Observing structural integrity of nanoparticles is essential in bionanotechnology but not always straightforward to measure in situ and in real-time. Fluorescent labels used for tracking ...intrinsically nonfluorescent nanomaterials generally do not allow simultaneous observation of integrity. Consequently, structural changes like degradation and disassembly cannot easily be followed in situ using fluorescence signals. We show that thioflavin T (ThT), a fluorophore and molecular rotor known to tag specific fibril structures in amyloids, can “label” the structural integrity of widely used and intrinsically nonfluorescent, silica nanoparticles (SiNPs). Entrapment of ThT in SiNPs controls the fluorohphore’s relaxation pathway and leads to a red-shifted fluorescence spectrum providing real time information on SiNP integrity. The dynamic change of ThT fluorescence during degradation of doped SiNPs is found much higher than that of common labels fluorescein and rhodamine. Degradation kinetics of core–shell structures recorded by ThT fluorescence and light scattering prove the capability to clearly distinguish structural features during SiNPs degradation and allow obtaining degradation kinetics in vitro, in biological media, in serum, and in cells. The effect is transferable to different types of materials, here shown for ThT incorporated SiNPs with tightly tailorable sizes (9–100 nm), poly(lactic-co-glycolic acid) (PLGA) nanoparticles, poly(9-vinylcarbazole) (PVK) nanoparticles, and iron-doped-SiNPs (FeSiNPs). We thus suggest molecular rotors such as ThT as additional labels to effectively and easily sense nanoparticle structural status in situ and to enhance understanding and development of programmed nanoparticle disassembly in bionanotechnology.
Biopharmaceuticals are large, complex and labile therapeutic molecules prone to instability due to various factors during manufacturing. To ensure their safety, quality and efficacy, a wide range of ...critical quality attributes (CQAs) such as product concentration, aggregation, particle size, purity and turbidity have to be met. Size exclusion chromatography (SEC) is the gold standard to measure protein aggregation and degradation. However, other techniques such as dynamic light scattering (DLS) are employed in tandem to measure the particle size distribution (PSD) and polydispersity of biopharmaceutical formulations. In this study, the application of multi-angle dynamic light scattering (MADLS) was evaluated for the determination of particle size, particle concentration and aggregation in 3 different protein modalities, namely bovine serum albumin (BSA) and two biopharmaceuticals including a monoclonal antibody (mAb) and an enzyme. The obtained calibration curve (
R
2
> 0.95) for the particle number concentration of the 3 proteins and the observed correlation between MADLS and SEC (
R
2
= 0.9938) for the analysis of aggregation in the enzyme can be employed as a 3-in-1 approach to assessing particle size, concentration and aggregation for the screening and development of products while also reducing the number of samples and experiments required for analysis prior to other orthogonal tests.
Graphical Abstract
Nanoparticle constructs for oral peptide delivery at a minimum must protect and present the peptide at the small intestinal epithelium in order to achieve oral bioavailability. In a reproducible, ...scalable, surfactant-free process, a core was formed with insulin in ratios with two established excipients and stabilizers, zinc chloride and l-arginine. Cross-linking was achieved with silica, which formed an outer shell. The process was reproducible across several batches, and physicochemical characterization of a single batch was confirmed in two independent laboratories. The silica-coated nanoparticles (SiNPs) entrapped insulin with high entrapment efficiency, preserved its structure, and released it at a pH value present in the small intestine. The SiNP delivered insulin to the circulation and reduced plasma glucose in a rat jejunal instillation model. The delivery mechanism required residual l-arginine in the particle to act as a permeation enhancer for SiNP-released insulin in the jejunum. The synthetic process was varied in terms of ratios of zinc chloride and l-arginine in the core to entrap the glucagon-like peptide 1 analogue, exenatide, and bovine serum albumin. SiNP-delivered exenatide was also bioactive in mice to some extent following oral gavage. The process is the basis for a platform for oral peptide and protein delivery.
Fibrous peptide networks, such as the structural framework of self-assembled fluorenylmethyloxycarbonyl diphenylalanine (Fmoc–FF) nanofibrils, have mechanical properties that could successfully mimic ...natural tissues, making them promising materials for tissue engineering scaffolds. These nanomaterials have been determined to exhibit shear piezoelectricity using piezoresponse force microscopy, as previously reported for FF nanotubes. Structural analyses of Fmoc–FF nanofibrils suggest that the observed piezoelectric response may result from the noncentrosymmetric nature of an underlying β-sheet topology. The observed piezoelectricity of Fmoc–FF fibrous networks is advantageous for a range of biomedical applications where electrical or mechanical stimuli are required.
The objective of this work was to probe the internal morphology, porosity, and polarity of conjugated polymer poly9,9-di(2-ethylhexyl)-fluorenyl-2,7-diyl (PF2/6) nanoparticles (NPs) to assess their ...feasibility as drug-delivery vehicles. To this end, conjugated polymer PF2/6 NPs of varying size were prepared using a reprecipitation method; size was controlled using different injection speed and agitation power. Dynamic light scattering and fluorescence-based maximum entropy method Fluorescence correlation spectroscopy measurements indicate that the hydrodynamic diameters of NPs increase from method 1 to method 3 (40, 60, 80 nm, respectively). However, atomic force microscopy measurements indicate that NPs shrink to ca. 10 nm upon drying. Further experiments suggest that this occurs due to NP porosity, which increases with increasing hydrodynamic size as progressively more water becomes trapped within the microenvironment of the NP. Single NP fluorescence images show that fluorescence brightness for NPs of differing hydrodynamic size does not change, indicating that an increase in size may not be due to additional polymer chains. The solvatochromic fluorescent dyes Nile red and 4-dicy-anomethylene-2-methyl-6(p-dimethylamino-styryl)-4H-pyran were used to precisely probe the internal polarity of NPs. They were separately doped into the hydrophobic core of PF2/6 NPs with different hydrodynamic sizes. The photophysical properties of both of the two dyes agrees that the polarity of the NPs increase with increasing PF2/6 NP size, providing further evidence of trapped water in NPs of larger size. Furthermore, the restricted motion of the dye molecules in the NPs has been investigated by time-resolved anisotropy. The anisotropy of dyes in PF2/6 NPs with different sizes is the same, indicating that the randomness, rotational motion restriction, and rigidity of the encapsulated dyes does not change as NP size varies. The Lippert–Mataga polarity of PF2/6 NPs was calculated for three different methods by applying the two fluorescent dyes.
The objective of this work is to demonstrate that conjugated polymer:fullerene hybrid nanoparticles encapsulated in the hydrophobic cores of triblock copolymer micelles may successfully act as ...spatially confined donor–acceptor systems capable of facilitating photoinduced charge carrier separation. To this end, aqueous dispersions of poly2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene (MEH-PPV) nanoparticles were first prepared by solubilization of the polymer in the cores of poly(oxyethylene)–poly(oxypropylene)–poly(oxyethylene) triblock copolymer, Pluronic F-127 micelles. A number of significant optical spectroscopic changes were observed on transfer of the conjugated polymer from a nonaqueous solvent to the aqueous micellar environment. These were primarily attributed to increased interchain interactions due to conjugated polymer chain collapse during encapsulation in the micellar cores. When prepared in buffer solution, the micelles exhibited good long-term collodial stability. When MEH-PPV micelles were blended by the addition of controlled amounts of 6,6-phenyl-C61-butyric acid methyl ester (PCBM), the observed correspondence of photoluminescence emission quenching, quantum yield decreases, and emission lifetime shortening with increasing PCBM concentration indicated efficient photoinduced donor-to-acceptor charge transfer between MEH-PPV and the fullerenes in the cores of the micelles, an assignment that was confirmed by transient absorption spectroscopic monitoring of carrier photogeneration and recombination.
The functionalization of polyfluorene (PFO) nanoparticles by coprecipitation of the conjugated polymer with an amphiphilic comb polymer, consisting of a hydrophobic polystyrene backbone with ...hydrophilic, carboxylic acid-terminated polyethylene oxide side-chains (PS-PEG-COOH), is investigated. The comb polymer affects the properties of the formed hybrid nanoparticles. Non-functionalized particles are typically larger (28 nm) than functionalized ones (20 nm); peak molar extinction coefficients are found to differ in a similar trend. Zeta potentials are negative, consistent with negative surface charge on PFO particles due to chemical defect formation, with additional charge on functionalized particles due to the pendant carboxylic acid groups. Emission quantum yields of functionalized particles are typically larger, consistent with lower efficiency of energy transfer to quenchers in smaller particles and weaker PFO interchain interactions due to chain dilution. The trend in per-particle fluorescence brightness values, as confirmed by single particle fluorescence imaging, reflects the nanoparticle extinction coefficients. Photostability studies on aqueous dispersions of hybrid particles indicate mild photobrightening under continuous illumination while PFO particles exhibit slow exponential emission decay. Functionalized particles are also resistant to aggregation during exposure to adenocarcinoma cells. Generally, the hybrid particles exhibit more favorable time-, pH- and medium-dependent stabilities, likely due to steric and electrostatic stabilization by PEG-carboxylic acid functionalities. Overall, the functionalized particles exhibit attractive properties: Reasonably small size, tight size distribution, high absorption cross section, radiative rate and emission quantum yield, excellent brightness and photostability, and good colloidal stability.
The restoration of habitats degraded by industrial disturbance is essential for achieving conservation objectives in disturbed landscapes. In boreal ecosystems, disturbances from seismic exploration ...lines and other linear features have adversely affected biodiversity, most notably leading to declines in threatened woodland caribou. Large‐scale restoration of disturbed habitats is needed, yet empirical assessments of restoration effectiveness on wildlife communities remain rare.
We used 73 camera trap deployments from 2015 to 2019 and joint species distribution models to investigate how habitat use by the larger vertebrate community (>0.2 kg) responded to variation in key seismic line characteristics (line‐of‐sight, width, density and mounding) following restoration treatments in a landscape disturbed by oil and gas development in northeastern Alberta.
The proportion of variation explained by line characteristics was low in comparison to habitat type and season, suggesting short‐term responses to restoration treatments were relatively weak. However, we found that lines with characteristics consistent with restored conditions were predicted to support an altered community composition, with reduced use by wolf and coyote, thereby indicating that line restoration will result in reduced contact rates between caribou and these key predators.
Synthesis and applications. Our analysis provides a framework to assess and predict wildlife community responses to emerging restoration efforts. With the growing importance of habitat restoration for caribou and other vertebrate species, we recommend longer‐term monitoring combined with landscape‐scale comparisons of different restoration approaches to more fully understand and direct these critical conservation investments. Only by combining rigorous multispecies monitoring with large‐scale restoration, will we effectively conserve biodiversity within rapidly changing environments.
Our analysis provides a framework to assess and predict wildlife community responses to emerging restoration efforts. With the growing importance of habitat restoration for caribou and other vertebrate species, we recommend longer‐term monitoring combined with landscape‐scale comparisons of different restoration approaches to more fully understand and direct these critical conservation investments. Only by combining rigorous multispecies monitoring with large‐scale restoration, will we effectively conserve biodiversity within rapidly changing environments.
Vegetation phenology and productivity drive resource use by wildlife. Vegetation dynamics also reveal patterns of habitat disturbance and recovery. Monitoring these fine‐scale vegetation patterns ...over large spatiotemporal extents can be difficult, but camera traps (CTs) commonly used to survey wildlife populations also collect data on local habitat conditions. We used CTs (n = 73) from 2016 to 2019 to assess impacts of habitat change in a boreal landscape of northern Canada, where seismic lines for petroleum exploration disturbed wildlife habitat and prompted vegetation restoration efforts. First, we quantified vegetation dynamics from CTs, comparing them to satellite‐based estimates that are typically used to monitor vegetation at broad spatial scales. We then used understory phenology and productivity estimated from CT time‐lapse images to assess vegetation recovery on seismic lines. Finally, we related vegetation dynamics with the habitat use of three wildlife species: sandhill cranes Grus canadensis, woodland caribou Rangifer tarandus, and white‐tailed deer Odocoileus virginianus. CTs provided unique insight into vegetation dynamics that were different from signals measured by satellites, with temporally inconsistent and even some negative correlations between CT and satellite metrics. We found some indication of vegetation recovery on seismic lines that had received restoration treatment, with understory patterns more similar to undisturbed habitat than to seismic lines that did not receive restoration treatment. CTs also provided inferences about wildlife activity related to vegetation resources, which approaches using satellite data failed to detect. Wildlife habitat use tracked vegetation phenology, but did not always increase with vegetation productivity at weekly, 16‐day, or annual intervals. Instead, associations with vegetation productivity depended on species, temporal scale, and productivity metrics. Given the widespread and growing use of CTs to monitor terrestrial wildlife, we recommend their use to simultaneously monitor habitat conditions to better understand the mechanisms that govern wildlife habitat use in changing environments.
We used time‐lapse images from n = 73 camera traps from 2016 to 2019 to quantify the phenology and productivity of understory vegetation in a boreal landscape of northern Canada, where seismic lines for petroleum exploration disturbed wildlife habitat and prompted vegetation restoration efforts. Camera traps revealed vegetation dynamics different from signals measured by satellites, showed evidence of ongoing but slow vegetation recovery after restoration treatments, and provided inferences about wildlife activity related to vegetation patterns that satellites failed to detect. Our results demonstrate how habitat monitoring and biodiversity conservation can capitalize on the currently underutilized ability of camera traps to simultaneously monitor fine‐scale vegetation dynamics and wildlife activity in order to assess the impacts of habitat change.
Human disturbance directly affects animal populations and communities, but indirect effects of disturbance on species behaviors are less well understood. For instance, disturbance may alter predator ...activity and cause knock‐on effects to predator‐sensitive foraging in prey. Camera traps provide an emerging opportunity to investigate such disturbance‐mediated impacts to animal behaviors across multiple scales. We used camera trap data to test predictions about predator‐sensitive behavior in three ungulate species (caribou Rangifer tarandus; white‐tailed deer, Odocoileus virginianus; moose, Alces alces) across two western boreal forest landscapes varying in disturbance. We quantified behavior as the number of camera trap photos per detection event and tested its relationship to inferred human‐mediated predation risk between a landscape with greater industrial disturbance and predator activity and a “control” landscape with lower human and predator activity. We also assessed the finer‐scale influence on behavior of variation in predation risk (relative to habitat variation) across camera sites within the more disturbed landscape. We predicted that animals in areas with greater predation risk (e.g., more wolf activity, less cover) would travel faster past cameras and generate fewer photos per detection event, while animals in areas with less predation risk would linger (rest, forage, investigate), generating more photos per event. Our predictions were supported at the landscape‐level, as caribou and moose had more photos per event in the control landscape where disturbance‐mediated predation risk was lower. At a finer‐scale within the disturbed landscape, no prey species showed a significant behavioral response to wolf activity, but the number of photos per event decreased for white‐tailed deer with increasing line of sight (m) along seismic lines (i.e., decreasing visual cover), consistent with a predator‐sensitive response. The presence of juveniles was associated with shorter behavioral events for caribou and moose, suggesting greater predator sensitivity for females with calves. Only moose demonstrated a positive behavioral association (i.e., longer events) with vegetation productivity (16‐day NDVI), suggesting that for other species bottom‐up influences of forage availability were generally weaker than top‐down influences from predation risk. Behavioral insights can be gleaned from camera trap surveys and provide complementary information about animal responses to predation risk, and thus about the indirect impacts of human disturbances on predator–prey interactions.
Camera traps provide an emerging opportunity to investigate variation in animal behaviors across gradients of disturbance. We used camera trap data to show that caribou, moose, and white‐tailed deer exhibited behavioural patterns consistent with predator‐sensitive foraging induced by anthropogenic habitat disturbance in northern boreal forests.
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