Macrophage Delivery of Nanoformulated Antiretroviral Drug
to the Brain in a Murine Model of NeuroAIDS1
Huanyu Dou,* Cassi B. Grotepas,* JoEllyn M. McMillan,* Christopher J. Destache,‡
Mahesh Chaubal,§Jane Werling,§James Kipp,§Barrett Rabinow,§
and Howard E. Gendelman2*†
Antiretroviral therapy (ART) shows variable blood-brain barrier penetration. This may affect the development of neurological com-
plications of HIV infection. In attempts to attenuate viral growth for the nervous system, cell-based nanoformulations were developed
with the focus on improving drug pharmacokinetics. We reasoned that ART carriage could be facilitated within blood-borne macro-
phages traveling across the blood-brain barrier. To test this idea, an HIV-1 encephalitis (HIVE) rodent model was used where HIV-
1-infected human monocyte-derived macrophages were stereotactically injected into the subcortex of severe combined immunodeficient
mice. ART was prepared using indinavir (IDV) nanoparticles (NP, nanoART) loaded into murine bone marrow macrophages (BMM,
IDV-NP-BMM) after ex vivo cultivation. IDV-NP-BMM was administered i.v. to mice resulting in continuous IDV release for 14 days.
Rhodamine-labeled IDV-NP was readily observed in areas of HIVE and specifically in brain subregions with active astrogliosis, mi-
crogliosis, and neuronal loss. IDV-NP-BMM treatment led to robust IDV levels and reduced HIV-1 replication in HIVE brain regions.
We conclude that nanoART targeting to diseased brain through macrophage carriage is possible and can be considered in develop-
mental therapeutics for HIV-associated neurological disease. The Journal of Immunology, 2009, 183: 661–669.
and immune deterioration (1). A substantive pathogenic event for
disease is the infiltration of blood-borne mononuclear phagocytes
(MP;3monocytes, tissue macrophages, and microglia) into affected
brain tissue. This accelerates viral dissemination in brain precipi-
tating productive HIV replication and the subsequent formation of
macrophage-derived multinucleated giant cells (MGC) (2–4). We
reasoned that as the vehicle for virus carriage into the nervous
system, MP could also be harnessed as an antiretroviral drug car-
rier (5, 6). In this way, drug-loaded blood-borne macrophages
would cross the blood-brain barrier (BBB) into diseased brain sub-
regions and release antiretroviral drugs serving to improve its ef-
ficacy. The importance of this strategy is bolstered by antiretroviral
therapy (ART) known to reduce HIV-associated neurocognitive
n its most significant form, HIV-associated neurocognitive
disorders, are defined as cognitive, motor, and/or behavioral
impairments. These are linked to progressive viral infection
disorder severity. Indeed, HIV patients on ART live longer and
neurological dysfunctions are reduced, showing a mixture of more
mild disease with reduced viral replication (7–11). All together,
improving ART BBB penetration could positively affect disease
outcomes and, as such, be an integral part of HIV treatments tar-
geting the nervous system (8, 12–15).
Current ART limitations are due to its inabilities to combat viral
mutation and achieve continuous, effective drug levels in virus
target tissues (12, 16–18). Indeed, resistance to antiretroviral com-
pounds can and often does develop and when present HIV-1 levels
can rapidly rebound to pretreatment concentrations if ART is dis-
continued (19–22). Such effects might be attenuated if optimal
ART transport across tissue barriers could be achieved. One im-
pediment in reaching this goal is the BBB. This tissue barrier
serves to restrict macromolecular drug transport and as such ef-
fective drug concentrations (23–26).
A means to facilitate ART passage through the BBB is by using
circulating monocyte-derived macrophages (MDM) as drug de-
pots. Previously, our laboratory used laboratory and animal sys-
tems to pursue this idea. The research demonstrated that macro-
phages can deliver drugs to sites of viral infection and show
sustained antiretroviral activities (5, 27). Recently, we also showed
that bone marrow macrophages (BMM) can cross the BBB into
HIV-1-infected brain regions (6). Based on these findings, a BMM
pharmacological nanoparticle (NP) delivery system (nanoART)
was developed to test whether blood-borne macrophages could
deliver ART directly to the brain. Our results demonstrate that
BMM can serve as vehicles for indinavir (IDV) NP delivery.
BMM showed consistent uptake and release of IDV-NP and free
IDV while targeting areas of viral replication in a severe combined
immunodeficient (SCID) model of HIV-1 encephalitis (HIVE).
These data support the notion that nanoART brain penetration,
drug distribution, and therapeutic responses can be achieved
through cell-based nanoformulation and as such lower drug-dosing
intervals, adherence, and bioavailability.
*Department of Pharmacology and Experimental Neuroscience and†Department of
Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68198;
‡School of Pharmacy and Health Professions Creighton University, Omaha, NE
68178; and§Baxter Healthcare Corporation, Round Lake, IL 60073
Received for publication January 26, 2009. Accepted for publication April 27, 2009.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by Grants 2R01 NS034239, 2R37 NS36126, P01
NS31492, P20RR 15635, P20RR 21937, P01 MH64570, and P01 NS43985 (to
H.E.G.) from the National Institutes of Health.
2Address correspondence and reprint requests to Dr. Howard E. Gendelman, Depart-
ment of Pharmacology and Experimental Neuroscience, University of Nebraska Med-
ical Center, 985880 Nebraska Medical Center, Omaha, NE 68198-5880. E-mail ad-
3Abbreviations used in this paper: MP, mononuclear phagocyte; MGC, multinucle-
ated giant cell; BBB, blood-brain barrier; ART, antiretroviral therapy; MDM, mono-
cyte-derived macrophage; BMM, bone marrow macrophage; NP, nanoparticle; IDV,
indinavir; HIVE, HIV-1 encephalitis; Vim, vimentin; GFAP, glial fibrillary acidic
protein; NF, neurofilament; p-NF, phosphorylated NF; CSF, cerebrospinal fluid; RP-
HPLC, reverse phase HPLC.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
The Journal of Immunology
Materials and Methods
NP preparation and characterization
IDV-NP suspensions were prepared using high-pressure homogenization.
The surfactant coating of the IDV crystals was made with 1.2% (w/v)
Lipoid E80, an egg phosphatide mixture of phosphatidylcholine, phos-
phatidylethanoloamine, and the hydrolyzed lyso-forms (single aliphatic
chain) of each phospholipid. Lipoid E80 coated the actual particles. The
nanosuspension was made at an alkaline pH of 8.5. IDV-free base (1.2 g)
was added to the phospholipid dispersion and a presuspension manufac-
tured using an Ultraturrax rotor-stator mixer for 4 min was used to reduce
the particle size. An isotonic buffer solution was prepared by dissolving
1.8 g of sodium chloride and 0.28 g of sodium phosphate dibasic in 200 ml
of water. The presuspension was homogenized at 15,000 psi for 40 passes.
The final mean NP size of the suspension was 1.6 ?m, with 99% of the
particles ?8.4 ?m. The process was optimized for temperature, pressure,
and homogenization cycles. Particle size was optimized to minimize dis-
solution before and during macrophage uptake and measured using light
scattering and suspension stability assays were assessed by stress and
short-term stability tests. The NP suspension was made at a concentration
at 10?2M. Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-
phosphoethanolamine, triethylammonium salt (rDHPE; Invitrogen) was
used to label IDV-NP and appeared as red fluorescence (rDHPE-IDV-NP).
Monocyte isolation, cultivation, and viral infection
Human monocytes were obtained from leukopaks of HIV-1, HIV-2, and
hepatitis B-seronegative donors and purified by countercurrent centrifugal
elutriation (28). The University of Nebraska Medical Center Institutional
Review Board approved the procedure. Cells were cultured with DMEM
with 10% heat-inactivated pooled human serum, 1% glutamine, 10 mg/ml
ciprofloxacin (Sigma-Aldrich), and 1000 U/ml highly purified recombinant
human M-CSF (a generous gift from Wyeth). Seven days after plating,
MDM were infected with HIV-1ADAat a multiplicity of infection of 0.1
infectious viral particles per target cell (28). Culture medium was half-
exchanged every 2–3 days. All viral stocks were tested and found to be free
of Mycoplasma and endotoxin contamination (Gen-Probe II; Gen-Probe).
Murine HIVE model
Four- to 6-wk-old male C.B.-17 SCID mice were purchased from The
Charles River Laboratory. BALB/c-Rag2?/??c
University of Nebraska Medical Center for parallel studies of cell migra-
tion (29). Animal experiments were performed under strict observance of
the National Institutes of Health and University of Nebraska guidelines for
animal care. Animals were maintained in sterile microisolator cages.
Briefly, all animals were anesthetized and placed in a stereotaxic apparatus
(Stoetling) for intracranial injection. The animal’s head was secured with
ear bars and mouthpiece. An injector with a 10-?l syringe was used for cell
injections. The left hemisphere of each sham-operated animal (sham) re-
ceived a total of 5 ?l of saline. Cell suspensions (5 ? 105) with uninfected
or HIV-1ADA-infected MDM were injected into the brain’s left hemisphere
to induce HIVE in mice (30).
?/?mice were bred at the
BMM isolation and cultivation
Male BALB/c mice (Charles River Laboratory), 4–5 wk of age were used
as BMM donors. Briefly, the femur was removed, the bone marrow cells
were dissociated into single-cell suspensions, and were cultured for 10 days
supplemented with 1000 U/ml M-CSF (Wyeth). Cultured BMM proved to
be 98% CD11b?by flow cytometric analysis using a FACSCalibur flow
cytometer (BD Biosciences).
Super paramagnetic iron oxide (SPIO)
HIVE SCID mice were injected with BMM containing SPIO particles
(Feridex; Berlex). BMM were incubated at a SPIO concentration of 2 mg/
107cells/ml for 2 h. This resulted in ?95% labeled cells as determined by
Prussian blue stain. Cells were washed twice with DMEM and each recip-
ient mouse was injected i.v. through the tail vein with 150 ?l containing
1 ? 107BMM loaded with SPIO (SPIO-BMM).
BMM were incubated with rDHPE-IDV-NP at a concentration of 5 ? 10?4
M for 12 h and BMM packaged rDHPE-IDV-NP (rDHPE-IDV-NP-BMM)
were washed twice with DMEM. A single dose of rDHPE-IDV-NP-BMM
or IDV-NP was injected into each mouse i.v. through the tail vein.
IDV-NP-BMM-treated HIVE SCID mice (five in total per time point per
group) were used to evaluate blood and brain tissue IDV levels at 1, 3, 7,
and 14 days after treatment. Macroscopic resections of the injected brain
regions (regions including HIV-1-infected MDM), control hemispheres
and whole blood were homogenized by sonication in 95% methanol (1
ml/2 g of tissue and 1 ml/0.5 ml of blood). Prepared tissue lysates were
maintained at 4°C overnight and clarified by centrifugation at 14,000 ? g
for 10 min at 4°C. Supernatants were collected and analyzed by reverse
phase HPLC (RP-HPLC) (Waters) for determination of drug levels. Trip-
licate 20-?l aliquots of each sample were injected for RP-HPLC analysis.
IDV was separated from other tissue components using a mobile phase of
(60/40) 25 mM potassium phosphate (pH 4.15):acetonitrile at 0.4 ml/min
and a Waters YMC Octyl C8 column (3.0 ? 150 mm). IDV was quanti-
tated by comparison of peak area to that of a series of known IDV stan-
dards. Data are expressed as ?g of IDV per 100 mg of tissue or ?g/ml in
blood. Processing and analyses were validated using known concentrations
of IDV and spiking drug into homogenized tissue samples from naive
For fluorescence evaluation of rDHPE-IDV-NP-BMM-targeted migration
to the regions of viral infection, brain tissue was collected on posttreatment
day 3 after perfusion fixation with 4% paraformaldehyde in PBS. Immu-
nofluorescent staining was performed on sucrose-processed 25-?m frozen
brain sections. Abs to human specific vimentin (Vim)-intermediate fila-
ments (clone 3B4; DakoCytomation) were used for detection of human
macrophages in the mouse brain. Ab to HIV-1p24 Ag (DakoCytomation)
was used to determine the number of HIV-1-infected MDM. Rabbit poly-
clonal Abs to ionized calcium-binding adaptor molecule 1 (Iba-1, 1/500;
Wako) was used to identify both MDM and murine microglia. Astrocytes
were detected with Abs against glial fibrillary acidic protein (GFAP;
DakoCytomation). Abs to H chain (200-kDa) neurofilament (NF) Ags
(DakoCytomation) were used to detect neurons. Fluorescent images were
visualized with an LSM 410 confocal laser-scanning microscope (Zeiss)
with argon/krypton at 488/568/647 nm. Quantification of rDHPE-IDV-NP-
BMM levels was analyzed by using Image-Pro Plus (version 4.0; Media-
Cybernetics). The red fluorescence area of rDHPE-IDV-NP-BMM was de-
termined as a percentage of the total image area per microscopy field and
calculated for a 0.1-mm window of tissue immediately surrounding the
Immunohistochemistry and image analyses
Sham, MDM, and HIVE with or without IDV-NP-BMM-treated mice were
sacrificed at 7 and 14 days after treatment. Each brain was paraffin pro-
cessed and cut into 5-?m slices to identify the injection site. Immunohis-
tochemistry was performed with the above Abs. For location of SPIO-
BMM, paraffin brain sections were stained with Prussian blue.
Quantification of GFAP-, Iba-1-, and NF-positive staining was achieved on
serial coronal brain sections as a percentage of the total image area per
microscopy field with a total of 30 fields (six sections per mouse, five mice
in each group) using Image-Pro Plus (MediaCybernetics). The absolute
number of Vim?and HIV-1p24?cells and MGC and MGC nuclei were
counted under microscopy with six sections per mouse, five mice in each
The data were analyzed and comparisons were performed using five mice
per time point per group by a two-tailed unpaired t test using Prism sta-
tistical software for MacIntosh (version 4.0; GraphPad Software). Values
of p ? 0.05 were deemed significant.
Uptake and cell release of IDV-NP
Our overarching idea is to use monocyte-macrophages as both car-
riers and extended depots for antiretroviral drugs for delivery to
reservoirs for HIV and particularly the CNS. In a first step to test
this idea, we analyzed BMM uptake and release of IDV-NP using
confocal microscopy and RP-HPLC tests. We used successive
washes of adherent ?99% pure CD68?BMM cultures to displace
surface-bound NP and prove such displacement by confocal Z-
scan analysis (27). NP visualized by fluorescence microscopy were
seen within the cytoplasm of BMM and provided clear evidence
662NANOFORMULATED ANTIRETROVIRAL DRUG DELIVERY TO BRAIN
that rDHPE-IDV-NP (red) were readily phagocytized within the
macrophage (green, Fig. 1A). rDHPE-IDV-NP (red, Fig. 1A) were
observed in ?98% of BMM. This was supported by HPLC tests
performed after rDHPE-IDV-NP treatment (Fig. 1B). Following
sequential medium changes, drug was released continuously as
shown by HPLC tests and demonstrated both intracellular and ex-
tracellular levels of IDV. These progressively diminished over 7
days (Fig. 1C).
Tracking BMM migration to diseased brain subregions
The next series of experiments examined the distributions of
monocyte-macrophages after i.v. cell injections. To determine dif-
ferences for BMM migration as a consequence of HIVE, we per-
formed replicate experiments with virus-infected and uninfected
human MDM and sham-operated injections into subcortical (cau-
date and putamen) brain regions. In these experiments, the sub-
cortical injection of HIV-1-infected human MDM induced a focal
HIVE reflective of human HIVE (30). This included astrogliosis
and microgliosis, loss of neurons, and ongoing viral replication in
affected brain regions as demonstrated by the presence of HIV-
1p24?cells (Fig. 2A). MDM and saline sham-operated mice were
controls. Into these animals BMM-carrying IDV NP were admin-
istered through the tail vein 24 h after brain injections. Mice were
sacrificed on days 1, 3, 7, and 14 for histopathological analyses and
assay of IDV drug levels. We reasoned that the neuroinflammatory
responses induced by viral infection and, in particular, HIVE, pro-
vided a biological system wherein blood-borne monocytes-macro-
phages carrying NP would ingress to diseased brain sites. Thus,
BMM migration in diseased brain regions was measured. Initial
experiments performed with BMM loaded with SPIO and admin-
istered i.v. to HIVE mice showed that the macrophages readily
migrated to areas of HIVE as seen by Prussian blue staining (Fig.
2, A and C). This paralleled sites of reactive gliosis and HIV-
1p24?cells. No Prussian blue-stained BMM were obtained in the
contralateral hemispheres of either HIVE or sham-operated ani-
mals (Fig. 2, B and D). Neuropathological examinations confirmed
that BMM target sites of ongoing viral replication (Fig. 2, A and
C). The migration patterns of BMM were highly specific to sites of
tissue injury, inflammation, and viral growth.
HIV-1-infected macrophage neuroinflammatory responses elicit
BMM brain transmigration
HIV-1 infection of brain macrophages is associated with ongoing
viral infection, astrogliosis, and microgliosis. This is seen where
HIV-1p24-, GFAP?-, and Iba-1?-stained cells are linked with
each other (Fig. 3, A–C). In Fig. 2, it was demonstrated that BMM
migration occurs readily toward neuroinflammatory sites. In mu-
rine HIVE, the association between such cell migration and neu-
roinflammation involves activation of astrocytes and microglia and
consequent proinflammatory CNS responses (31–36). To deter-
mine whether BMM carrying ART can migrate into HIVE- af-
fected brain regions, we determined NP levels in brain 3 days
following i.v. injection of rDHPE-IDV-NP-BMM. For these ex-
periments 1 day after brain stereotactic injection with infected
MDM, rDHPE-IDV-NP-loaded BMM were injected i.v. through
the tail vein. Brain tissue was examined in areas around the human
MDM injection site. In HIVE mice, significant GFAP?astroglio-
sis (green, Fig. 3A) and Iba-1?microglial responses (green, Fig.
3B) were observed. Importantly, such neuroinflammatory re-
sponses were observed colocalized with rDHPE-IDV-NP-BMM
(Fig. 3C). In regard to specificity of these responses, BMM levels
were reduced in MDM mice when compared in HIV mice. More-
over, few red fluorescence cells were detected in sham-operated
brains (Fig. 3, A and B). In all animal groups, no rDHPE-IDV-
NP-BMM were found in the contralateral hemisphere. Few num-
bers of rDHPE-IDV-NP-BMM were seen in brains injected with
uninfected MDM (supplemental Fig. 14).
Cell-based NP delivery affect IDV brain levels
To determine brain distribution of BMM loaded with nanoformu-
lated IDV, the optical properties of red fluorescent rDHPE-
IDV-NP were used. The images of brain sections reflect robust
levels of BMM-rDHPE-IDV-NP (red) targeted in the areas of
4The online version of this article contains supplemental material.
copy imaging of rDHPE-IDV-NP (red)-treated BMM (green, CD68?)
demonstrating intracellular localization of IDV-NP (A). Ingested NP ap-
pears as red fluorescent dots and is located within the cytoplasm of green
CD68?BMM. BMM-released IDV was assayed by RP-HPLC from cell
lysates (B). With subsequent medium changes (wash, arrowheads), extra-
cellular (medium) and intracellular (BMM) IDV levels diminished over
Cellular NP uptake and IDV release. Fluorescence micros-
were injected i.v. into HIVE or sham-operated mice. Histology assay ex-
amined BMM migration to diseased brain sites. Sections of brain from
HIVE (A and C) and sham (B and D) mice were immunostained for HIV-
1p24 (A and B, brown) and GFAP for astrocytes (C and D, brown). Prus-
sian blue staining (blue) revealed SPIO-labeled BMM (A and C, blue)
migration into the area of HIV-1-infected MDM 5 days after adoptive
transfer of BMM to recipient mice. In sham-operated mice (B and D), no
significant BMM migration was detected. Original magnification, ?200.
BMM migration to HIVE-affected brain tissue. SPIO-BMM
663 The Journal of Immunology
HIV-1 infection. Mouse-specific BMM CD68?cells (green, Fig.
4A) were vehicles for IDV-NP delivery to the brain. BMM mi-
grated to sites of HIVE. The rDHPE-IDV-NP-BMM (red) were
concentrated around the virus-infected sites (Fig. 4B) and colocal-
ized with CD68?immunostaining (Fig. 4A). Higher levels of
CD68?BMM and rDHPE-IDV-NP-BMM were in HIVE brains
when compared with uninfected MDM (supplemental figures) and
sham controls (Fig. 4, A and B). Associations between productive
viral infection and the presence of rDHPE-IDV-NP-BMM were
easily seen, indicating that viral infection and inflammatory re-
sponses induced BMM brain migration. The xenogenic responses
to human MDM (supplemental Fig. 2) showed, in part, that
rDHPE-IDV-NP entered the brain as a result of even modest neu-
roinflammatory responses. However, BMM migration was en-
hanced significantly by HIV-1 infection. These results suggest that
ongoing HIV-1 infection of macrophages affected activation of
astrocytes and microglia that in turn induced BMM CNS migra-
tion. In support of this idea was the fact that large numbers of
rDHPE-IDV-NP-BMM were found in association with HIV-
1p24?MDM (Fig. 4B, green). Furthermore, HIV-1 infection of
MDM served to increase the levels of GFAP?-reactive astrocytes
in the injected hemisphere of HIVE mice (Fig. 4B, blue) when
mice was used to induce HIVE, resulting in neuroinflammation by astrocytes and microglia activation. Immunostaining was performed on frozen
brain sections from sham-operated and HIVE mice with or without rDHPE-IDV-NP-BMM. Confocal imaging of Iba-1 (green, B) and GFAP (green,
A) immunoreactivity reflect astrogliosis and microgliosis responses. The red fluorescence spots were around and within the site of injection in HIVE
mice 3 days after rDHPE-IDV-NP-BMM treatment. rDHPE-IDV-NP-BMM are colocated with GFAP- reactive astrocytes in HIVE mice when
compared with sham-operated animals (A). Confocal imaging of Iba-1 (green) reflects microgliosis (B). The Vim (blue) staining was used to
distinguish between the human MDM and murine microglia in B with composite of IDV, Vim, and Iba-1 immunostaining in C. The red rDHPE-
IDV-NP-BMM differentiated microglia from migrated BMM. However, Iba-1 also labeled native circulating monocytes in the mice. Moreover, sham
mice showed minimal glial reactions as visualized by GFAP and Iba-1 (green) in response to needle trauma with few rDHPE-IDV-NP-BMM. The
photomicrograph shows BMM migration and brain immune responses in the infected animals. IDV* denotes rDHPE-IDV-NP-BMM. Original
Neuroinflammatory responses affect BMM migration. Stereotactic injection of HIV-1-infected MDM into the caudate/putamen of SCID
brain sections were stained with Ab to CD68, HIV-1p24, and GFAP (A and B). Confocal microscopy was used to assess the distribution of CD68?cells
(green) in and around injection areas of the brain in both sham and HIVE animals 3 days after IDV-NP-BMM treatment. CD68?cells were located in and
surrounding the injection line in all animals seen in A. The intensity of CD68?cells was associated with rDHPE-IDV-NP-BMM (red, A) in treated mice
compared with untreated mice in all groups. A substantial increase of rDHPE-IDV-NP-BMM (red, B) was observed in viral infection areas in HIVE mice
compared with sham and MDM animals. Confocal imaging of brain sections with double immunostaining to HIV-1p24 (green, B) and GFAP (blue, B)
showed increased red fluorescence (rDHPE-IDV-NP-BMM) in brain areas where there was active viral infection (HIV-1p24?MDM). Greater levels of
rDHPE-IDV-NP-BMM were in HIVE compared with sham animals. GFAP (blue, B) and rDHPE-IDV-NP-BMM (red, B) were linked to HIV-1 infection
and GFAP astrogliosis (blue, B and C); however, there was no correlation between rDHPE-IDV-NP-BMM levels and GFAP expression in treated animals
compared with untreated mice in sham and HIVE mouse groups. Composite of rDHPE, HIV-1p24, and GFAP stainings are in C. IDV* denotes rDHPE-
IDV-NP-BMM. Original magnification, ?200.
Brain tissue distributions of rDHPE-IDV-NP-BMM. Sham and HIVE mice were injected with rDHPE-IDV-NP-BMM for 3 days and frozen
664 NANOFORMULATED ANTIRETROVIRAL DRUG DELIVERY TO BRAIN
compared with uninfected MDM (supplemental Fig. 2B) and
sham-injected animals. The area of rDHPE-IDV-NP-BMM, deter-
mined by digital image analysis, was increased in HIVE mice (p ?
0.01) compared with both uninfected MDM and sham controls
(Fig. 5A). Likewise, numbers of CD68?BMM were also increased
in HIVE mice (p ? 0.01) compared with sham-operated controls
(Fig. 5B). Altogether, these findings support targeted migration of
IDV-NP-BMM into areas of active HIV-1 infection and
To validate these findings of selective drug-carried BMM into
brain-diseased areas, we administered by i.v. injection a single
dose of IDV-NP-BMM to HIVE mice and determined IDV levels
in tissues from the caudate and putamen on days 1, 3, 7, and 14
after treatment. Quantifiable amounts of IDV were obtained in
blood (Fig. 5C) and comparable levels of IDV in diseased (ipsi-
lateral) and control (contralateral) hemispheres (Fig. 5D) were as-
sayed by RP-HPLC. One IDV-NP-BMM treatment elicited sus-
tained drug levels in blood for up to 14 days (Fig. 5C). More
importantly, IDV-NP-BMM delivery attained higher drug levels at
day 14 in the ipsilateral than in the contralateral hemisphere of
HIVE mice (Fig. 5D). These results confirmed that NP-IDV was
successfully delivered into the brain by packaging into BMM, thus
providing proof-of-concept for therapeutic drug delivery in animal
models of human disease.
Antiretroviral responses of IDV-NP-BMM
We previously demonstrated that after IDV-NP-BMM administra-
tion long-term viral suppression and increased CD4?T cell levels
were achieved (5). A single administration of IDV-NP-BMM
achieved drug concentrations 4- to 10-fold higher in plasma and
lymph tissues and were more sustained than those attained with a
single bolus of nonformulated IDV. To validate our results, we
graftment of human MDM in the murine environment (29). To
reach a therapeutic IDV concentration and determine antiviral ef-
ficacy in brains, IDV-NP-BMM was administered to HIVE mice;
replicate animals were untreated. After 7 and 14 days, the extent of
HIV-1 infection in the brains was determined. Immunostaining of
brain sections showed that human Vim?MDM (Fig. 6A) colocated
with activation of GFAP?astrocytes in HIVE mice. Immunohis-
tochemistry determined the levels of MDM reconstitution and viral
infection by counting the absolute number of Vim?and HIV-
1p24?MDM in brain sections. The absolute number of Vim?and
HIV-1p24?MDM was counted as cells per section in six sections
per mouse as shown in Table I. To determine the effects of IDV-
NP-BMM administration on long-term antiviral responses, HIV-
1p24?cells were assessed as a percentage of total human MDM
(Vim?). With a single treatment of IDV-NP-BMM, decreased
numbers of HIV-1-infected cells were observed in IDV-NP-BMM-
treated HIVE mice compared with untreated animals (Fig. 6B).
This was significantly apparent in brain sections (p ? 0.01) on
days 7 and 14, reflecting a long-term robust antiretroviral response
elicited by IDV-NP-BMM. Based on the observation of a reduc-
tion in HIV-1p24?cells in the IDV-NP-BMM-treated HIVE mice,
we studied MGC formation in brain sections found exclusively in
injection sites where HIV-1p24?cells were seen. Brain histopa-
thology of untreated HIVE mice is shown (Fig. 6A). Visualization
of MGC showed large numbers of nuclei within cells shown by
arrowheads. MGC and nuclei were counted using absolute num-
bers in brain sections at days 7 and 14. The numbers of MGC were
11.9 ? 2.7 and 7.2 ? 4.08 (counts per section) on day 7 and 5.1 ?
1.6 and 1.7 ? 1.2 (counts per section) on day 14 in untreated and
IDV-NP-BMM-treated HIVE mice, respectively. Mean numbers
of nuclei within MGC were 13.8 ? 2.8 and 7.1 ? 2.0 on day 7 and
7.4 ? 0.7 and 4.9 ? 0.6 on day 14 in untreated and IDV-NP-
BMM-treated HIVE mice, respectively. Significantly decreased
numbers of MGC (both p ? 0.01) and nuclei (p ? 0.05 and p ?
0.01) on days 7 and 14 were observed following IDV-NP-BMM
treatment. Fig. 6A also demonstrated changes in the MDM phe-
notype (Vim?). A stalk containing nucleus became elongated in
untreated HIVE mice (Fig. 6A). IDV-NP-BMM treatment limited
MGC formation. Based on a significantly reduced HIV-1p24 ex-
pression in the treated group, we determined that MGC formation
was linked to HIV-1 infection. The levels of MGC was signifi-
cantly decreased when assayed by ratios of MGC:total Vim?
MDM (Fig. 6C) in IDV-NP-BMM-treated HIVE brains.
?/?mice which provided long-term en-
Preliminary toxicology studies
To investigate the potential toxicity of IDV-NP-BMM at the
delivery site and more extensively throughout the brain, his-
topathological analysis of brain sections was used to examine
neuronal integrity in SCID mice injected in the brain with hu-
man MDM (supplemental Fig. 2C) or HIV-1-infected MDM.
Sham-operated animals served as controls. Neuronal injury in-
duced as a consequence of viral infection and/or inflammation
caused by xenogenic MDM was limited (supplemental Fig. 2).
Three groups of mice (sham control, MDM, and HIVE) were
used to determine whether any neurotoxicity was induced by
the nanoformulations themselves. MDM are known to induce
inflammatory responses and are capable of promoting BMM
migration into the brain. Immunostaining for NF was performed
in brain sections to identify neuronal loss. Confocal microscopy
sis assayed the distribution of rDHPE-IDV-NP-BMM and CD68?cells
(A). The levels of rDHPE-IDV-NP-BMM (?) were significantly increased
in HIVE compared with both sham-operated (?) and MDM mice (#).
CD68?cells were assayed in the same brain slides by quantitative image
analysis of untreated (f) or treated (?) with rDHPE-IDV-NP-BMM (B).
Mean (?SEM) percent area distribution of rDHPE-IDV-NP-BMM was
determined for five mice per group. ?, p ? 0.05 compared with untreated.
IDV levels were assayed by HPLC from blood in HIVE mice treated with
IDV-NP-BMM at days 1, 3, 7, and 14 (C and D). With a single dosage of
treatment, IDV levels were testable by HPLC until reaching day 14. Blood
IDV levels were obtained in treated HIVE mice at experimental time points
after i.v. injection via the tail vein. Mean (?SEM) ?g of IDV per ml of
blood was determined for five mice per group. IDV were assayed by HPLC
from lysates of brain tissues (D). The diseased (ipsilateral) brain tissue
levels of IDV were analyzed and compared with the contralateral hemi-
sphere at the experimental time points. Mean (?SEM) ?g of IDV per 100
mg of brain tissue was determined for five mice per group. ?, p ? 0.01
compared with the contralateral hemisphere.
Brain tissue IDV concentration. Quantitative image analy-
665 The Journal of Immunology
images demonstrated IDV-NP-BMM (red) migration into areas
of MDM with or without HIV-1 infection (Figs. 3–7). HIV-1
infection and ongoing inflammatory responses are shown as
HIV-1p24 expressions and GFAP?astrogliosis and Iba-1 mi-
crogliosis (Figs. 3, 4, and 6) were revealed in response to the
needle track in sham-operated animals. Immunostaining for NF
(green, Fig. 7A) was performed in brain sections to identify
neuronal loss in HIVE-diseased areas with or without IDV-NP-
BMM treatment (Fig. 7A). Indeed, abnormal accumulation of
NF?neuron bodies was located in the diseased areas where
ongoing inflammatory and viral infection was occurring. Con-
focal microscopy images demonstrated IDV-NP-BMM (red)
migration into diseased areas with ongoing HIV-1 infection
(Fig. 7A) and/or inflammatory responses (supplemental Fig.
2C). Sham controls revealed few rDHPE-positive spots. NF
staining loss was seen in HIV-1- infected MDM-occupied sites
and surrounding areas. Indeed, abnormal accumulation of phos-
phorylated NF?(p-NF) in the neuron body was observed in
both MDM and HIVE mice with or without IDV-NP-BMM
treatment. NF expression (Fig. 7, A–C) and p-NF?neuron bod-
ies were evaluated. Image quantitation of neuronal damage
(NF?axons) and p-NF?neuron bodies demonstrates that NF
expression (Fig. 7B) was no different in the IDV-NP-BMM-
treated HIVE mice compared with the untreated group. The
of IDV-NP-BMM in HIVE mice. Ste-
reotactic injection of HIV-1-infected
MDM into the caudate/putamen of
SCID mice was used to induce HIVE
with resulting neuroinflammation, viral
infection, and neuronal injury. Immuno-
sections from HIVE mice (those ani-
mals injected with
MDM) with or without IDV-NP-BMM
treatment (designated as HIVE and
HIVE and IDV, respectively). Staining
for Vim, HIV-1p24, and GFAP (brown)
reflect the presence of human MDM, vi-
rus-infected cells, and neuroinflamma-
tory responses (A). Microscopic images
of human Vim?and HIV-1p24?MDM
and GFAP?astrocytes showing similar
human MDM reconstitution and astro-
gliosis were observed in the brains of all
groups. Original magnification, ?200.
Numbers of virus-infected MDM were
assayed by percentage of HIV-1p24?/
Vim?-stained cells (B). A significant
decrease in the numbers of virus-in-
fected cells was observed in IDV-NP-
BMM-treated HIVE mice. MGC, a
known pathological hallmark of HIVE,
were found in injection sites where
HIV-1p24?cells were seen (A and C).
duced size, were seen in antiretroviral-
treated HIVE mice. MGC formation
was determined by the number of MGC
per total number of Vim?MDM
(mean ? SEM) (C). IDV* represents
IDV-NP-BMM. ?, p ? 0.01 and ??,
p ? 0.01 compared with untreated
Table I. Antiretroviral activities of IDV-NP-BMMa
Day 7Day 14
HIVE and BMM-IDV-NP
368.88 ? 43.54
419.11 ? 45.64
342.26 ? 18.88
0 93.5 ? 12.24
103.17 ? 12.53
81.63 ? 19
128.5 ? 31.35
52.67 ? 20.12*
62 ? 5.02
22.88 ? 9.86*
aAbsolute numbers of Vim?and HIV-1p24?MDM were determined from serial immunohistological sections. These were
used to assess levels of HIV-1 infection. Means (?SEM) are absolute numbers of Vim?and HIV-1p24?MDM determined for
five animals per group. IDV-NP-BMM i.v. administration significantly reduced levels of HIV-1p24?MDM when compared to
untreated HIVE mice. Uninfected MDM injected into the subcortex of immunodeficient mice served as a control.
?, p ? 0.05 compared with IDV-NP-BMM-treated to untreated HIVE mice.
666NANOFORMULATED ANTIRETROVIRAL DRUG DELIVERY TO BRAIN
numbers of accumulated p-NF?neuron bodies along with
GFAP?and Iba-1?astrocytes and microglia showed (Fig. 7,
C–E) no changes among treated and untreated mice.
Invasion of HIV into the CNS occurs early after viral exposure and
during the development of a seroconversion reaction (37, 38). Dis-
ease, however, occurs years later as a consequence of chronic viral
infection of brain MP including blood-borne perivascular macro-
phages and microglia, culminating in neuronal injury and death
(39–43). Interestingly, these same MP cells carry the virus from
the periphery into the brain and serve as sources of neuroinflam-
matory mediators. Such an inflammatory response generates che-
mokine gradients, encouraging additional monocyte-macrophages
to enter the brain as well as providing a rich source of neurotoxins
(42–46). Cognitive, motor, and behavioral abnormalities occur as
a consequence of such pathogenic events and are fueled by con-
tinuous viral growth in the face of damaged or lost adaptive im-
munity (47–49). We reasoned that improving brain penetration of
ART would affect the tempo and progression of disease by con-
trolling viral growth. To accomplish this, we took advantage of the
ingress of monocytes-macrophages from the blood to the brain
operative in disease. Such cell ingress correlates with disease se-
verity (50–52) and could be harnessed for therapeutic gain. By
using BMM as ART carriers, the actual entry of disease-causing
cells could be used to improve disease outcomes. BMM loaded
with IDV-NP readily penetrate the BBB, enter brain subregions,
and migrate to disease sites of continuous viral replication and
neuroinflammation. These results provide further validation for the
use of macrophage-drug delivery systems to combat HIV infection
(5, 27, 53, 54).
ART can restore cognitive function while limiting neural dam-
age in HIV-1-infected individuals (11, 55, 56). Indeed, the HIV
load present in cerebrospinal fluid (CSF) and the number of im-
mune-competent macrophages correlate with the degree of cogni-
tive deficits and most notably, the numbers of CD4?T lympho-
cytes (57–61). This supports the idea that sustained penetration of
ART across the BBB improves clinical neurological outcomes (41,
62). Indeed, ART can prolong life expectancy and restore immune
activities, resulting in improved surveillance of virus and reduc-
tions of opportunistic infections and primary CNS lymphomas
(63–66). In contrast, ineffective use of ART or its reduced brain
penetration could contribute to viral mutation and sustained HIV
replication within the brain sanctuary (67). Significant evidence
shows that viral resistance patterns within the CSF compartment
are distinct from that found in plasma (68, 69). Moreover, viro-
logical CSF suppression is associated with ART brain penetrance
(70). Nonetheless, the BBB limits the numbers of drugs that
readily enter the CNS. Therefore, drugs that enter the CNS and
suppress ongoing viral replication are believed to provide the best
clinical outcomes. These observations, taken together, indicated
that the development of a novel antiretroviral drug delivery system
to improve the CNS penetration and ART efficacy is important.
Our laboratory and those of others developed macrophage-based
nanoformulations to treat neuroAIDS and other neurodegenerative
diseases (5, 27, 53, 54, 71). Such macrophage drug carriage was
shown to enhance local drug concentration, elicit limited systemic
side effects, and affect ART efficacy in rodent models of HIV in-
fection (5). Our previous works with the HIV-1 protease inhibitor
IDV showed that IDV-NP carried in BMM could positively affect
pharmacokinetic drug delivery and improve tissue distribution in
laboratory and animal models of HIV disease (5, 27). The current
results extend these observations significantly by demonstrating
the biodistribution and antiretroviral activity of IDV-NP-BMM
within CNS tissue compartments exhibiting active HIV-1-induced
disease. Levels of IDV in HIV-1-infected brain areas were signif-
icantly increased and extended to 14 days with a single dosage of
IDV-NP-BMM treatment in comparison to i.v. administered IDV.
Compared with control hemispheres, a significantly high level of
IDV was obtained in diseased hemispheres on day 14.
Nanotechnology has revolutionized modern-day pharmacology
(72–76). The ability to alter carrier size, shape, and composition
nal immunostaining for NF, which included both the NF and p-NF forms,
was performed in brain tissue sections of SCID mice 3 days after a single
i.v. injection of rDHPE-IDV-NP-BMM (A). SCID mice were intracranially
injected with saline, MDM, or HIV-1- infected MDM. Serial 25-?m frozen
brain tissue sections were stained with Abs to NF (green). Spatial relation-
ships between NF?axon loss and p-NF neuronal body accumulation in
viral infection were determined by confocal image analysis. The local
rDHPE-IDV-NP-BMM (red) distribution showed no changes in NF?axon
loss and p-NF accumulation compared with three groups of animals that
did not receive treatment. Original magnification, ?200. Quantitative im-
age analyses was used to assess immunostaining of NF (B), p-NF (C),
GFAP (D), and Iba-1 (E). Absolute number of p-NF?neuronal bodies was
counted in HIVE mice with or without IDV-NP-BMM treatment. These
results showed that there was no relationship between IDV-NP-BMM lev-
els to either neuronal injury or neuroinflammatory responses. IDV* repre-
IDV-NP-BMM does not affect neural morphology. Neuro-
667 The Journal of Immunology
allows incorporation of drugs with a broad range of physical and
biochemical properties (77). Nanoformulations have a number of
advantages over conventional oral or i.v. drug systems in their
capacity to increase systemic bioavailability and solubility and to
slow drug degradation. Our macrophage-based system expands
these observations even further in a number of divergent ways.
First, monocytes-macrophages can carry drugs across the BBB to
target disease areas and improve local drug distribution. Second,
the macrophage delivery system relies on natural pathogenic pro-
cesses elicited during inflammatory responses. These responses
serve to target disease sites of active HIV-1 replication. In this
way, there is a natural control for drug penetration that is based on
disease severity. Third, monthly dosing positively affects thera-
peutic outcomes by prolonging the presence of local drug and, in
so doing, reducing opportunities for viral mutation and disease (5).
Macrophages have received significant attention for their role as
drug carriers (78, 79). However, relatively few in vivo studies have
assessed the ability of the macrophage-drug delivery system to
target migration to disease sites. We developed a novel method
using macrophages for delivery of IDV-NP across the BBB to
improve antiviral efficacy and enhance brain drug distribution. The
advantages of BMM as a carrier of NP for antiretroviral drugs
include an effective and systemic delivery system in vivo to track
cell migration and to use therapeutic activities. The significance of
this work is reflected by its interdisciplinary approaches to strat-
egizing crossing of the BBB, targeting migration, improving brain
drug levels, and assessing antiretroviral responses. Based on the
numbers of blood-borne macrophages that have entered affected
brain regions and taking into account that ?98% of the cells carry
drugs (5), the IDV levels observed in brain were lower than would
be expected. Although measures of the drug in wedge brain dis-
sections provide proof-of-concept, absolute drug levels are diluted
by the necessary inclusion of surrounding unaffected tissues in
drug analysis. Thus, the precise amount of drug delivered into
areas of active disease will require microdissection of encephalitic
brain subregions. This remains a major and ongoing focus of our
own research efforts. Improvements of CNS drug penetration, tar-
geted delivery, single dosage administration, economy, sustained
release, and drug bioavailability can assuredly make nanoART at-
tractive for human use. This study is certainly important because it
represents a new direction for effective treatment of one of the
most debilitating complications of HIV-1 infection, namely, cog-
We gratefully thank Robin Taylor and Lana Reinhardt for critical read-
ing of this manuscript and outstanding computer support and Michael
T. Jacobsen and Janice Taylor, who provided confocal microscopic assistance.
Baxter Healthcare employees are coauthors on this manuscript.
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669The Journal of Immunology