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Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.
The selective delivery of drugs and therapeutic macromolecules (peptides, proteins and nucleic acids) to cellular and tissue sites of active disease and ongoing microbial infections will improve pharmaceutical responses during disease1-3. One particular cellular site is the macrophage that is both highly mobile and immune engaging and is a consistent principal target for the human immunodeficiency virus (HIV).4 Importantly, macrophage engaged inflammation also underlies a broad range of disorders that include degenerative, inflammatory, infectious and cancerous diseases; and the cell's mobility to disease sites underlies progression of tissue injuries5-9. Importantly, the use of blood borne macrophages as drug, macromolecule, and signal carriers has gained recent attention for its translational potential. However, a significant obstruction in realizing therapeutic potentials is the blood brain barrier (BBB) amongst other tissue barriers that are impermeable to a spectrum of macromolecules and proteins. These, barriers, nonetheless, do permit cell passage. All together it is projected that in the natural course of disease peripheral macrophages that bypass barriers can carry formulated drugs, markers, and peptides to sites of infection or inflammation. Nonetheless, such technologies remain only in development. It is through our works that cell-mediated delivery can be developed for diagnostic and therapeutic applications and such applications are supported by laboratory and animal models of human disease10-12.
Preparation of nanomaterials for drug delivery and biodistribution studies is the topic of a parallel manuscript in this issue (reference parallel manuscript). All procedures for crystalline nanoparticle manufacture are carried out in a laminar flow hood. All surfaces are disinfected prior to use with 70% alcohol. This includes working surface, the exterior of gloves and any spills. All are covered with solution of replicate 70% alcohol immediately with wipes. Gloves are discarded after use and are not worn when entering any other laboratory area. Excipient, drug, sterile water with/containing any/all reagents for manufacture of drug-laden particles are only brought into work areas when needed for procedures. Sterile wrapped pipettes are used only and discarded after use into a biohazard waste container. The wet willing apparatus is disinfected with alcohol prior to and following use. Work area is cleaned immediately before and after with 70% alcohol. Nanoparticle solution is tested for pyrogen in accordance with FDA guidelines to assess the absence of bacterial endotoxin in drug particle solutions used for animals. Briefly,
Examples of DTI and 1HMRS are shown in figures 1 and 2. Additional examples of 1H MRS24-26 and DTI27 results can be seen in our previous publications. Examples of preinjection T2* weighted MRI with an overlay of the location of labeled cells in yellow is shown in Figure 3. The mouse had labeled monocytes derived macrophages injected into the tail vein. Five days later, T2* weighted MRI was acquired and processed as described, above. The mouse was prepared by injection of HIV infected human macrophages into the brain, which is seen as a line of detected mouse monocytes derived macrophages. Further examples of both detection of labeled cells and coregistration with histology can be seen in our previous publications10,12.
Figure 1. Depiction of regions analyzed for DTI metrics.
Figure 2. Spectroscopic fitting using QUEST in the jMRUI signal processing suite.
Figure 3. Detection of SPIO labeled cells migrating from the peripheral blood into a brain region with focal encephalitis. Cell positions (yellow) overlay representative slices from a T2* weighted MRI acquisition as detailed in the text.
No conflicts of interest declared.
The accurate registration of histology with in-vivo imaging results is a critical step in the development of imaging biomarkers for detection and staging of neuronal disease. Some imaging metrics are likely to be correlated with gross morphological changes including changes in magnetic relaxation properties of tissue used for detecting the presence of white matter disease and cancers. Other more subtle methods, such as DTI, are likely to detect early cellular changes that may not be detectable as histological changes caused by the disease do not appear until later the stages of disease. Still other markers, such as spectroscopic markers, may be indicators of early and reversible changes, which precede even the subtlest cellular alterations.
Biodistribution can be determined non-invasively using a variety of methods. The primary non-invasive methods are positron emission tomography (PET), single photon emission computed tomography (SPECT), optical imaging, and MRI. Nuclear medicine based imaging (PET and SPECT) have been used over the years for many biodistribution, but these methods are limited by the half life of the radiotracers used for labeling the compounds or nanomaterials, especially for PET tracers. Optical imaging can be used for small rodents but cannot be translated to human use except for regions easily accessible such as surface tumors due to light absorption and light scattering. In addition, it is difficult to quantify the optical signals for these same reasons. MRI uses persistent tags such as SPIO that can be tracked in the body over a period of weeks. This, too, must be used with caution, as the label can be transferred to different cells or be reabsorbed by the body.
Detection specificity for SPIO in MRI can be provided by a variety of methods. Detection methods, which provide positive as well as negative signals, are used for improving specificity of the MRI for detecting the presence of SPIO in tissue. The subtraction method used in this work has been used by others, as well28. Other approaches include off resonance detection29-31, phase sensitive imaging that produces a particular pattern near SPIO voids32, and zero echo time image that uses T1 weighting to produce a positive signal intensity in the region of SPIO33. The advancement of these methods for improving quantitation of label, sensitivity and specificity is an area of active research today.
The work was supported by grants 1K25MH089851, 1P01DA028555-01A1, 2R01 NS034239, 2R37 NS36126, P01 NS31492, P20RR 15635, P01MH64570, and P01 NS43985 from the National Institutes of Health. The authors thank Ms. Robin Taylor for critical reading of the manuscript and outstanding graphic and literary support. The authors would also like to thank Erin McIntyre, Melissa Mellon, and Lindsay Rice for their technical support.