Distribution and Activity of Macrophage-Targeting Immunomodulatory Nanogels in Whole Blood

Introduction:: Macrophages adopt a spectrum of inflammatory or regenerative phenotypes in response to different environmental cues. Through antigen presentation, co-stimulatory, and effector functions they are key regulators of both the innate and adaptive immune responses. Macrophages’ diverse immune functions make them attractive therapeutic targets for immune diseases, such as cancer and autoimmunity. Cytokines regulate macrophage phenotype, but systemic infusion or delivery of cytokines for therapeutic purposes is limited by cytokines’ short half-life, instability, and systemic pleiotropy. Targeted delivery of cytokines to macrophages could overcome the limitations of cytokine therapeutics and enable new treatments for a variety of inflammatory diseases. Here, we hypothesized that cytokine conjugation to hydrogel nanoparticles (nanogels) would increase their macrophage colocalization, while retaining the cytokine’s immunostimulatory or suppressive activity. To test this hypothesis, we must optimize the biological activity of the nanogel-cytokine conjugates and evaluate the nanoparticle distribution within bloodstream circulation or peripheral tissue at the cellular level. Here, we developed and characterized synthetic poly(acrylamide-co-methacrylic acid) (P(AAm-co-MAA)) nanogels as cytokine delivery vehicles. We assessed the inflammatory activity of a nanogel conjugate with recombinant interferon gamma (rIFN-γ) to macrophages in vitro, and quantified nanogel distribution with whole blood components ex vivo.

Materials and Methods:: To establish a nanogel-cytokine platform for immune therapy, we conjugated mouse rIFN-γ to P(AAm-co-MAA) nanogels using a two-step NHS/EDC carbodiimide reaction at 1 wt%. We assessed the inflammatory activity of these conjugates by examining the expression of pro-inflammatory markers (MHC II, CD86) on Raw264.7 macrophages using flow cytometry after a 24-hour treatment with the conjugates. The activity of cytokine bioconjugates was compared to free rIFN-γ at 10 ng/ml and control nanogels at 0.1 mg/mL. The co-localization of cytokine-free Rhodamine B-conjugated nanogels with Raw264.7 macrophages was assessed using fluorescence microscopy and flow cytometry. To evaluate the interactions of the nanogels with other immune cells in whole blood, we incubated fluorescent nanogels (0.3 mg/mL) with healthy, gender pooled, heparinized mouse whole blood for three hours at 37°C on a rotator. The co-localization of nanogels with packed red blood cells (RBCs) and different immune cell subtypes was quantified using flow cytometry. To evaluate the influence of RBCs on the interaction between the nanogels and white blood cells (WBCs), we lysed RBCs prior to or after the incubation of the nanogels with the whole blood and quantified nanogel distribution using flow cytometry. Several quality control experiments were performed. The hydrodynamic diameter of nanogels was determined by dynamic light scattering (DLS). In vitro safety was established using LDH and MTS assays after incubation with Raw264.7 cells at concentrations up to 3 mg/mL for 24 hours. Statistical analysis was performed using GraphPad Prism.

Results, Conclusions, and Discussions:: Results and Discussion: The hydrodynamic diameter and zeta-potential of the nanogels were measured to be 97.59 ± 32.58 nm and -27.77 ± 7.01 mV, respectively. LDH and MTS assays did not show any significant dose-dependent toxicity to the cells treated with the nanogels (p-value: < 0.01). Immune phenotyping performed on Raw264.7 cells treated with the nanogel- rIFN-γ conjugates showed that rIFN-γ retained its activity after conjugation. However, the conjugates were less efficient than the free cytokine in inducing the expression MHC II and CD86. This observation was demonstrated by a 10-fold lower expression of CD86 and 3-fold lower expression of MHC II, at an estimated concentration of 1 µg/ml of conjugated rIFN-γ vs 10 ng/ml of free rIFN-γ. These results demonstrated the feasibility of using our nanogel-cytokine conjugates for modulating macrophage phenotype, with the potential to maximize their activity by modulating different reaction conditions. Fluorescence microscopy and flow cytometry confirmed the co-localization of the nanogels with Raw264.7 cells, with 99% of the cells showing a positive signal for Rhodamine B. Ex vivo cellular distribution studies revealed that nanogels do not co-localize any WBC subtype. This outcome was unaffected by the presence or absence of RBCs during nanogel incubation. Moreover, packed RBCs also did not co-localize with fluorescent nanogels. Together, these results indicate that our nanogels do not interact with blood cells ex vivo, remaining stably dispersed in plasma, but exhibit significant co-localization with adherent macrophages.

Conclusions: Our study demonstrated the successful synthesis of a nanogel-cytokine delivery system that retains inflammatory activity on Raw264.7 macrophages without evident cellular interactions in whole blood. Our proof of concept with the nanogel-IFN-γ conjugate shows promise in retaining cytokine activity after conjugation. Our ex vivo results highlighted the complex and variable interactions between nanoparticles and cellular components that are expected in vivo. Overall, our study contributes to the development of a potential new platform for immune therapy with improved stability and targeting ability, which may have translational potential for the treatment of inflammatory diseases.


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