Oxidative Degradation of Sequence-Defined Peptoid Oligomers.

Due to their N-substitution, peptoids are generally regarded as resistant to biological degradation, such as enzymatic and hydrolytic mechanisms. This stability is an especially attractive feature for therapeutic development and is a selling point of many previous biological studies. However, another key mode of degradation remains to be fully explored, namely oxidative degradation mediated by reactive oxygen and nitrogen species (ROS/RNS). ROS and RNS are biologically relevant in numerous contexts where biomaterials may be present, thus, improving understanding of peptoid oxidative susceptibility is crucial to exploit their full potential in the biomaterials field, where an oxidatively-labile but enzymatically stable molecule can offer attractive properties. Toward this end, we demonstrate a fundamental characterization of sequence-defined peptoid chains in the presence of chemically generated ROS, as compared to ROS-susceptible peptides such as proline and lysine oligomers. Lysine oligomers showed the fastest degradation rates to ROS and the enzyme trypsin. Peptoids degraded in metal catalyzed oxidation conditions at rates on par with poly(prolines), while maintaining resistance to enzymatic degradation. Furthermore, lysine-containing peptide-peptoid hybrid molecules showed tunability in both ROS-mediated and enzyme-mediated degradation, with rates intermediate to lysine and peptoid oligomers. When lysine-mimetic side-chains were incorporated into a peptoid backbone, the rate of degradation matched that of the lysine peptide oligomers, but remained resistant to enzymatic degradation. These results expand understanding of peptoid degradation to oxidative and enzymatic mechanisms, and demonstrate the potential for peptoid incorporation into materials where selectivity towards oxidative degradation is necessary, or directed enzymatic susceptibility is desired.

Peptomer substrates for quantitative pattern-recognition sensing of proteases.

The utility of active proteases as biomarkers is often limited by overlapping substrate specificity. Here, this feature is leveraged to develop a quantitative pattern-recognition sensing system driven by the degradation patterns of peptide-peptoid hybrid substrates to classify proteases and estimate their concentration by multivariate data analysis.

Fluorescent Peptomer Substrates for Differential Degradation by Metalloproteases.

Proteases, especially MMPs, are attractive biomarkers given their central role in both physiological and pathological processes. Distinguishing MMP activity with degradable substrates, however, is a difficult task due to overlapping substrate specificity profiles. Here, we developed a system of peptomers (peptide-peptoid hybrids) to probe the impact of non-natural residues on MMP specificity for an MMP peptide consensus sequence. Peptoids are non-natural, N-substituted glycines with a large side-chain diversity. Given the presence of a hallmark proline residue in the P3 position of MMP consensus sequences, we hypothesized that peptoids may offer N-substituted alternatives to generate differential interactions with MMPs. To investigate this hypothesis, peptomer substrates were exposed to five different MMPs, as well as bacterial collagenase, and monitored by fluorescence resonance energy transfer and liquid chromatography-mass spectrometry to determine the rate of cleavage and the composition of degraded fragments, respectively. We found that peptoid residues are well tolerated in the P3 and P3' substrate sites and that the identity of the peptoid in these sites displays a moderate influence on the rate of cleavage. However, peptoid residues were even better tolerated in the P1 substrate site where activity was more strongly correlated with side-chain identity than side-chain position. All MMPs explored demonstrated similar trends in specificity for the peptomers but exhibited different degrees of variability in proteolytic rate. These kinetic profiles served as "fingerprints" for the proteases and yielded separation by multivariate data analysis. To further demonstrate the practical application of this tunability in degradation kinetics, peptomer substrates were tethered into hydrogels and released over distinct timescales. Overall, this work represents a significant step toward the design of probes that maximize differential MMP behavior and presents design rules to tune degradation kinetics with peptoid substitutions, which has promising implications for diagnostic and prognostic applications using array-based sensors.

Assessing the range of enzymatic and oxidative tunability for biosensor design.

Development of multi-functional materials and biosensors that can achieve an in situ response designed by the user is a current need in the biomaterials field, especially in complex biological environments, such as inflammation, where multiple enzymatic and oxidative signals are present. In the past decade, there has been extensive research and development of materials chemistries for detecting and monitoring enzymatic activity, as well as for releasing therapeutic and diagnostic agents in regions undergoing oxidative stress. However, there has been limited development of materials in the context of enzymatic and oxidative triggers together, despite their closely tied and overlapping mechanisms. With research focusing on enzymatically and oxidatively triggered materials separately, these systems may be inadequate in monitoring the complexity of inflammatory environments, thus limiting in vivo translatability and diagnostic accuracy. The intention of this review is to highlight a variety of enzymatically and oxidatively triggered materials chemistries to draw attention to the range of synthetic tunability available for the construction of novel biosensors with a spectrum of programmed responses. We focus our discussion on several types of macromolecular sensors, generally classified by the causative material response driving ultimate signal detection. This includes sensing based on degradative processes, conformational changes, supramolecular assembly/disassembly, and nanomaterial interactions, among others. We see each of these classes providing valuable tools toward coalescing current gaps in the biosensing field regarding specificity, selectivity, sensitivity, and flexibility in application. Additionally, by considering the materials chemistry of enzymatically and oxidatively triggered biomaterials in tandem, we hope to encourage synthesis of new biosensors that capitalize on their synergistic roles and overlapping mechanisms in inflammatory environments for applications in disease diagnosis and monitoring.

Poly-d-lysine coated nanoparticles to identify pro-inflammatory macrophages.

Identifying pro-inflammatory macrophages (M1) is of immense importance to diagnose, monitor, and treat various pathologies. In addition, adoptive cell therapies, where harvested cells are isolated, modified to express an M1-like phenotype, then re-implanted to the patient, are also becoming more prevalent to treat diseases such as cancer. In a step toward identifying, labeling, and monitoring macrophage phenotype for adoptive cell therapies, we developed a reactive oxygen species (ROS)-sensitive, gold nanoparticle (AuNP) that fluorescently labels M1 macrophages. AuNPs are electrostatically coated with a proteolysis resistant, fluorescein isothiocyanate-conjugated, poly-d-lysine (PDL-FITC) that is susceptible to backbone cleavage by ROS. When PDL-FITC is bound to AuNPs, fluorescence is quenched via a combination of nanoparticle surface (NSET) and Forster resonance (FRET) energy transfer mechanisms. Upon ROS-induced cleavage of PDL-FITC, up to a 7-fold change in fluorescence is demonstrated. PDL-FITC AuNPs were loaded into RAW 264.7 macrophages (RAWs) and primary bone marrow- derived macrophages (BMDMs) prior to in vitro polarization. For both cell types, detectable differences in intracellular fluorescence were observed between M1 polarized and non-stimulated (M0) control groups after 24 h using both confocal imaging and flow cytometry. PDL-FITC AuNPs can potentially be useful in identifying M1 macrophages within diverse cell populations and provide longitudinal macrophage response data to external cues.

Formation of self-assembled gold nanoparticle supercrystals with facet-dependent surface plasmonic coupling.

Metallic nanoparticles, such as gold and silver nanoparticles, can self-assemble into highly ordered arrays known as supercrystals for potential applications in areas such as optics, electronics, and sensor platforms. Here we report the formation of self-assembled 3D faceted gold nanoparticle supercrystals with controlled nanoparticle packing and unique facet-dependent optical property by using a binary solvent diffusion method. The nanoparticle packing structures from specific facets of the supercrystals are characterized by small/wide-angle X-ray scattering for detailed reconstruction of nanoparticle translation and shape orientation from mesometric to atomic levels within the supercrystals. We discover that the binary diffusion results in hexagonal close packed supercrystals whose size and quality are determined by initial nanoparticle concentration and diffusion speed. The supercrystal solids display unique facet-dependent surface plasmonic and surface-enhanced Raman characteristics. The ease of the growth of large supercrystal solids facilitates essential correlation between structure and property of nanoparticle solids for practical integrations.

Pressure compression of CdSe nanoparticles into luminescent nanowires.

Oriented attachment (OA) of synthetic nanocrystals is emerging as an effective means of fabricating low-dimensional nanoscale materials. However, OA relies on energetically favorable nanocrystal facets to grow nanostructured materials. Consequently, nanostructures synthesized through OA are generally limited to a specific crystal facet in their final morphology. We report our discovery that high-pressure compression can induce consolidation of spherical CdSe nanocrystal arrays, leading to unexpected one-dimensional semiconductor nanowires that do not exhibit the typical crystal facet. In particular, in situ high-pressure synchrotron x-ray scattering, optical spectroscopy, and high-resolution transmission electron microscopy characterizations indicate that by manipulating the coupling between nanocrystals through external pressure, a reversible change in nanocrystal assemblies and properties can be achieved at modest pressure. When pressure is increased above a threshold, these nanocrystals begin to contact one another and consolidate, irreversibly forming one-dimensional luminescent nanowires. High-fidelity molecular dynamics (MD) methods were used to calculate surface energies and simulate compression and coalescence mechanisms of CdSe nanocrystals. The MD results provide new insight into nanowire assembly dynamics and phase stability of nanocrystalline structures.