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Chukwuazam Nwasike,1 Erin Purr,1 Jaspreet Singh Nagi,2 Gretchen J Mahler,1 Amber L Doiron2 1Department of Biomedical Engineering, Binghamton University (SUNY), Binghamton, NY, USA; 2Department of Electrical and Biomedical Engineering, University of Vermont, Burlington, VT, USACorrespondence: Amber L Doiron, Email amber.doiron@uvm.eduIntroduction: Inflammatory diseases are the leading cause of death in the world, accounting for 3 out of 5 deaths. Despite the abundance of diagnostic tools for detection, most screening and diagnostic methods are indirect and insufficient as they are unable to reliably discriminate between high-risk or low-risk stages of inflammatory diseases. Previously, we showed that the selective activation of interpolymer complexed superparamagnetic iron oxide nanoparticles (IPC-SPIOs) under oxidative conditions can be detected by a change in T2 magnetic resonance (MR) contrast. In this work, IPC-SPIOs were further modified by incorporating mannose as a targeting biomolecule to enhance nanoparticle delivery to M2 macrophages at inflammatory sites.Methods: Uncoated SPIOs were synthesized via coprecipitation from a mixture of FeCl2 and FeCl3, PEGylated by adsorbing PEG 300 kDa (40 mg/mL in water) to SPIOs (3 mg/mL in water) over 24 hours, and complexed by mixing 0.25 mg/mL aqueous poly(gallol) with 2 mg/mL PEG-SPIOs and adding 1 M of phosphate buffer in a 9:9:2 ratio. Mannose-PEG attachment was accomplished conducting a second complexation of mannose-PEG to IPC-SPIOs. M2 macrophages were treated with 150, 100, and 75 μg/mL of IPC-SPIOs and mannose-IPC-SPIOs to investigate activation of T2 MRI signals.Results and Discussion: Surface modification resulted in a slight reduction in ROS scavenging capacity; however, nanoparticle uptake by M2 macrophages increased by over 50%. The higher uptake did not cause a reduction in cellular viability. In fact, mannose-IPC-SPIOs induced significant T2 MR contrast in M2 macrophages compared to IPC-SPIOs and nanoparticles exposed to M1 macrophages. M2 macrophages activated over 30% of mannose-IPC-SPIOs after 6 hours of exposure compared to M1 macrophages and untargeted M2 macrophages. These findings demonstrated that mannose-IPC-SPIOs specifically targeted M2 macrophages and scavenged cellular ROS to activate T2 MR signal, which can be used to detect inflammation.Graphical Abstract: Keywords: targeted nanoparticles, inflammatory diseases, MRI, contrast agents, mannose biomolecules

A theranostic refers to the concept of integrating diagnostic and therapeutic capabilities into a single platform. In this work, a layer of gold was reduced around superparamagnetic iron oxide (SPIO) nanoparticles to be used as a theranostic for magnetic resonance imaging and photoablation. In addition, a gold shell was included in the particle design to improve biocompatibility, stabilise nanoparticles in suspension, and allow robust surface functionalisation. Gold-coated SPIO nanoparticles of 8 nm in diameter exhibited surface plasmon resonance while also maintaining their superparamagnetic nature. Energy dispersive spectroscopy confirmed the existence of gold at the surface of the nanoparticles, the thickness of which was 0.5 nm based on transmission electron microscopy measurements. To demonstrate their potential for targeted therapies, nanoparticles were targeted to VCAM-1 on human umbilical vein endothelial cells and detected via darkfield microscopy.

The formation of biofilms is a developmental process initiated by planktonic cells transitioning to the surface, which comes full circle when cells disperse from the biofilm and transition to the planktonic mode of growth. Considering that pyruvate has been previously demonstrated to be required for the formation of P. aeruginosa biofilms, we asked whether pyruvate likewise contributes to the maintenance of the biofilm structure, with depletion of pyruvate resulting in dispersion. Here, we demonstrate that the enzymatic depletion of pyruvate coincided with the dispersion of established biofilms by S. aureus and laboratory and clinical P. aeruginosa isolates. The dispersion response was dependent on pyruvate fermentation pathway components but independent of proteins previously described to contribute to P. aeruginosa biofilm dispersion. Using porcine second-degree burn wounds infected with P. aeruginosa biofilm cells, we furthermore demonstrated that pyruvate depletion resulted in a reduction of biofilm biomass in vivo. Pyruvate-depleting conditions enhanced the efficacy of tobramycin killing of the resident wound biofilms by up to 5-logs. Our findings strongly suggest the management of pyruvate availability to be a promising strategy to combat biofilm-related infections by two principal pathogens associated with wound and cystic fibrosis lung infections.

The emergence of activatable magnetic resonance (MR) contrast agents has prompted significant interest in the detection of functional markers of diseases, resulting in the creation of a plethora of nanoprobes capable of detecting these biomarkers. These markers are commonly dysregulated in several chronic diseases, specifically select cancers and inflammatory diseases. Recently, the development of redox-sensitive nanoparticle-based contrast agents has gained momentum given advances in medicine linking several inflammatory diseases to redox imbalance. Researchers have pinpointed redox dysregulation as an opportunity to use activatable MR contrast agents to detect and stage several diseases as well as monitor the treatment of inflammatory diseases or conditions. These new classes of agents represent an advancement in the field of MR imaging as they elicit a response to stimuli, creating contrast while providing evidence of biomarker changes and commensurate disease state. Most redox-sensitive nanoparticle-based contrast agents are sensitive to reductive glutathione or oxidative reactive oxygen species. In this review, we will explore recent investigations into redox-activatable, nanoparticle-based MR contrast agent candidates.

Reactive oxygen species (ROS) are key markers of inflammation, with varying levels of superoxide indicating the degree of inflammation. Inflammatory diseases remain the leading cause of death in the developed world. Previously, we showed that interpolymer complexed superparamagnetic iron oxide nanoparticles (IPC-SPIOs) are capable of decomplexing and activating T2 magnetic resonance (MR) contrast in superoxide-rich environments. Here, we investigate the ability of IPC-SPIOs to scavenge ROS in immune and endothelial cells which should activate the superparamagnetic core. In exogenously generated superoxide, ROS scavenging by the nanoparticles was concentration dependent and ranged from 5% to over 50% of available ROS. A statistically significant reduction in ROS was observed in the presence of IPCSPIOs compared to poly(ethylene glycol)-coated SPIOs (PEG-SPIOs). During in vitro cellular assays, a reduction in ROS was observed in macrophages, monocytes, and human endothelial cells. Macrophages and endothelial cells experienced significantly higher ROS reduction compared to monocytes. ROS scavenging peaked 12 hours post-exposure to IPC-SPIOs in most studies, with some cell samples experiencing extended scavenging with increasing IPC-SPIO concentration. At the tested concentrations, particles were not cytotoxic, and confocal imaging showed localization of particles within cells. These findings demonstrate the potential of IPC-SPIOs as activatable MR contrast agents capable of activating under inflammation-induced cellular redox conditions as reporters of inflammatory disease severity or staging.

The effect of nanoparticle surface coating characteristics on colloidal stability in solution is a critical parameter in understanding the potential applications of nanoparticles, especially in biomedicine. Here we explored the modification of the surface of poly(ethylene glycol)-coated superparamagnetic iron oxide nanoparticles (PEG-SPIOs) with the synthetic pseudotannin polygallol via interpolymer complexation (IPC). Changes in particle size and zeta potential were indirectly assessed via differences between PEG-SPIOs and IPC-SPIOs in particle velocity and scattering intensity using near-field light scattering. The local scattering intensity is correlated with the distance between the particle and waveguide, which is affected by the size of the particle (coating thickness) as well as the interactions between the particle and waveguide (related to the zeta potential of the coating). Therefore, we report here the use of near-field light scattering using nanophotonic force microscopy (using a NanoTweezerTM instrument, Halo Labs) to determine the changes that occurred in hydrated particle characteristics, which is accompanied by an analytical model. Furthermore, we found that altering the salt concentration of the suspension solution affected the velocity of particles due to the change of dielectric constant and viscosity of the solution. These findings suggest that this technique is suitable for studying particle surface changes and perhaps can be used to dynamically study reaction kinetics at the particle surface.

Chronic infections have posed a tremendous burden on health care systems worldwide. Approximately 60% of chronic infections are estimated to be related to biofilms, in large part due to the extraordinary antibiotic resistance of biofilm bacteria. Nanoparticle (NP)-based therapies are viable approaches to treat biofilm-associated infections due to NPs’ unique chemical and physical properties, granted by their high surface area to volume ratio. The mechanism underlying the anti-biofilm activity of various types of NPs is actively under investigation. Simply comparing biofilm disruption or reduction rates is not adequate to describe the effectiveness of NPs; many other factors need to be taken into account, such as the NP type, bacterial strain, concentration of NPs, quantification methods, and the biofilm culture environment. This review focuses on recent research on the creation, characterization, and evaluation of NPs for the prevention or treatment of biofilm infections.

Magnetic resonance contrast agents that can be activated in response to specific triggers hold potential as molecular biosensors that may be of great utility in non-invasive disease diagnosis. We developed an activatable agent based on superparamagnetic iron oxide nanoparticles (SPIOs) that is sensitive to oxidative stress, a factor in the pathophysiology of numerous diseases. SPIOs were coated with poly(ethylene glycol) (PEG) and complexed with poly(gallol), a synthetic tannin. Hydrogen bonding between PEG and poly(gallol) creates a complexed layer around the SPIO that decreases the interaction of solute water with the SPIO, attenuating its magnetic resonance relaxivity. The complexed interpolymer nanoparticle is in an OFF state (decreased T2 contrast), where the contrast agent has a low T2 relaxivity of 7 ± 2 mM−1 s−1. In the presence of superoxides, the poly(gallol) is oxidized and the polymers decomplex, allowing solute water to again interact with the SPIO, representing an ON state (increased T2 contrast) with a T2 relaxivity of 70 ± 10 mM−1 s−1. These contrast agents show promise as effective sensors for diseases characterized in part by oxidative stress such as atherosclerosis, diabetes, and cancer.

Novel dye-linked zinc oxide nanoparticles (NPs) hold potential as photosensitizers for biomedical applications due to their excellent thermal- and photo-stability. The particles produced reactive oxygen species (ROS) upon irradiation with 850 nm near infrared (NIR) light in a concentration- and time-dependent manner. Upon irradiation, ROS detected in vitro in human umbilical vein endothelial cells (HUVEC) and human carcinoma MCF7 cells positively correlated with particle concentration and interestingly, ROS detected in MCF7 was higher than in HUVEC. Preferential cytotoxicity was also exhibited by the NPs as cell killing was higher in MCF7 than in HUVEC. In the absence of irradiation, dye-linked ZnO particles minimally affected the viability of cell (HUVEC) at low concentrations (&lt, 30 &mu, g/mL), but viability significantly decreased at higher particle concentrations, suggesting a need for particle surface modification with poly (ethylene glycol) (PEG) for improved biocompatibility. The presence of PEG on particles after dialysis was indicated by an increase in size, an increase in zeta potential towards neutral, and spectroscopy results. Cell viability was improved in the absence of irradiation when cells were exposed to PEG-coated, dye-linked ZnO particles compared to non-surface modified particles. The present study shows that there is potential for biological application of dye-linked ZnO particles in photodynamic therapy.

The rapid development of modern nanotechnology has resulted in nanomaterial being use in nearly all applications of life, raising the potential risk of nanomaterial exposure alongside the need to design safe and effective materials. Previous work has demonstrated a specific effect of gold nanoparticles (GNPs) of approximately 20 nm on endothelial barrier function in vitro. To expand our understanding of this size-specific effect, titanium dioxide, silicon dioxide, and polystyrene nanoparticles (NPs) in this similar size range were studied. All tested nanoparticles were found to have minimal effects on cell viability, but exhibited a significant detrimental effect on endothelial barrier function. Nanoparticles in the size range of 20 to 30 nm were internalized by endothelial cells through caveolae/raft-mediated endocytosis, causing intracellular calcium elevation by approximately 30% at 2 h after administration, and triggering myosin light chain kinase (MLCK)-regulated actomyosin contraction. These effects culminated in an increase in endothelial monolayer permeability across all particle types within the 20–30 nm range. This nanoparticle exposure-induced endothelial barrier dysfunction may provide valuable information for designing safer nanomaterials or potential applications of this nanoparticle exposure-induced permeability effect in biomedicine.

Computed tomography (CT) is among the most popular medical imaging modalities due to its high resolution images, fast scan time, low cost, and compatibility with all patients. CT scans of soft tissues require the localization of imaging contrast agents (CA) to create contrast, revealing anatomic information. Gold nanoparticles (AuNP) have attracted interest recently for their use as CT CA due to their high X-ray attenuation, simple surface chemistry, and biocompatibility. Targeting molecules may be attached to the particles to allow for the targeting of specific cell types and disease states. AuNP can also be readily designed to incorporate other imaging contrast agents such as rare earth metals and dyes. This review summarizes the current state-of-the-art knowledge in the field of AuNP used as X-ray and multimodal contrast agents. Primary research is analyzed through the lens of structure-property-function to best explain the design of a particle for a given application. Design specification of particles includes size, shape, surface functionalization, composition, circulation time, and component synergy. Key considerations include delivery of a CA payload to the site of interest, nontoxicity of particle components, and contrast enhancement compared to the surrounding tissue. Examples from literature are included to illustrate the strategies used to address design considerations.

In this work, we evaluated the impact of gold nanoparticles on endothelial cell behavior and function beyond the influence on cell viability. Five types of gold nanoparticles were studied: 5 nm and 20 nm bare gold nanoparticles, 5 nm and 20 nm gold nanoparticles with biocompatible polyethylene glycol (PEG) coating and 60 nm bare gold nanoparticles. We found that all tested gold nanoparticles did not affect cell viability significantly and reduced the reactive oxygen species (ROS) level in endothelial cells. Only 20 nm bare gold nanoparticles caused an over 50% increase in endothelial barrier permeability and slow recovery of barrier function was observed after the gold nanoparticles were removed. This impairment in endothelial barrier function was caused by unbalanced forces between intracellular tensions and paracellular forces, actin microfilament rearrangement, which occurred through a Rho/ROCK kinase-dependent pathway and broke the force balance between intracellular tensions and paracellular forces. The size-specific effect of gold nanoparticles on endothelial cells may have important implications regarding the behavior of nanoparticles in the biological system and provide valuable guidance in nanomaterial design and biomedical applications.

To determine the initial feasibility of using magnetic resonance (MR) imaging to detect early atherosclerosis, we investigated inflammatory cells labeled with a positive contrast agent in an endothelial cell–based testing system. The human monocytic cell line THP-1 was labeled by overnight incubation with a gadolinium colloid (Gado CELLTrack) prior to determination of the in vitro release profile from T1-weighted MR images. Next, MR signals arising from both a synthetic model of THP-1/human umbilical vein endothelial cell (HUVEC) accumulation and the dynamic adhesion of THP-1 cells to activated HUVECs under flow were obtained. THP-1 cells were found to be successfully—but not optimally—labeled with gadolinium colloid, and MR images demonstrated increased signal from labeled cells in both the synthetic and dynamic THP-1/HUVEC models. The observed THP-1 contrast release profile was rapid, suggesting the need for an agent that is optimized for retention in the target cells for use in further studies. Detection of labeled THP-1 cells was accomplished with no signal enhancement from unlabeled cells. These achievements demonstrate the feasibility of targeting early atherosclerosis with MR imaging, and suggest that using an in vitro system like the one described provides a rapid, efficient, and cost-effective way to support the development and evaluation of novel MR contrast agents.

Accurate imaging of atherosclerosis is a growing necessity for timely treatment of the disease. Magnetic resonance imaging (MRI) is a promising technique for plaque imaging. The goal of this study was to create polymeric particles of a small size with high loading of diethylenetriaminepentaacetic acid gadolinium (III) (Gd-DTPA) and demonstrate their usefulness for MRI. A water-in-oil-in-oil double emulsion solvent evaporation technique was used to encapsulate the MRI agent in a poly(lactide-co-glycolide) (PLGA) or polylactide-poly(ethylene glycol) (PLA-PEG) particle for the purpose of concentrating the agent at an imaging site. PLGA particles with two separate average sizes of 1.83 μm and 920 nm, and PLA-PEG particles with a mean diameter of 952 nm were created. Loading of up to 30 wt % Gd-DTPA was achieved, and in vitro release occurred over 5 h. PLGA particles had highly negative zeta potentials, whereas the particles incorporating PEG had zeta potentials closer to neutral. Cytotoxicity of the particles on human umbilical vein endothelial cells (HUVEC) was shown to be minimal. The ability of the polymeric contrast agent formulation to create contrast was similar to that of Gd-DTPA alone. These results demonstrate the possible utility of the contrast agent-loaded polymeric particles for plaque detection with MRI.

With the broadening field of nanomedicine poised for future molecular level therapeutics, nano- and microparticles intended for the augmentation of either single- or multimodal imaging are created with PLGA as the chief constituent and carrier.Emulsion techniques were used to encapsulate hydrophilic and hydrophobic imaging contrast agents in PLGA particles. The imaging contrast properties of these PLGA particles were further enhanced by reducing silver onto the PLGA surface, creating a silver cage around the polymeric core.The MRI contrast agent Gd-DTPA and the exogenous dye rhodamine 6G were both encapsulated in PLGA and shown to enhance MR and fluorescence contrast, respectively. The silver nanocage built around PLGA nanoparticles exhibited strong near infrared light absorbance properties, making it a suitable contrast agent for optical imaging strategies such as photoacoustic imaging.The biodegradable polymer PLGA is an extremely versatile nano- and micro-carrier for several imaging contrast agents with the possibility of targeting diseased states at a molecular level.