Pharmacokinetics of Single Domain Antibodies and Conjugated Nanoparticles Using a Hybrid near Infrared Method.

Iron oxide nanoparticles and single domain antibodies from camelids (VHHs) have been increasingly recognized for their potential uses for medical diagnosis and treatment. However, there have been relatively few detailed characterizations of their pharmacokinetics (PK). The aim of this study was to develop imaging methods and pharmacokinetic models to aid the future development of a novel family of brain MRI molecular contrast agents. An efficient near-infrared (NIR) imaging method was established to monitor VHH and VHH conjugated nanoparticle kinetics in mice using a hybrid approach: kinetics in blood were assessed by direct sampling, and kinetics in kidney, liver, and brain were assessed by serial in vivo NIR imaging. These studies were performed under "basal" circumstances in which the VHH constructs and VHH-conjugated nanoparticles do not substantially interact with targets nor cross the blood brain barrier. Using this approach, we constructed a five-compartment PK model that fits the data well for single VHHs, engineered VHH trimers, and iron oxide nanoparticles conjugated to VHH trimers. The establishment of the feasibility of these methods lays a foundation for future PK studies of candidate brain MRI molecular contrast agents.

Voxel-Based Dosimetry of Iron Oxide Nanoparticle-Conjugated 177Lu-Labeled Folic Acid Using SPECT/CT Imaging of Mice.

Several radiolabeled folic acid conjugates have been developed for targeted imaging and therapy. However, the therapeutic concept with radiolabeled folate conjugates has not yet been applied to clinical applications owing to the high renal absorbed dose. The effectiveness of targeted radionuclide therapy (TRT) depends primarily on the absorbed dose rate and on the total absorbed dose delivered to the tumor and to normal tissue. Owing to various limitations associated with organ level dosimetry, voxel-based dosimetry has become essential for the assessment of a more  accurate absorbed dose during TRT. In this study, we synthesized iron oxide nanoparticle (IONP)-conjugated radiolabeled folate (177Lu-IONP-Folate) and performed voxel-based dosimetry using SPECT/CT images of normal mice through direct Geant4 application for emission tomography (GATE) Monte Carlo (MC) simulation. We also prepared 177Lu-Folate and 177Lu-IONPs for the comparison of absorbed doses with that of 177Lu-IONP-Folate. In addition, we calculated the mean absorbed dose at the organ-level using the medical internal radiation dose (MIRD) schema. The radioactivities of all three radiotracers were mainly accumulated in the liver and kidneys immediately after injection. For the kidneys, the voxel-based absorbed doses obtained with 177Lu-IONP-Folate, 177Lu-Folate, and 177Lu-IONPs were 1.01 ± 0.17, 2.46 ± 0.50, and 0.52 ± 0.08 Gy/MBq, respectively. The renal absorbed dose decreased significantly (∼half) when 177Lu-IONP-Folate was used compared with when the 177Lu-Folate only was used. The mean absorbed dose values obtained at organ-level using the MIRD schema were comparable to voxel-based absorbed doses estimated with GATE MC. The voxel-based absorbed dose values obtained in this study of individualized activity show that the renal absorbed dose could be reduced to almost half with 177Lu-IONP-Folate. Therefore, 177Lu-IONP-Folate could be clinically applicable in the TRT of folate receptor-positive cancers in a personalized manner when using the voxel-based dosimetry method.

The impact of myelination on axon sparing and locomotor function recovery in spinal cord injury assessed using diffusion tensor imaging.

The dysmyelinated axons of shiverer mice exhibit impaired conduction characteristics, similar to early postnatal axons before myelination, whereas the patterns of neuronal activity and connectivity are relatively comparable with those of wild-type myelinated axons. This unique dysmyelination pattern is exploited in the present study to determine the role of compact myelin in the loss and recovery of function following traumatic spinal cord injury (SCI). We applied in vivo diffusion tensor imaging (DTI) and post-mortem immunohistochemistry analysis to examine changes in myelin and axonal integrity, and evaluated these changes in concert with the analysis of locomotor function from 1 to 4 weeks following a mid-thoracic contusion injury in homozygous shiverer and heterozygous littermate mice. The DTI biomarkers, axial and radial diffusivities, are noninvasive indicators of axon and myelin integrity in response to SCI of both myelinated and dysmyelinated spinal cord. We show that myelin is critical for normal hind limb function in open field locomotion. However, when the functional outcome is limited during chronic SCI, the extent of recovery is associated with residual axonal integrity and independent of the extent of intact myelin at the lesion epicenter.

Diffusion tensor imaging detects treatment effects of FTY720 in experimental autoimmune encephalomyelitis mice.

Fingolimod (FTY720) is an orally available sphingosine-1-phosphate (S1P) receptor modulator reducing relapse frequency in patients with relapsing-remitting multiple sclerosis (RRMS). In addition to immunosuppression, neuronal protection by FTY720 has also been suggested, but remains controversial. Axial and radial diffusivities derived from in vivo diffusion tensor imaging (DTI) were employed as noninvasive biomarkers of axonal injury and demyelination to assess axonal protection by FTY720 in experimental autoimmune encephalomyelitis (EAE) mice. EAE was induced through active immunization of C57BL/6 mice using myelin oligodendrocyte glycoprotein peptide 35-55 (MOG(35-55)). We evaluated both the prophylactic and therapeutic treatment effect of FTY720 at doses of 3 and 10 mg/kg on EAE mice by daily clinical scoring and end-point in vivo DTI. Prophylactic administration of FTY720 suppressed the disease onset and prevented axon and myelin damage when compared with EAE mice without treatment. Therapeutic treatment by FTY720 did not prevent EAE onset, but reduced disease severity, improving axial and radial diffusivity towards the control values without statistical significance. Consistent with previous findings, in vivo DTI-derived axial and radial diffusivity correlated with clinical scores in EAE mice. The results support the use of in vivo DTI as an effective outcome measure for preclinical drug development.

Bone marrow transplantation augments the effect of brain- and spinal cord-directed adeno-associated virus 2/5 gene therapy by altering inflammation in the murine model of globoid-cell leukodystrophy.

Globoid-cell leukodystrophy (GLD) is an inherited demyelinating disease caused by the deficiency of the lysosomal enzyme galactosylceramidase (GALC). A previous study in the murine model of GLD (twitcher) demonstrated a dramatic synergy between CNS-directed adeno-associated virus 2/5 (AAV2/5) gene therapy and myeloreductive bone marrow transplantation (BMT). However, the mechanism by which these two disparate therapeutic approaches synergize is not clear. In addition, the therapeutic efficacy may have been limited since the CNS-directed gene therapy was restricted to the forebrain and thalamus. In the current study, intrathecal and intracerebellar injections were added to the therapeutic regimen and the mechanism of synergy between BMT and gene therapy was determined. Although AAV2/5 alone provided supraphysiological levels of GALC activity and reduced psychosine levels in both the brain and spinal cord, it significantly increased CNS inflammation. Bone marrow transplantation alone provided essentially no GALC activity to the CNS and did not reduce psychosine levels. When AAV2/5 is combined with BMT, there are sustained improvements in motor function and the median life span is increased to 123 d (range, 92-282 d) compared with 41 d in the untreated twitcher mice. Interestingly, addition of BMT virtually eliminates both the disease and AAV2/5-associated inflammatory response. These data suggest that the efficacy of AAV2/5-mediated gene therapy is limited by the associated inflammatory response and BMT synergizes with AAV2/5 by modulating inflammation.

Sensitive detection of extremely small iron oxide nanoparticles in living mice using MP2RAGE with advanced image co-registration.

Magnetic resonance imaging (MRI) is a widely used non-invasive methodology for both preclinical and clinical studies. However, MRI lacks molecular specificity. Molecular contrast agents for MRI would be highly beneficial for detecting specific pathological lesions and quantitatively evaluating therapeutic efficacy in vivo. In this study, an optimized Magnetization Prepared-RApid Gradient Echo (MP-RAGE) with 2 inversion times called MP2RAGE combined with advanced image co-registration is presented as an effective non-invasive methodology to quantitatively detect T1 MR contrast agents. The optimized MP2RAGE produced high quality in vivo mouse brain T1 (or R1 = 1/T1) map with high spatial resolution, 160 × 160 × 160 µm3 voxel at 9.4 T. Test-retest signal to noise was > 20 for most voxels. Extremely small iron oxide nanoparticles (ESIONPs) having 3 nm core size and 11 nm hydrodynamic radius after polyethylene glycol (PEG) coating were intracranially injected into mouse brain and detected as a proof-of-concept. Two independent MP2RAGE MR scans were performed pre- and post-injection of ESIONPs followed by advanced image co-registration. The comparison of two T1 (or R1) maps after image co-registration provided precise and quantitative assessment of the effects of the injected ESIONPs at each voxel. The proposed MR protocol has potential for future use in the detection of T1 molecular contrast agents.

Proteolytic fluorescent signal amplification on gold nanoparticles for a highly sensitive and rapid protease assay.

A new strategy for highly sensitive and rapid protease assay is developed by mediating proteolytic formation of oligonucleotide duplexes and using the duplexes for signal amplification. In the presence of matrix metalloprotease-2 (MMP-2), fragmentation of the intact DNA-peptide on gold nanoparticles (GNP) by hydrolytic cleavage of a peptide bond within the substrate allows diffusion of DNA away from the GNP and the formation of a DNA/RNA heteroduplex, leading to digestion of RNA by RNase H. Because of the high quenching efficacy of GNP to the fluorophore in RNA and multiple digestions of the RNA, the fluorescence signal recovery is amplified. This method permits the assessment of the activity of MMP-2 at concentrations as low as 10 pM within 4 h. Compared with the reported protease nanosensors using quantum dots, GNP, and magnetic nanoparticles with the same peptide sequence, the assay time of this method is sixfold faster and the limit of detection is 100-fold more sensitive. The formulations for proteolytic formations of oligonucleotides duplexes for signal amplification on GNP could lead to the development of more sensitive and rapid protease assay techniques, thus extending the role of proteases as therapeutic targets and disease indicators.

Automatic segmentation of rodent spinal cord diffusion MR images.

MRI, is a key tool for noninvasive spinal cord lesion analysis; however, accurate, quantitative methods for this analysis are lacking. A new, multistep, multidimensional approach, utilizing the classification expectation maximization algorithm, is proposed for MRI segmentation of spinal cord tissues. Diffusion tensor imaging is used to generate multiple images of each spinal slice, with different diffusion direction weightings. The maximum likelihood tissue classifications are then jointly estimated to produce a binary classification image, corresponding to voxels containing either spinal cord or background. Edge detection is employed to find a nonparametric curve encapsulating the entire spinal cord. The algorithm is evaluated using data from in vivo diffusion tensor imaging of control and injured mouse spinal cords. The algorithm is shown to remain accurate for whole spinal cord, white matter, and hemorrhage segmentation in the presence of significant injury. The results of the method are shown to be at least on par with expert manual segmentation.

Specific and sensitive detection of nucleic acids and RNases using gold nanoparticle-RNA-fluorescent dye conjugates.

Gold nanoparticles were modified with RNA and utilized to detect specific DNA sequences and various RNA nucleases.

Test-retest reliability of high spatial resolution diffusion tensor and diffusion kurtosis imaging.

We assessed the test-retest reliability of high spatial resolution diffusion tensor imaging (DTI) and diffusion kurtosis imaging (DKI). Diffusion MRI was acquired using a Siemens 3 Tesla Prisma scanner with 80 mT/m gradients and a 32-channel head coil from each of 3 concussive traumatic brain injury (cTBI) patients and 4 controls twice 0 to 24 days apart. Coefficients of variation (CoV) for DTI parameters were calculated in each DTI Studio parcellated white matter tract at 1.25 mm and 1.75 mm isotropic voxel resolution, as well as DKI parameters at 1.75 mm isotropic. Overall, fractional anisotropy had the best reliability, with mean CoV at 5% for 1.25 mm and 3.5% for 1.75 mm isotropic voxels. Mean CoV for the other DTI metrics were <7.0% for both 1.25 and 1.75 mm isotropic voxels. The mean CoV was ≤4.5% across the DKI metrics. In the commonly injured orbitofrontal and temporal pole regions CoV was <3.5% for all parameters. Thus, with appropriate processing, high spatial resolution advanced diffusion MRI has good to excellent test-retest reproducibility in both human cTBI patients and controls. However, further technical improvements will be needed to reliably discern the most subtle diffusion abnormalities, especially at high spatial resolution.

Comprehensive locomotor outcomes correlate to hyperacute diffusion tensor measures after spinal cord injury in the adult rat.

In adult rats, locomotor deficits following a contusive thoracic spinal cord injury (SCI) are caused primarily by white matter loss/dysfunction at the epicenter. This loss/dysfunction decreases descending input from the brain and cervical spinal cord, and decreases ascending signals in long propriospinal, spinocerebellar and somatosensory pathways, among many others. Predicting the long-term functional consequences of a contusive injury acutely, without knowledge of the injury severity is difficult due to the temporary flaccid paralysis and loss of reflexes that accompany spinal shock. It is now well known that recovery of high quality hindlimb stepping requires only 12-15% spared white matter at the epicenter, but that forelimb-hindlimb coordination and precision stepping (grid or horizontal ladder) require substantially more trans-contusion communication. In order to translate our understanding of the neural substrates for functional recovery in the rat to the clinical arena, common outcome measures and imaging modalities are required. In the current study we furthered the exploration of one of these approaches, diffusion tensor magnetic resonance imaging (DTI), a technique now used commonly to image the brain in clinical research but rarely used diagnostically or prognostically for spinal cord injury. In the adult rat model of SCI, we found that hyperacute (<3h post-injury) DTI of the lateral and ventral white matter at the injury epicenter was predictive of both electrophysiological and behavioral (locomotor) recovery at 4 weeks post-injury, despite the presence of flaccid paralysis/spinal shock. Regions of white matter with a minimum axial diffusivity of 1.5 µm(2)/ms at 3h were able to conduct action potentials at 4 weeks, and axial diffusivity within the lateral funiculus was highly predictive of locomotor function at 4 weeks. These observations suggest that acute DTI should be useful to provide functional predictions for spared white matter following contusive spinal cord injuries clinically.

Full tensor diffusion imaging is not required to assess the white-matter integrity in mouse contusion spinal cord injury.

In vivo diffusion tensor imaging (DTI) derived indices have been demonstrated to quantify accurately white-matter injury after contusion spinal cord injury (SCI) in rodents. In general, a full diffusion tensor analysis requires the acquisition of diffusion-weighted images (DWI) along at least six independent directions of diffusion-sensitizing gradients. Thus, DTI measurements of the rodent central nervous system are time consuming. In this study, diffusion indices derived using the two-direction DWI (parallel and perpendicular to axonal tracts) were compared with those obtained using six-direction DTI in a mouse model of SCI. It was hypothesized that the mouse spinal cord ventral-lateral white-matter (VLWM) tracts, T8-T10 in this study, aligned with the main magnet axis (z) allowing the apparent diffusion coefficient parallel and perpendicular to the axis of the spine to be derived with diffusion-weighting gradients in the z and y axes of the magnet coordinate respectively. Compared with six-direction full tensor DTI, two-direction DWI provided comparable diffusion indices in mouse spinal cords. The measured extent of spared white matter after injury, estimated by anisotropy indices, using both six-direction DTI and two-direction DWI were in close agreement and correlated well with histological staining and behavioral assessment. The results suggest that the two-direction DWI derived indices may be used, with significantly reduced imaging time, to estimate accurately spared white matter in mouse SCI.

Diffusion tensor imaging at 3 hours after traumatic spinal cord injury predicts long-term locomotor recovery.

Accurate diagnosis of spinal cord injury (SCI) severity must be achieved before highly aggressive experimental therapies can be tested responsibly in the early phases after trauma. These studies demonstrate for the first time that axial diffusivity (lambda||), derived from diffusion tensor imaging (DTI) within 3 h after SCI, accurately predicts long-term locomotor behavioral recovery in mice. Female C57BL/6 mice underwent sham laminectomy or graded contusive spinal cord injuries at the T9 vertebral level (5 groups, n = 8 for each group). In-vivo DTI examinations were performed immediately after SCI. Longitudinal measurements of hindlimb locomotor recovery were obtained using the Basso mouse scale (BMS). Injured and spared regions of ventrolateral white matter (VLWM) were reliably separated in the hyperacute phase by threshold segmentation. Measurements of lambda|| were compared with histology in the hyperacute phase and 14 days after injury. The spared normal VLWM determined by hyperacute lambda|| and 14-day histology correlated well (r = 0.95). A strong correlation between hindlimb locomotor function recovery and lambda||-determined spared normal VLWM was also observed. The odds of significant locomotor recovery increased by 18% with each 1% increase in normal VLWM measured in the hyperacute phase (odds ratio = 1.18, p = 0.037). The capability of measuring subclinical changes in spinal cord physiology and murine genetic advantages offer an early window into the basic mechanisms of SCI that was not previously possible. Although significant obstacles must still be overcome to derive similar data in human patients, the path to clinical translation is foreseeable and achievable.

Efficient photoconductive terahertz source using line excitation.

We report significant improvement in terahertz (THz) power and efficiency using photoconductive sources by use of a spatially extended line source excitation and the trap enhanced field effect that occurs in sources made on semi-insulating GaAs. The combination of high electric fields and reduced screening effects allows 10 microW of THz power to be generated with 14 mW of absorbed optical power, demonstrating nearly 0.1% optical-to-THz conversion efficiency.