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Quantitative magnetic resonance and SPECT imaging
for macrophage tissue migration and nanoformulated
drug delivery
Santhi Gorantla,*
,†
Huanyu Dou,*
,†
Michael Boska,*
,†,‡
Chris J. Destache,
§
Jay Nelson,*
,†,‡
Larisa Poluektova,*
,†
Barett E. Rabinow,
¶
Howard E. Gendelman,*
,†,1
and R. Lee Mosley*
,†
*Center for Neurovirology and Neurodegenerative Disorders, Departments of
†
Pharmacology and Experimental
Neuroscience and
‡
Radiology, University of Nebraska Medical Center, Omaha;
§
School of Pharmacy and Health
Professions, Creighton University, Omaha, Nebraska; and
¶
Baxter Healthcare Corporation, Round Lake, Illinois
Abstract: We posit that the same mononuclear
phagocytes, bone marrow (BM) and blood mono-
cytes, tissue macrophages, microglia, and den-
dritic cells, which serve as targets, reservoirs, and
vehicles for HIV dissemination, can be used as
vehicles for antiretroviral therapy (ART). Toward
this end, BM macrophages (BMM) were used as
carriers for nanoparticle-formulated indinavir
(NP-IDV), and the cell distribution was monitored
by single photon emission computed tomography
(SPECT), T
2
* weighted magnetic resonance imag-
ing (MRI), histology, and ␥-scintillation spectrom-
etry. BMM labeled with super paramagnetic iron
oxide and/or
111
indium oxine were infused i.v. into
naı¨ve mice. During the first 7 h, greater than 86%
of cell label was recorded within the lungs. On Days
1, 3, 5, and 7, less than 10% of BMM were in
lungs, and 74 – 81% and 13–18% were in liver and
spleen, respectively. On a tissue-volume basis, as
determined by SPECT and MRI, BMM densities in
spleen and liver were significantly greater than
other tissues. Migration into the lymph nodes on
Days 1 and 7 accounted for 1.5–2% of the total
BMM. Adoptive transfer of BMM loaded with NP-
IDV produced drug levels in lymphoid and nonlym-
phoid tissues, which exceeded reported therapeu-
tic concentrations by 200- to 350-fold on Day 1
and remained in excess of 100- to 300-fold on Day
14. These data show real-time kinetics and desti-
nations of macrophage trafficking and demonstrate
the feasibility of monitoring macrophage-based,
nanoformulated ART. J. Leukoc. Biol. 80:
000 –000; 2006.
Key Words: monocytes 䡠 cell trafficking 䡠 mouse 䡠 indinavir
INTRODUCTION
Peripheral blood mononuclear phagocytes (MP), bone marrow
(BM) and blood monocytes, tissue macrophages, microglia, and
dendritic cells (DC) serve as vehicles for dissemination and
reservoirs for the HIV-1 infection [1–3]. The ability of BM-
derived macrophages (BMM) to migrate to the sites of infection
makes them attractive candidates for use as vehicles to deliver
antiretroviral agents. Indeed, the tissue distribution of circu-
lating monocytes and notably, subpopulations of monocytes
(CD14/CD16), which emerge during inflammatory conditions,
closely parallels sites of active viral replication [4, 5].
The question we asked is whether the same MP, which
disseminate virus, could be used to traffic antiretroviral drugs
to tissue sites of active viral replication. Precedent is provided
by cell-based systems already operative for DNA vaccines,
viral gene, and drug delivery systems developed for a variety of
degenerative and neoplastic diseases [6 –9]. However, obsta-
cles in realizing this goal center on drug cell uptake and
accurate real-time longitudinal study of monocyte trafficking to
tissues known to harbor HIV-1 [10]. The first was demonstrated
in our laboratories in cell culture experiments [11]. The second
involves cross-sectional studies of macrophage trafficking and
cell distribution kinetics, which are limited in number [10, 12].
Over 20 years ago, one study, combining radioactive tracer and
histological examinations, demonstrated solely that 45% of
splenic macrophages are locally produced, and 55% are de-
rived from monocytic extravasation [13]. Moreover, a single,
“theoretical “report of mathematical models for the path of
macrophages and their potential as vehicles for drug delivery
was reported without biologic support [14]. Thus, we strove to
use two sensitive methods, magnetic resonance imaging (MRI)
and single photon emission computer tomography (SPECT), to
examine BMM trafficking to test the feasibility of macrophage-
based drug delivery systems. Demonstrable numbers of BMM
in lymphoid tissues by MRI and SPECT analyses compelled us
to examine whether macrophage delivery of nanoformulated
antiretroviral agents could provide rapid and prolonged anti-
retroviral, therapeutic concentrations in tissues, wherein HIV
is harbored and actively replicates.
1
Correspondence: Department of Pharmacology and Experimental Neuro-
science, University of Nebraska Medical Center, 985880 Nebraska Medical
Center, Omaha, NE 68198-5880. E-mail: hegendel@unmc.edu
Received February 21, 2006; revised June 19, 2006; accepted June 20,
2006; doi: 10.1189/jlb.0206110.
0741-5400/06/0080-0001 © Society for Leukocyte Biology Journal of Leukocyte Biology Volume 80, November 2006 1
Uncorrected Version. Published on August 14, 2006 as DOI:10.1189/jlb.0206110
Copyright 2006 by The Society for Leukocyte Biology.
MATERIALS AND METHODS
Animals
Male BALB/c mice (Charles River Laboratory, Inc., Wilmington, MA), 5– 8
weeks old, were used for all experiments. Animals were housed in sterile
microisolator cages and maintained in accordance with ethical guidelines for
the care of laboratory animals of University of Nebraska Medical Center
(Omaha) and National Institutes of Health (NIH; Bethesda, MD).
Preparation of BMM
To obtain BM-derived mononuclear cells, femurs were excised and flushed
with HBSS. RBC were lysed with ammonium chloride potassium lysis buffer,
and clumps were removed by passing the cell suspension through a 40-m cell
strainer. BM cells were cultured for 10 days in Teflon flasks [15] at 2 ⫻ 10
6
cells/ml in DMEM supplemented with 10% FCS, 2 mM L-glutamine, 1%
penicillin/streptomycin, and 2 g/ml M-CSF (complete medium). All reagents
for cell culture were obtained from Invitrogen (Carlsbad, CA), except for
M-CSF, which was a generous gift from Wyeth, Inc. (Cambridge, MA). Half of
the medium was exchanged for fresh medium every 2 days. The purity of
monocytes from culture was determined by flow cytometry using PE-conjugated
antibody to CD11b (BD PharMingen, San Diego, CA) and analyzed with a
FACSCalibur (BD Biosciences, San Jose, CA). CD11b
⫹
cells within the BMM
cultures were always detected in excess of 98%.
Magnetic resonance tracking of Feridex-labeled
macrophages
BMM were labeled with super paramagnetic iron oxide (SPIO) particles (Feri-
dex, Berlex Inc., Wayne, NJ) by culturing at 2.5 mg Feridex/10
7
cells/ml
complete media for1hat37°C. Cells were washed twice with DMEM, and
each recipient mouse received 1 ⫻ 10
7
SPIO-labeled BMM in 200 l i.v. via
the tail vein. Greater than 95% cells were labeled with SPIO particles as
determined by enumeration of Prussian blue-stained cells from cytological
preparations. The presence of SPIO-labeled BMM in tissues was evaluated by
MRI. Accumulation of SPIO particles in tissue causes an increase in the
magnetic relaxivity of tissue water, which is strongly field-dependent. Mea-
sures of relaxivity in our laboratories using a 7T system (Bruker 21 cm Biospec
operating Paravision 3.0.2) demonstrated that relaxivity is related directly to
cell density, assuming uniform labeling and no cell death leading to loss of
SPIO to native macrophage activity. This approach was used to track the
migration of peripheral monocytes within the liver and spleen after i.v. injec-
tion [16, 17]. High-resolution, three-dimensional (3D) gradient-recalled echo
MRI scans of mouse body were acquired using a 25-mm birdcage volume coil
covering a region from the neck to the hips with acquisition parameters of TE
⫽ 3 ms, TR ⫽ 50 ms, 30% echo, flip angle ⫽ 35 degrees, NA ⫽ 2, field of
view ⫽ 35 ⫻ 25 ⫻ 50 mm with a resolution of 256 ⫻ 128 ⫻ 128 (voxel
size⫽137⫻195⫻390 m), reconstructed to 256 ⫻ 256 ⫻ 128, total acqui-
sition time ⫽ 30 min. Signal intensity was normalized to an external standard
to account for signal drift over time. BMM accumulation was determined by
signal loss over time within selected regions of interest. After the injection of
SPIO-labeled cells, 3D gradient-recalled echo images were acquired every 30
min for 6.5 h, at 24 h, and on Days 3, 5, and 7 thereafter. Signal intensity,
normalized to an external standard, was measured within anatomical regions of
interest to determine the rate of labeled cell influx or efflux. R
2
* (1/T
2
*)
decreases in direct proportion to the density of labeled cells in the tissue. We
used this to calculate the average cell density from normalized signal loss using
the equation: Cell density ⬀ ln(M
unlabeled
) – ln(M
SPIO
), whereby cell density is
proportional to the normalized signal intensity before injection of SPIO-labeled
cells (M
unlabeled
) minus the normalized signal intensity at each time-point after
injection (M
SPIO
).
Monocyte tracking using SPECT
To assess cell migration by SPECT, BMM were labeled with 111indium (
111
In)
oxyquinoline (Indium oxine, Amersham Healthcare, Arlington Heights, IL) at
a dose of 600 Ci per 30 ⫻ 106 cells in 1 ml RPMI 1640/10 mM HEPES for
45 min at 37°C. Cells were washed extensively and resuspended in HBSS.
Labeling efficiency, as determined by ␥-scintillation spectrometry (Packard
Instrument Co., Meriden, CT), was routinely 70 – 80% of total input isotope.
Each recipient received 5–10 ⫻ 10
6 111
In-labeled BMM i.v. Mice were
anesthetized with 0.5–1% isoflurane delivered in a 2:1 mixture of nitrous oxide
and oxygen. Image acquisition was accomplished with a ␥-scintillation camera
detector fitted with a 1-mm pinhole collimator and interfaced with image
acquisition software (A-SPECT, Gamma Medica, Northridge, CA). Briefly for
each animal, 64 1-min, equiangular exposures over a 360° axis of rotation were
acquired at each time-point. Acquired exposures were reconstructed into a
single 3D tomogram. Regions of interest (ROI) within the processed tomograms
were circumscribed by electronic bit maps to contain the lungs, liver, or
spleen, and relative activities for each were determined. After acquisition of
SPECT images for final time-point, animals were killed, and tissues excised,
weighed, and submitted for ␥-scintillation spectrometry to determine the
intensity of
111
In signal in each tissue. After scintillation spectrometry, tissue
was processed for autoradiography. Frozen sections of 30 m were obtained
and exposed to X-ray film. Autoradiographic images were digitized, and
intensities of
111
In-labeled BMM were assessed by digital image analysis
(MCID Image Analysis System, Imaging Research, Inc., St. Catherines, On-
tario, Canada).
Histology
Tissues were collected from mice after MRI tracking, fixed in 4% parafarm-
aldehyde for 24 h, and embedded in paraffin, and 5 m-thick sections were cut
for analysis. Slide-mounted sections were deparaffinized, rehydrated, and
reacted for 30 min in 2% potassium ferrocyanide in 3.7% hydrochloric acid to
visualize ferric iron particles by Prussian blue. Stained sections were washed
and counter-stained with nuclear fast red. Replicate serial sections were
stained with H&E to provide histological cell distributions. Images of stained
sections were imported into Image-Pro Plus, Version 4.0 (Media Cybernetics,
Silver Spring, MD) for quantifying Prussian blue-stained cells. As a control for
tissue distributions obtained by cell-free Feridex, Feridex alone was adminis-
tered i.v. to naı¨ve recipients at 0.25 mg for each mouse, equivalent to the
amount used to load 10 ⫻ 10
6
. Spleen, liver, lung, and lymph nodes were
collected on Eay 5 after Feridex administration, paraffin-embedded, sectioned,
and stained for Prussian blue.
Indinavir (IDV) loading of BMM, adoptive transfer,
and detection in tissues
BMM were incubated with 5 ⫻ 10
⫺4
M nanoparticle-formulated IDV (NP-IDV;
Baxter Healthcare Corp., Round Lake, IL) in complete medium for 12 h of
incubation at 37°C. Cells were washed and adoptively transferred into recip-
ient mice via tail vein. Animals were killed on Days 1 and 14, and tissues were
collected for analysis of IDV concentrations by reverse phase (RP)-HPLS, as
modified by the method of Jayewardene et al. [18]. Briefly, tissues were
homogenized in 60% methanol, maintained at 4°C overnight, and clarified by
centrifugation at 20,000 g for 15 min at 4°C. Supernatants were collected and
added to glass tubes containing 1 ml diethyl ether. The tubes were mixed for
30 s and maintained at –20°C for 30 min. The ether layer was evaporated to
dryness under a nitrogen stream at room temperature-to-dryness. The residue
was reconstituted in 150 L mobile phase [10 mM ammonium dihydrogen
phosphate with 1 mM 1-heptanesulfonic acid at pH 4.8 mixed with acetonitrile
65:35 (v/v)]. The rehydrated samples were clarified by centrifugation at 20,000
g for 5 min. Triplicate 35 l aliquots of each sample were injected for
RP-HPLC analysis (Shimadzu Corp., Columbia, MD). A C
4
RP column with 5
m particle size packing (Phenomenex, Torrance, CA) was used, and analytes
were measured at 210 nm. The data were analyzed using chromatographic
software (EZStart, Shimadzu Corp.), and peak area was integrated, concentra-
tions of IDV were determined compared with a standard concentration curve of
IDV in mobile phase. Processing and analyses were validated by spiking
known concentrations of IDV in homogenized tissue samples from naı¨ve
animals.
NP-IDV was tagged with Lissamine™ rhodamine B 1,2-dihexadecanoyl-sn-
glycero-3-phosphoethanolamine, triethylammonium salt (rDHPE; Invitrogen).
BMM were loaded with rhodamine-labeled NP-IDV (rh-NP-IDV) and adop-
tively transferred i.v. to naı¨ve recipient mice. Frozen sections of lymphoid and
nonlymphoid tissues were reacted with anti-CD11b (Serotec, Raleigh, NC) and
Alexa Fluor 488 (Molecular Probes, Eugene, OR)-labeled secondary antibody
and evaluated by fluorescence microscopy to detect rh-NP-IDV (red) and
CD11b
⫹
(green) cells.
2 Journal of Leukocyte Biology Volume 80, November 2006 http://www.jleukbio.org
Statistical analyses
Normative data from SPECT, MRI, ␥-scintillation spectrometry, and counts
from histology were evaluated by Student’s t-test and ANOVA with least
significant difference post-hoc test using the statistical software packages,
SPSS, v13.0 (SPSS, Inc., Chicago, IL) and Statistica, v7.1 (StatSoft, Inc., Tulsa,
OK). Data are expressed as means ⫾ SEM for 4 – 6 animals per group.
Significance levels were chosen as P ⱕ 0.05.
RESULTS
Tracking
111
In-labeled BMM migration by SPECT
To assess real-time migration of macrophages, 5–7 ⫻ 10
6
111
In-labeled BMM were adoptively transferred to naı¨ve recip-
ients, and migration was assessed by SPECT between 5 and 8 h
post-transfer and on 1, 3, 5, and 7 days thereafter. Within 5 h
post-transfer (Day 0), the majority of radiolabeled BMM was
detected on tomographic images within the lungs with lower
intensity signals emanating from the spleen and liver (Fig.
1A). By Day 1 post-transfer and times thereafter, the most
intense signals were found within the spleen and liver. Con-
gruous signal intensities were demonstrated by all animals.
The progressively decreasing signal intensities exhibited on
Days 3, 5, and 7 reflect the decay characteristics of
111
In
(t
1/2
⫽2.8 days). To quantify the density of BMM within each
tissue, ROI were drawn with electronic bit maps, and the
relative counts and volumes were determined and corrected for
radioactive decay. In agreement with tomographic images, the
number of counts/cm
3
in lungs was significantly greater at 5– 8
h post-transfer compared with those in spleen and liver; how-
ever, by Day 1 and thereafter, counts decreased in lungs and
were increased significantly in spleen and liver, but no signif-
icant differences in densities of BMM were discernible be-
tween liver and spleen (Fig. 1B). Evaluation of percent distri-
bution of BMM indicated that after 5 h post-transfer, 66% ⫾
2% of the labeled BMM were in the lung, 25% ⫾ 2% in the
liver, and 9% ⫾ 1% in the spleen (Fig. 1C). Later, significantly
greater levels of BMM (74 – 81%) remained in the liver, 13–
17% in spleen, and 6 –9% in the lungs.
Tracking Feridex-labeled BMM by MRI and
coregistration with SPECT
To evaluate BMM migration by MRI, Feridex-loaded BMM
were adoptively transferred i.v. to naı¨ve recipients, and migra-
tion was tracked over a 7-day period. BMM density exponen-
tially increased in the spleen (Fig. 2A) at a rate of 6.4 h
compared with 8.7 h for liver (Fig. 2, B and C). Cell density in
liver and spleen was significantly higher by Day 1 after transfer
compared with MRI signals at initiation of image acquisition
(time⫽0) and remained significantly higher throughout the
study. A higher but insignificant density of cells was demon-
strated in the spleen compared with liver; however, the relative
concentration of cells within the spleen on Day 7 appears to be
underestimated in MRI compared with SPECT, histology, and
tissue scintillation measures. As a control for migration into a
highly vascular but nonlymphoid tissue, kidneys were evalu-
ated and showed negligible signals, especially after Day 1, and
only a slight increase in BMM density was detected over the
7-day time-course, a finding also appreciated in SPECT im-
ages, indicating little BMM migration into the kidneys. Tissue
movement during vital examination precluded analysis by MRI
in the lungs of viable animals.
To validate MRI and SPECT analyses, BMM were dual-
labeled with Feridex and
111
In and extensively washed, and
10 ⫻ 10
6
were adoptively transferred to recipient mice. Re-
constructed SPECT images were coregistered to MRI scans
using the Analyze package (AnalyzeDirect, Inc.). SPECT data
were interpolated to the resolution of the MRI data
(256⫻256⫻128), and fiducial markers were used for scaling
and alignment. Regions of interest were drawn on MRI and
transferred to interpolated SPECT images to obtain region
counts in fiducial markers and signal intensity in T
2
* weighted
MRI. Coregistered images showed the existence of dual-la-
beled BMM in spleen and liver with increased intensities in
SPECT images (green) as a result of
111
In label, which corre-
sponded to Feridex-induced loss of signal in MRI images (red;
Fig. 2D and Supplemental Video 1). Light-green intensities
around the areas of kidney demonstrated that although signal
loss is negligible by MRI, migration of BMM to kidney is
detectable by SPECT. Coregistration with strong signal-to-
noise ratios at optimal operating efficiencies provided strong
correlation between MRI and SPECT data for spleen (r⫽0.66)
and liver (r⫽0.77). These data confirmed the separate MRI and
SPECT analyses preformed previously (Figs. 1 and 2, A–C),
which indicated that the majority of BMM migrated to spleen
and liver after Day 1 post-transfer and was retained in those
tissues at times thereafter.
Tissue distribution of
111
In-labeled BMM
To validate SPECT analysis, we assessed tissue distribution
and density of
111
In-labeled macrophages by ␥-scintillation
spectrometry of lymphoid (spleen and lymph nodes) and non-
lymphoid (liver, lung, and kidney) tissues harvested immedi-
ately after the last SPECT data acquisition on Days 1 and 7.
After i.v. administration of BMM, spleen, liver, and lung re-
tained 96% of the tissue counts (62.5, 21.3, and 12.1%,
respectively) after Day 1 and were essentially unchanged by
Day 7 (68.2, 20.6, and 6.7%, respectively). Rank order anal-
ysis of BMM density (relative counts/mg tissue weight) and
distribution of counts from spleen and liver indicated signifi-
cant differences (P⬍0.002) between those two tissues and from
other tissues; however, no significant differences were detected
among other tissues. As the densities and tissue distributions
of BMM were similar on Days 1 and 7 post-transfer, this
suggested that after initial migration into tissues, the bulk of
macrophages is retained in those tissues. On Days 1 and 7,
distribution of BMM in lymph nodes (brachial, cervical, and
inguinal) accounted for 2% ⫾ 1.6% and 1.5% ⫾ 0.7%, re-
spectively, of the total radioactive signal, suggesting that mac-
rophages possess the capacity to migrate to and remain within
lymph nodes. Despite negligible signals in MRI tracking,
␥-scintillation spectrometry indicated that kidneys retained
1.9% ⫾ 1.4% of total
111
In-labeled BMM measured in tissues,
demonstrating that BMM are capable of migrating through
nonlymphoid tissues as well.
Gorantla et al. Macrophage migration and delivery of antiretroviral drugs 3
Days
07531
Lg
Sp
Lv
Lv
Sp
Lv
B
A
C
Sp
Sp
Sp
Lv
Lv
Lv
Lv
Lv Lv
Fig. 1. SPECT analysis to track BMM after adoptive transfer to naı¨ve mice. Mouse BMM were labeled with
111
In oxine [half-life (t
1/2
)⫽2.8 days] and washed,
and 1 ⫻ 10
7
were adoptively transferred by i.v. injection to naı¨ve, syngeneic recipients. Trafficking of BMM was evaluated by SPECT analysis for each animal
after 6 h (Day 0) and 1, 3, 5, and 7 days post-transfer. (A) SPECT images of all four mice on Days 0, 1, 3, 5, and 7, showing congruous intensity of
111
In-labeled
BMM in lung (Lg), liver (Lv), and spleen (Sp). (B) For each lung, liver, and spleen within a tomographic image, a ROI was circumscribed by electronic bit maps
to include each tissue and saved as a subimage. The tomographic subimage was sectioned transaxially, and the relative number of counts and volumes was
determined by image analysis software provided by the manufacturer. Relative counts were corrected for decay, and the mean counts/cm
3
⫾ SEM (n⫽4) for each
tissue are plotted as a function of time. (D) The distribution of labeled BMM was determined by normalizing the total relative counts as a percentage of total counts
within all tissues for each animal, and the mean distribution ⫾
SEM (n⫽4) was evaluated as a function of time after transfer of BMM.
a
, P ⱕ 0.05, for spleen and
liver compared with lung.
4 Journal of Leukocyte Biology Volume 80, November 2006 http://www.jleukbio.org
Histological confirmations of Feridex-labeled
BMM
To validate migration of Feridex-labeled macrophages after
adoptive transfer, tissues were harvested from recipient mice
after the last MRI examination at 24 h and from another set
after the last MRI examination on Day 7 post-transfer. Sections
from lung, liver, spleen, and lymph node were processed and
stained to detect Feridex-labeled BMM as Prussian blue-
stained cells. Tissues from uninjected control animals without
Feridex were stained for Prussian blue to detect endogenous
ferrum (data not shown), and this baseline stain was used for
comparison and analysis. Histological examination of tissues
following acquisition of MRI images confirmed that the major-
ity of macrophages migrated to spleen, liver, lung, (Fig. 3), and
lymph nodes (Fig. 4). When quantified using Image-Pro soft-
ware, spleens showed the greatest density of Prussian blue-
stained cells on Days 1 and 7 without detectable, significant
differences between days (555⫾25 and 473⫾35 Prussian blue
cells/mm
2
, respectively; P⫽0.07). Prussian blue-stained BMM
were distributed primarily within the resident macrophage
areas surrounding the germinal centers (Fig. 3). Lungs showed
an initial accumulation of Feridex-labeled cells by 24 h post-
transfer (237⫾19 cells/mm
2
), but virtually no cells could be
detected by Day 7 (1.1⫾0.3 cells/mm
2
), demonstrating a sig-
nificant reduction in BMM in the lung over the final 6 days
after transfer (P⬍0.0001). Feridex-labeled cells were detected
in the sinusoids of the liver. The density of BMM in the liver
on Day 1 (81⫾9 cells/mm
2
) was significantly less than densi-
ties in the spleen and lung at the same time (P⬍0.0001) but
significantly increased by Day 7 (121⫾10 cells/mm
2
,
P⫽0.009) exceeding levels in lung (P⫽0.001), although re-
mained less than BMM density in spleen at that time
(P⬍0.0001).
Feridex-labeled cells were detected in the lymph nodes from
animals on Day 1 after cell transfer within afferent lymphatics.
Increased magnifications (Fig. 4, C and D) of cortical and
paracortical areas of a representative lymph node (Fig. 4A)
show Feridex-labeled BMM in the interfollicular regions. Mac-
rophages, which are lightly stained for Prussian blue (Fig. 4D),
may represent macrophages with less Feridex as a result of
partial expulsion or death upon reaching the lymph nodes. By
Day 7, significantly higher amounts of Feridex-loaded macro-
phages (19.5⫾3.5 cells/mm
2
, P⫽0.004) were detected in the
lymph nodes (Fig. 4, E, G, and H), indicating that macrophages
were capable of reaching lymph nodes, tissues that the drug
must reach for the greatest efficacy against HIV-1 infection and
reservoirs.
Animals were also injected with Feridex alone and tissues
collected on Day 5. When stained for Prussian blue, tissues
from mice administered Feridex alone showed uniquely differ-
Spl
Liv
100%
0%
013
5
Days
B
C
D
A
Spl
Spl Spl
Liv
Liv
Liv
Kid
Bwl
Fig. 2. Tracking BMM migration by MRI and coreg-
istration with SPECT images. (A) Time series of spleen
(Spl), kidneys (Kid), and bowel (Bwl; upper panels) and
liver (Liv; lower panels) from mice before (Day 0) and
on 1, 3, and 5 days after i.v. injection of SPIO (Feri-
dex)-labeled BMM, demonstrating visible signal loss
after injection of Feridex-labeled BMM. Quantitation of
in vivo tracking of mouse BMM labeled with Feridex
using T
2
* weighted MRI during (B) the initial 6.5 h
after adoptive transfer and (C) on Days 1, 3, 5, and 7
thereafter. Results shown are measures of Feridex-
labeled cell densities in spleen, liver, and kidney
(mean⫾
SEM from n⫽4 mice per group).
a
, P ⱕ 0.05,
compared with kidney. (D) BMM were dual-labeled
with Feridex and
111
In and transferred i.v. to recipient
mice. Recipients were anesthetized and positioned in
custom-built, MRI/SPECT-compatible holders with at-
tached fiducial markers (not shown), and serial acqui-
sitions of MRI and SPECT scans were performed on
Day 1 after transfer. SPECT data were interpolated to
the resolution of the MRI data (256⫻256⫻128), and
fiducial markers were used for scaling and alignment.
Reconstructed SPECT images (green) were coregis-
tered and overlaid to MRI scans (red) using the Analyze
package (AnalyzeDirect, Inc., Lenexa, KS). Displayed,
coregistered images are 390 m slices containing
spleen and kidney (left panel) and liver (right panel)
and show areas of radioactive intensities of
111
In-la-
beled BMM in SPECT images (green), which corre-
spond to loss of MRI signal as a result of Feridex-
labeled BMM.
Gorantla et al. Macrophage migration and delivery of antiretroviral drugs 5
ent patterns of Feridex distribution compared with tissues from
mice administered Feridex-labeled BMM (Fig. 5). Livers from
mice injected with Feridex showed higher accumulations of
Prussian blue-staining cells than those injected with Feridex-
loaded BMM. Spleens showed robust staining only from ani-
mals treated with Feridex-labeled BMM.
Tissue distribution of dual-labeled BMM
To validate measurements of BMM tissue distribution, we
evaluated tissues from mice treated with BMM, which were
dual-labeled with Feridex and
111
In. Spleen, liver, lungs, and
lymph nodes were excised from mice on Day 5 after treatment
with dual-labeled BMM. Tissues were fixed, paraffin-embed-
ded, sectioned, and subjected to autoradiography and histolog-
ical analysis. Digital image analysis (Fig. 6A) and quantifica-
tion (Fig. 6B) of the autoradiographic images show that spleen
retained the highest amount of radioactivity compared with
liver and lung. In spleen, the highest intensity signals were
observed to be concentrated in macrophage-rich areas sur-
rounding the germinal centers. Measurement of radioactivity in
lymph nodes by ␥-scintillation spectrometry confirmed the
migration and presence of
111
In-labeled BMM in lymph nodes
(Fig. 6C).
Uptake and distribution of BMM NP-IDV
IDV was readily taken up by BMM as evident by the intense
black NP inclusions visualized by light microscopy in BMM
cocultured in the presence of NPs (Fig. 7B) compared with
those cultured in the absence of NPs (Fig. 7A). Virtually 100%
of the BMM retained the NPs. RP-HPLC analysis of BMM
lysates revealed an IDV concentration of ⬃37.5 M or 75% of
the initial NP-IND concentration [21]. To evaluate the poten-
tial for delivery of NP-IDV by macrophages and assess whether
therapeutic concentrations of IDV can be attained in immune
tissues, we loaded BMM with NP-IDV, as shown in Figure 7B,
and adoptively transferred the NP-IDV-loaded BMM i.v. to
naı¨ve, recipient mice. IDV concentrations were evaluated by
RP-HPLC in spleen, lymph nodes, lung, liver, and kidney on
Days 1 and 14 after transfer (Fig. 7C). Of foremost significance
is the finding that for all tissues examined, IDV concentrations,
on a tissue-weight basis, were at least one order of magnitude
in excess of the reported therapeutic concentration range in
human plasma [19, 20] and were at least 2 and 3 orders of
magnitude higher, respectively, in the lymph nodes and spleen;
both lymphoid tissues implicated in harboring HIV-1 reser-
voirs. One day after transfer of NP-IDV-loaded BMM, IDV
concentrations were significantly highest in the lung compared
with other tissues, reaching levels that were in excess of 3.5
orders of magnitude greater than the therapeutic concentration
range of IDV in plasma. It is notable that significant levels of
IDV were detected in spleen and lymph nodes. By Day 14 after
transfer, IDV concentrations were significantly higher in lym-
phoid tissues of the spleen and lymph nodes compared with
other nonlymphoid tissues of lung, liver, and kidney. Although
IDV concentrations were not changed significantly in lymph
nodes between Days 1 and 14, levels increased significantly in
spleen by Day 14 and diminished in lungs, liver, and kidney.
DISCUSSION
Macrophages have been of great interest as gene and drug
delivery vehicles [9, 22]. However, relatively few in vivo stud-
ies have assessed the ability of ex vivo-transferred macro-
phages to migrate to the target sites. For optimal use of mac-
rophage delivery systems, such studies are necessary to accu-
rately assess whether cells and drugs reach a targeted
distribution. In this study, we explored the feasibility of using
macrophages for delivery of nanoformulations of the anti-
HIV-1 protease, IDV, with the expressed intent to improve the
efficacy of the drug by targeted distribution. A necessary step
toward implementing this strategy required the measurement of
rh-NP-IDV
CD11b
Prussian
Blue
Spleen
H & E
LungLiver
Germinal center
Interfollicular area
50
50
µm
5 µm
Fig. 3. Feridex or NP-IDV-loaded macrophages within
lymphoid and nonlymphoid tissues after transfer to naı¨ve
recipients. BMM were loaded with Feridex or rhodamine-
labeled NP-IDV (rh-NP-IDV) and injected i.v. into naı¨ve
recipients. Spleen, lung, and liver were harvested and
evaluated by immunohistological analysis. After acquisi-
tion of final MRI images after Day 1 post-transfer, tissues
were stained to detect Feridex-labeled macrophages by
Prussian blue staining (blue) and counter-stained with nu-
clear fast red. Serial sections were stained with H&E for
histological comparison. Feridex-labeled BMM were de-
tected in areas of resident interfollicular macrophages sur-
rounding the germinal centers. Tissues from mice receiving
rh-NP-IDV-loaded BMM were collected at Day 5 after
adaptive transfer, processed as frozen sections for immu-
nohistology, and examined by fluorescent microscopic
analysis. CD11b (green)-positive macrophages containing
rhodamine-labeled NPs (red) are shown in spleen, liver,
and lung. Original, large micrograph magnifications, 200
m; original bar, 50 m; original inset magnifications,
⫻1000; bar, 5 m.
6 Journal of Leukocyte Biology Volume 80, November 2006 http://www.jleukbio.org
macrophage migration and homing to ensure the adequacy of
cellular penetration into tissues wherein HIV is harbored and
replicates.
For these studies, we used syngeneic BMM as sources
originating from normal progenitor cells, which were expanded
by in vitro cultivation. For tracking studies, we used BMM as
radiolabeled (
111
In) macrophages or macrophages laden with
Feridex. The former were tracked by SPECT and the latter by
T
2
* MRI. Labeled BMM were adoptively transferred i.v. to
naı¨ve recipient mice, and recipients were subjected to MRI
and SPECT analyses. Images were initially obtained every
30 – 60 min during the first 8 h post-transfer and then, 24 h and
every other day thereafter. Clearly, as assessed by MRI and
SPECT, the majority of BMM initially remained in the lung but
infiltrated to the spleen by 24 h and remained within lymphoid
tissue for up to 7 days. ␥-Scintillation spectrometry and his-
tological examination of spleen confirmed the presence of
labeled BMM and further demonstrated their presence in
lymph nodes. Although the majority of BMM was retained in
the liver as a result of the mass of the tissue, the densities of
BMM in spleen and liver on a volume basis were not signifi-
cantly different from each other, as determined by SPECT and
MRI. Several studies reported similar pattern of distribution of
systemically administered macrophages [10, 12]. Migration of
macrophages into the spleen was shown to be dependent on
their activation state. Murine peritoneal macrophages elicited
by Brewer’s thioglycollate medium when injected i.v. localized
in the lung with minimal migration to spleen even after 72 h
[10]. Macrophage-like cell lines were shown to have no migra-
tion capability but were rapidly cleared from the circulation.
SPECT and MRI provide invaluable tools for noninvasive
longitudinal assessment of migration and distribution of cell
trafficking after transfer of leukocytes in animals and humans.
SPECT is a sensitive method to follow the time-course of
migration of transplanted cells using
111
In-oxine-labeled cells
[23].
111
In has been used in studies to find biodistribution of
the cells after transplantation, but animals typically had to be
killed at each time-point for autoradiography or radioactivity
measurement [24]. SPECT scanning allows in vivo real-time
tracking of cell trafficking in the same animal or patient.
111
In-oxine labeling of peripheral blood leukocytes to assess
foci of inflammation has long been used in clinical studies of
inflammatory disorders [25, 26]. Moreover, the use of
111
In-
labeled macrophages to study the distribution of mononuclear
cells in tumors was demonstrated as an elegant procedure for
tracking the distribution of macrophages and DC [27–30].
50 µm 50 µm
Fig. 5. Tissue distributions from mice treated with BMM loaded with Feridex
or Feridex alone. Animals were injected i.v. with 10 ⫻ 10
6
BMM loaded with
0.25 mg Feridex or 0.25 mg Feridex alone. Tissues were obtained on Day 5
postadministration and paraffin-embedded, and sections were subjected to
Prussian blue staining. Feridex was distributed uniquely when injected as a
cell-free formulation compared with BMM-loaded Feridex showing greater
accumulation of Feridex in liver and less in spleen. Original micrograph
magnifications, ⫻100; bar, 50 m.
100 µm
100 µm
100 µm
100 µm
10 µm
10 µm 10 µm
25 µm
Fig, 4. BMM migration to the lymph nodes, which were extracted from mice
at (A–D) Day1 and (E–H) Day 7 after adaptive transfer of Feridex-labeled
BMM. (A, C–E, G, H) Sections were stained to detect Feridex using Prussian
blue staining and counterstained with nuclear fast red. (B, F) Serial sections
were stained with H&E for histological comparison to show the afferent
lymphatics marked by the arrow. (A, B, E, F) Micrographs are ⫻100 original
magnifications. (C, D) Micrographs are ⫻1000 original magnifications of the
areas marked in A showing (C) Feridex-labeled cells and (D) many degener-
ating macrophages faintly labeled with Feridex. Panels are (G) ⫻400 original
magnification and (H) ⫻1000 original magnification of the boxed area shown
in E containing released Feridex and Feridex-labeled cells in the afferent
lymphatics.
Gorantla et al. Macrophage migration and delivery of antiretroviral drugs 7
Similarly, MRI provides another noninvasive method for lon-
gitudinally studying in vivo, the fate of transplanted cells
labeled with a paramagnetic probe such as Feridex [31], there-
fore rendering cell-specific imaging an increasingly important
field of MRI [16, 32–34].
Having shown by SPECT, MRI, ␥-scintillation spectrometry,
and histological examination that adoptive transfer of unlad-
ened and NP-ladened BMM provided detectable and prolonged
levels of macrophage migration into lymphoid tissues, whether
adoptive transfer of macrophages ladened with antiretroviral
agents can be used for efficacious, antiretroviral drug delivery
has not been assessed. Adoptive transfer of macrophages has
been shown to be a relatively risk-free procedure in humans
with only mild side effects [35, 36] and should provide a
unique, efficacious delivery platform by which to introduce
antiretroviral agents deeper into lymphoid tissues. To assess
the efficacies of antiretroviral distribution into tissues where
HIV replicates, strategies were initiated to deliver antiretrovi-
ral drugs in macrophage vehicles, cells that naturally migrate
to tissues associated with HIV infection and harboring HIV
reservoirs. Our data demonstrated that with one administration
of macrophages loaded with NP-IDV, therapeutic levels of IDV
in lymphoid and nonlymphoid tissues were attained rapidly by
1-day postadministration and were maintained in the spleen,
lung, and lymph nodes for up to 14 days thereafter. These
observations are supported by investigations in humanized
mice infected with HIV-1. Here, NP-IDV administered in
BMM demonstrates antiretroviral responses and protection of
human CD4⫹ T cells [21]. It is interesting that although IDV
levels were highest in the lung on Day 1 compared with other
tissues, BMM in the lung were relatively low as determined by
SPECT for
111
In-labeled BMM and by MRI using Feridex-
labeled BMM. This suggested that by Day 1 after transfer, a
sufficient number of NP-IDV-loaded BMM remained in the
lung to release such high levels of IDV or high levels of
NP-IDV or free IDV remained in the lung after cells had
immigrated, possibly as a result of release from BMM and
subsequent uptake by endogenous lung macrophages. The
kinetics and biodistribution of free IDV given orally have been
shown to be short-lived and require multiple doses per day to
attain and maintain therapeutic levels [37]; nanoformulated
IDV delivered via macrophage vehicles afforded rapid and
prolonged attainment of therapeutic levels of IDV in tissues
where HIV is harbored and replicates. Although the levels
reported here resulted from a single administration of NP-IDV-
loaded IDV, the potential for higher concentrations remains
possible upon multiple doses. Therefore, these findings
strongly support the feasibility of using macrophages as drug
Germinal
center
100 µm 100 µm100 µm
Fig. 6. Distribution of
111
In/Feridex-labeled BMM,
which were dual-labeled with Feridex and
111
In, and 10 ⫻
10
6
-labeled cells were adoptively transferred to recipient
mice i.v. Tissues were collected on Day 5 after BMM
administration, and 30 m frozen sections were obtained
and exposed to X-ray film for autoradiography. Autoradio-
graphic images were digitized and assessed by digital
image analysis. (A) Original autoradioraphic images (top
panels) and enlarged areas (center panels) show radioac-
tive signal intensities from
111
In-labeled BMM in spleen,
liver, and lung. Higher radioactive signal is observed in
splenic interfollicular areas around germinal centers. The
same sections used for autoradiography were visualized for
Feridex by Prussian blue staining (bottom panels) and
nuclear fast red counterstain. A splenic germinal center is
depicted (circle) surrounded by Prussian blue-stained
BMM in interfollicular areas (arrows). Original magnifica-
tions for bottom panels are ⫻100. (B) Intensities of radio-
active signals in tissues from the images collected from six
animals were quantified and expressed as mean ⫾
SEM. (C)
Radioactivity in lymph nodes collected from animals on
Day 5 after BMM administration was measured by ␥-scin-
tillation spectrometry and graphed as cpm/mg tissue
weight (mean⫾SEM of six animals). In LN, Inguinal lymph
node; Br LN, brachial lymph node; Cer LN, cervical lymph
node.
8 Journal of Leukocyte Biology Volume 80, November 2006 http://www.jleukbio.org
delivery vehicles for nanoformulated drugs to enhance the
efficacy of antiretroviral therapeutics. This strategy has been
shown to reduce markers of infectious HIV-1 in serum and
lymphoid tissues of a human/mouse chimera model of HIV-1
infection [21].
Antiretroviral therapy (ART), which includes IDV as a po-
tent protease inhibitor, has been effective in reducing plasma
viral load in HIV-infected patients. The efficacy of ART is
compromised typically as a result of the nature of complex
regimens requiring multiple daily dosing, diligent adherence to
the regimen, and the limited biodistribution of an orally ad-
ministered drug [38]. The efficacy of IDV use is also compro-
mised, as administration of an unformulated drug results in low
levels in deep lymphoid organs [39], where HIV infection is
prevalent as a result of trapped HIV particles on the follicular
DC in the germinal centers [40 – 42]. Viral replication in
lymphoid tissues has been shown to be ten- to100-fold higher
than that in PBMC [41]. Using higher doses of antiretroviral
agents can increase the drug concentration within lymphoid
tissues; however, the drawback is that this strategy for attain-
ment of therapeutically effective concentrations produces toxic
side effects by many antiretroviral drugs and often complicates
HIV drug therapies [38, 43]. Such adverse effects could be
minimized by nanoformulated drug delivery modalities that
result in selective uptake of drug by macrophages [9, 44, 45]
and provide site-specific delivery to the lymphoid tissues, thus
preventing HIV replication and the establishment of HIV
reservoirs during a clinical latency period.
ACKNOWLEDGMENTS
This work was supported by NIH Grants P01 NS31492, R01
NS34239, R37 NS36136, and P01 MH64570 (to H. E. G.) and
P01 NS43985 (to M. B. and H. E. G.). The authors thank Robin
Taylor of the University of Nebraska Medical Center for ad-
ministrative assistance.
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