Phagocytosed Polyhedrin-Cytokine Cocrystal Nanoparticles Provide Sustained Secretion of Bioactive Cytokines from Macrophages

Abstract

Many cells possess the ability to engulf and incorporate particles by phagocytosis. This active process is characteristic of microorganisms as well as higher order species. In mammals, monocytes, macrophages, and microglia are among the so-called professional phagocytes. In addition, cells such as fibroblast and chondrocytes are classified as nonprofessional phagocytes. Professional phagocytes play important roles in both the innate and adaptive immune responses, wound healing, and tissue homeostasis. Consequently, these cells are increasingly studied as targets and vectors of therapeutic intervention to treat a range of diseases. Professional phagocytes are notoriously difficult to transfect limiting their study and manipulation. Consequently, efforts have shifted towards the development of nanoparticles to deliver a cargo to phagocytic cells via phagocytosis. However, this approach carries significant technical challenges, particularly for protein cargos. We have focused on the development of nanoscale cocrystalline protein depots, known as PODS®, that contain protein cargos, including cytokines. Here, we show that PODS are readily phagocytosed by nonprofessional as well as professional phagocytic cells and have attributes, such as highly sustained release of cargo, that suggest potential utility for the study and exploitation of phagocytic cells for drug delivery. Monocytes and macrophages that ingest PODS retain normal characteristics including a robust chemotactic response. Moreover, the PODS-cytokine cargo is secreted by the loaded cell at a level sufficient to modulate the behavior of surrounding nonphagocytic cells. The results presented here demonstrate the potential of PODS nanoparticles as a novel molecular tool for the study and manipulation of phagocytic cells and for the development of Trojan horse immunotherapy strategies to treat cancer and other diseases.

Low-dose cytokine immunotherapy of solid cancers enabled by phagocytic-competent protein co-crystals

“…There was a direct relationship between the number of PODS and the amount of accumulated IL-2 detected by the ELISA assay. As had been observed in our previous study 5 , where human IL-6 release was measured from phagocytosed PODS, ingestion by macrophages significantly reduces the amount of free cargo protein accumulating in the media. In the absence of macrophages, (measured only on day 6) the amount of IL-2 that accumulated was about 10-fold higher.…”

“…As may be predicted from their physical characteristics 12 (size, shape, surface charge and elasticity), we previously reported 5 that PODS are efficiently taken up by phagocytic cells in-vitro. We also demonstrated that, following phagocytic uptake, the PODS cargo proteins are sustainably secreted in a bioactive form that is able to modulate the phenotype of adjacent heterogenous cells.…”

“…Measurement of the release of hIL-2 from PODS-hIL-2, with and without macrophages THP-1 cells were seeded at a density of 2x10 5 cells/ml in 96-well plates and differentiated into M0 macrophages. M0 cells were then incubated with 1x10 5 , 2x10 5 , or 4x10 5 PODS IL-2 per ml for 24 h in complete medium (resulting in 5, 10, or 20 PODS/cell). Additionally, the same number of PODS containing human IL-2 (PODS-IL-2) were added to the wells of a 96-well plate without cells and spun down at 3000 x g for 25 min.…”

“…To address this challenge, a drug delivery mechanism that (1) stabilizes the protein (2) maintains low serum levels (3) actively delivers to and targets the diseased tissue and (4) provides sustained release at the target tissue, can be expected to reduce drug-related toxicity and improve therapeutic efficacy. PODS® (Polyhedrin Delivery System) 5 is a natural mimetic technology that incorporates target proteins into nano-to-micron-scale crystal structures to form PODS co-crystals (PODS). Purified PODS are non-brittle, durable, and transparent.…”

Exploiting Nanomedicine for Cancer Polychemotherapy: Recent Advances and Clinical Applications

“…They showed that PODS particles are very efficiently phagocytosed and that cargo cytokines avoid or resist the harsh conditions of phagolysosomes and enable secretion by macrophages. These results suggest the potential utility of PODS in Trojan horse drug delivery strategies for the treatment of cancer [ 206 ].…”