Optimized gadolinium-DO3A loading in RAFT-polymerized copolymers for superior MR imaging of aging blood-brain barrier.

The development of gadolinium-based contrast agents (GBCAs) has been pivotal in advancing magnetic resonance imaging (MRI), offering enhanced soft tissue contrast without ionizing radiation exposure. Despite their widespread clinical use, the need for improved GBCAs has led to innovations in ligand chemistry and polymer science. We report a novel approach using methacrylate-functionalized DO3A ligands to synthesize a series of copolymers through direct reversible addition-fragmentation chain transfer (RAFT) polymerization. This technique enables precise control over the gadolinium content within the polymers, circumventing the need for subsequent conjugation and purification steps, and facilitates the addition of other components such as targeting ligands. The resulting copolymers were analysed for their relaxivity properties, indicating that specific gadolinium-DO3A loading contents between 12-30 mole percent yield optimal MRI contrast enhancement. Inductively coupled plasma (ICP) measurements corroborated these findings, revealing a non-linear relationship between gadolinium content and relaxivity. Optimized copolymers were synthesized with the claudin-1 targeting peptide, C1C2, to image BBB targeting in aged mice to show imaging utility. This study presents a promising pathway for the development of more efficient GBCA addition to copolymers for targeted drug delivery and bioimaging application.

Restoration of normal blood flow in atherosclerotic arteries promotes plaque stabilization.

Blood flow is a key regulator of atherosclerosis. Disturbed blood flow promotes atherosclerotic plaque development, whereas normal blood flow protects against plaque development. We hypothesized that normal blood flow is also therapeutic, if it were able to be restored within atherosclerotic arteries. Apolipoprotein E-deficient (ApoE-/-) mice were initially instrumented with a blood flow-modifying cuff to induce plaque development and then five weeks later the cuff was removed to allow restoration of normal blood flow. Plaques in decuffed mice exhibited compositional changes that indicated increased stability compared to plaques in mice with the cuff maintained. The therapeutic benefit of decuffing was comparable to atorvastatin and the combination had an additive effect. In addition, decuffing allowed restoration of lumen area, blood velocity, and wall shear stress to near baseline values, indicating restoration of normal blood flow. Our findings demonstrate that the mechanical effects of normal blood flow on atherosclerotic plaques promote stabilization.

Sex-based differences of antioxidant enzyme nanoparticle effects following traumatic brain injury.

Following traumatic brain injury (TBI), reactive oxygen species (ROS) are released in excess, causing oxidative stress, carbonyl stress, and cell death, which induce the additional release of ROS. The limited accumulation and retention of small molecule antioxidants commonly used in clinical trials likely limit the target engagement and therapeutic effect in reducing secondary injury. Small molecule drugs also need to be administered every several hours to maintain bioavailability in the brain. Therefore, there is a need for a burst and sustained release system with high accumulation and retention in the injured brain. Here, we utilized Pro-NP™ with a size of 200 nm, which was designed to have a burst and sustained release of encapsulated antioxidants, Cu/Zn superoxide dismutase (SOD1) and catalase (CAT), to scavenge ROS for >24 h post-injection. Here, we utilized a controlled cortical impact (CCI) mouse model of TBI and found the accumulation of Pro-NP™ in the brain lesion was highest when injected immediately after injury, with a reduction in the accumulation with delayed administration of 1 h or more post-injury. Pro-NP™ treatment with 9000 U/kg SOD1 and 9800 U/kg CAT gave the highest reduction in ROS in both male and female mice. We found that Pro-NP™ treatment was effective in reducing carbonyl stress and necrosis at 1 d post-injury in the contralateral hemisphere in male mice, which showed a similar trend to untreated female mice. Although we found that male and female mice similarly benefit from Pro-NP™ treatment in reducing ROS levels 4 h post-injury, Pro-NP™ treatment did not significantly affect markers of post-traumatic oxidative stress in female CCI mice as compared to male CCI mice. These findings of protection by Pro-NP™ in male mice did not extend to 7 d post-injury, which suggests subsequent treatments with Pro-NP™ may be needed to afford protection into the chronic phase of injury. Overall, these different treatment effects of Pro-NP™ between male and female mice suggest important sex-based differences in response to antioxidant nanoparticle delivery and that there may exist a maximal benefit from local antioxidant activity in injured brain.

Antioxidant theranostic copolymer-mediated reduction in oxidative stress following traumatic brain injury improves outcome in a mouse model.

Following a traumatic brain injury (TBI), excess reactive oxygen species (ROS) and lipid peroxidation products (LPOx) are generated and lead to secondary injury beyond the primary insult. A major limitation of current treatments is poor target engagement, which has prevented success in clinical trials. Thus, nanoparticle-based treatments have received recent attention because of their ability to increase accumulation and retention in damaged brain. Theranostic neuroprotective copolymers (NPC3) containing thiol functional groups can neutralize ROS and LPOx. Immediate administration of NPC3 following injury in a controlled cortical impact (CCI) mouse model provides a therapeutic window in reducing ROS levels at 2.08-20.83 mg/kg in males and 5.52-27.62 mg/kg in females. This NPC3-mediated reduction in oxidative stress improves spatial learning and memory in males, while females show minimal improvement. Notably, NPC3-mediated reduction in oxidative stress prevents the bilateral spread of necrosis in male mice, which was not observed in female mice and likely accounts for the sex-based spatial learning and memory differences. Overall, these findings suggest sex-based differences to oxidative stress scavenger nanoparticle treatments, and a possible upper threshold of antioxidant activity that provides therapeutic benefit in injured brain since female mice benefit from NPC3 treatment to a lesser extent than male mice.

Smooth muscle cells affect differential nanoparticle accumulation in disturbed blood flow-induced murine atherosclerosis.

Atherosclerosis is a lipid-driven chronic inflammatory disease that leads to the formation of plaques in the inner lining of arteries. Plaques form over a range of phenotypes, the most severe of which is vulnerable to rupture and causes most of the clinically significant events. In this study, we evaluated the efficacy of nanoparticles (NPs) to differentiate between two plaque phenotypes based on accumulation kinetics in a mouse model of atherosclerosis. This model uses a perivascular cuff to induce two regions of disturbed wall shear stress (WSS) on the inner lining of the instrumented artery, low (upstream) and multidirectional (downstream), which, in turn, cause the development of an unstable and stable plaque phenotype, respectively. To evaluate the influence of each WSS condition, in addition to the final plaque phenotype, in determining NP uptake, mice were injected with NPs at intermediate and fully developed stages of plaque growth. The kinetics of artery wall uptake were assessed in vivo using dynamic contrast-enhanced magnetic resonance imaging. At the intermediate stage, there was no difference in NP uptake between the two WSS conditions, although both were different from the control arteries. At the fully-developed stage, however, NP uptake was reduced in plaques induced by low WSS, but not multidirectional WSS. Histological evaluation of plaques induced by low WSS revealed a significant inverse correlation between the presence of smooth muscle cells and NP accumulation, particularly at the plaque-lumen interface, which did not exist with other constituents (lipid and collagen) and was not present in plaques induced by multidirectional WSS. These findings demonstrate that NP accumulation can be used to differentiate between unstable and stable murine atherosclerosis, but accumulation kinetics are not directly influenced by the WSS condition. This tool could be used as a diagnostic to evaluate the efficacy of experimental therapeutics for atherosclerosis.

Claudin-1-Targeted Nanoparticles for Delivery to Aging-Induced Alterations in the Blood-Brain Barrier.

Aging-induced alterations to the blood-brain barrier (BBB) are increasingly being seen as a primary event in chronic progressive neurological disorders that lead to cognitive decline. With the goal of increasing delivery into the brain in hopes of effectively treating these diseases, a large focus has been placed on developing BBB permeable materials. However, these strategies have suffered from a lack of specificity toward regions of disease progression. Here, we report on the development of a nanoparticle (C1C2-NP) that targets regions of increased claudin-1 expression that reduces BBB integrity. Using dynamic contrast enhanced magnetic resonance imaging, we find that C1C2-NP accumulation and retention is significantly increased in brains from 12 month-old mice as compared to nontargeted NPs and brains from 2 month-old mice. Furthermore, we find C1C2-NP accumulation in brain endothelial cells with high claudin-1 expression, suggesting target-specific binding of the NPs, which was validated through fluorescence imaging, in vitro testing, and biophysical analyses. Our results further suggest a role of claudin-1 in reducing BBB integrity during aging and show altered expression of claudin-1 can be actively targeted with NPs. These findings could help develop strategies for longitudinal monitoring of tight junction protein expression changes during aging as well as be used as a delivery strategy for site-specific delivery of therapeutics at these early stages of disease development.

Interactive processes in an instance model of memory: A computational analysis of Jacoby's (1983) dissociation between perception and recognition.

In a classic and well-cited work, Jacoby (1983) demonstrated an important dissociation in which conceptual processing at study resulted in high performance on a standard memory test but low performance on a perceptual test. Perceptual processing at study resulted in the opposite pattern. I simulated the dissociation in MINERVA2, a classic instance model of memory. I assumed that stimulus representations are composed of perceptual and contextual features and that different study tasks favor the encoding of some features over others. I also assumed that different test tasks utilize some features more than others. The model successfully produced the dissociation. The simulations provide a formal account of a core principle of memory: Performance is determined by the appropriateness of processing at encoding given the demands of retrieval. In conjunction with previous work, I conclude that the result emerged from the same mechanisms that underlie empirical regularities from other areas of memory research (e.g., the production effect) and argue in favor of incorporating converging evidence across multiple modelling frameworks to provide stronger theoretical foundations. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Computational and empirical simulations of selective memory impairments: Converging evidence for a single-system account of memory dissociations.

Current theory has divided memory into multiple systems, resulting in a fractionated account of human behaviour. By an alternative perspective, memory is a single system. However, debate over the details of different single-system theories has overshadowed the converging agreement among them, slowing the reunification of memory. Evidence in favour of dividing memory often takes the form of dissociations observed in amnesia, where amnesic patients are impaired on some memory tasks but not others. The dissociations are taken as evidence for separate explicit and implicit memory systems. We argue against this perspective. We simulate two key dissociations between classification and recognition in a computational model of memory, A Theory of Nonanalytic Association. We assume that amnesia reflects a quantitative difference in the quality of encoding. We also present empirical evidence that replicates the dissociations in healthy participants, simulating amnesic behaviour by reducing study time. In both analyses, we successfully reproduce the dissociations. We integrate our computational and empirical successes with the success of alternative models and manipulations and argue that our demonstrations, taken in concert with similar demonstrations with similar models, provide converging evidence for a more general set of single-system analyses that support the conclusion that a wide variety of memory phenomena can be explained by a unified and coherent set of principles.

False recognition of instruction-set lures.

False remembering has been examined using a variety of procedures, including the Deese-Roediger-McDermott procedure, the false fame procedure and the two-list recognition procedure. We present six experiments in a different empirical framework examining false recognition of words included in the experimental instructions (instruction-set lures). The data show that participants' false alarm rate to instruction-set lures was twice their false alarm rate to standard lures. That result was statistically robust even when (1) the relative strength of targets to instruction-set lures was increased, (2) participants were warned about the instruction-set lures, (3) the instruction-set lures were camouflaged in the study instructions and (4) the instruction-set lures were presented verbally at study but visually at test. False recognition of instruction-set lures was only mitigated when participants were distracted between encountering the instruction-set lures and studying the training list. The results confirm the ease with which recognition succumbs to familiarity and demonstrate the robustness of false recognition.

Near-infrared optical imaging for monitoring the regeneration of osteogenic tissue-engineered constructs.

Millions of cases of bone injury or loss due to trauma, osteoporosis, and cancer occur in the United States each year. Because bone is limited in its ability to regenerate, alternative therapy approaches are needed. Bone tissue engineering has the potential to correct musculoskeletal disorders through the development of cell-based substitutes for osteogenic tissue replacement. Multiple medical imaging techniques such as magnetic resonance microscopy (MRM) were investigated recently; these techniques are able to provide useful information on the anatomical and structural changes of developing bone. However, there is a need for noninvasive approaches to evaluate biochemical constituents and consequent compositional development associated with growing osteogenic constructs. In this study, near-infrared (NIR) optical imaging with a bone-specific NIR-targeted probe, IRDye(®) 800CW BoneTag™ (800CW BT), was applied in this study to longitudinally visualize regions of mineralization of tissue-engineered bone constructs in vivo. A fluorescent cell-based assay was performed to confirm the preferential binding of 800CW BT to the mineralized matrix of differentiated osteogenically driven human mesenchymal stem cells (hMSCs) in vitro. The hMSCs were seeded onto a biocompatible gelatin scaffold, allowed to develop, and implanted into a mouse model. Engineered constructs were examined in vivo using NIR imaging for bone mineralization, paired with MRM for verification of developing tissue. Results showed that NIR imaging with 800CW BT labeling can effectively assess the calcification of the developing osteogenic constructs, which is consistent with the analysis of excised tissue using NIR microscopy and histology. In conclusion, this study evaluated bone-like function of regenerating bone through tracking calcium deposition via NIR optical imaging with a fluorophore-labeled probe in a noninvasive manner.

Curcumin-loaded magnetic nanoparticles for breast cancer therapeutics and imaging applications.

BACKGROUND: The next generation magnetic nanoparticles (MNPs) with theranostic applications have attracted significant attention and will greatly improve nanomedicine in cancer therapeutics. Such novel MNP formulations must have ultra-low particle size, high inherent magnetic properties, effective imaging, drug targeting, and drug delivery properties. To achieve these characteristic properties, a curcumin-loaded MNP (MNP-CUR) formulation was developed. METHODS: MNPs were prepared by chemical precipitation method and loaded with curcumin (CUR) using diffusion method. The physicochemical properties of MNP-CUR were characterized using dynamic light scattering, transmission electron microscopy, and spectroscopy. The internalization of MNP-CUR was achieved after 6 hours incubation with MDA-MB-231 breast cancer cells. The anticancer potential was evaluated by a tetrazolium-based dye and colony formation assays. Further, to prove MNP-CUR results in superior therapeutic effects over CUR, the mitochondrial membrane potential integrity and reactive oxygen species generation were determined. Magnetic resonance imaging capability and magnetic targeting property were also evaluated. RESULTS: MNP-CUR exhibited individual particle grain size of ~9 nm and hydrodynamic average aggregative particle size of ~123 nm. Internalized MNP-CUR showed a preferential uptake in MDA-MB-231 cells in a concentration-dependent manner and demonstrated accumulation throughout the cell, which indicates that particles are not attached on the cell surface but internalized through endocytosis. MNP-CUR displayed strong anticancer properties compared to free CUR. MNP-CUR also amplified loss of potential integrity and generation of reactive oxygen species upon treatment compared to free CUR. Furthermore, MNP-CUR exhibited superior magnetic resonance imaging characteristics and significantly increased the targeting capability of CUR. CONCLUSION: MNP-CUR exhibits potent anticancer activity along with imaging and magnetic targeting capabilities. This approach can be extended to preclinical and clinical use and may have importance in cancer treatment and cancer imaging in the future. Further, if these nanoparticles can functionalize with antibody/ligands, they will serve as novel platforms for multiple biomedical applications.

Magnetic resonance elastography methodology for the evaluation of tissue engineered construct growth.

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development. The method proposed in the following protocol uses microscopic magnetic resonance elastography (µMRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner. Recently, µMRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map. The resulting measurements at each voxel have been shown to strongly correlate (R(2)>0.9914) with data collected using dynamic mechanical analysis. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

MR elastography monitoring of tissue-engineered constructs.

The objective of tissue engineering (TE) is to create functional replacements for various tissues; the mechanical properties of these engineered constructs are critical to their function. Several techniques have been developed for the measurement of the mechanical properties of tissues and organs; however, current methods are destructive. The field of TE will benefit immensely if biomechanical models developed by these techniques could be combined with existing imaging modalities to enable noninvasive, dynamic assessment of mechanical properties during tissue growth. Specifically, MR elastography (MRE), which is based on the synchronization of a mechanical actuator with a phase contrast imaging pulse sequence, has the capacity to measure tissue strain generated by sonic cyclic displacement. The captured displacement is presented in shear wave images from which the complex shear moduli can be extracted or simplified by a direct measure, termed the shear stiffness. MRE has been extended to the microscopic scale, combining clinical MRE with high-field magnets, stronger magnetic field gradients and smaller, more sensitive, radiofrequency coils, enabling the interrogation of smaller samples, such as tissue-engineered constructs. The following topics are presented in this article: (i) current mechanical measurement techniques and their limitations in TE; (ii) a description of the MRE system, MRE theory and how it can be applied for the measurement of mechanical properties of tissue-engineered constructs; (iii) a summary of in vitro MRE work for the monitoring of osteogenic and adipogenic tissues originating from human adult mesenchymal stem cells (MSCs); (iv) preliminary in vivo studies of MRE of tissues originating from mouse MSCs implanted subcutaneously in immunodeficient mice with an emphasis on in vivo MRE challenges; (v) future directions to resolve current issues with in vivo MRE in the context of how to improve the future role of MRE in TE.

Multi-functional magnetic nanoparticles for magnetic resonance imaging and cancer therapy.

We have developed a multi-layer approach for the synthesis of water-dispersible superparamagnetic iron oxide nanoparticles for hyperthermia, magnetic resonance imaging (MRI) and drug delivery applications. In this approach, iron oxide core nanoparticles were obtained by precipitation of iron salts in the presence of ammonia and provided ß-cyclodextrin and pluronic polymer (F127) coatings. This formulation (F127250) was highly water dispersible which allowed encapsulation of the anti-cancer drug(s) in ß-cyclodextrin and pluronic polymer for sustained drug release. The F127250 formulation has exhibited superior hyperthermia effects over time under alternating magnetic field compared to pure magnetic nanoparticles (MNP) and ß-cyclodextrin coated nanoparticles (CD200). Additionally, the improved MRI characteristics were also observed for the F127250 formulation in agar gel and in cisplatin resistant ovarian cancer cells (A12780CP) compared to MNP and CD200 formulations. Furthermore, the drug-loaded formulation of F127250 exhibited many folds of imaging contrast properties. Due to the internalization capacity of the F127250 formulation, its curcumin-loaded formulation (F127250-CUR) exhibited almost equivalent inhibition effects on A2780CP (ovarian), MDA-MB-231 (breast), and PC-3 (prostate) cancer cells even though curcumin release was only 40%. The improved therapeutic effects were verified by examining molecular effects using Western blotting and transmission electron microscopic (TEM) studies. F127250-CUR also exhibited haemocompatibility, suggesting a nanochemo-therapeutic agent for cancer therapy.