Nanoparticle Internalization Promotes the Survival of Primary Macrophages

Abstract

Macrophages, a class of tissue resident innate immune cells, are responsible for sequestering foreign objects through the process of phagocytosis, making them a promising target for immune-modulation via particulate engineering. Here, we report that nanoparticle (NP) dosing and cellular internalization via phagocytosis significantly enhances survival of ex vivo cultures of primary bone marrow-derived, alveolar, and peritoneal macrophages over particle-free controls. The enhanced survival is attributed to suppression of caspase-dependent apoptosis and is linked to phagocytosis and lysosomal signaling, which was also found to occur in vivo. Uniquely, poly(ethylene glycol)-based NP treatment does not alter macrophage polarization or lead to inflammatory effects. The enhanced survival phenomenon is also applicable to NPs of alternative chemistries, indicating the potential universality of this phenomenon with relevant drug delivery particles. These findings provide a framework for extending the lifespan of primary macrophages ex vivo for drug screening, vaccine studies, and cell therapies and has implications for any in vivo particulate immune-engineering applications.

Hydrogel nanoparticle degradation influences the activation and survival of primary macrophages

“…In all of the tested formulations, NP treatment enhanced the survival of ex vivo BMMs in a concentration-dependent manner. This trend agrees with results from our previous study with other PEGDA-based NPs, 18 even with the notably different additions to the PEGDA NP compositions. Treatment of NPs to BMMs at a concentration of 100 mg mL À1 resulted in statistically significantly higher % viability than the untreated (UT) cells for the three tested NP formulations as early as 72 h following treatment (p o 0.05 using Tukey's multiple comparisons tests as part of a two-way ANOVA).…”

“…44 Following the synthesis of the 0%, 10%, and 20% NPs, we characterized the NPs via DLS, cryo-SEM, zeta potential, and EDS to obtain NP sizes (DLS, cryo-SEM), overall surface charge (zeta potential), and relative sulfur content (XEDS). Similar to our previous syntheses of PEGDA-based NPs, 18,41 the synthesized NPs were typically B500 nm in diameter, as measured via DLS (Fig. 2A-C) and confirmed via cryo-SEM (examples shown in Fig.…”

“…We previously demonstrated that treatment of inert 17 poly(ethylene glycol) (PEG) diacrylate (PEGDA)-based nanoparticles (NPs) drives pro-survival signaling following NP internalization in a range of ex vivo and in vivo macrophages. 18 Combined with supporting studies of pro-survival signaling centered in the lysosome, 19,20 this prior work highlights an untapped opportunity for intelligently-designed NP platforms to further modulate this response. Upon phagocytosis, the phagosome undergoes compartment acidification and fusion with the lysosome to form the phagolysosome, a strongly acidic and hydrolytic environment enriched with a wide range of enzymes and signaling molecules that are responsible for breakdown of the internalized material and the triggering of subsequent cell activation signaling.…”

“…HDDMA and a higher amount of CEA were included to improve the resulting hydrogel NP modulus and surface charge, which are notable differences to our previously used formulations in studying NP-macrophage interactions. 18 1 mg of photoinitiator diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (PI) (Millipore Sigma) and 0.05 mg fluorescent label cyanine 5 (Cy5) maleimide (AAT Bioquest) were added and the formulations were diluted 1 : 1 by mass in methanol (Fisher Scientific) to arrive at 50 wt% mixtures. 100 mL of the mixture was emulsified in 1 mL of silicone oil AP1000 (Millipore Sigma) by vortex mixing for 1 minute followed by sonicating for 30 seconds.…”

“…We began this exploration by investigating the effect of NP degradation rate on pro-apoptotic effectors. We have previously demonstrated that NP internalization by BMMs enhances survival through the upregulation of anti-apoptotic Bcl-2 family genes and proteins, 18 which have been shown to suppress caspase-dependent apoptotic pathways. 55 Caspase-3/7 activity was measured in BMMs treated with 100 mg mL À1 , 50 mg mL À1 , and 10 mg mL À1 of 0%, 10%, and 20% NPs and caspase-3/7 activity was normalized to the corresponding cell count in each group (Fig.…”