Macrophages sequester clofazimine in an intracellular liquid crystal-like supramolecular organization.
ABSTRACT Clofazimine is a poorly-soluble but orally-bioavailable small molecule drug that massively accumulates in macrophages when administered over prolonged periods of time. To determine whether crystal-like drug inclusions (CLDIs) that form in subcellular spaces correspond to pure clofazimine crystals, macrophages of clofazimine-fed mice were elicited with an intraperitoneal thioglycollate injection. Inside these cells, CLDIs appeared uniform in size and shape, but were sensitive to illumination. Once removed from cells, CLDIs were unstable. Unlike pure clofazimine crystals, isolated CLDIs placed in distilled water burst into small birefringent globules, which aggregated into larger clusters. Also unlike pure clofazimine crystals, CLDIs fragmented when heated, and disintegrated in alkaline media. In contrast to all other organelles, CLDIs were relatively resistant to sonication and trypsin digestion, which facilitated their biochemical isolation. The powder x-ray diffraction pattern obtained from isolated CLDIs was consistent with the diffraction pattern of liquid crystals and inconsistent with the expected molecular diffraction pattern of solid, three dimensional crystals. Observed with the transmission electron microscope (TEM), CLDIs were bounded by an atypical double-layered membrane, approximately 20 nanometers thick. CLDIs were polymorphic, but generally exhibited an internal multilayered organization, comprised of stacks of membranes 5 to 15 nanometers thick. Deep-etch, freeze-fracture electron microscopy of unfixed snap-frozen tissue samples confirmed this supramolecular organization. These results suggest that clofazimine accumulates in macrophages by forming a membrane-bound, multilayered, liquid crystal-like, semi-synthetic cytoplasmic structure.
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ABSTRACT: The present study aims at elucidating the intricate nature of the drug release and absorption following intramuscular (i.m.) injection of sustained-release prodrug nanocrystals/microcrystals. A paliperidone palmitate (PPP) long-acting suspension was characterized with regard to particle size (Dv,50 = 1.09 μm) and morphology prior to i.m. injection in rats. The local disposition was rigorously investigated by means of (immuno)histochemistry and transmission electron microscopy while the concurrent multiphasic pharmacokinetics was linked to the microanatomy. A transient (24 h) trauma-induced inflammation promptly evolved into a subclinical but chronic granulomatous inflammatory reaction initiated by the presence of solid material. The dense inflammatory envelope (CD68+ macrophages) led to particle agglomeration with subsequent drop in dissolution rate beyond 24 h postinjection. This was associated with a decrease in apparent paliperidone (PP) absorption (near-zero order) until 96 h and a delayed time of occurrence of observed maximum drug plasma concentration (168 h). The infiltrating macrophages phagocytosed large fractions of the depot, thereby influencing the (pro)drug release. Radial angiogenesis (CD31+) was observed throughout the inflammatory rim from 72 h onwards and presumably contributed to the sustained systemic PP concentrations by maintaining a sufficient absorptive capacity. No solid-state transitions of the retrieved formulation were recorded with X-ray diffraction analysis. In summary, the initial formulation-driven prodrug (PPP) dissolution and drug (PP) absorption were followed by a complex phase determined by the relative contribution of formulation factors and dynamic physiological variables. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm SciJournal of Pharmaceutical Sciences 05/2014; 103(7). · 3.13 Impact Factor
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ABSTRACT: Uptake of dyes into living cells and organisms is of concern to several diverse groups of people. These include those not wishing dyes to enter cells (e.g. manufacturers and users of textile dyes, or laboratory workers using dyes as analytical reagents) and those requiring dye entry (e.g. biologists imaging cell contents, or clinicians using photoactive dyes as antitumour drugs). This diversity results in the need to consider an extremely wide range of dyes – and indeed of cells and organisms. An overview of methods for predicting uptake and intracellular localisation is provided, followed by a more detailed account of the concepts and procedures involved in decision-rule quantitative structure–activity relationship (QSAR) models. Some of these models permit the prediction of which dyes are likely to enter cells, and which dyes will be excluded. Other models predict where internalised dyes will localise within the live cells. Use of QSAR models to understand intracellular accumulation, redistribution, loss from the cell, and metabolic modification of dyes is also considered. In particular, the relationship of such predictions to toxicity is discussed. An extended case example is provided, describing the modelling of dye binding to nucleic acids in single-cell systems. A further case example then illustrates dye localisation in multicellular organisms. Finally, conclusions, critiques, and probable future directions concerning the QSAR modelling approach to dye uptake and localisation are given. A summary of key QSAR decision rules in the form of decision logic tabulations is provided.Coloration Technology 04/2014; · 1.17 Impact Factor
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ABSTRACT: New drugs and drugs with a novel mechanism of action are desperately needed to shorten the duration of tuberculosis treatment, to prevent the emergence of drug resistance, and to treat multiple drug resistant strains of Mycobacterium tuberculosis. Recently, there has been renewed interest in clofazimine (CFZ). In this study, we utilized the C3HeB/FeJ mouse model possessing highly organized, hypoxic pulmonary granulomas with caseous necrosis to evaluate CFZ monotherapy in comparison to BALB/c mice which only form multifocal, coalescing cellular aggregates devoid of caseous necrosis. While CFZ treatment was highly effective in BALB/c mice, its activity was attenuated in the lungs of C3HeB/FeJ mice. This lack of efficacy was directly related to the pathological progression of disease in these mice, as administration of CFZ prior to the formation of hypoxic, necrotic granulomas reconstituted bactericidal activity in this mouse strain. These results support the continued use of mouse models of tuberculosis infection which exhibit a granulomatous response in the lungs that more closely resembles the pathology found in human disease.Antimicrobial Agents and Chemotherapy 05/2014; · 4.57 Impact Factor
Macrophages Sequester Clofazimine in an Intracellular
Liquid Crystal-Like Supramolecular Organization
Jason Baik, Gus R. Rosania*
Department of Pharmaceutical Sciences, University of Michigan College of Pharmacy, Ann Arbor, Michigan, United States of America
Clofazimine is a poorly-soluble but orally-bioavailable small molecule drug that massively accumulates in macrophages
when administered over prolonged periods of time. To determine whether crystal-like drug inclusions (CLDIs) that form in
subcellular spaces correspond to pure clofazimine crystals, macrophages of clofazimine-fed mice were elicited with an
intraperitoneal thioglycollate injection. Inside these cells, CLDIs appeared uniform in size and shape, but were sensitive to
illumination. Once removed from cells, CLDIs were unstable. Unlike pure clofazimine crystals, isolated CLDIs placed in
distilled water burst into small birefringent globules, which aggregated into larger clusters. Also unlike pure clofazimine
crystals, CLDIs fragmented when heated, and disintegrated in alkaline media. In contrast to all other organelles, CLDIs were
relatively resistant to sonication and trypsin digestion, which facilitated their biochemical isolation. The powder x-ray
diffraction pattern obtained from isolated CLDIs was consistent with the diffraction pattern of liquid crystals and
inconsistent with the expected molecular diffraction pattern of solid, three dimensional crystals. Observed with the
transmission electron microscope (TEM), CLDIs were bounded by an atypical double-layered membrane, approximately
20 nanometers thick. CLDIs were polymorphic, but generally exhibited an internal multilayered organization, comprised of
stacks of membranes 5 to 15 nanometers thick. Deep-etch, freeze-fracture electron microscopy of unfixed snap-frozen
tissue samples confirmed this supramolecular organization. These results suggest that clofazimine accumulates in
macrophages by forming a membrane-bound, multilayered, liquid crystal-like, semi-synthetic cytoplasmic structure.
Citation: Baik J, Rosania GR (2012) Macrophages Sequester Clofazimine in an Intracellular Liquid Crystal-Like Supramolecular Organization. PLoS ONE 7(10):
Editor: Arto Urtti, University of Helsinki, Finland
Received March 19, 2012; Accepted September 17, 2012; Published October 11, 2012
Copyright: ? 2012 Baik, Rosania. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health grants GM007767 (to J.B.), and RO1GM078200 (to G.R.R.). The contents are sole responsibilities
of authors, and do not represent the official views of the NIH. J.B. was also supported by American Foundation for Pharmaceutical Education. The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Clofazimine is an antibiotic and anti-inflammatory drug that is
very poorly soluble yet orally bioavailable [1,2,3,4]. It is clinically
approved to treat leprosy and skin inflammation associated with
Mycobacterium leprae infection [2,4,5]. Clofazimine possesses three
ionizable amine groups that become protonated and charged at
acidic pH. It is a highly hydrophobic molecule, with a logP .7.
Thus, clofazimine’s solubility increases in acidic environments, but
it is virtually insoluble in aqueous media at neutral or alkaline pH
. Therefore, in the gastrointestinal tract, clofazimine could form
supersaturated solutions as it passes from the acidic pH of the
stomach to the more alkaline pH of the intestine. Clofazimine
possesses a large volume of distribution and its elimination half-life
is more than 70 days [1,2,3,4]. However, the drug’s biodistribu-
tion pathways are not known. Clofazimine could bind to proteins
and form complexes with intracellular membranes . It could
also precipitate out as particulate aggregates or crystals that may
be actively phagocytosed by cells of the mononuclear phagocyte
Discovered in the 1950s, clofazimine is active against drug
resistant strains of mycobacteria and possesses a unique spectrum
of anti-inflammatory activities. It remains clinically useful to this
day. However, clofazimine accumulates to very high levels in
tissues [4,7,8,9] resulting in visible changes in the pigmentation of
skin and other organs. In patients, clofazimine has been reported
to form crystal-like drug inclusions (CLDIs) in macrophages
[4,10,11]. Clofazimine bioaccumulation is associated with various
other side effects, but the drug is well-tolerated and side effects
disappear upon discontinuation of treatment [10,12,13,14,15].
Because of its complex pharmacokinetics, clofazimine has been
relegated to a category 5 agent.
Since the number of mycobacterial infections resistant to first
line antibiotic therapy has been increasing, there is renewed
interest in developing a new generation of clofazimine derivatives
active against drug resistant mycobacterial strains, but with
a decreased propensity to bioaccumulate. Well-informed mod-
ifications of the chemical structure of clofazimine could provide
a good starting point for the development of a second generation
of improved clofazimine derivatives . This is particularly
timely and important since phenazines are highly effective against
multidrug resistant mycobacteria  that are responsible for drug
resistant tuberculosis and leprosy epidemics which are spreading in
Africa and Asia [3,18,19].
CLDIs found in vivo have been generally assumed to correspond
to solid clofazimine crystals similar to the crystals that precipitate
out in pure clofazimine solutions. However, CLDIs found in
biological samples are too small for single crystal X-ray diffraction
structural studies. Therefore, we decided to directly probe the
physical and chemical properties of isolated CLDIs, using
PLOS ONE | www.plosone.org1 October 2012 | Volume 7 | Issue 10 | e47494
a materials science inspired approach in combination with various
microscopic imaging techniques.
1. Long term clofazimine bioaccumulation and retention
For long term, continuous administration, mice were fed with
powdered rodent feed supplemented with clofazimine, ad libitum
[4,9,20,21]. This resulted in an approximate daily intake of
20 mg/kg, well below clofazimine’s LD50 and comparable to the
human daily dose of 4.3 mg/kg . As in humans , mice skin
gradually turned red after three weeks (Fig. 1A). Otherwise, the
treated animals gained weight and behaved similarly to their
untreated counterparts (Fig. 1B). From 3 to 8 weeks of dietary
supplementation, continuous, non-steady state accumulation
occurred in spleen, liver and lung; in other organs, like the
kidney, accumulation was minimal (Fig. 1C). In spleen, the mass of
clofazimine almost reached 1% of the wet organ weight (Fig. 1C).
After discontinuation of supplementation, mice skin gradually
returned to a paler color of untreated skin, over a period of two
months. However, clofazimine concentration only decreased by
76% and 73% in liver and lung, while changes in concentration in
the spleen were statistically insignificant even after two months of
clofazimine withdrawal. This confirmed a specific clofazimine
sequestration and retention mechanism in this organ (Fig. 1C).
Consistent with previous studies [3,20], LC/MS analysis of
spleen, liver and plasma samples revealed a single drug-associated
peak with a molecular weight matching clofazimine’s, indicating
clofazimine was present in tissues in metabolically intact form
(data not shown). Examination of unfixed, unstained frozen tissue
sections by transmitted light microscopy revealed dark, ruby red
CLDIs present in all organs exhibiting continuous accumulation
and retention of the drug (Fig. 1D). CLDIs were most numerous in
the spleen and lymph nodes, followed by the liver, small intestine
and lungs. In treated mice, splenomegaly (mass increase by .3.4
fold (N=5), P,0.01; t-test) and swollen mesenteric lymph nodes
were apparent . At 3 week treatment, the average size of the
inclusions was 2.260.78 (SD) mm in width and 3.962.6 mm in
length. After continued feeding until 8 week, the individual
inclusions were only slightly more elongated to 6.063.6 mm
(P,0.01) but increased in numbers. Thus, the growth of drug
inclusions appeared to be constrained by the size of the cells. After
clofazimine diet was switched to a clofazimine-free, regular diet for
two months to let drugs be washout out (Fig. 1C, wash), CLDIs
were still present only in those organs that retained clofazimine.
2. CLDIs are exclusively present inside macrophage-like
CLDIs appeared exclusively inside macrophage-like cells
(Fig. 1E), consistent with prior reports [4,11,20]. In liver,
hepatocytes (Fig. 1E, H) clearly lacked CLDIs, whereas macro-
phages (Fig. 1E, M) with CLDIs formed small clusters resembling
microgranulomas, which are normally formed by liver macro-
phages under stress . This was confirmed by immunohisto-
chemical analysis (Fig. 1F). These clusters were adjacent to blood
vessels (Fig. 1F, V) and, most cells in these clusters expressed the
F4/80 antigen (macrophage marker; Fig. 1F) which also labeled
the Kupffer cells (Fig. 1F, K). The cell clusters were embedded in
extracellular matrix (Masson’s trichrome staining, MTS; Fig. 1F)
and they did not stain with von Willebrand factor (endothelial cell
marker, vWF; Fig. 1F) or smooth muscle actin (smooth muscle
marker, aSMA; Fig. 1F).
To determine if CLDIs were associated with viable, chemotac-
tic, adherent cells, bone marrow macrophages (BMM) were
isolated from femurs and peritoneal macrophages (PM) were
elicited by intraperitoneal injection of 4% thioglycollate 
(Fig. 2). BMM and PM cells containing CLDIs were able to attach
and spread on tissue culture plastic (Fig. 2A). The size and shape of
the CLDIs of the elicited macrophages resembled those observed
in tissue cryosections (Fig. 1). PMs containing CLDIs were viable
and motile, and migrated out from clusters onto the tissue culture
plastic (Fig. 2B). In these cells, CLDIs maintained their shape and
size without any disintegration, degradation or dissolution. In-
terestingly, with peritoneal macrophages isolated in vitro, illumi-
nating CLDIs with 490 nm light induced release of clofazimine
which could be detected using the standard TRITC filter set of an
epifluorescence microscope (Fig. 2C). Soon after illumination,
clofazimine’s fluorescence filled the cell. Upon continued illumi-
nation, clofazimine’s fluorescence visibly diffused to the neighbor-
From the spleen, CLDIs could be removed from the cells by
forcefully grinding the tissue homogenate and passing the
homogenate through a cell strainer. Initially, CLDIs appeared
morphologically homogeneous. However, CLDIs gradually trans-
formed into irregular, rhomboidal shapes resembling typical, pure
clofazimine crystals, which grew larger than cells in size (from
9.763.1 (SD) mm on day 0, to 1564.7 mm on day 7; Fig. 2D).
Similar results were obtained with bone marrow macrophages
which exhibited varying number of intracellular CLDIs that
appeared as rigid, prism-like structures after cells were rounded
and detached from the plastic (Fig. 2E).
3. CLDIs possess physicochemical properties different
from those of pure clofazimine crystals
We proceeded to compare some of the physical and chemical
properties of isolated CLDIs directly with those of pure
clofazimine crystals [7,10,14,15,25]. Clofazimine crystals were
irregular in shape and size (Fig. 3A). The pure clofazimine crystal
powder obtained from the manufacturer fluoresced in the
standard, green (eGFP) channel and in the red (Cy3) channel of
the epifluorescence microscope. Their fluorescence or morphology
did not change when they were placed in water and heated to
100uC, placed in 1N NaOH or trypsinized (the melting point of
pure clofazimine crystals is 212uC).
For comparison, CLDIs were isolated by mincing and
disaggregating spleens with 0.125% trypsin-EDTA, ultrasonicat-
ing them for 30 minutes after passing them through a cell strainer.
CLDIs were the only microscopic structure visibly remaining in
the filtrates, and could be concentrated by centrifugation. Isolated
CLDIs in the pellets could be resuspended in 10% sucrose in
water, and appeared stable when examined within a couple of
hours after isolation. They were dark, ruby red in color, and
prism-like in appearance when viewed with transmitted light.
They were birefringent when viewed with polarized light. Unlike
pure clofazimine crystals, the isolated CLDIs were homogenous in
shape and size, and polarized light as a single domain. They
appeared bright red and monolithic when viewed using cross-
polarizers, in contrast to the heterogeneous, yellow-orange
fragmented birefringence of pure clofazimine crystals. Unlike
pure clofazimine crystals, isolated CLDIs did not fluoresce in the
green eGFP channel, yet they were brightly fluorescent in the red
Cy3 channel (Fig. 3B).
Unlike clofazimine crystals, isolated CLDIs were highly re-
sponsive to changes in the environment. Upon exposure to
distilled water (Fig. 3C), they burst into smaller birefringent
globules which aggregated into large masses and became
Intracellular Clofazimine Sequestration
PLOS ONE | www.plosone.org2October 2012 | Volume 7 | Issue 10 | e47494
fluorescent in the green eGFP channel. Upon treatment with 1N
NaOH (Fig. 3D), they underwent localized changes in structure:
parts of the elongated CLDIs fragmented and transformed to the
green eGFP fluorescent form. Other CLDIs treated in this manner
became fluorescent at the tips (arrows). Isolated CLDIs in
suspension disintegrated when heated to 100uC for 15 minutes
(Fig. 3E). When heated, they turned yellow, became fluorescent in
the green eGFP channel, burst into small globules that remained
attached without aggregating to each other. Morphologically,
CLDIs appeared relatively resistant to 0.125% trypsin-EDTA
treatment for 1 hour (Fig. 3F).
To determine whether CLDIs possessed subnanometer molec-
ular features associated with the three-dimensional lattice structure
of solid clofazimine crystals, powder X-ray diffraction analysis was
performed on isolated CLDI samples. Based on the powder X-ray
diffraction pattern of isolated CLDIs (Fig. 3G), a single peak was
observed at a small diffraction angle. The absence of other
diffraction peaks at higher angles was noteworthy, as those peaks
correspond to the subnanometer features of the three dimensional
lattice structure of pure, solid clofazimine crystals .
4. Electron microscopy reveals the internal organization
In transmission electron microscope images, CLDIs generally
appeared as empty, featureless polyhedral cavities. Superficially,
the outline of these cavities resembled the faceted outline of pure
Figure 1. Clofazimine inclusions formed in macrophage-like cells in vivo. (A) Mice fed with clofazimine (above) showed reddish
pigmentation visible in the ear, tail, and skin when compared to mice treated with vehicle only (below). (B) Weight gains in mice fed with and
without clofazimine were comparable (N=40, &, vehicle; #, treated; *, P,0.01, end-point T-test). (C) Biochemical analysis of various organs revealed
differences in the accumulation and retention of clofazimine after wash out (*, P,0.01, N=5 per group, ANOVA). (D) Ruby red inclusions appeared in
frozen sections of spleen, lung and liver, but not in kidneys of 8 wk supplemented diet. H, hepatocyte; V, blood vessel; M, microgranulomas. (E)
Intracellular inclusions were extracted in perfusion-fixed liver upon ethanol-dehydration and staining with toluidine blue. Arrows indicate needle-like
cavities remaining after extraction. (F) Histological sections revealed cellular changes in liver of mice fed with clofazimine. H&E staining, F4/80
macrophage specific marker, Masson’s trichrome staining (MTS, collagen fibers), von Willebrand factor (vWF, endothelium) and alpha smooth muscle
actin (aSMA). K, Kupffer cells. Scale bar =10 mm unless otherwise indicated.
Intracellular Clofazimine Sequestration
PLOS ONE | www.plosone.org3 October 2012 | Volume 7 | Issue 10 | e47494
crystals, as has been previously reported [4,11]. Nevertheless,
some CLDIs observed in our samples were filled with osmiophilic
material (Fig. 4A). These osmiophilic bodies of filled CLDIs were
also elongated and polyhedral in shape, and bounded by a double
membrane (Fig. 4B). In some cells, other atypical transitional
organelles were observed. These putative, transitional organelles
appeared as heterogeneous granular or multivesicular bodies
deformed by an internal, elongated CLDI-like structure, or
multilamellar bodies that appeared to be in the process of
transforming or fusing with granular or multivesicular bodies
At higher magnification, visual inspection of these CLDIs
revealed morphological details in the 1 to 20 nanometer scale. As
seen in medial cross-sections cut along the long axis of the object
(Fig. 4D and 4E), the filled CLDI contained a multilamellar core
comprised of planar sheets separated by parallel array of clear
‘‘channels’’. These channels were spaced 18 nm apart, and aligned
parallel to the long axis of the structure (Fig. 4F and 4G). In other
regions, the CLDI appeared as a lattice with periodically-spaced
elements repeating every 10 or 18 nm (Fig. 4F).
Transversal cross-sections cut perpendicularly to the long axis
(Fig. 4H) revealed a multilamellar core comprised of planar stack
of trilayer membranes of 10 nm thickness (Fig. 4I). The entire
structure was bounded by an outermost double membrane, about
20 nm thick (Fig. 4H). The core was surrounded by concentric
lamellae of cortical, trilayer membranes which were observed in all
the CLDIs and the transitional structures (Fig. 4C). The trilayer
membrane consisted of a thick, dark osmiophilic band at the
center (Fig. 4J, arrows) flanked by two thinner osmiophilic bands
on either side (Fig. 4J, triangles). A clear, 5 nm layer separated the
central band and the flanking bands (Fig. 4K). These trilayer
membranes sometimes merged with membrane-free regions
(Fig. 4H, *).
5. Deep-etch freeze-fracture electron microscopy on
Remarkably, this intracellular multilayered structure appeared
different from all other multilamellar organelles previously
reported inside cells . Thus, we also considered the possibility
that the observed morphological features could be an artifact of
the transmission electron microscopy sample preparation tech-
nique. To study the morphology of CLDIs as closely to their native
state as possible, we turned to a completely different sample
preparation technique: deep-etch freeze-fracture electron micros-
copy . This technique creates a platinum replica of a snap-
frozen tissue sample after surface layers of frozen water molecules
Figure 2. Macrophages containing intracellular CLDIs were collected, plated and studied in vitro. (A) Bone marrow macrophage (BMM)
and thioglycollate elicited peritoneal macrophages (PM) were obtained from mice fed with clofazimine, attached and spread on tissue culture plastic,
and were stained with Hoechst 33342 to show nuclei. (B) Peritoneal macrophages with CLDIs migrated away from large clusters when plated on
tissue culture dishes. (C) Illuminating peritoneal macrophages with blue (490 nm) light triggers clofazimine release (observed in TRITC channel) from
CLDIs. (D) Once removed from cells, extracellular CLDIs grew in size and became irregular in morphology, unlike intracellular CLDIs. Red blood cells
(d=8 mm) in the background serve as size markers, for reference. (E) CLDIs inside bone marrow-derived cells in suspension, stained with Trypan Blue.
Scale bars =10 mm unless otherwise indicated.
Intracellular Clofazimine Sequestration
PLOS ONE | www.plosone.org4 October 2012 | Volume 7 | Issue 10 | e47494
are sublimated in vacuum. By eliminating the fixation, de-
hydration and polymerization steps used in transmitted electron
microscopy, the deep-etch freeze-fracture technique preserves the
topography of cellular membranes with high fidelity. Remarkably,
in the sample of unfixed liver of 6 wk treated mouse, CLDIs
appeared prominently and clearly stood out from the rest of the
cytoplasm (Fig. 5). Their size, shape, location and general
morphology was consistent with the results obtained with TEM
studies. Viewed from the outside, CLDIs appeared surrounded by
cellular membrane with protein-like structural features similar to
those present in the other cellular membranes in the cytoplasm of
the cell (white triangle, Fig. 5).
Inside the CLDI, deep-etch freeze-fracture EM images revealed
evidence of a multilayered structure at the core surrounded by
a double membrane (Fig. 6A). Zooming into the core (Fig. 6B), the
multilayered structure appeared stacked in the z direction,
perpendicular to the long axis of the CLDI. No lateral
organization was visible in the xy plane. The lamellar spacing
and orientation of the layered planes was not always regular due to
uneven fracture, and ranged from 6 nm to 14 nm at different
points inside the structure (Fig. 6C). Consistent with a liquid
crystal-like structure, there was no obvious lateral organization
along the planar surface of each lamellae (Fig. 6D). We also noted
that the core of the structure did not seem to possess protein-like
globular features (Fig. 6D). Protein-like globular features were only
observed in the outer or inner face of the outer membrane
covering the entire structure (Fig. 6E, arrowheads). As in TEM
images (Fig. 4B) the entire structure was surrounded by a double
membrane (Fig. 6F). However the intermembrane space was in the
range of 20 to 30 nanometers thick, with ‘‘pillars’’ bridging the
outer and inner membrane (Fig. 6F, arrows).
To confirm that the structures observed in tissue samples
corresponded to the biochemically isolated, purified CLDIs, we
performed deep-etch freeze-fracture electron microscopy on
isolated CLDI preparations (Fig. 7). In these preparations, CLDIs
were readily apparent based on their shape and multilayered
internal organization from cytosolic debris in the background
(Fig. 7A). Nevertheless, unlike CLDIs observed in tissue samples
(Fig. 5, 6), isolated CLDIs lacked the outer double membrane
(Fig. 7B, C). We also noticed that, unlike CLDIs from intact tissues
(Fig. 6), the internal layers of isolated CLDIs appeared to be
peeling off (Fig. 7C), suggesting a partial disassembly.
This is the first study to directly probe the physical and chemical
properties of CLDIs, and to directly reveal the morphology of
CLDIs formed in clofazimine-treated animals. Initially, we
considered the possibility that clofazimine accumulation in
macrophages would result from the phagocytosis of extracellular
clofazimine crystals by macrophages. However, extracellular
clofazimine crystals were not observed in vivo. Chemically and
physically, the intracellular CLDIs appeared very different from
chemically-pure clofazimine crystals: they were uniform in size
and shape, and were highly responsive to changes in medium
tonicity, pH, temperature and illumination. While CLDIs have
different birefringence pattern from pure crystals, this is a general
property shared by many different kinds of anisotropic supramo-
lecular organizations. Based on their stimulus-responsiveness,
morphological characteristics and powder X-ray diffraction
Figure 3. CLDIs exhibited different chemical and physical
properties from pure clofazimine crystals. Polarized light and
epifluorescence microscopy (using the eGFP or Cy3 fluorescence
channels) showed that pure clofazimine crystals (control, A) were
unchanged by different treatments. These crystals appeared birefrin-
gent and fluoresced in the standard eGFP and Cy3 channels of the
epifluorescence microscope. (B) Isolated CLDIs remained intact in
isotonic solution of 10% sucrose in water, did not fluoresce in the eGFP
channel but fluoresced in the Cy3 channel. (C) Isolated CLDIs burst and
aggregated in distilled water, and became fluorescent in the eGFP
channel. (D) After exposure to 1N NaOH, isolated CLDIs partially
disintegrated in different parts. Arrows point to the tips of a CLDI that
were fluorescent in the eGFP channel. (E) After 15 min at 100uC, CLDIs
fragmented and changed to a pale orange color. (F) CLDIs appeared to
remain partly intact when viewed after 30 min sonication and 1 hour
trypsin treatment. Scale bars =10 mm. (G) Powder X-ray diffractogram
for isolated CLDI and 8 wk treated mouse spleen homogenate showed
a single peak at 2-theta =7.2uControl spleen homogenate from vehicle-
only treated mouse did not show this peak. As a reference, pure, solid
clofazimine crystals (monoclinic and triclinic) showed many peaks at
higher angle indicative of a three dimensional, molecular lattice
Intracellular Clofazimine Sequestration
PLOS ONE | www.plosone.org5 October 2012 | Volume 7 | Issue 10 | e47494
Figure 4. TEM and deep-etch freeze fracture electron microscopy analysis of CLDIs. (A) Intact CLDIs were observed in the cells of the
lamina propria of 8.5 wk treated mouse jejunum using TEM. P, extracted polyhedral cavities. M, mitochondria. (B) CLDIs were delimited by a lipid
double layer. (C) CLDIs appeared to form from heterogeneous granular domains transforming into a lamellar structure, observed in 4.5 wk treated
jejunum (D). (E) Zoomed image of the CLDI from (A) showed the lattice-like lamellar structure. (F) and (G), Discrete Fourier Transforms confirmed the
regular, periodic structure of CLDIs. (H) Transversal cross section of CLDIs from 8.5 wk jejunum showed an internal organization of parallel bands and
some amorphous regions indicated by (*). (I) Zoomed image revealed the trilayer membrane of 10 nm in width separated by inter-laminar space
continuous with amorphous region. (J) Zoomed image of rectangle in (H), showing trilayer membrane structure comprised of a central dark band
(arrows) flanked by a pair of less prominent, dark bands (triangles) on either side, separated by clear 5 nm space. The trilayer membranes were
regularly spaced and formed planar stacks, with the central bands exhibiting the 18 nm spacing in the Discrete Fourier Transform (K).
Intracellular Clofazimine Sequestration
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pattern, CLDIs do not resemble the three dimensional molecular
arrangement of clofazimine molecules present in pure, solid,
clofazimine crystals . CLDIs are best described as a new kind
of semi-synthetic biomaterial: a membrane-bound, stimulus re-
sponsive, macrophage-dependent, liquid crystal-like, supramolec-
Interestingly, the intracellular location and growth of CLDIs
appears to be exclusively constrained to the cytoplasm of
macrophages. In the liver, CLDIs never appeared in association
with hepatocytes. Accordingly, we hypothesize that the cell type-
specific localization of CLDIs likely reflects the presence of an
active intracellular sequestration mechanism that is present in
macrophages and absent in other cells of the host. The presence of
CLDIs in thioglycollate-elicited macrophages confirmed that these
structures are found inside viable, functioning, chemotactic cells.
Once removed from these cells, CLDIs were unstable: they
transformed to irregular shapes and began to grow in size like
a typical clofazimine crystal. Nevertheless, inside the organism,
CLDIs remained uniform in shape and smaller than cells in size.
This suggests an active role of the macrophages in terms of
controlling the size and shape of the CLDIs.
CLDIs appeared different from all the other cellular organelles.
Mechanically, their core structure was rigid and resisted sonica-
tion. In addition, three different lines of evidence suggest that their
internal organization corresponded to that of a supramolecular
liquid crystal: 1) transmission electron microscopy of fixed and
stained samples revealed and highly organized multilamellar
structure; 2) powder X-ray diffraction patterns revealed a single
low angle diffraction peak consistent with a planar organization in
a single spatial dimensions and no evidence of higher angle
diffraction peaks that would be consistent with a three di-
mensional, pure crystal; 3) deep-etch freeze-fracture microscopy
revealed the presence of a 2D, layered structure, with no evidence
of lateral organization along the plane of each layer. The thickness
of the layers observed by TEM and freeze-fracture microscopy
were in the order of 5 to 15 nanometers, which is too large for the
expected subnanometer features of a pure clofazimine crystal .
Interestingly, in deep-etch freeze-fracture preparations, there were
no indications of protein-sized globular features present inside the
CLDIs. Instead, the scale of supramolecular features observed by
freeze-fracture microscopy, together with their response to
changes in temperature, osmolarity and pH is most analogous to
the supamolecular structure and phase transition behaviors of
liquid crystalline mesophases adopted by concentrated phospho-
lipids in aqueous media [29,30,31,32,33].
Based on the absence of acute toxicity in vivo, we propose that
the sequestration of clofazimine in CLDIs may primarily serve as
a defense mechanism. By sequestering clofazimine, CLDIs may
have a net cytoprotective effect, reducing the concentration of
soluble clofazimine molecules that would be toxic to the host. Like
clofazimine , other compounds that induce the formation of
autophagosome-like membrane complexes have also been found
to possess beneficial, cytoprotective effects [6,39,40]. When
assayed in vitro, clofazimine disrupts mitochondrial membrane
potential and inhibits the growth of cells in tissue culture [6,34]. In
solution, clofazimine can generate superoxide anions upon in-
teraction with isolated rat peritoneal macrophages  and
human neutrophils  which may be related to its in vitro
cytotoxic activity [34,37]. Superoxide production has been
proposed to account for clofazimine’s broad bactericidal activity
against many different microorganisms including Mycobacterium
tuberculosis , Staphylococcus aureus, and Escherichia coli .
However, when mice were treated with clofazimine, there were
no obvious toxicological manifestations. In humans, clofazimine is
well tolerated, with gastrointestinal disorders being the major
toxicological side effect manifested after long term treatment
[10,15,25]. Nevertheless, this side effect is reversible and subsides
after treatment is discontinued.
In relation to other chemotherapeutic agents in clinical use,
clofazimine has many unique pharmacokinetic properties. In
humans, clofazimine exhibits a very long half-life. Because of its
high logP, clofazimine would be expected to be distributed mostly
in association with body fat. Thus, the local accumulation of
clofazimine in tissue macrophages most likely reflects an active
transport mechanism that promotes the influx or retention of
clofazimine in these cells. Because macrophages are actively
involved in the body’s defense against bacterial infection, the
accumulation of clofazimine in macrophages could effectively
serve to mobilize clofazimine to its site of action. Therefore,
although excessive bioaccumulation in macrophages may be
related to some of the drug’s undesirable side effects, the
accumulation of clofazimine in macrophages could be therapeu-
tically advantageous. This observation has important implications
for the design of macrophage-targeted chemotherapeutic agents.
To the extent that CLDIs massively sequester clofazimine inside
macrophages, our results also suggest a potential role of the
immune system as a determinant of drug distribution and
disposition. The specific accumulation of clofazimine in some,
but not all macrophages suggests there may be a specialized
subpopulation of macrophages involved in xenobiotic sequestra-
tion. Interestingly, clofazimine possesses potent anti-inflammatory
activity in the clinic which makes it especially useful in the
treatment of erythema nodosum, a skin inflammation that
accompanies M. leprae infection. Thus, clofazimine’s bioaccumula-
tion in macrophages may also be associated with downstream
immunomodulatory activity. By monitoring changes in immune
system-related signaling molecules, it should be possible to
determine whether bioaccumulation of clofazimine in macro-
phages activates a natural anti-inflammatory pathway that may
serve to protect the host from bioaccumulation-related injury.
Figure 5. Deep-etch freeze-fracture microscopy of the in-
tracellular CLDI from 6 wk treated mouse liver. Note the outer
membrane of the CLDI is studded with globular protein-like features
(white triangle). The CLDI was broken during freeze-fracture, revealing
a lamellar internal structure at the top.
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Previously, many in vitro QSAR studies have been published
exploring the relationship between the chemical structure of
clofazimine and its antimycobacterial properties. Most of these
studies have focused on assaying the properties of phenazine
molecules in solution: For example, probing how the redox
properties of clofazimine depend on the type of alkylimino group
Figure 6. Outer membrane and internal organization of a CLDI from 6 wk treated mouse liver. (A) Zoomed out image, revealing the
exposed, inner multilamellar core with the outer double membrane layers peeled back towards the cytoplasm of the cell. The CLDI was broken open
during the sample preparation process. Regions of interest (corresponding to panels B to F) are marked with letters. (B) Zoomed image of the inner
lamellar surface. (C) Zoomed image of the multilamellar core region, showing variable spacings in the order of ranging from 6 to 14 nanometers. (D)
Zoomed image of the innermost face of the inner bounding double membrane without the globular, protein-like features. (E) Zoomed image of the
innermost face of the outer bounding double membrane. Protein-sized features are observed on the inner surface of the outer membrane
(arrowheads). (F) Zoomed image of the outer double membrane. Note the large space between the double membranes, with ‘‘pillars’’ bridging the
Intracellular Clofazimine Sequestration
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at position 2 of the phenazine ring structure [34,41]. Only
recently, structure-activity relationship studies have been aimed at
identifying phenazine compounds that inhibit the growth of
Mycobacterium tuberculosis while possessing reduced potential for
bioaccumulation . Interestingly, the lipophilicity of clofazimine
derivatives (clogP)  does not appear to correlate with their
serum half-life, suggesting that topological features may be as
important as physicochemical properties in terms of determining
clofazimine’s bioaccumulation and biodistribution. Using intracel-
lular crystal formation as an endpoint, we are currently performing
QSAR studies to elucidate how the physicochemical and
topological features of clofazimine impact its cellular pharmaco-
kinetics. By screening these compounds for activity against M.
tuberculosis, these QSAR studies should facilitate the design of new
phenazine derivatives with different tissue distribution and
bioaccumulation potential, and may help identify an improved
drug candidate with increased efficacy against M. tuberculosis.
To conclude, the results presented in this study constitute
evidence that macrophages sequester clofazimine by forming
a complex, multilayered supramolecular organization. This
organization bears many unique structural features that are unlike
those of natural organelles of eukaryotic cells and unlike those of
chemically-pure clofazimine crystals. The distinctive physical,
chemical and biological properties of CLDIs set them in a class of
their own. Based on the presence of organelles with transitional
morphologies (Fig. 4C), we propose CLDIs may be derived from
multilamellar drug-membrane aggregates that have been observed
to form in the presence of clofazimine and other drugs [6,42].
More direct insights into CLDI structure may be possible in the
near future, with higher resolution, single particle microdiffraction
studies. Because CLDI formation may be an important mecha-
nistic determinant of both clofazimine’s efficacy and toxicity
properties, ongoing and future studies will aim to establish the
extent to which different topological features and physicochemical
Figure 7. Deep-etch freeze-fracture electron microscopy of isolated CLDIs. (A) Pure isolated CLDIs stood out from surrounding ice and
cytosolic debris based on their elongated polyhedral shape and internal layered structure. (B) Isolated CLDIs clearly lacked the outer double
membrane covering. (C) Biochemically-isolated CLDI often showed outer layers of material that appeared to be peeling off from the structure.
Intracellular Clofazimine Sequestration
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properties of clofazimine and related phenazine compounds lead
to intracellular CLDI formation. Understanding the upstream and
downstream effects of macrophages on clofazimine bioaccumula-
tion and distribution, and the role of CLDI formation on
clofazimine’s pharmaco- and toxico-kinetics, should facilitate
development of next generation clofazimine derivatives against
multidrug resistant mycobacterial infections.
Materials and Methods
All animal studies and procedures were performed as approved
by University of Michigan Committee of Use and Care of
Dosing protocol and TEM imaging
Mice (4–5 wk male Balb/c, Jackson Labs, Maine) were fed with
drug with powder feed (3 mg/ml clofazimine (Sigma, C8895) in
sesame oil, mixed at 0.01% oil to feed). Blood was collected from
euthanized mice and fixed by perfusing 0.1M Sorensen’s buffer
and Karnovsky’s fixative (3% paraformaldehyde, 2.5% glutaral-
dehyde) infused to left ventricle and egressed to vena cava (2.5 ml/
min). Tissues were minced smaller than 1 mm in each dimension
followed by TEM sample preparation and imaging, as previously
described . Control mice were fed with 0.01% oil to feed, and
wash out mice were fed drug- and oil-free diet.
Biochemical analysis of clofazimine in tissues
At predetermined time points, mice were euthanized using
CO2, and blood was removed through cardiac puncture. Next, the
organs were collected, washed in cold DPBS, and kept at 220uC
until further analysis. Tissue (0.05–0.1 g/ml water) was homog-
enized with Tissumizer (TekmarH, Cincinnati, OH), extracted
with dichloromethane twice and the solvent was evaporated .
For measurement, clofazimine was dissolved again in methanol
and its absorbance was measured at 490 nm. Concentration was
calculated using a standard curve generated by spiking extracted
tissue of the control (vehicle-only treated) mice tissue with known
amounts of drug. Extraction yield was 60–80%.
Tissues were perfusion fixed as in TEM imaging, paraffin-
embedded and stained with the standard H&E and Masson’s
trichrome technique. Horse Radish Peroxidase and intelliPATH
FLX DAB chromogen kit (IPK5010, Biocare Medical, Concord,
CA) was used for anti-F4/80 (1/100, ab6640, abcamH), a-SMA
(1/200, ab5694, abcamH), and vWF (1/500, ab7356, Millipore)
antibody staining. All staining was performed by the Pathology
Core for Animal Research (PCAR) in the Unit for Laboratory
Animal Medicine (ULAM) at the University of Michigan.
Primary cell isolation and culture
4% Brewer’s thioglycollate medium was prepared sterile as
described . A 2 ml volume of solution was injected IP. Cells
were collected 4 days later using cold DPBS. Bone marrow
macrophages were flushed out from mice femurs using a fine
needle syringe with DPBS . Cells from spleen and lymph
nodes were collected by mincing small tissue using a cell strainer
with 100 mm mesh size. Cells were seeded in tissue culture plates
and kept 7 days in 37uC, 5% CO2with DMEM supplemented
with 10% FBS, Pen/Strep, and non-essential amino acids.
Tissue homogenate was sonicated for 30 minutes, centrifuged
(100 g 61 min) to remove large cell debris. Supernatant was
resuspended in 0.125% Trypsin-EDTA solution (Gibco) and kept
at 37uC for 1 hour, followed by centrifugation at 100 g to remove
large cell debris. The drug inclusions in supernatant were then
pelleted by centrifugation (21,000 g 61 min), and resuspended in
water. Protein content was determined with the BCA assay (Pierce
23227, Thermo Scientific) and clofazimine content was de-
termined spectrophotometrically. For protein assay, equal volume
of 5% SDS solution was mixed with the samples and the protein
content was measured following the BCA kit instructions. The
calculated clofazimine content normalized to the protein content
indicated that the isolation procedure enriched as much as 16-fold
for 8 wk treated spleen homogenate. Greater than 90% of the total
clofazimine mass in the homogenates was recovered in the CLDI
fraction while 95% of proteins were removed.
Powder X-ray diffraction of clofazimine crystals and
PXRD of dried samples of isolated CLDIs and 8 wk treated (or
control) mouse tissue homogenate were carried out with benchtop
Rigaku Miniflex X-ray diffractometer (Danvers, MA). CuKa
radiation (l=1.54A˚), tube voltage =30 kV, tube current
=15 mA. Data were collected at 2 theta from 2.5 to 40 at
a continuous scan at the rate of 2.5u/min. Diffractograms of
triclinic and monoclinic form of clofazimine crystals were
imported from Cambridge Structural Database (CSD) and positive
control of clofazimine crystal from the bottle was used for
Deep-etch, freeze-fracture EM
Unfixed liver was collected after exsanguination and processed
for freeze-etch EM analysis, as reported previously  with
minor modifications. In brief, all samples were kept at 4uC after
removal from the animals. Samples were quickly frozen against
a copper block, cooled with liquid helium using slam freezing and
kept in liquid nitrogen. The sample was fractured with Balzers 400
nitrogen cooled vacuum evaporator and freeze-etched for 2 min at
2100uC. Rotary replica was generated with 2 nm platinum and
backed with 10 nm carbon film support. It was cleaned with
chromo-sulfuric cleaning solution (Fisher Scientific, cat# SC88)
for 12 hours and rinsed with DI water. The sample was picked up
on formvar coated grids for viewing on a JEOL 1400 electron
microscope with AMT camera.
We thank Dorothy Sorenson (MIL, Univ. of Michigan), Paula Arrowsmith
(PCAR, Univ. of Michigan), Dr. Gerald Hish (ULAM, Univ. of Michigan),
Dr. Charles Evans (Core Services, Univ. of Michigan) and Dr. Robyn Roth
(Lab of Electron Microscopy Sciences, Washington Univ. School of
Medicine) for technical support. We thank Dr. Charles Burant, Dr. Nair
Rodriguez-Hornedo, and Dr. David E. Smith for helpful suggestions.
Conceived and designed the experiments: JB GRR. Performed the
experiments: JB. Analyzed the data: JB GRR. Contributed reagents/
materials/analysis tools: JB GRR. Wrote the paper: JB GRR.
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