Macrophages represent an important therapeutic target, because their activity has been implicated in the progression of debilitating diseases such as cancer and atherosclerosis. In this work, we designed and characterized pH-responsive polymeric micelles that were mannosylated using ‘click’ chemistry in order to achieve CD206 (mannose receptor)-targeted siRNA delivery. CD206 is primarily expressed on macrophages and dendritic cells, and upregulated in tumor-associated macrophages, a potentially useful target for cancer therapy. The mannosylated nanoparticles improved siRNA delivery into primary macrophages by 4-fold relative to a non-targeted version of the same carrier (p < 0.01). Further, 24h of treatment with the mannose-targeted siRNA carriers achieved 87±10% knockdown of a model gene in primary macrophages, cell type that is typically difficult to transfect. Finally, these nanoparticles were also avidly recognized and internalized by human macrophages and facilitated the delivery of 13-fold more siRNA into these cells relative to model breast cancer cell lines. We anticipate that these mannose receptor-targeted, endosomolytic siRNA delivery nanoparticles will become an enabling technology to target macrophage activity in various diseases, especially those where CD206 is up-regulated in macrophages present within the pathologic site. This work also establishes a generalizable platform that could be applied for click functionalization with other targeting ligands to direct siRNA delivery.

Abstract:The assessment of macrophage response to nanoparticles is a central component in the evaluation of new nanoparticle designs for future in vivo application. This work investigates which feature, nanoparticle size or charge, is more predictive of non-specific uptake of nanoparticles by macrophages. This was investigated by synthesizing a library of polymer-coated iron oxide micelles, spanning a range of 30-100 nm in diameter and −23 mV to +9 mV, and measuring internalization into macrophages in vitro. Nanoparticle size and charge both contributed towards non-specific uptake, but within the ranges investigated, size appears to be a more dominant predictor of uptake. Based on these results, a protease-responsive nanoparticle was synthesized, displaying a matrix metalloproteinase-9 (MMP-9)-cleavable polymeric corona. These nanoparticles are able to respond to MMP-9 activity through the shedding of 10-20 nm of hydrodynamic diameter. This MMP-9-triggered decrease in nanoparticle size also led to up to a six-fold decrease in nanoparticle internalization by macrophages and is observable by T 2 -weighted magnetic resonance imaging. These findings guide the design of imaging or therapeutic nanoparticles for in vivo targeting of macrophage activity in pathologic states.

In this study, a microgel composed of chitosan and inorganic phosphates was used to deliver poly(lactic-co-glycolic acid) (PLAGA) microspheres loaded with sphingolipid growth factor FTY720 to critical size cranial defects in Sprague Dawley rats. We show that sustained release of FTY720 from injected microspheres used alone or in combination with recombinant human bone morphogenic protein-2 (rhBMP2) improves defect vascularization and bone formation in the presence and absence of rhBMP2 as evaluated by quantitative microCT and histological measurements. Moreover, sustained delivery of FTY720 from PLAGA and local targeting of sphingosine 1-phosphate (S1P) receptors reduces CD45+ inflammatory cell infiltration, promotes endogenous recruitment of CD29+CD90+ bone progenitor cells and enhances the efficacy of rhBMP2 from chitosan microgels. Companion in vitro studies suggest that selective activation of sphingosine receptor subtype-3 (S1P3) via FTY720 treatment induces smad-1 phosphorylation in bone-marrow stromal cells. Additionally, FTY720 enhances stromal cell-derived factor-1 (SDF-1) mediated chemotaxis of CD90+CD11B-CD45- bone progenitor cells in vitro after stimulation with rhBMP2. We believe that use of such small molecule delivery formulations to recruit endogenous bone progenitors may be an attractive alternative to exogenous cell-based therapy.

Tumor-associated macrophages (TAMs) represent a potentially promising therapeutic target in cancer because they have been shown to facilitate tumor growth, invasiveness, and metastasis. However, methods to specifically target therapies to TAMs are lacking. To address this problem, we designed and synthesized mannosylated micellar nanoparticles (ManNPs), composed of tri-block co-polymers. The three polymer blocks include (1) an azido-displaying block for functionalization with biomolecules via azide-alkyne ‘click’ chemistry, (2) a cationic block for the condensation of polyanions such as siRNA, and (3) a pH-responsive terpolymer block that facilitates endosomal disruption. This terpolymer is hydrophobic at pH 7.4, allowing these polymers to self-assemble into 25 nm micellar nanoparticles under physiologic conditions. However, they become protonated at lower pH ranges representative of endosomal compartments (5.8 - 6.2), leading to disassembly of the nanoparticles, and increased ability to disrupt endosomal membranes and enable cytoplasmic delivery. This pH-dependent behavior has been validated using a red blood cell hemolysis assay. This environmentally-responsive behavior facilitates improved cytoplasmic delivery of siRNA, access to the intracellular silencing machinery, and consequently, knockdown of target gene expression. Further, we demonstrate that mannosylation of these nanoparticles via ‘click’ chemistry significantly enhances their ability to deliver siRNA into murine bone marrow-derived primary macrophages (BMDMs), relative to control, untargeted nanoparticles. Targeted nanoparticle uptake is mediated specifically through the macrophage mannose receptor (CD206), as validated through competition experiments with free mannose, or by pre-incubating BMDMs with lipopolysaccharide to downregulate CD206 expression. This is particularly important for cancer applications because CD206 is upregulated in tumor-suppressed and non-activated macrophages, enabling more specific targeting of TAMs versus healthy macrophages in other tissues. The ManNPs described here present new opportunities to target TAMs in various cancers, providing an enabling technology for the modification of the immunosuppressive tumor environment by targeting TAM activity. Studies are pending to demonstrate this behavior in an in vivo murine model of metastatic breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2894. doi:1538-7445.AM2012-2894

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