Multivalent Gd-DOTA Decorated Oligopeptide as Sensitive MRI Molecular Probes for In Vivo Imaging of Brain Connectivity.

One of the most important goals of brain imaging is to define the anatomical connections within the brain. In addition to revealing normal circuitry, studies of neural connections and neuronal transport can show rewiring and degeneration following brain injury and diseases. In this work, a highly sensitive magnetic resonance imaging (MRI)-visible neural tracer that can be used to visualize brain connectivity in vivo is developed. It is based on an oligopeptide with gadolinium chelates appended to the peptide backbone. This peptide construct is a sensitive MRI contrast agent that was conjugated to the classical neurotracer, Cholera-toxin Subunit-B. Injection of this probe enabled it to be used to trace neural connections in vivo. This complements other MRI tracing techniques such as diffusion tensor imaging and manganese-enhanced MRI for neural tracing.

Early detection of cerebrovascular pathology and protective antiviral immunity by MRI.

Central nervous system (CNS) infections are a major cause of human morbidity and mortality worldwide. Even patients that survive, CNS infections can have lasting neurological dysfunction resulting from immune and pathogen induced pathology. Developing approaches to noninvasively track pathology and immunity in the infected CNS is crucial for patient management and development of new therapeutics. Here, we develop novel MRI-based approaches to monitor virus-specific CD8+ T cells and their relationship to cerebrovascular pathology in the living brain. We studied a relevant murine model in which a neurotropic virus (vesicular stomatitis virus) was introduced intranasally and then entered the brain via olfactory sensory neurons - a route exploited by many pathogens in humans. Using T2*-weighted high-resolution MRI, we identified small cerebral microbleeds as an early form of pathology associated with viral entry into the brain. Mechanistically, these microbleeds occurred in the absence of peripheral immune cells and were associated with infection of vascular endothelial cells. We monitored the adaptive response to this infection by developing methods to iron label and track individual virus specific CD8+ T cells by MRI. Transferred antiviral T cells were detected in the brain within a day of infection and were able to reduce cerebral microbleeds. These data demonstrate the utility of MRI in detecting the earliest pathological events in the virally infected CNS as well as the therapeutic potential of antiviral T cells in mitigating this pathology.

Correction to "Luteinizing Hormone Releasing Hormone-Targeted Cisplatin-Loaded Magnetite Nanoclusters for Simultaneous MR Imaging and Chemotherapy of Ovarian Cancer".

[This corrects the article PMC10317193.].

Neural precursor cells form integrated brain-like tissue when implanted into rat cerebrospinal fluid.

There is tremendous interest in transplanting neural precursor cells for brain tissue regeneration. However, it remains unclear whether a vascularized and integrated complex neural tissue can be generated within the brain through transplantation of cells. Here, we report that early stage neural precursor cells recapitulate their seminal properties and develop into large brain-like tissue when implanted into the rat brain ventricle. Whereas the implanted cells predominantly differentiated into glutamatergic neurons and astrocytes, the host brain supplied the intact vasculature, oligodendrocytes, GABAergic interneurons, and microglia that seamlessly integrated into the new tissue. Furthermore, local and long-range axonal connections formed mature synapses between the host brain and the graft. Implantation of precursor cells into the CSF-filled cavity also led to a formation of brain-like tissue that integrated into the host cortex. These results may constitute the basis of future brain tissue replacement strategies.

Magnetic resonance imaging of odorant activity-dependent migration of neural precursor cells and olfactory bulb growth.

Neural progenitors or neuroblasts are produced by precursor cells in the subventricular zone (SVZ) and migrate along the rostral migratory stream (RMS) to the olfactory bulbs (OB) throughout life. In the OB, these adult born neurons either die or replace existing olfactory interneurons, playing a critical role in the stabilization of OB circuitry. Although several aspects of the addition of new neurons into the OB have been studied, it is unclear whether long-distance activity from the OB can regulate the influx of migrating neuroblasts along the RMS. In this study, iron oxide-assisted MRI was used to track the migration of neuroblasts in combination with reversible naris occlusion to manipulate odorant-induced activity. It was found that decreasing olfactory activity led to a decrease in the rate of neuroblast migration along the RMS. Removal of the naris occlusion led to an increase in migratory rate back to control levels, indicating that olfactory activity has regulatory function on neuroblast migration in the RMS. Blocking odorant activity also led to an arrest in OB growth and re-opening the block led to a rapid re-growth returning the bulb size to control levels. Furthermore, pharmacogenetic elimination of the neuroblasts demonstrated that they were required for re-growth of the bulb following sensory deprivation. Together, these results show that sensory activity, neural migration and OB growth are tightly coupled in an interdependent manner.

Remote Actuation of Magnetic Nanoparticles For Cancer Cell Selective Treatment Through Cytoskeletal Disruption.

Motion of micron and sub-micron size magnetic particles in alternating magnetic fields can activate mechanosensitive cellular functions or physically destruct cancer cells. However, such effects are usually observed with relatively large magnetic particles (>250 nm) that would be difficult if at all possible to deliver to remote sites in the body to treat disease. Here we show a completely new mechanism of selective toxicity of superparamagnetic nanoparticles (SMNP) of 7 to 8 nm in diameter to cancer cells. These particles are coated by block copolymers, which facilitates their entry into the cells and clustering in the lysosomes, where they are then magneto-mechanically actuated by remotely applied alternating current (AC) magnetic fields of very low frequency (50 Hz). Such fields and treatments are safe for surrounding tissues but produce cytoskeletal disruption and subsequent death of cancer cells while leaving healthy cells intact.

Luteinizing Hormone Releasing Hormone-Targeted Cisplatin-Loaded Magnetite Nanoclusters for Simultaneous MR Imaging and Chemotherapy of Ovarian Cancer.

Given the superior soft tissue contrasts obtained by MRI and the long residence times of magnetic nanoparticles (MNPs) in soft tissues, MNP-based theranostic systems are being developed for simultaneous imaging and treatment. However, development of such theranostic nanoformulations presents significant challenges of balancing the therapeutic and diagnostic functionalities in order to achieve optimum effect from both. Here we developed a simple theranostic nanoformulation based on magnetic nanoclusters (MNCs) stabilized by a bisphosphonate-modified poly(glutamic acid)-b-(ethylene glycol) block copolymer and complexed with cisplatin. The MNCs were decorated with luteinizing hormone releasing hormone (LHRH) to target LHRH receptors (LHRHr) overexpressed in ovarian cancer cells. The targeted MNCs significantly improved the uptake of the drug in cancer cells and decreased its IC50 compared to the nontargeted formulations. Also, the enhanced LHRHr-mediated uptake of the targeted MNCs resulted in enhancement in the T2-weighted negative contrast in cellular phantom gels. Taken together, the LHRH-conjugated MNCs show good potential as ovarian cancer theranostics.

Self-organized Mn2+-Block Copolymer Complexes and Their Use for In Vivo MR Imaging of Biological Processes.

Manganese-block copolymer complexes (MnBCs) that contain paramagnetic Mn ions complexed with ionic-nonionic poly(ethylene oxide-b-poly(methacrylate) have been developed for use as a T1-weighted MRI contrast agent. By encasing Mn ion within ionized polymer matrices, r1 values could be increased by 250-350 % in comparison with free Mn ion at relative high fields of 4.7 to 11.7 T. MnBCs were further manipulated by treatment with NaOH to achieve more stable complexes (iMnBCs). iMnBCs delayed release of Mn2+ which could be accelerated by low pH, indeed by cellular uptake via endocytosis into acidic compartments. Both complexes exhibited good T1 contrast signal enhancement in liver following intravenous infusion. The contrast was observed in gallbladder due to the clearance of Mn ion from liver to biliary process. iMnBCs, notably, showed a delayed contrast enhancement profile in gallbladder, which was interpreted to be due to degradation and excretion of Mn2+ ions into the gallbladder. Intracortical injection of iMnBCs into the rat brain also led to delayed neuronal transport to thalamus. The delayed enhancement feature may have benefits for targeting MRI contrast to specific cells and surface receptors that are known to be internalized by endocytosis.

Live nephron imaging by MRI.

The local sensitivity of MRI can be improved with small MR detectors placed close to regions of interest. However, to maintain such sensitivity advantage, local detectors normally need to communicate with the external amplifier through cable connections, which prevent the use of local detectors as implantable devices. Recently, an integrated wireless amplifier was developed that can efficiently amplify and broadcast locally detected signals, so that the local sensitivity was enhanced without the need for cable connections. This integrated detector enabled the live imaging of individual glomeruli using negative contrast introduced by cationized ferritin, and the live imaging of renal tubules using positive contrast introduced by gadopentetate dimeglumine. Here, we utilized the high blood flow to image individual glomeruli as hyperintense regions without any contrast agent. These hyperintense regions were identified for pixels with signal intensities higher than the local average. Addition of Mn(2+) allowed the simultaneous detection of both glomeruli and renal tubules: Mn(2+) was primarily reabsorbed by renal tubules, which would be distinguished from glomeruli due to higher enhancement in T1-weighted MRI. Dynamic studies of Mn(2+) absorption confirmed the differential absorption affinity of glomeruli and renal tubules, potentially enabling the in vivo observation of nephron function.

Manganese graft ionomer complexes (MaGICs) for dual imaging and chemotherapy.

Novel manganese graft ionomer complexes (MaGICs) that contain Mn ions complexed with a polyaminobisphosphonate-g-poly(ethylene oxide) (PEO) copolymer were developed for use as T1-weighted contrast agents for MRI. The complexes exhibited good colloidal stability without release of free manganese and did not result in any in vitro toxicity against mouse hepatocytes. T1 relaxivities of the MaGICs at physiological pH were 2-10 times higher than that of a commercial manganese-based positive contrast agent. Anticancer drugs including doxorubicin, cisplatin and carboplatin were successfully encapsulated into the MaGICs with high efficiency. Drug release behavior was sustained and depended on pH (faster in acidic environments), drug structures and drug concentration (faster with high concentration). The anticancer drug-loaded manganese nanocarriers exhibited excellent anticancer activity against MCF-7 breast cancer cells together with high relaxivity. Thus, these drug-loaded MaGICs could potentially be utilized for simultaneous diagnosis and treatment of cancer.

Wireless amplified nuclear MR detector (WAND) for high-spatial-resolution MR imaging of internal organs: preclinical demonstration in a rodent model.

PURPOSE: To assess the feasibility of imaging deep-lying internal organs at high spatial resolution by imaging kidney glomeruli in a rodent model with use of a newly developed, wireless amplified nuclear magnetic resonance (MR) detector. MATERIALS AND METHODS: This study was approved by the Animal Care and Use Committee at the National Institutes of Health/National Institute of Neurologic Disorder and Stroke. As a preclinical demonstration of this new detection technology, five different millimeter-scale wireless amplified nuclear MR detectors configured as double frequency resonators were chronically implanted on the medial surface of the kidney in five Sprague-Dawley rats for MR imaging at 11.7 T. Among these rats, two were administered gadopentetate dimeglumine to visualize renal tubules on T1-weighted gradient-refocused echo (GRE) images, two were administered cationized ferritin to visualize glomeruli on T2*-weighted GRE images, and the remaining rat was administered both gadopentetate dimeglumine and cationized ferritin to visualize the interleaved pattern of renal tubules and glomeruli. The image intensity in each pixel was compared with the local tissue signal intensity average to identify regions of hyper- or hypointensity. RESULTS: T1-weighted images with 70-µm in-plane resolution and 200-µm section thickness were obtained within 3.2 minutes to image renal tubules, and T2*-weighted images of the same resolution were obtained within 5.8 minutes to image the glomeruli. Hyperintensity from gadopentetate dimeglumine enabled visualization of renal tubules, and hypointensity from cationic ferritin enabled visualization of the glomeruli. CONCLUSION: High-spatial-resolution images have been obtained to observe kidney microstructures in vivo with a wireless amplified nuclear MR detector.

Magnetic Nanoclusters with Hydrophilic Spacing for Dual Drug Delivery and Sensitive Magnetic Resonance Imaging.

Magnetic Block Ionomer Clusters (MBIClusters) with hydrophilic ionic cores and nonionic coronas have been prepared that have ultrahigh transverse NMR relaxivities together with capacities for incorporating high concentrations of polar antibiotic payloads. Magnetite-polymer nanoparticles were assembled by adsorbing the polyacrylate block of an aminofunctional poly(ethylene oxide-b-acrylate) (H2N-PEO-b-PAA) copolymer onto magnetite nanoparticles. The PEO blocks extended into aqueous media to keep the nanoparticles dispersed. Amines at the tips of the H2N-PEO corona were then linked through reaction with a PEO diacrylate oligomer to yield MBIClusters where the metal oxide in the precursor nanoparticles were distinctly separated by the hydrophilic polymer. The intensity average spacing between the magnetite nanoparticles within the clusters was estimated to be ~50 nm. These MBIClusters with hydrophilic intra-cluster space had transverse relaxivities (r2's) that increased from 190 to 604 s-1 mM Fe-1 measured at 1.4 T and 37 °C as their average sizes increased. The clusters were loaded with up to ~38 wt% of the multi-cationic drug gentamicin. MRI scans focused on the livers of mice demonstrated that these MBIClusters are sensitive contrast agents.

Nanomedicine for intracellular therapy.

Intracellular pathogens like Salmonella evade host phagocytic killing by various mechanisms. Classical antimicrobial therapy requires multiple dosages and frequent administration of drugs for a long duration. Intracellular delivery of antimicrobials using nanoparticle may effectively devise therapies for bacterial infections. This review will address the mechanisms used by Salmonella to avoid host pathogenic killing, reasons for therapeutic failure and advances in nanoparticle drug delivery technology for efficient intracellular bacterial clearance.

Targeting Brucella melitensis with polymeric nanoparticles containing streptomycin and doxycycline.

Treatment and eradication of intracellular pathogens such as Brucella is difficult because infections are localized within phagocytic cells and most antibiotics, although highly active in vitro, do not actively pass through cellular membranes. Thus, an optimum strategy to treat these infections should address targeting of active drugs to the intracellular compartment where the bacteria replicate, and should prolong the release of the antibiotics so that the number of doses and associated toxicity can be reduced. We incorporated streptomycin and doxycycline into macromolecular nanoplexes with anionic homo- and block copolymers via cooperative electrostatic interactions among the cationic drugs and anionic polymers. The approach enabled simultaneous binding of both antibiotics into the nanoplexes, and their use resulted in an improvement in performance as compared with the free drugs. Administration of two doses of the nanoplexes significantly reduced the Brucella melitensis load in the spleens and livers of infected BALB/c mice. The nanoplexes were more effective than free drugs in the spleens (0.72-log and 0.51-log reductions, respectively) and in the livers (0.79-log and 0.42-log reductions, respectively) of the infected mice. Further research regarding the design of optimum nanoplex structures will be directed towards alterations in both the core and the shell properties to investigate the effects of the rates and pathways of entry into immune cells where the brucellae replicate.

Antibacterial efficacy of core-shell nanostructures encapsulating gentamicin against an in vivo intracellular Salmonella model.

Pluronic based core-shell nanostructures encapsulating gentamicin were designed in this study. Block copolymers of (PAA(+/-)Na-b-(PEO-b-PPO-b-PEO)-b-PAA(+/-)Na) were blended with PAA(-) Na(+) and complexed with the polycationic antibiotic gentamicin to form nanostructures. Synthesized nanostructures had a hydrodynamic diameter of 210 nm, zeta potentials of -0.7 (+/-0.2), and incorporated approximately 20% by weight of gentamicin. Nanostructures upon co-incubation with J774A.1 macrophage cells showed no adverse toxicity in vitro. Nanostructures administered in vivo either at multiple dosage of 5 microg g(-1) or single dosage of 15 microg g(-1) in AJ-646 mice infected with Salmonella resulted in significant reduction of viable bacteria in the liver and spleen. Histopathological evaluation for concentration-dependent toxicity at a dosage of 15 microg g(-1) revealed mineralized deposits in 50% kidney tissues of free gentamicin-treated mice which in contrast was absent in nanostructure-treated mice. Thus, encapsulation of gentamicin in nanostructures may reduce toxicity and improve in vivo bacterial clearance.

Synthesis and colloidal properties of polyether-magnetite complexes in water and phosphate-buffered saline.

Biocompatible magnetic nanoparticles show great promise for many biotechnological applications. This paper addresses the synthesis and characterization of magnetite nanoparticles coated with poly(ethylene oxide) (PEO) homopolymers and amphiphilic poly(propylene oxide-b-ethylene oxide) (PPO-b-PEO) copolymers that were anchored through ammonium ions. Predictions and experimental measurements of the colloidal properties of these nanoparticles in water and phosphate-buffered saline (PBS) as functions of the polymer block lengths and polymer loading are reported. The complexes were found to exist as primary particles at high polymer compositions, and most formed small clusters with equilibrium sizes as the polymer loading was reduced. Through implementation of a polymer brush model, the size distributions from dynamic light scattering (DLS) were compared to those from the model. For complexes that did not cluster, the experimental sizes matched the model well. For complexes that clustered, equilibrium diameters were predicted accurately through an empirical fit derived from DLS data and the half-life for doublet formation calculated using the modified Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. Deviation from this empirical fit provided insight into possible additional interparticle hydrophobic interactions for select complexes for which the DLVO theory could not account. While the polymers remained bound to the nanoparticles in water, most of them desorbed slowly in PBS. Desorption was slowed significantly at high polymer chain densities and with hydrophobic PPO anchor blocks. By tailoring the PPO block length and the number of polymer chains on the surface, flocculation of the magnetite complexes in PBS was avoided. This allows for in vitro experiments where appreciable flocculation or sedimentation will not take place within the specified time scale requirements of an experiment.