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 supramolecular organizations. Based on their stimulus-responsiveness, morphological characteristics and powder X-ray diffraction 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 responsive, macrophage-dependent, liquid crystal-like, supramolecular organization.
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 sonication. 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 dimensional, 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 phospholipids in aqueous media 
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 
. When assayed in vitro
, clofazimine disrupts mitochondrial membrane potential and inhibits the growth of cells in tissue culture 
. In solution, clofazimine can generate superoxide anions upon interaction with isolated rat peritoneal macrophages 
and human neutrophils 
which may be related to its in vitro
cytotoxic activity 
. 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 
. 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 therapeutically 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 sequestration. 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 bioaccumulation 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 macrophages activates a natural anti-inflammatory pathway that may serve to protect the host from bioaccumulation-related injury.
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 at position 2 of the phenazine ring structure 
. 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 intracellular crystal formation as an endpoint, we are currently performing QSAR studies to elucidate how the physicochemical and topological features of clofazimine impact its cellular pharmacokinetics. 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 (), 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 
. 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 mechanistic 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 properties of clofazimine and related phenazine compounds lead to intracellular CLDI formation. Understanding the upstream and downstream effects of macrophages on clofazimine bioaccumulation 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.