In this work, we demonstrate the targeted diagnosis of immunomarker programmed death ligand 1 (PD-L1) and simultaneous detection of epidermal growth factor receptor (EGFR) in breast cancer tumors in vivo using gold nanostars (AuNS) with multiplexed surface enhanced Raman spectroscopy (SERS). Real-time longitudinal tracking with SERS demonstrated maximum accumulation of AuNS occurred 6 h post intravenous (IV) delivery, enabling detection of both biomarkers simultaneously. Raman signal correlating to both PD-L1 and EGFR decreased by ∼30% in control tumors where receptors were pre-blocked prior to AuNS delivery, indicating both the sensitivity and specificity of SERS in distinguishing tumors with different levels of PD-L1 and EGFR expression. Our in vivo study was combined with the first demonstration of ex vivo SERS spatial maps of whole tumor lesions that provided both a qualitative and quantitative assessment of biomarker status with near cellular-level resolution. High resolution SERS maps also provided an overview of AuNS distribution in tumors which correlated well with the vascular density. Mass spectrometry showed AuNS accumulation in tumor and liver, and clearance via spleen, and electron microscopy revealed AuNS were endocytosed in tumors, Kupffer cells in the liver, and macrophages in the spleen. This study demonstrates that SERS-based diagnosis mediated by AuNS provides an accurate measure of multiple biomarkers both in vivo and ex vivo, which will ultimately enable a clinically-translatable platform for patient-tailored immunotherapies and combination treatments.
Targeting of drug carriers to endocytic cell-receptors facilitates intracellular drug delivery. Carrier size and number of targeting moieties (valency) influence cell binding and uptake. However, how these parameters influence receptor-mediated cell-signaling (the link between binding and uptake) remains uncharacterized. We studied this using polymer carriers of different sizes and valencies, targeted to endothelial intercellular adhesion molecule-1 (ICAM-1), a marker overexpressed in many pathologies. Unexpectedly, induction of cell-signals (ceramide and PKC enrichment and activation) and uptake, were independent of: carrier avidity, total number of carriers bound per cell, cumulative cell-surface area occupied by carriers, number of targeting antibodies at the carrier-cell contact, and cumulative receptor engagement by all bound carriers. Instead, “valency density” (number of antibodies per carrier surface area) ruled signaling, and carrier size independently influenced uptake. These results are key to understanding the interplay between carrier design parameters and receptor-mediated signaling conducive to endocytosis, paramount for intracellular drug delivery.
Precise monitoring of specific biomarkers in biological fluids with accurate biodiagnostic sensors is critical for early diagnosis of diseases and subsequent treatment planning. In this work, we demonstrated an innovative biodiagnostic sensor, portable reusable accurate diagnostics with nanostar antennas (PRADA), for multiplexed biomarker detection in small volumes (~50 μl) enabled in a microfluidic platform. Here, PRADA simultaneously detected two biomarkers of myocardial infarction, cardiac troponin I (cTnI), which is well accepted for cardiac disorders, and neuropeptide Y (NPY), which controls cardiac sympathetic drive. In PRADA immunoassay, magnetic beads captured the biomarkers in human serum samples, and gold nanostars (GNSs) "antennas" labeled with peptide biorecognition elements and Raman tags detected the biomarkers via surface-enhanced Raman spectroscopy (SERS). The peptide-conjugated GNS-SERS barcodes were leveraged to achieve high sensitivity, with a limit of detection (LOD) of 0.0055 ng/ml of cTnI, and a LOD of 0.12 ng/ml of NPY comparable with commercially available test kits. The innovation of PRADA was also in the regeneration and reuse of the same sensor chip for~14 cycles. We validated PRADA by testing cTnI in 11 de-identified cardiac patient samples of various demographics within a 95% confidence interval and high precision profile. We envision low-cost PRADA will have tremendous translational impact and be amenable to resourcelimited settings for accurate treatment planning in patients.
In this study, we demonstrate the
theranostic capability of actively
targeted, site-specific multibranched gold nanoantennas (MGNs) in
triple-negative breast cancer (TNBC) cells in vitro. By utilizing
multiplexed surface-enhanced Raman scattering (SERS) imaging, enabled
by the narrow peak widths of Raman signatures, we simultaneously targeted
immune checkpoint receptor programmed death ligand 1 (PDL1) and the
epidermal growth factor receptor (EGFR) overexpressed in TNBC
cells. A 1:1 mixture of MGNs functionalized with anti-PDL1 antibodies
and Raman tag 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) and MGNs
functionalized with anti-EGFR antibodies and Raman tag para-mercaptobenzoic acid (pMBA) were incubated with
the cells. SERS imaging revealed a cellular traffic map of MGN localization
by surface binding and receptor-mediated endocytosis, enabling targeted
diagnosis of both biomarkers. Furthermore, cells incubated with anti-EGFR–pMBA–MGNs and illuminated with an 808 nm laser for
15 min at 4.7 W/cm2 exhibited photothermal cell death only
within the laser spot (indicated by live/dead cell fluorescence assay).
Therefore, this study not only provides an optical imaging platform
that can track immunomarkers with spatiotemporal control but also
demonstrates an externally controlled light-triggered therapeutic
approach enabling receptor-specific treatment with biocompatible theranostic
nanoprobes.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.