Recent Advances in Optimizing Radiation Therapy Decisions in Early Invasive Breast Cancer.

Adjuvant whole breast irradiation after breast-conserving surgery is a well-established treatment standard for early invasive breast cancer. Screening, early diagnosis, refinement in surgical techniques, the knowledge of new and specific molecular prognostic factors, and now the standard use of more effective neo/adjuvant systemic therapies have proven instrumental in reducing the rates of locoregional relapses. This underscores the need for reliably identifying women with such low-risk disease burdens in whom elimination of radiation from the treatment plan would not compromise oncological safety. This review summarizes the current evidence for radiation de-intensification strategies and details ongoing prospective clinical trials investigating the omission of adjuvant whole breast irradiation in molecularly defined low-risk breast cancers and related evidence supporting the potential for radiation de-escalation in HER2+ and triple-negative clinical subtypes. Furthermore, we discuss the current evidence for the de-escalation of regional nodal irradiation after neoadjuvant chemotherapy. Finally, we also detail the current knowledge of the clinical value of stromal tumor-infiltrating lymphocytes and liquid-based biomarkers as prognostic factors for locoregional relapse.

Cucurbituril-mediated quantum dot aggregates formed by aqueous self-assembly for sensing applications.

Self-assembled nanoparticles have important applications in energy systems, optical devices and sensors, via the formation of aggregates with controlled interparticle spacing. Here we report aqueous self-assembly of rigid macrocycle cucurbit[7]uril (CB[7]) and fluorescent quantum dots (QDs), and demonstrate the potential of the system for efficient energy transfer and sensing of small molecules.

Small Surface, Big Effects, and Big Challenges: Toward Understanding Enzymatic Activity at the Inorganic Nanoparticle-Substrate Interface.

Enzymes are important biomarkers for molecular diagnostics and targets for the action of drugs. In turn, inorganic nanoparticles (NPs) are of interest as materials for biological assays, biosensors, cellular and in vivo imaging probes, and vectors for drug delivery and theranostics. So how does an enzyme interact with a NP, and what are the outcomes of multivalent conjugation of its substrate to a NP? This invited feature article addresses the current state of the art in answering this question. Using gold nanoparticles (Au NPs) and semiconductor quantum dots (QDs) as illustrative materials, we discuss aspects of enzyme structure-function and the properties of NP interfaces and surface chemistry that determine enzyme-NP interactions. These aspects render the substrate-on-NP configurations far more complex and heterogeneous than the conventional turnover of discrete substrate molecules in bulk solution. Special attention is also given to the limitations of a standard kinetic analysis of the enzymatic turnover of these configurations, the need for a well-defined model of turnover, and whether a "hopping" model can account for behaviors such as the apparent acceleration of enzyme activity. A detailed and predictive understanding of how enzymes turn over multivalent NP-substrate conjugates will require a convergence of many concepts and tools from biochemistry, materials, and interface science. In turn, this understanding will help to enable rational, optimized, and value-added designs of NP bioconjugates for biomedical and clinical applications.

Mimicking Cell Surface Enhancement of Protease Activity on the Surface of a Quantum Dot Nanoparticle.

Nature has evolved mechanisms to increase the specificity of enzymes and enhance their activity to support particular biological functions. Mimicking these mechanisms with artificial systems, such as nanoparticles, would greatly benefit bioanalysis. Here, we have taken inspiration from platelet cells to create a fluorescent nanoparticle probe with enhanced sensitivity toward its protease target. Platelets use protease-activated receptor 1 (PAR1) to enhance thrombin activity that initiates their aggregation in the early stages of blood clotting. We therefore coconjugated multiple copies each of a peptide substrate and a fragment of PAR1 to a semiconductor quantum dot (QD) to mimic this behavior. Thrombin activity toward conjugates with and without the PAR1 fragment were tracked via Förster resonance energy transfer (FRET). The co-conjugated PAR1 increased thrombin-catalyzed hydrolysis of the substrate by several-fold up to multiple orders of magnitude, albeit dependent on the surface chemistry of the QD. The enhancement effect arose from a combination of selective binding between the PAR1 fragment and thrombin, and from colocalization of the substrate and the PAR1 fragment at the QD interface. Substrate-receptor co-conjugation is thus a promising strategy for the rational design of nanoparticle bioconjugates with optimized sensitivity and specificity for biosensing and imaging.

Exploiting exciton coupling of ligand radical intervalence charge transfer transitions to tune NIR absorption.

We detail the rational design of a series of bimetallic bis-ligand radical Ni salen complexes in which the relative orientation of the ligand radical chromophores provides a mechanism to tune the energy of intense intervalence charge transfer (IVCT) bands in the near infrared (NIR) region. Through a suite of experimental (electrochemistry, electron paramagnetic resonance spectroscopy, UV-vis-NIR spectroscopy) and theoretical (density functional theory) techniques, we demonstrate that bimetallic Ni salen complexes form bis-ligand radicals upon two-electron oxidation, whose NIR absorption energies depend on the geometry imposed in the bis-ligand radical complex. Relative to the oxidized monomer [1˙]+ (E = 4500 cm-1, ε = 27 700 M-1 cm-1), oxidation of the cofacially constrained analogue 2 to [2˙˙]2+ results in a blue-shifted NIR band (E = 4830 cm-1, ε = 42 900 M-1 cm-1), while oxidation of 5 to [5˙˙]2+, with parallel arrangement of chromophores, results in a red-shifted NIR band (E = 4150 cm-1, ε = 46 600 M-1 cm-1); the NIR bands exhibit double the intensity in comparison to the monomer. Oxidation of the intermediate orientations results in band splitting for [3˙˙]2+ (E = 4890 and 4200 cm-1; ε = 26 500 and 21 100 M-1 cm-1), and a red-shift for [4˙˙]2+ using ortho- and meta-phenylene linkers, respectively. This study demonstrates for the first time, the applicability of exciton coupling to ligand radical systems absorbing in the NIR region and shows that by simple geometry changes, it is possible to tune the energy of intense low energy absorption by nearly 400 nm.

Polyacrylamide gel electrophoresis of semiconductor quantum dots and their bioconjugates: materials characterization and physical insights from spectrofluorimetric detection.

Colloidal semiconductor quantum dot (QD) nanocrystals have ideal fluorescence properties for bioanalysis and bioimaging, but these materials must be functionalized with an inorganic shell, organic ligand or polymer coating, and conjugated with biomolecules to be useful in such applications. Several different analytical techniques are used to characterize QDs and their multiple layers of functionalization. Here, we revisit poly(acrylamide) gel electrophoresis (PAGE), which has been scarcely used for the characterization of QDs and their bioconjugates in deference to the routine use of agarose gel electrophoresis. We implemented PAGE in a novel "stubby" capillary format with spectrofluorimetric detection, the combination of which enabled more rapid and more detailed characterization of QDs than was possible with both poly(acrylamide) and agarose slab gels. Correlations between the peak photoluminescence (PL) emission wavelength and electropherogram peaks, especially when combined with Ferguson analysis, provided new and significant insight into the key factors that determine the electrophoretic mobility of QDs, and helped to resolve heterogeneity and sub-populations in ensembles of QDs. The method was useful for characterization of the inorganic core/shell nanocrystals, their organic ligand and polymer coatings, and their final bioconjugates, the latter of which were in the form of peptide and protein conjugates. With further development and optimization, we anticipate that capillary PAGE with spectrofluorimetric detection will become a valuable addition to the toolbox of characterization techniques suitable for QDs, their bioconjugates, and other nanoparticle materials as well.

Optimization and Changes in the Mode of Proteolytic Turnover of Quantum Dot-Peptide Substrate Conjugates through Moderation of Interfacial Adsorption.

Enzymes have many important roles in biology and industry, and proteases are one of the most important classes of enzymes. Semiconductor quantum dots (QDs) are attractive materials for developing protease activity probes because of their advantageous physical and optical properties; however, interactions between a protease and a QD conjugated with its substrate can affect the turnover of that substrate. Here, we study the turnover of multivalent QD-peptide substrate conjugates as a function of multiple parameters: (i) the ligand coating on the QD, including dihydrolipoic acid (DHLA), glutathione (GSH), DHLA-poly(ethylene glycol) (DHLA-PEG), and DHLA-zwitterionic sulfobetaine (DHLA-SB); (ii) the identity of the protease, including trypsin, thrombin, and plasmin; and (iii) the number of substrate and nonsubstrate biomacromolecules conjugated per QD. We show that limiting protease adsorption on QDs is critical for optimizing the turnover of conjugated peptide substrates. Protease adsorption is inhibitory, and very strong adsorption leads to an apparent "scooting" mode of activity with limited turnover. In contrast, with weaker adsorption, enhancements in the turnover rate likely result from a "hopping" mode of activity. The putative hopping mode is thought to feature processive turnover of all substrates in multivalent conjugates with a rate-limiting step of diffusion between individual conjugates, and the magnitude of such enhancements increases with decreases in adsorption. Although it was possible to passivate DHLA- and GSH-coated QDs with high densities of conjugated biomacromolecules, the most effective strategy for reducing adsorption was the substitution of these ligands. Whereas passivation incrementally increased turnover, DHLA-PEG and DHLA-SB ligands converted the mode of turnover with plasmin from scooting to hopping and the DHLA-SB enhanced the turnover rates with thrombin and trypsin by approximately an order of magnitude relative to GSH ligands. The new insights from the broad scope of this study provide an important framework for designing optimized QD conjugates as probes and sensors for enzyme activity.