Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

The overall goal of this procedure is to image the distribution and assess the therapeutic efficacy of magnetically labeled or drug formulated nanoparticles. This is accomplished by first manufacturing the particles, testing their biologic properties for cell uptake in laboratory model systems, then injecting the cell laden formulations and assessing their tissue distribution via bioimaging. The affected brain regions have visualized before and after injection of virus infected monocyte derived macrophages.

The second step of the procedure is to image brain sub regions containing cell laden, magnetically labeled, or therapeutic nanoparticles. This is done at various times after the injections of cells containing magnetically labeled nanoparticles or nano formulated drugs. The third step of the procedure is to perform endpoint histological analysis of nanoparticle, biodistribution and cellular alterations.

Co-registration of the imaging results are done with histology. The final step of the procedure is to quantify and display the bio distribution of the nanoparticles, determine spectroscopic abnormalities and assess diffusion tensor abnormalities as a function of time after placement of cell injection. Ultimately, results can be obtained that show the biodistribution and effects on brain chemistry and cell morphology occurring as a consequence of therapeutic and diagnostic nanomaterial treatment through magnetic resonance imaging and spectroscopy that are coregistered to histological analyses.

Hi, I'm Dr.Michael Bosca, director of the Bioimaging Program at the University of Nebraska Medical Center. This work is part of an ongoing collaboration between my laboratory and Dr.Howard Gendelman's laboratory. The main advantage of non-invasive imaging over commonly used methods like confocal microscopy, fluorescent nanoparticles is that the whole brain can be covered in a single exam, and longitudinal studies are possible on the same animal.

This reduces biological variability, requires fewer animals, and in many cases can be translated to human use. This method can help answer key issues regarding newly derived nanoparticles, including biodistribution pharmacokinetic profiles and therapeutic efficacy. The combination of robust imaging procedures combined with histological co-registration is unique and required to perform the time resolved bio distribution and drug efficacy studies in complex animal models of disease demonstrating these procedures will be the technicians in my laboratory, Lindsey and Melissa and postdoctoral fellow Dr.P.They're responsible for acquiring imaging data from animals in the laboratory.

Aaron and Mariano are responsible for image processing, including diffusion tensor imaging and histological co-registration. Hi, my name is Dr.Howard Delman and I'm the chief of the Carol Schwartz Emerging Neuroscience Laboratory at the University of Nebraska Medical Center. This interdisciplinary collaboration with Dr.Basca and his laboratory has broad implications for therapy and diagnosis of a range of neurodegenerative diseases.

Magnetic resident spectroscopy and diffusion tenor imaging abnormalities are used to monitor neuronal and neuro process injury during disease. For the current project, the methods insight into biodistribution efficacy of nano medications such as whole body drug distributions and therapeutic efficacy for the nervous system. A range of particle manufacturing techniques are being developed that could improve detection of cell distribution during disease and include alterations of particle size coating and charge.

These will all remain active areas of investigations within our laboratories. First, nanomaterials for drug delivery and biodistribution should be prepared, which is a topic of a parallel manuscript in this issue. First, replicate candidate nano formulations for in vivo use by replacing the drug core or droplet with an identical sized particle or milled piece of super paramagnetic iron oxide before coating with the appropriate surfactant.

Then measure size, charge shape and cytotoxicity to determine whether the SPIO model system has the same properties as the candidate nano formulated drug. Finally perform cell loading assays by incubation with the candidate SPIO model nano formulation to determine the activity within the cells using phantoms composed of labeled cells suspended in agar gel phantoms should be prepared in triplicate at a series of concentrations in order to quantify the activity due to the SPIO uptake in cells. This provides an index of sensitivity and determines whether the nano formulations may affect the oxidation state, and hence the visibility of the SPIO in magnetic resonance imaging scans.

Prior to scanning, place the animal in an anesthesia chamber prefilled with the anesthetic gasses. This will speed the onset of anesthesia and minimize the amount of time required to ensure that the animal will not wake up upon removal from the chamber. Once fully anesthetized, remove the animal from the chamber and place into the stereotactic holder equipped to monitor breath rate and temperature.

Place the animal's head into the head holder, secure the ear barss and tape the body with gauze. The animal holder should be equipped with an adjustable tooth bar, allowing for vertical and horizontal alignment of the head, ensure that the head is properly positioned in the cordal rostral direction and that the head is not rotated. Check that the surface coil is properly placed on the head, allowing for rotation of the animal holder.

When in the magnet will provide compensation for minor rotations, which may occur from animal to animal. Once the animal is secure in the holder place the holder in the MRI scanner. Now move the holder with the surface coil into the center of the scanner.

An actively decoupled 72 millimeter resonator is used to transmit RF energy to create the signal while the head coil is used for signal reception. Now determine the initial position using a realtime one dimensional readout in the cordal rostral direction. Use of surface coil reception restricts signal to the area around the surface coil limiting the need for interpretation of the observed wave forms.

Then acquire a three plane localizer to determine the precise position of the animal in the scanner. Next shimming or adjustment to the homogeneity of the magnetic field is necessary. To do this, we use a multi gradient echo sequence and mapping software developed by Dr.Hobie Heatherington, which matches regions of homogeneity to the region examined by each individual imaging method.

Once shimming is complete, the scans of interest can be acquired. Biodistribution of SPIO containing nanoparticles can be detected as regions of signal loss in high resolution three DT two star weighted MRI first, prior to injecting the animal, determine the region of the brain to be scanned from the LOCALIZER scans. Then prescribe a high resolution 3D gradient, recalled echo sequence with a 150 micron isotropic resolution.

Acquire a baseline scan, then inject the animal and acquire the same sequence again. Parallel studies for determining therapeutic efficacy of nano medications can be performed using diffusion tensor imaging and hydrogen magnetic resonance spectroscopy examinations. First acquire S scout images and shim the region to be scanned.

Then acquire DTI using a respiratory gated spin echo diffusion, weighted echo planar imaging sequence in our scans. The diffusion encoding used was a balanced rotationally and variant and alternating polarity ISO cathedral scheme with 12 directions, and the encoding scheme was designed to reduce background diffusion gradient couplings. Total acquisition time was 20 to 40 minutes depending on respiration rate.

Next, acquire Mr.Spectroscopy. Locate the region of interest on previously acquired images, perform shimming on the selected region, then optimize the power of the water suppression pulses. Measure water frequency to ensure on resonance water signal and acquire a short test spectrum to provide quality control if, if spectra are of insufficient quality.

Check system settings including radio frequency, power, and shim settings. Finally, acquire the spectra in short blocks with resetting of the system frequency between acquisitions to eliminate effects of magnetic field drift. At the end of the acquisition.

Use a single pulse water spectrum as a predefined pre amplifier gain as a quantitative signal amplitude reference. After the final MRI scan in the series, perfuse the mouse, remove the brain and embed it into a block of OCT compound, which has been darkened using a drop of India ink. Then place this block in a cryostat for slicing and histological analysis.

Acquire block face images using a digital camera mounted to the front of the cryostat. Images should be taken every 50 micrometers through the entire brain volume number, the slices to allow registration within the volume. After histological processing and staining, align individual block face slices to reconstruct the 3D volume.

Using the block outlines to account jitter in the position of the cryostat head, then automatically segment the brain volume using a seed based region growing algorithm in the analyze software package. Super paramagnetic iron oxide causes signal loss in T two star weighted MRI and as such, the MRI signal void is a sensitive but not specific marker for its presence. To provide both sensitivity and specificity for the presence of SPIO in the brain.

Subtraction images should be created from the pre and post-injection 3D MRI scans for our study. The scans were sub imaged using a constrained level set method developed in our laboratory. Sub imaged brain volumes were then co registered signal intensity normalized and the volume subtracted to detect regions within the brain with signal loss, which was not along the edges to eliminate force positive signal from any sub pixel registration errors.

Next, co-registration between histology and MRI is accomplished by using the block face image as a common reference. Accurate colocalization of these two signals provides one measure of the accuracy of co-registration and warping of histology back to the original shapes of the slices. DTI scans can be analyzed by first selecting and anatomical region of interest to determine the average diffusion properties of the tissue in a particular anatomical substructure.

In our studies, analyses of the diffusion weighted data are then performed using custom programs written in interactive data language provided by Dr.Cada Hassan. San analyses produce maps of the tensor diffusivity, average diffusivity and fractional anes atrophy. Transverse and longitudinal components of the diffusion tensor can also be obtained once the maps are constructed.

Draw regions of interest on the T two weighted MRI overlaid with color encoded directionality maps. Examples of the region's chosen in our experiments for analysis in the HIV mouse model are displayed here. Data from each region of interest are downloaded as a text file to be input into a database at a later time.

An example MRS curve fit result from a single spectrum is shown here in our laboratory. We employ a time domain fitting method called Quest. In the J-M-R-U-I signal processing package.

Quest fits spectra using a linear combination of individual metabolite spectra to perform at least squares minimization between the stimulated and actual spectrum. We use a basis set of 22 individual metabolites as potential contributing factors. Basis spectra are simulated and validated using spectra of solutions of individual metabolites.

Examples of the bio distribution of SPIO labeled macrophages using these methods are seen here with an overlay of the location of labeled cells detected by MRI in red location of labeled cells using coregistered histology in blue and the overlapping yellow. The mouse had labeled monocyte derived macrophages injected into the tail vein. Five days later, T two star weighted MRI was acquired and processed as described.

The mouse was prepared by injection of HIV infected human macrophages into the brain, which is seen as a line of detected mouse monocyte derived macrophages. Further examples of both detection of labeled cells and co-registration with histology can be seen in our previous publications. The manufacturer of the particles and the testing of their biological properties for cell uptake in laboratory model systems is required and needs to be performed under sterile procedures for use in these animal investigations.

The injections of the cell laden formulations must be done without compromise of vein access as it will affect brain and other tissue regions visualized the integration of the cell biologic with bioimaging techniques and the strong cross collaboration scene proved vital to the success of the overall project. Once mastered, the procedures can be completed in two hours contingent on the availability of trained personnel to set up position and calibrate the systems during performance of the procedure. It's important to remember to maintain sterility so that the fragile immune compromised mice are kept viable for the time course of serial studies, some of which can last for months.

These methods have set the stage for how researchers can explore the relationship between cellular alterations visualized using histology and the morphological physiological and biochemical abnormalities detected. Using neuroimaging methods in brain co-registration of histology is challenging and it's been accomplished by only a few groups. After watching this video.

You should understand how brain imaging experiments are performed and coregistered with histology on a rodent model of disease. This can be used for measuring biodistribution of super paramagnetic iron oxide particles. Alternatively, MRI can and localized Mr.Spectroscopy can be used to track neurodegeneration seen during the disease processes.

Therapeutic effects to ameliorate brain damage can then be serially assessed. Imaging can be used together with histology to validate experimental findings. Overall, these methods will provide a dynamic and precise set of measures for nanoparticle, biodistribution and therapeutic efficacy.