Shann S. Yu

Emerging roles of lymphatic endothelium in regulating adaptive immunity

Emerging research on the roles of stromal cells in modulating adaptive immune responses has included a new focus on lymphatic endothelial cells (LECs). LECs are presumably the first cells that come into direct contact with peripheral antigens, cytokines, danger signals, and immune cells travelling from peripheral tissues to lymph nodes. LECs can modulate dendritic cell function, present antigens to T cells on MHC class I and MHC class II molecules, and express immunomodulatory cytokines and receptors, which suggests that their roles in adaptive immunity are far more extensive than previously realized. This Review summarizes the emergent evidence that LECs are important in maintaining peripheral tolerance, limiting and resolving effector T cell responses, and modulating leukocyte function.

Size- and charge-dependent non-specific uptake of PEGylated nanoparticles by macrophages

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 T2-weighted magnetic resonance imaging. These findings guide the design of imaging or therapeutic nanoparticles for in vivo targeting of macrophage activity in pathologic states.

Engineering complement activation on polypropylene sulfide vaccine nanoparticles.

The complement system is an important regulator of both adaptive and innate immunity, implicating complement as a potential target for immunotherapeutics. We have recently presented lymph node-targeting, complement-activating nanoparticles (NPs) as a vaccine platform. Here we explore modulation of surface chemistry as a means to control complement deposition, in active or inactive forms, on polypropylene sulfide core, block copolymer Pluronic corona NPs. We found that nucleophile-containing NP surfaces activated complement and became functionalized in situ with C3 upon serum exposure via the alternative pathway. Carboxylated NPs displayed a higher degree of C3b deposition and retention relative to hydroxylated NPs, upon which deposited C3b was more substantially inactivated to iC3b. This in situ functionalization correlated with in vivo antigen-specific immune responses, including antibody production as well as T cell proliferation and IFN-γ cytokine production upon antigen restimulation. Interestingly, inactivation of C3b to iC3b on the NP surface did not correlate with NP affinity to factor H, a cofactor for protease factor I that degrades C3b into iC3b, indicating that control of complement protein C3 stability depends on architectural details in addition to factor H affinity. These data show that design of NP surface chemistry can be used to control biomaterials-associated complement activation for immunotherapeutic materials.

Ex vivo red blood cell hemolysis assay for the evaluation of pH-responsive endosomolytic agents for cytosolic delivery of biomacromolecular drugs.

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this barrier, a number of synthetic drug carriers have been engineered to actively disrupt the endosomal membrane and deliver cargo into the cytoplasm. Here, we describe the hemolysis assay, which can be used as rapid, high-throughput screen for the cytocompatibility and endosomolytic activity of intracellular drug delivery systems. In the hemolysis assay, human red blood cells and test materials are co-incubated in buffers at defined pHs that mimic extracellular, early endosomal, and late endo-lysosomal environments. Following a centrifugation step to pellet intact red blood cells, the amount of hemoglobin released into the medium is spectrophotometrically measured (405 nm for best dynamic range). The percent red blood cell disruption is then quantified relative to positive control samples lysed with a detergent. In this model system the erythrocyte membrane serves as a surrogate for the lipid bilayer membrane that enclose endo-lysosomal vesicles. The desired result is negligible hemolysis at physiologic pH (7.4) and robust hemolysis in the endo-lysosomal pH range from approximately pH 5-6.8.

Physiologically Relevant Oxidative Degradation of Oligo(proline) Cross-Linked Polymeric Scaffolds

Chronic inflammation-mediated oxidative stress is a common mechanism of implant rejection and failure. Therefore, polymer scaffolds that can degrade slowly in response to this environment may provide a viable platform for implant site-specific, sustained release of immunomodulatory agents over a long time period. In this work, proline oligomers of varying lengths (P(n)) were synthesized and exposed to oxidative environments, and their accelerated degradation under oxidative conditions was verified via high performance liquid chromatography and gel permeation chromatography. Next, diblock copolymers of poly(ethylene glycol) (PEG) and poly(ε-caprolactone) (PCL) were carboxylated to form 100 kDa terpolymers of 4%PEG-86%PCL-10%cPCL (cPCL = poly(carboxyl-ε-caprolactone); i% indicates molar ratio). The polymers were then cross-linked with biaminated PEG-P(n)-PEG chains, where P(n) indicates the length of the proline oligomer flanked by PEG chains. Salt-leaching of the polymeric matrices created scaffolds of macroporous and microporous architecture, as observed by scanning electron microscopy. The degradation of scaffolds was accelerated under oxidative conditions, as evidenced by mass loss and differential scanning calorimetry measurements. Immortalized murine bone-marrow-derived macrophages were then seeded on the scaffolds and activated through the addition of γ-interferon and lipopolysaccharide throughout the 9-day study period. This treatment promoted the release of H(2)O(2) by the macrophages and the degradation of proline-containing scaffolds compared to the control scaffolds. The accelerated degradation was evidenced by increased scaffold porosity, as visualized through scanning electron microscopy and X-ray microtomography imaging. The current study provides insight into the development of scaffolds that respond to oxidative environments through gradual degradation for the controlled release of therapeutics targeted to diseases that feature chronic inflammation and oxidative stress.

Enzymatic- and temperature-sensitive controlled release of ultrasmall superparamagnetic iron oxides (USPIOs)

BackgroundDrug and contrast agent delivery systems that achieve controlled release in the presence of enzymatic activity are becoming increasingly important, as enzymatic activity is a hallmark of a wide array of diseases, including cancer and atherosclerosis. Here, we have synthesized clusters of ultrasmall superparamagnetic iron oxides (USPIOs) that sense enzymatic activity for applications in magnetic resonance imaging (MRI). To achieve this goal, we utilize amphiphilic poly(propylene sulfide)-bl-poly(ethylene glycol) (PPS-b-PEG) copolymers, which are known to have excellent properties for smart delivery of drug and siRNA.ResultsMonodisperse PPS polymers were synthesized by anionic ring opening polymerization of propylene sulfide, and were sequentially reacted with commercially available heterobifunctional PEG reagents and then ssDNA sequences to fashion biofunctional PPS-bl-PEG copolymers. They were then combined with hydrophobic 12 nm USPIO cores in the thin-film hydration method to produce ssDNA-displaying USPIO micelles. Micelle populations displaying complementary ssDNA sequences were mixed to induce crosslinking of the USPIO micelles. By design, these crosslinking sequences contained an EcoRV cleavage site. Treatment of the clusters with EcoRV results in a loss of R2 negative contrast in the system. Further, the USPIO clusters demonstrate temperature sensitivity as evidenced by their reversible dispersion at ~75°C and re-clustering following return to room temperature.ConclusionsThis work demonstrates proof of concept of an enzymatically-actuatable and thermoresponsive system for dynamic biosensing applications. The platform exhibits controlled release of nanoparticles leading to changes in magnetic relaxation, enabling detection of enzymatic activity. Further, the presented functionalization scheme extends the scope of potential applications for PPS-b-PEG. Combined with previous findings using this polymer platform that demonstrate controlled drug release in oxidative environments, smart theranostic applications combining drug delivery with imaging of platform localization are within reach. The modular design of these USPIO nanoclusters enables future development of platforms for imaging and drug delivery targeted towards proteolytic activity in tumors and in advanced atherosclerotic plaques.

The Multistrata Nanoparticle: an FeOx/Au Core/Shell Enveloped in a Silica–Au Shell

nanoparticles, have been clinically assessed for tissue-specifi c photothermal therapy [ 12 ] optimized for in vivo use by design of the surface plasmonic properties of the nanomaterial. Unlike FeOx/Au nanoparticles, which have plasmonic extinction peaks in the visible spectrum, extinction peaks in the NIR (700‐1200 nm) can be achieved by control of the core/ shell thickness ratio of the silica/gold layers. Extinction peaks in the NIR allow for the optimal heating of subdermal tissue for photothermal therapy [ 19 ] and effi cient optical imaging. Harnessing the surface plasmon resonance properties of core/shell materials, the nanosphere-in-a-nanoshell (the “gold nanomatryushka”) [ 20 ] was synthesized and demonstrated

Abstract 3981: Reprogramming tumor associated macrophages toward an anti-tumor phenotype by targeting the NF-κB pathway using novel targeted nanotherapeutics.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Tumor associated macrophages (TAMs) can modify the tumor microenvironment to create an inflammatory, pro-tumor niche. Activation of the NF-κB pathway has been implicated in creating a pro-tumor phenotype in TAMs. Manipulation of TAM phenotype is a new approach to engage anticancer immunity, but has been limited by a lack of methods capable of therapeutic delivery to TAMs in vivo. We have successfully utilized mannosylated polymer nanoparticles capable of disrupting the endosomal compartment to deliver siRNA for RNAi of NF-κB proteins into bone marrow derived macrophages (BMDMs) derived from transgenic mice with a GFP/Luciferase reporter of NF-κB activity (NGL). In in vitro studies, the nanoparticles are comparable to commercial transfection agents using both gene and protein level readouts for knockdown. The transfection protocol utilizing these novel vehicles has been optimized with respect to transfection time, siRNA dose, and siRNA:polymer ratio with the intent to inform in vivo experiments. The presence of serum does not significantly affect transfection efficiency in vitro, presumably due to an almost neutral particle surface charge. Preliminary in vivo studies have revealed no significant particle toxicity. Delivering siRNA specific to the p52/p100 protein in the alternative pathway achieves knockdown of total NF-κB activity by approximately 80% in NGL BMDMs stimulated by TNF-α. By targeting proteins in the classical pathway, we have decreased total NF-κB activity by approximately 50% in the same model. While inhibition of NF-κB activity may be desirable in some contexts, we recently reported that induced activation of NF-κB in macrophages can result in anti-tumor activity. We have delivered a liposomal formulation of muramyl tripeptide (Mifamurtide), a synthetic derivative of a bacterial cell wall peptide and an activator of macrophages, to NGL BMDMs. Mifamurtide delivery increases both NF-κB activity, and the production of reactive oxygen species, indicating a preliminary mechanistic explanation for the therapeutic potential of NF-κB activation. Mifamurtide is used clinically in the European Union to treat osteosarcoma, potentially providing an avenue for rapid clinical translation of NF-κB modulating therapy for other tumor types. However, Mifamurtide has the potential to activate multiple pathways simultaneously. A more elegant approach would be to target knockdown of an NF-κB inhibitor to macrophages to mediate pathway specific activation. In preliminary studies we have demonstrated the ability to increase total NF-κB activity by treating NGL macrophages with nanoparticles carrying siRNA against the IκBα inhibitor of NF-κB. Our data provides evidence that delivering siRNA specifically to macrophages to modulate their functions using nanoparticles has potential as a therapeutic approach to cancer treatment. Citation Format: Ryan A. Ortega, Whitney Barham, Bharat Kumar, Shann S. Yu, Fiona Yull, Todd D. Giorgio. Reprogramming tumor associated macrophages toward an anti-tumor phenotype by targeting the NF-κB pathway using novel targeted nanotherapeutics. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3981. doi:10.1158/1538-7445.AM2013-3981

Abstract 2894: Environmentally responsive nanoparticles for the intracellular delivery of RNAi therapeutics into tumor-associated macrophages

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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