primary human-derived macrophages were used as relevant in vitro cell model. Alamar Blue assay, ELISA, PCR and flow cytometry were used to determine macrophage viability, polarization, chemokine secretion and uptake of nanocomplexes. The cytotoxic activity of pre-treated macrophages against PANC-1 cancer cells was assessed by flow cytometry. Results: The final poly(I:C) nanocomplexes presented sizes lower than 200 nm, with surface charges ranging from +40 to -20 mV, depending on the envelopment. They all presented high poly(I:C) loading values, from 12 to 50%, and great stability in cell culture media. In vitro, poly(I:C) nanocomplexes were highly taken up by macrophages, in comparison to the free molecule. Macrophage treatment with these nanocomplexes did not reduce their viability and efficiently stimulated the secretion of the T-cell recruiter chemokines CXCL10 and CCL5, of great importance for an effective anti-tumor immune response. Finally, poly(I:C) nanocomplexes significantly increased the ability of treated macrophages to directly kill cancer cells. Conclusion: Overall, these enveloped poly(I:C) nanocomplexes might represent a therapeutic option to fight cancer through the induction of cytotoxic M1-polarized macrophages.

Keywords: arginine-rich peptides; cancer immunotherapy; nanocomplexes; poly(I:C); toll-like receptor (TLR) 3; tumor-associated macrophages (TAMs).

Figure 1

Screening of different arginine-rich polymers to form nanocomplexes with poly(I:C). Values of (A) particle size, PDI and (B) zeta potential of the nanocomplexes obtained for the different ratios tested. Values represent mean ± SD (n ≥ 3). C12r8, laurate-octaarginine; pArg, polyarginine; PDI, polydispersity index; pIC, poly(I:C); w/w, weight/weight.

Figure 2

Physicochemical properties and poly(I:C) binding affinity of the selected nanocomplexes. (A) FESEM images of each of the four developed nanosystems. Values represent mean ± SD (n ≥ 12). Size bars represent 200 nm, and all images present a 50 K magnification. (B) Agarose gel retardation assay to evaluate the poly(I:C) binding capacity of the nanocomplexes. Lanes: (1) free poly(I:C), (2,4,6,8) are pArg:pIC, pArg:pIC/PEG–PGA, pArg:pIC/HA and C12r8:pIC/PEG–PGA nanocomplexes, respectively; and (3,5,7,9) are the corresponding nanocomplexes incubated with heparin. (C) Agarose gel retardation assay to evaluate the release and integrity of poly(I:C) after 4 h of incubation in cell culture media at 37°C. Lanes: (1) free poly(I:C) in solution and (2) in cell culture media; (3,5,7,9) are pArg:pIC, pArg:pIC/PEG–PGA, pArg:pIC/HA and C12r8:pIC/PEG–PGA nanocomplexes in cell culture media; and (4,6,8,10) are the same conditions incubated with heparin. C12r8, laurate-octaarginine; FESEM, field emission scanning electron microscopy; HA, hyaluronic acid; pArg, poly-arginine; PEG–PGA, pegylated polyglutamic acid; PDI, polydispersity index; pIC, poly(I:C); w/w, weight/weight.

Figure 3

Toxicity, uptake and cellular localization of poly(I:C) and nanocomplexes. Toxicity toward (A) M0 and (B) M2 primary human monocyte-derived macrophages after 24 h of incubation. (C) FACS evaluation of rhodamine-labeled-poly(I:C) uptake by primary human monocyte-derived macrophages when included in the different nanocomplexes after 4 and 24 h of incubation, expressed as the fold increase in comparison to free poly(I:C), for a final poly(I:C) dose of 5 μg/mL. (D) Co-localization with the endosome of rhodamine-labeled pArg:pIC nanocomplexes after 2 and 8 h of incubation (100x magnification, size bars of 10 μm) evaluated by confocal microscopy. (E) Quantification of the co-localization of rhodamine-labeled pArg:pIC nanocomplexes with the endosome after 2 and 8 h of incubation, with a poly(I:C) dose of 5 μg/mL. Values represent mean ± SD (n ≥ 3). Statistical comparison was done using a two-way ANOVA followed by a Tukey's multiple comparison test. Statistically significant differences are represented as **p < 0.01, ***p < 0.005, and ****p < 0.001. C12r8, laurate-octaarginine; HA, hyaluronic acid; pArg, poly-arginine; PEG–PGA, pegylated polyglutamic acid; pIC, poly(I:C).

Figure 4

Polarization of M0 and M2 macrophages after treatment with free and nanocomplexed poly(I:C). Expression of the M2 markers (A–D) CD206 and (E–H) CD163 in ENCP-treated M0 and M2 macrophages, in comparison to the prototypic phenotypes evaluated by FACS. M2 M1 represents M2 macrophages that were treated with LPS + IFN-γ for their M1 polarization. Macrophages were incubated with the treatments for 48 h, and the poly(I:C) dose used was 5 μg/mL. Each symbol shape represents a different donor. Values are shown as mean ± SD (n ≥ 3). Statistical comparison was done using an ordinary one-way ANOVA followed by a Tukey's comparison test between groups; or a paired t-test for (C,G). Statistically significant differences are represented as **p < 0.01. C12r8, laurate-octaarginine; HA, hyaluronic acid; pArg, poly-arginine; PEG–PGA, pegylated polyglutamic acid; pIC, poly(I:C).

Figure 5

mRNA production of different M1/M2 associated factors. Fold change in the mRNA levels of IRF7 in (A) prototypic M1/M2 macrophages and in (B) M0 macrophages treated with the different nanocomplexes after 8 h of incubation. The dose of poly(I:C) was 5 μg/mL. Values represent mean ± SD (N = 4). Statistical comparison was done using an ordinary one-way ANOVA followed by a Tukey's comparison test between groups. Statistically significant differences are represented as ***p < 0.005. C12r8, laurate-octaarginine; HA, hyaluronic acid; pArg, poly-arginine; PEG–PGA, pegylated polyglutamic acid; pIC, poly(I:C).

Figure 6

Secretion of the T cell attracting chemokines upon treatment with the poly(I:C) nanocomplexes. (A,B) CXCL10 secretion in (A) prototypic macrophages and in (B) M0 macrophages treated with the different nanocomplexes after 24 h of incubation. (C,D) CCL5 secretion in (C) prototypic macrophages and in (D) M0 macrophages treated with the different nanocomplexes after 24 h of incubation. Poly(I:C) was used at the final dose of 5 μg/mL. Each symbol shape represents a different donor. Values represent mean ± SD (n ≥ 3). Statistical comparison was done using an ordinary one-way ANOVA followed by a Tukey's comparison test between groups. Statistically significant differences are represented as *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001. C12r8, laurate-octaarginine; HA, hyaluronic acid; pArg, poly-arginine; PEG–PGA, pegylated polyglutamic acid; pIC, poly(I:C).

Figure 7

Macrophage cytotoxicity toward PANC-1 cancer cells after pre-treatment with the different nanocomplexes. (A) Schematic representation of the in vitro model for the determination of the killing capacity of pre-treated macrophages. (B,C) % of cancer cell death caused by (B) the prototypic macrophages or (C) M0 macrophages pre-treated with free or nanocomplexed poly(I:C). Poly(I:C) was used at the final dose of 5 μg/mL. Each symbol shape represents a different donor. Values represent mean ± SD (n ≥ 3). Statistical comparison was done using an ordinary one-way ANOVA followed by a Tukey's multiple comparison test between groups. Statistically significant differences are represented as ***p < 0.005. C12r8, laurate-octaarginine; HA, hyaluronic acid; pArg, poly-arginine; PANC-1, pancreatic cancer cells; PEG–PGA, pegylated polyglutamic acid; pIC, poly(I:C).

Figure 8

Schematic illustration of the in vitro effects of the poly(I:C) ENCPs developed in this study. (A) Upon interaction with macrophages, poly(I:C) nanocomplexes are taken up. (B) This process allows poly(I:C) to reach its target receptor TLR3, found in the endosomes. It is expected that this interaction activates the TLR3 and the TRIF pathway, stimulating the upregulation of type I IFN genes. (C) The expression of M2 (CD206 and CD163) surface markers was slightly decreased, while M1 (CD80 and MHC II) markers are not substantially modified. Nevertheless, (D) CXCL10 and CCL5 chemokines, involved in the attraction of CD8 T cells to the tumor microenvironment, are secreted. (E) The direct cytotoxicity of macrophages toward cancer cells is also enhanced. Images were reproduced from Servier Medical Art under a Creative Commons Attribution 3.0 Unported License https://creativecommons.org/licenses/by/3.0.