Ralph Weissleder

Multiplexed bead assays for solution‐phase biosensing often encounter cross‐over reactions during signal amplification, leading to false positives and high background signals. Current solutions involve complex, custom‐designed equipment or costly setups, limiting their application in simple laboratory environments. In this study, we introduce a straightforward protocol to adapt a multiplexed single‐bead assay to standard fluorescence imaging plates, enabling the simultaneous analysis of thousands of reactions per plate. Our approach focuses on the design and synthesis of bright fluorescent and magnetic microspheres (MagSiGlow) with multiple fluorescent wavelengths serving as detection markers. This imaging‐based single‐bead assay, combined with a scripted algorithm, allows the detection, segmentation, and co‐localization on average of 7500 microspheres per field of view across five imaging channels …

Macrophages are critical regulators of the tumor microenvironment and often present an immuno-suppressive phenotype, supporting tumor growth and immune evasion. Promoting a robust pro-inflammatory macrophage phenotype has emerged as a therapeutic modality that supports tumor clearance, including through synergy with immune checkpoint therapies. Polyglucose nanoparticles (macrins), which possess high macrophage affinity, are useful vehicles for delivering drugs to macrophages, potentially altering their phenotype. Here, we examine the potential of functionalized macrins, synthesized by crosslinking carboxymethyl dextran with L-lysine, as effective carriers of immuno-stimulatory drugs to tumor-associated macrophages (TAMs). Azide groups incorporated during particle synthesis provided a handle for click-coupling of propargyl-modified β-cyclodextrin to macrins under mild conditions. Fluorescence-based competitive binding assays revealed the ability of β-cyclodextrin to non-covalently bind to hydrophobic immuno-stimulatory drug candidates (Keq ~ 103 M-1), enabling drug loading within nanoparticles. Furthermore, transcriptional profiles of macrophages indicated robust pro-inflammatory reprogramming (elevated Nos2 and Il12; suppressed Arg1 and Mrc1 expression levels) for a subset of these immuno-stimulatory agents (UNC2025 and R848). Loading of R848 into the modified macrins improved the drug’s effect on primary murine macrophages by three-fold in vitro. Intravital microscopy in IL-12-eYFP reporter mice (24 h post-injection) revealed a two-fold enhancement in mean YFP fluorescence intensity in macrophages …

Clinical treatment of cancer commonly incorporates X‐ray radiation therapy (XRT), and developing spatially‐precise radiation‐activatable drug delivery strategies may improve XRT efficacy while limiting off‐target toxicities associated with systemically administered drugs. Nevertheless, achieving this has been challenging thus far because strategies typically rely on radical species with short lifespans, and the inherent nature of hypoxic and acidic tumor microenvironments may encourage spatially heterogeneous effects. We hypothesized that the challenge could be bypassed by using scintillating nanoparticles that emit light upon X‐ray absorption, locally forming therapeutic drug depots in tumor tissues. We thus developed a nanoparticle platform ( Scintillating nanoparticle Drug Depot; SciDD) that enables the local release of cytotoxic payloads only after activation by XRT, thereby limiting off‐target toxicity. As a …

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Tumor‐associated macrophages (TAM) are a diverse population of myeloid cells that are often abundant and immunosuppressive in human cancers. CXCL9Hi TAM has recently been described to have an antitumor phenotype and is linked to immune checkpoint response. Despite the emerging understanding of the unique antitumor TAM phenotype, there is a lack of TAM‐specific therapeutics to exploit this new biological understanding. Here, the discovery and characterization of multiple small‐molecule enhancers of chemokine ligand 9 (CXCL9) and their targeted delivery in a TAM‐avid systemic nanoformulation is reported. With this strategy, it is efficient encapsulation and release of multiple drug loads that can efficiently induce CXCL9 expression in macrophages, both in vitro and in vivo in a mouse tumor model. These observations provide a window into the molecular features that define TAM‐specific states …

Disclosed herein are nanoparticles that include one or more cyclodextrin moieties crosslinked by a linker. The cyclodextrin moieties can complex therapeutic (eg, anticancer) agents, and can be used to treat diseases such as cancer.

Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) are a class of cancer drugs that enzymatically inhibit PARP activity at sites of DNA damage. Yet, PARPi function mainly by trapping PARP1 onto DNA with a wide range of potency among the clinically relevant inhibitors. How PARPi trap and why some are better trappers remain unknown. Here, we show trapping occurs primarily through a kinetic phenomenon at sites of DNA damage that correlates with PARPi koff. Our results suggest PARP trapping is not the physical stalling of PARP1 on DNA, rather the high probability of PARP re-binding damaged DNA in the absence of other DNA-binding protein recruitment. These results clarify how PARPi trap, shed new light on how PARPi function, and describe how PARPi properties correlate to trapping potency.

Exosomes and extracellular vesicles (EV) are increasingly being explored as circulating biomarkers, but their heterogenous composition will likely mandate the development of multiplexed EV technologies. Iteratively multiplexed analyses of near single EVs have been challenging to implement beyond a few colors during spectral sensing. Here we developed a multiplexed analysis of EV technique (MASEV) to interrogate thousands of individual EVs during 5 cycles of multi-channel fluorescence staining for 15 EV biomarkers. Contrary to the common belief, we show that: several markers proposed to be ubiquitous are less prevalent than believed; multiple biomarkers concur in single vesicles but only in small fractions; affinity purification can lead to loss of rare EV subtypes; and deep profiling allows detailed analysis of EV, potentially improving the diagnostic content. These findings establish the potential of MASEV …