ABSTRACT
Recombinant retroviruses provide highly efficient gene delivery and the potential for sustained gene expression, but suffer from significant disadvantages including low titer, expensive production, poor stability and limited flexibility for modification of tropism. In contrast, polymer-based vectors are more robust and allow cell- and tissue-specific deliveries via conjugation of ligands, but are comparatively inefficient. The design of hybrid gene delivery agents comprising both virally derived and synthetic materials (nanobiovectors) represents a promising approach to development of safe and efficient gene therapy vectors. Non-infectious murine leukemia virus-like particles (M-VLPs) were electrostatically complexed with chitosan (χ) to replace the function of the viral envelope protein. At optimal fabrication conditions and compositions, ranging from 6 to 9 μg chitosan/10^9 M-VLPs at 10 × 10^9 M-VLPs/ml to 40 μg chitosan/10^9 M-VLPs at 2.5 × 10^9 M-VLPs/ml, χ/M-VLPs were ~ 300–350 nm in diameter and exhibited efficient transfection similar to amphotropic MLV vectors. In addition, these nanobiovectors were non-cytotoxic and provided sustained transgene expression for at least three weeks in vitro. This combination of biocompatible synthetic agents with inactive viral particles to form a highly efficient hybrid vector is a significant extension in the development of novel gene delivery platforms.
- RK Keswani, IM Pozdol & DW Pack, 2013, “Design of Hybrid Lipid/Retroviral-Like Particle Gene Delivery Vectors”, Molecular Pharmaceutics, 10(5):1725-1735 (PMC pdf) (link).
- Can inactive retroviral particles lacking the envelope protein required for viral transduction be revived using a synthetic lipid mixture comprising of cationic lipid and other helper lipids?
ABSTRACT
Recombinant retroviruses provide highly efficient gene delivery and the potential for stable gene expression. The retroviral envelope protein, however, is the source of significant disadvantages such as immunogenicity, poor stability (half-life of transduction activity of 5–7 h at 37 °C for amphotropic murine leukemia virus), and difficult production and purification. To address these problems, we report the construction of efficient hybrid vectors through the association of murine leukemia virus (MLV)-like particles (M-VLP) with synthetic liposomes comprising DOTAP, DOPE, and cholesterol (φ/M-VLP). We conclude that the lipid composition is a significant determinant of the transfection efficiency and uptake of φ/M-VLP in HEK293 cells with favorable compositions for transfections being those with low DOTAP, low DOPE, and high cholesterol content. Cellular uptake, however, was dependent on DOTAP content alone. By extrusion of liposomes prior to vector assembly, the size of these hybrid vectors could also be decreased to ≈300 nm, as confirmed via DLS and TEM. φ/M-VLP were also robust on storage in terms of vector size and transfection efficiency and provided stable transgene expression over a period of three weeks. We conclude that the noncovalent combination of biocompatible synthetic lipids with inactive retroviral particles to form a highly efficient hybrid vector is a significant extension to the development of novel gene delivery platforms.
- DM Drake, RK Keswani, DW Pack, 2010, “Effect of serum on transfection by Polyethylenimine/Virus-Like Particle Hybrid Gene Delivery Vectors”, Pharmaceutical Research, 27(11):2457-2465 (link).
- Are serum proteins responsible for the reduction in transduction measured when hybrid vectors are formulated using polyethylenimine and retroviral particles?
ABSTRACT
PURPOSE:Murine leukemia virus-like particles (M-VLP) complexed with polymers to promote cellular uptake and endosomal escape represent a new class of effective gene delivery vectors. Building upon recent studies of viral-synthetic hybrid vectors, we report the effects of serum on the formation, activity and stability of PEI/M-VLP complexes.
METHODS:M-VLP were produced by cells grown in serum-supplemented media (M-VLP-S), serum-free media (M-VLP-SF) or serum-free Opti-MEM® I (M-VLP-OM). PEI/M-VLP stoichiometry was varied to investigate complex formation and optimal transfection conditions. The effects of prolonged storage, freeze-thaw cycles, and ultracentrifugation of M-VLP on the stability of vector transduction efficiency were also observed.
RESULTS:M-VLP-S required more PEI to form infective complexes than M-VLP-SF and M-VLP-OM. The stoichiometry of PEI/M-VLP-S was dependent on total PEI concentration (7-8 μg/100 μL M-VLP supernatant), while optimal infectivity of PEI/M-VLP-SF and PEI/M-VLP-OM depended on PEI/M-VLP ratios (12-17 μg and 10-14 μg PEI/10(9) M-VLP, respectively). PEI/M-VLP-SF and PEI/M-VLP-OM complexes were significantly more efficient than PEI/M-VLP-S. Stability of the hybrid vectors was not significantly affected by serum.
CONCLUSIONS:PEI/M-VLP complexes exhibiting increased efficiency were constructed by producing M-VLP in serum-free media. M-VLP could be stored by freezing or refrigeration and concentrated by ultracentrifugation without unacceptable loss of infectivity.
METHODS:M-VLP were produced by cells grown in serum-supplemented media (M-VLP-S), serum-free media (M-VLP-SF) or serum-free Opti-MEM® I (M-VLP-OM). PEI/M-VLP stoichiometry was varied to investigate complex formation and optimal transfection conditions. The effects of prolonged storage, freeze-thaw cycles, and ultracentrifugation of M-VLP on the stability of vector transduction efficiency were also observed.
RESULTS:M-VLP-S required more PEI to form infective complexes than M-VLP-SF and M-VLP-OM. The stoichiometry of PEI/M-VLP-S was dependent on total PEI concentration (7-8 μg/100 μL M-VLP supernatant), while optimal infectivity of PEI/M-VLP-SF and PEI/M-VLP-OM depended on PEI/M-VLP ratios (12-17 μg and 10-14 μg PEI/10(9) M-VLP, respectively). PEI/M-VLP-SF and PEI/M-VLP-OM complexes were significantly more efficient than PEI/M-VLP-S. Stability of the hybrid vectors was not significantly affected by serum.
CONCLUSIONS:PEI/M-VLP complexes exhibiting increased efficiency were constructed by producing M-VLP in serum-free media. M-VLP could be stored by freezing or refrigeration and concentrated by ultracentrifugation without unacceptable loss of infectivity.
- RK Keswani‡, HB Ghodke‡, D Sarkar‡, KC Khilar, RS Srinivasa, 2010, “Room Temperature Synthesis of Titanium Dioxide Nanoparticles of different phases in water in oil microemulsion”, Colloids and Surfaces A: Physicochemical and Engineering Aspects, 369:75-81 (‡ - equal contributions) (pdf) (link).
- The synthesis of Titanium dioxide nanoparticles requires prolonged exposure to very high temperatures (>600 ºC) or does it?
ABSTRACT
We report the synthesis of crystalline titanium dioxide nanoparticles of two different phases (rutile and anomalous pseudobrookite) at room temperature by a novel microemulsion technique. In this method, one of the reactants (titanium tetrachloride) is dissolved in the continuous organic phase, whereas the second reactant (ammonium hydroxide) is added as an aqueous solution. The site of the reaction has been controlled by using two different addition methods for the second reactant. The size and the crystalline phase of the titanium dioxide nanoparticles varied according to the site of the reaction between the two reactants. Reaction in the aqueous core gives rutile titanium dioxide nanoparticles having an average size of 4.2 nm verified by X-ray diffraction and FTIR. The reaction in the organic phase gives rise to crys- talline anomalous pseudobrookite titanium dioxide nanoparticles of much larger size (10–40 nm). This anomalous pseudobrookite structure is thermally unstable and converts to rutile on high temperature treatment.
ABSTRACT
Research on attraction of mosquitoes towards their hosts has recently received a boost with health organizations raising alarms on the spread of diseases through mosquito vectors. This report reviews the findings of current studies being conducted and presents useful analysis on the same. The present research shows that no particular attractant is the sole reason for the mosquitoes in locating their host. It is a blend of different odours and compounds like CO2, short chain carboxylic acids, ammonia and other sweat compounds that contribute to the olfaction and subsequent attraction of these pests. Various customized experimental models have been used throughout the world for carrying out research in this relatively new field. Experimental and technical aspects of mosquito research have been covered in great detail with particular importance on the use of new technology. Genomic studies have been briefed upon and certain useful aspects of mosquito research have been covered at the end of the report including the identification and location of odorant receptors in mosquitoes. Genomic results have only contributed to the further understanding of the exact mechanisms and bodily functions involved in location of potential hosts for blood feeding.
- RK Keswani, GS Yoon, S Sud, KA Stringer & GR Rosania, 2015, “A Far-Red Fluorescent Probe For Flow Cytometric Xenobiotic-Sequestering Cell Functional Studies”, Cytometry Part A, 87A(9):855-867 (PMC pdf) (link).
- Immunomodulatory cell-tracking can be useful in a variety of inflammatory diseases. As such, a cytometric approach to visualize and analyze macrophages using the associated specific fluorescence signatures of targeted drug bioaccumulation can provide a unique perspective on cellular homeostasis, particularly upon containing xenobiotic crystals
Abstract
Clofazimine (CFZ) is an optically active, red-colored chemotherapeutic agent that is FDA approved for the treatment of leprosy and is on the World Health Organization's list of essential medications. Interestingly, CFZ massively accumulates in macrophages where it forms crystal-like drug inclusions (CLDIs) after oral administration of the drug in animals and humans. The analysis of the fluorescence spectra of CLDIs formed by resident tissue macrophages revealed that CFZ, when accumulated as CLDIs, undergoes a red shift in fluorescence excitation (from Ex: 540–570 to 560–600 nm) and emission (Em: 560–580 to 640–700 nm) signal relative to the soluble and free-base crystal forms of CFZ. Using epifluorescence microscopy, CLDI(+) cells could be identified, relative to CLDI(−) cells, based on a >3-fold increment in mean fluorescence signal at excitation 640 nm and emission at 670 nm. Similarly, CLDI(+) cells could be identified by flow cytometry, based on a >100-fold increment in mean fluorescence signal using excitation lasers at 640 nm and emission detectors >600 nm. CLDI's fluorescence excitation and emission was orthogonal to that of cell viability dyes such as propidium iodide and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), cellular staining dyes such as Hoechst 33342 (nucleus) and FM 1-43 (plasma membrane), as well as many other fluorescently tagged antibodies used for immunophenotyping analyses. In vivo, >85% of CLDI(+) cells in the peritoneal exudate were F4/80(+) macrophages and >97% of CLDI(+) cells in the alveolar exudate were CD11c(+). Most importantly, the viability of cells was minimally affected by the presence of CLDIs. Accordingly, these results establish that CFZ fluorescence in CLDIs is suitable for quantitative flow cytometric phenotyping analysis and functional studies of xenobiotic sequestering macrophages.
- RK Keswani, J Baik, L Yeomans , C Hitzman, A Pawate, AM Johnson, PJA Kenis, N Rodriguez-Hornedo, KA Stringer & GR Rosania, 2015, “Chemical Analysis of Drug Biocrystals: A Role for Counterion Transport Pathways in Intracellular Drug Disposition”, Molecular Pharmaceutics, 12(7):2528-2536 (PMC pdf) (link).
- What is the chemical identity of these macrophage-specific biocrystals that accumulate upon prolonged drug uptake? Are they free drug crystals? Are they salts? Or are they a manifestation of a lipid-drug biocrystal? What intracellular chemistry could possibly direct such a massive crystallization event?
ABSTRACT
In mammals, highly lipophilic small molecule chemical agents can accumulate as inclusions within resident tissue macrophages. In this context, we characterized the biodistribution, chemical composition, and structure of crystal-like drug inclusions (CLDIs) formed by clofazimine (CFZ), a weakly basic lipophilic drug. With prolonged oral dosing, CFZ exhibited a significant partitioning with respect to serum and fat due to massive bioaccumulation and crystallization in the liver and spleen. The NMR, Raman, and powder X-ray diffraction (p-XRD) spectra of CLDIs isolated from the spleens of CFZ-treated mice matched the spectra of pure, CFZ hydrochloride crystals (CFZ-HCl). Elemental analysis revealed a 237-fold increase in chlorine content in CLDIs compared to untreated tissue samples and a 5-fold increase in chlorine content compared to CFZ-HCl, suggesting that the formation of CLDIs occurs through a chloride mediated crystallization mechanism. Single crystal analysis revealed that CFZ-HCl crystals had a densely packed orthorhombic lattice configuration. In vitro, CFZ-HCl formed at a pH of 4–5 only if chloride ions were present at sufficiently high concentrations (>50:1 Cl–/CFZ), indicating that intracellular chloride transport mechanisms play a key role in the formation of CLDIs. While microscopy and pharmacokinetic analyses clearly revealed crystallization and intracellular accumulation of the drug in vivo, the chemical and structural characterization of CLDIs implicates a concentrative, chloride transport mechanism, paralleling and thermodynamically stabilizing the massive bioaccumulation of a weakly basic drug.
- KA Min, WG Rajeswaran, R Oldenbourg, G Harris, RK Keswani, M Chiang, P Rzeczycki, M Hafeezma, R Horobin, SD Larsen, KA Stringer & GR Rosania, 2015, " Massive Bioaccumulation and Self-Assembly of Phenazine Compounds in Live Cells", Advanced Science, 2(8):1500025 (pdf) (link).
- How does the chemical structure of various phenazines affect their bioaccumulation and crystallization within macrophages? Could we harness such chemical information to crystallize other drugs within cells? What are the ramifications for such intense small molecule crystallization?
ABSTRACT
Clofazimine is an orally administered drug that massively bioaccumulates in macrophages, forming membrane-bound intracellular structures possessing nanoscale supramolecular features. Here, a library of phenazine compounds derived from clofazimine is synthesized and tested for ability to accumulate and form ordered molecular aggregates inside cells. Regardless of chemical structure or physicochemical properties, bioaccumulation is consistently greater in macrophages than in epithelial cells. Microscopically, some self-assembled structures exhibit a pronounced, diattenuation anisotropy signal, evident by the differential absorption of linearly polarized light, at the peak absorbance wavelength of the phenazine core. The measured anisotropy is well above the background anisotropy of endogenous cellular components, reflecting the self-assembly of condensed, insoluble complexes of ordered phenazine molecules. Chemical variations introduced at the R-imino position of the phenazine core lead to idiosyncratic effects on the compounds' bioaccumulation behavior as well as on the morphology and organization of the resulting intracellular structures. Beyond clofazimine, these results demonstrate how the self-assembly of membrane permeant, orally bioavailable small molecule building blocks can endow cells with unnatural structural elements possessing chemical, physical, and functional characteristics unlike those of other natural cellular components.
- GS Yoon, S Sud, RK Keswani, TJ Standiford, KA Stringer & GR Rosania, 2015, “Phagocytosed Clofazimine Biocrystals can Modulate Innate Immune Signaling by Inhibiting TNFα and Boosting IL-1RA Secretion”, Molecular Pharmaceutics, 12(7):2517-2527 (PMC pdf) (link).
- Soluble drug or cell-crystallized drug? Which one would be anti-inflammatory?
ABSTRACT
Clofazimine (CFZ) is an FDA-approved leprostatic and anti-inflammatory drug that massively accumulates in macrophages, forming insoluble, intracellular crystal-like drug inclusions (CLDIs) during long-term oral dosing. Interestingly, when added to cells in vitro, soluble CFZ is cytotoxic because it depolarizes mitochondria and induces apoptosis. Accordingly, we hypothesized that,in vivo, macrophages detoxify CFZ by sequestering it in CLDIs. To test this hypothesis, CLDIs of CFZ-treated mice were biochemically isolated and then incubated with macrophages in vitro. The cell biological effects of phagocytosed CLDIs were compared to those of soluble CFZ. Unlike soluble CFZ, phagocytosis of CLDIs did not lead to mitochondrial destabilization or apoptosis. Rather, CLDIs altered immune signaling response pathways downstream of Toll-like receptor (TLR) ligation, leading to enhanced interleukin-1 receptor antagonist (IL-1RA) production, dampened NF-κB activation and tissue necrosis factor alpha (TNFα) production, and ultimately decreased TLR expression levels. In aggregate, our results constitute evidence that macrophages detoxify soluble CFZ by sequestering it in a biocompatible, insoluble form. The altered cellular response to TLR ligation suggests that CLDI formation may also underlie CFZ’s anti-inflammatory activit
- RK Keswani, M Lazebnik, DW Pack, 2015, "Intracellular Trafficking of Hybrid Gene Delivery Vectors”, Journal of Controlled Release, 207:120-130 (PMC pdf) (link).
- While hybrid retroviral vectors can be engineered as conjugates between synthetic polymers and inactive retroviral particles, does their intracellular transport depend on the synthetic elements or does the retrovirus harness any innate mechanisms that are independent of the synthetic element involved?
ABSTRACT
Viral and non-viral gene delivery vectors are in development for human gene therapy, but both exhibit disadvantages such as inadequate efficiency, lack of cell-specific targeting or safety concerns. We have recently reported the design of hybrid delivery vectors combining retrovirus-like particles with synthetic polymers or lipids that are efficient, provide sustained gene expression and are more stable compared to native retroviruses. To guide further development of this promising class of gene delivery vectors, we have investigated their mechanisms of intracellular trafficking. Moloney murine leukemia virus-like particles (M-VLPs) were complexed with chitosan (Chi) or liposomes (Lip) comprising DOTAP, DOPE and cholesterol to form the hybrid vectors (Chi/M-VLPs and Lip/M-VLPs, respectively). Transfection efficiency and cellular internalization of the vectors were quantified in the presence of a panel of inhibitors of various endocytic pathways. Intracellular transport and trafficking kinetics of the hybrid vectors were dependent on the synthetic component and used a combination of clathrin- and caveolar-dependent endocytosis and macropinocytosis. Chi/M-VLPs were slower to transfect compared to Lip/M-VLPs due to the delayed detachment of the synthetic component. The synthetic component of hybrid gene delivery vectors plays a significant role in their cellular interactions and processing and is a key parameter for the design of more efficient gene delivery vehicles.
- ME Hwang, RK Keswani & DW Pack, 2015, "Dependence of PEI and PAMAM Gene Delivery on Active Clathrin and Caveolar Trafficking Pathways", Pharmaceutical Research, 32(6):2051-2059 (PMC pdf) (link).
- Gene therapy polyplexes are not as efficient as their viral counterparts. What are the predominant transport mechanisms in cancer cells that can be used to deliver plasmid DNA efficiently into the cytosol and eventually the nucleus?
ABSTRACT
Purpose
Non-viral gene delivery vehicles such as polyethylenimine and polyamidoamine dendrimer effectively condense plasmid DNA, facilitate endocytosis, and deliver nucleic acid cargo to the nucleus in vitro. Better understanding of intracellular trafficking mechanisms involved in polymeric gene delivery is a prerequisite to clinical application. This study investigates the role of clathrin and caveolin endocytic pathways in cellular uptake and subsequent vector processing.
Methods
We formed 25-kD polyethylenimine (PEI) and generation 4 (G4) polyamidoamine (PAMAM) polyplexes at N/P 10 and evaluated internalization pathways and gene delivery in HeLa cells. Clathrin- and caveolin-dependent endocytosis inhibitors were used at varying concentrations to elucidate the roles of these important pathways.
Results
PEI and PAMAM polyplexes were internalized by both pathways. However, the amount of polyplex internalized poorly correlated with transgene expression. While the caveolin-dependent pathway generally led to effective gene delivery with both polymers, complete inhibition of the clathrin-dependent pathway was also deleterious to transfection with PEI polyplexes. Inhibition of one endocytic pathway may lead to an overall increase in uptake via unaffected pathways, suggesting the existence of compensatory endocytic mechanisms.
Conclusions
The well-studied clathrin- and caveolin-dependent endocytosis pathways are not necessarily independent, and perturbing one mechanism of trafficking influences the larger trafficking network.
Non-viral gene delivery vehicles such as polyethylenimine and polyamidoamine dendrimer effectively condense plasmid DNA, facilitate endocytosis, and deliver nucleic acid cargo to the nucleus in vitro. Better understanding of intracellular trafficking mechanisms involved in polymeric gene delivery is a prerequisite to clinical application. This study investigates the role of clathrin and caveolin endocytic pathways in cellular uptake and subsequent vector processing.
Methods
We formed 25-kD polyethylenimine (PEI) and generation 4 (G4) polyamidoamine (PAMAM) polyplexes at N/P 10 and evaluated internalization pathways and gene delivery in HeLa cells. Clathrin- and caveolin-dependent endocytosis inhibitors were used at varying concentrations to elucidate the roles of these important pathways.
Results
PEI and PAMAM polyplexes were internalized by both pathways. However, the amount of polyplex internalized poorly correlated with transgene expression. While the caveolin-dependent pathway generally led to effective gene delivery with both polymers, complete inhibition of the clathrin-dependent pathway was also deleterious to transfection with PEI polyplexes. Inhibition of one endocytic pathway may lead to an overall increase in uptake via unaffected pathways, suggesting the existence of compensatory endocytic mechanisms.
Conclusions
The well-studied clathrin- and caveolin-dependent endocytosis pathways are not necessarily independent, and perturbing one mechanism of trafficking influences the larger trafficking network.
- M Lazebnik, RK Keswani & DW Pack, 2016, “Effective Endocytosis Pathways in siRNA polyplex and lipoplex delivery in HeLa cells”, Pharmaceutical Research, 33(12):2999-3011 (link).
- Polyplexes and Lipoplexes incorporating siRNA for cancer therapy are established vectors for gene knockdown, yet not as efficient to become therapeutic in character? Can their intracellular trafficking mechanisms suggest possible areas of improvement?
ABSTRACT
PURPOSE:siRNA may be delivered as electrostatic complexes with cationic lipids (lipoplexes) or polycations (polyplexes). The purpose of this project was to determine the effect of cellular internalization mechanism(s) on siRNA-mediated gene silencing efficiency.
METHODS:Lipoplexes were formed comprising siRNA and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP), cholesterol and dioleoyl phosphatidylethanolamine (DOPE), and polyplexes comprised siRNA with polyethylenimine (PEI). During transfections, specific uptake mechanisms were inhibited by pharmacological agents and RNAi-mediated knockdown of proteins involved in various endocytosis pathways. Confocal fluorescence microscopy further elucidated the predominant endocytic pathways of siRNA delivery via colocalization of vectors with endocytic vesicle markers.
RESULTS:Inhibition of macropinocytosis (MP), caveolin-mediated endocytosis (CvME), flotillin-mediated endocytosis (FME) and knockdown of ARF6 significantly decreased PEI/siRNA-mediated gene silencing. Inhibition of endocytosis pathways, however, had negligible effect on lipoplex uptake and gene silencing mediated by lipoplexes. Rather, internalization of lipoplexes and subsequent siRNA-mediated gene silencing occurred via an energy-independent process.
CONCLUSIONS:MP, CvME and FME, but not the acidified clathrin-mediated pathway, lead to effective gene silencing by PEI/siRNA polyplexes. Lipoplexes, in contrast, deliver siRNA primarily by direct fusion of the liposomal and cellular membranes. These results provide a new understanding of the mechanisms of siRNA delivery materials in HeLa cells and may aid in design of more effective RNAi strategies.
METHODS:Lipoplexes were formed comprising siRNA and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP), cholesterol and dioleoyl phosphatidylethanolamine (DOPE), and polyplexes comprised siRNA with polyethylenimine (PEI). During transfections, specific uptake mechanisms were inhibited by pharmacological agents and RNAi-mediated knockdown of proteins involved in various endocytosis pathways. Confocal fluorescence microscopy further elucidated the predominant endocytic pathways of siRNA delivery via colocalization of vectors with endocytic vesicle markers.
RESULTS:Inhibition of macropinocytosis (MP), caveolin-mediated endocytosis (CvME), flotillin-mediated endocytosis (FME) and knockdown of ARF6 significantly decreased PEI/siRNA-mediated gene silencing. Inhibition of endocytosis pathways, however, had negligible effect on lipoplex uptake and gene silencing mediated by lipoplexes. Rather, internalization of lipoplexes and subsequent siRNA-mediated gene silencing occurred via an energy-independent process.
CONCLUSIONS:MP, CvME and FME, but not the acidified clathrin-mediated pathway, lead to effective gene silencing by PEI/siRNA polyplexes. Lipoplexes, in contrast, deliver siRNA primarily by direct fusion of the liposomal and cellular membranes. These results provide a new understanding of the mechanisms of siRNA delivery materials in HeLa cells and may aid in design of more effective RNAi strategies.
- NP Gothe, RK Keswani & E McAuley, 2016, “Yoga Practice Improves Cognition by Attenuating Stress Levels”, Biological Psychology, 121(A):109-116 (link).
ABSTRACT
Background
Prolonged activation of the hypothalamus–pituitary–adrenal system is thought to have deleterious effects on brain function. Neuroendocrine studies suggest that brain exposure to higher cortisol concentrations contribute to cognitive deficits as we age. Mind-body techniques such as yoga have shown to improve stress levels by restoring the body’s sympathetic-parasympathetic balance. The objective of this study was to determine whether yoga practice moderated the stress response resulting in improved executive function.
Methods
Sedentary community dwelling older adults (N = 118, Mean age = 62.02) were randomized to an 8-week yoga intervention or a stretching control group. At baseline and following 8 weeks, all participants completed measures of executive function, self-reported stress and anxiety and provided saliva samples before and after cognitive testing to assess cortisol.
Results
Yoga participants showed improved accuracy on executive function measures and an attenuated cortisol response compared to their stretching counterparts who showed increased cortisol levels and poor cognitive performance at follow up. The change in cortisol levels as well as self-reported stress and anxiety levels predicted performance on the running span task, n-back working memory and task switching paradigm (β’s = 0.27–0.38, p’s ≤ 0.05 for yoga and β’s = −0.37–0.47, p’s ≤ 0.01 for stretching control).
Conclusion
Eight weeks of regular yoga practice resulted in improved working memory performance that was mediated by an attenuated response to stress as measured by self-report stress and objective salivary cortisol measurements. This trial offers evidence for non-traditional physical activity interventions such as yoga that may be helpful in restoring HPA balance in older adults, thereby preventing cognitive decline.
Prolonged activation of the hypothalamus–pituitary–adrenal system is thought to have deleterious effects on brain function. Neuroendocrine studies suggest that brain exposure to higher cortisol concentrations contribute to cognitive deficits as we age. Mind-body techniques such as yoga have shown to improve stress levels by restoring the body’s sympathetic-parasympathetic balance. The objective of this study was to determine whether yoga practice moderated the stress response resulting in improved executive function.
Methods
Sedentary community dwelling older adults (N = 118, Mean age = 62.02) were randomized to an 8-week yoga intervention or a stretching control group. At baseline and following 8 weeks, all participants completed measures of executive function, self-reported stress and anxiety and provided saliva samples before and after cognitive testing to assess cortisol.
Results
Yoga participants showed improved accuracy on executive function measures and an attenuated cortisol response compared to their stretching counterparts who showed increased cortisol levels and poor cognitive performance at follow up. The change in cortisol levels as well as self-reported stress and anxiety levels predicted performance on the running span task, n-back working memory and task switching paradigm (β’s = 0.27–0.38, p’s ≤ 0.05 for yoga and β’s = −0.37–0.47, p’s ≤ 0.01 for stretching control).
Conclusion
Eight weeks of regular yoga practice resulted in improved working memory performance that was mediated by an attenuated response to stress as measured by self-report stress and objective salivary cortisol measurements. This trial offers evidence for non-traditional physical activity interventions such as yoga that may be helpful in restoring HPA balance in older adults, thereby preventing cognitive decline.
- RK Keswani‡, C Tian‡, T Peryea, G Gandikota, X Wang, GR Rosania, 2016, "Repositioning Clofazimine as a Macrophage-Targeting Photoacoustic Contrast Agent", Scientific Reports, 6:23528 (‡ - equal contributions) (pdf) (link).
- Drug repurposing has gained considerable attention today to give new life to old approved drugs also lowering costs in bringing critical drugs to the market. Can such drugs be repositioned as diagnostic agents, perhaps even theranostic agents considering that they already are therapeutic and non-toxic?
ABSTRACT
Photoacoustic Tomography (PAT) is a deep-tissue imaging modality, with potential clinical applications in the diagnosis of arthritis, cancer and other disease conditions. Here, we identified Clofazimine (CFZ), a red-pigmented dye and anti-inflammatory FDA-approved drug, as a macrophage-targeting photoacoustic (PA) imaging agent. Spectroscopic experiments revealed that CFZ and its various protonated forms yielded optimal PAT signals at wavelengths −450 to 540 nm. CFZ’s macrophage-targeting chemical and structural forms were detected with PA microscopy at a high contrast-to-noise ratio (CNR > 22 dB) as well as with macroscopic imaging using synthetic gelatin phantoms. In vivo, natural and synthetic CFZ formulations also demonstrated significant anti-inflammatory activity. Finally, the injection of CFZ was monitored via a real-time ultrasound-photoacoustic (US-PA) dual imaging system in a live animal and clinically relevant human hand model. These results demonstrate an anti-inflammatory drug repurposing strategy, while identifying a new PA contrast agent with potential applications in the diagnosis and treatment of arthritis.
- C Tian‡, RK Keswani‡, G Girish, X Wang & GR Rosania, "Contrast-enhanced photoacoustic tomography of human joints", Photons Plus Ultrasound: Imaging and Sensing 2016, 2016;97084L. (‡ - equal contributions) (pdf) (link).
- Human joints suffer from a variety of stresses and diseases as we age. Important physiological information can be obtained by imaging them in the right context. Can we use an old FDA-approved pigment to potentially use it to coat the human joint as a contrast agent for photoacoustic tomography?
- Human joints suffer from a variety of stresses and diseases as we age. Important physiological information can be obtained by imaging them in the right context. Can we use an old FDA-approved pigment to potentially use it to coat the human joint as a contrast agent for photoacoustic tomography?
ABSTRACT
Photoacoustic tomography (PAT) provides a unique tool to diagnose inflammatory arthritis. However, the specificity and sensitivity of PAT based on endogenous contrasts is limited. The development of contrast enhanced PAT imaging modalities in combination with small molecule contrast agents could lead to improvements in diagnosis and treatment of joint disease. Accordingly, we adapted and tested a PAT clinical imaging system for imaging the human joints, in combination with a novel PAT contrast agent derived from an FDA-approved small molecule drug. Imaging results based on a photoacoustic and ultrasound (PA/US) dual-modality system revealed that this contrast-enhanced PAT imaging system may offer additional information beyond single-modality PA or US imaging system, for the imaging, diagnosis and assessment of inflammatory arthritis.
- GS Yoon, RK Keswani, S Sud, P Rzeczycki, M Murashov, T Koehn, TJ Standiford, KA Stringer & GR Rosania, 2016, “Clofazimine Biocrystal Accumulation in Macrophages Upregulates IL-1RA Production to Induce a Systemic Anti-Inflammatory State”, Antimicrobial Agents and Chemotherapy, 60(6): 3470-3479 (PMC pdf) (link).
- The biocrystallization of an anti-inflammatory drug results in an anti-inflammatory physiological state. How does a cell-specific accumulation phenomena result in a systemic immunomodulatory and anti-inflammatory state of the organism?
ABSTRACT
Clofazimine (CFZ) is a poorly soluble antibiotic and anti-inflammatory drug indicated for the treatment of leprosy. In spite of its therapeutic value, CFZ therapy is accompanied by the formation of drug biocrystals that accumulate within resident tissue macrophages, without obvious toxicological manifestations. Therefore, to specifically elucidate the off-target consequences of drug bioaccumulation in macrophages, we compared the level of inflammasome activation in CFZ-accumulating organs (spleen, liver and lung) in mice following 2 and 8 weeks of CFZ treatment when the drug exists in soluble and insoluble (biocrystalline) forms, respectively. Surprisingly, the results showed a drastic reduction of caspase 1 and IL-1β cleavage in the livers of 8 week CFZ-treated mice as compared to 2 week CFZ-treated and control mice, which was accompanied by a three-fold increase in hepatic interleukin 1 receptor antagonist (IL-1RA) production and a 21-fold increase in serum IL-1RA levels. In the lung and spleen, IL-1β cleavage and TNFα expression were unaffected by soluble or biocrystal CFZ forms. Functionally, there was a drastic reduction of carrageenan- and lipopolysaccharide-induced inflammation in the footpads and lungs, respectively, of 8 week CFZ-treated mice. This immunomodulatory activity of CFZ biocrystal accumulation was attributable to the upregulation of IL-1RA, as CFZ accumulation had minimal effect in IL-1RA knockout mice, or 2 week CFZ-treated mice. In conclusion, CFZ accumulation and biocrystal formation in resident tissue macrophages profoundly altered the host's immune system and prompted an IL-1RA-dependent, systemic anti-inflammatory response.
- EM Horstman‡, RK Keswani‡, B Frey, P Rzeczycki, V LaLone, JA Bertke, PJA Kenis & GR Rosania, 2017, “Elasticity in Macrophage-Synthesized Biocrystals", Angewandte Chemie, 56:1815-1819 (link) (‡ - equal authorship)
- Crystals with certain design rules and molecular packing features have recently been identified to be elastic. Do crystals that are synthesized within human cells such as in macrophages also capable of the same features, given that they share molecular packing features with elastic crystals?
ABSTRACT
Supramolecular crystalline assembly constitutes a rational approach to bioengineer intracellular structures. Here, biocrystals of clofazimine (CFZ) that form in vivo within macrophages were measured to have marked curvature. Isolated crystals, however, showed reduced curvature suggesting that intracellular forces bend these drug crystals. Consistent with the ability of biocrystals to elastically deform, the inherent
crystal structure of the principal molecular component of the biocrystals—the hydrochloride salt of CFZ (CFZ-HCl)—has a corrugated packing along the (001) face and weak dispersive bonding in multiple directions. These characteristics were previously found to be linked to the elasticity of other organic crystals. Internal stress in bent CFZ-HCl led to photoelastic effects on the azimuthal orientation of polarized light transmittance. We propose that elastic, intracellular crystals can serve as templates to construct functional microdevices with different applications.
crystal structure of the principal molecular component of the biocrystals—the hydrochloride salt of CFZ (CFZ-HCl)—has a corrugated packing along the (001) face and weak dispersive bonding in multiple directions. These characteristics were previously found to be linked to the elasticity of other organic crystals. Internal stress in bent CFZ-HCl led to photoelastic effects on the azimuthal orientation of polarized light transmittance. We propose that elastic, intracellular crystals can serve as templates to construct functional microdevices with different applications.
- J Trexel‡, GS Yoon‡, RK Keswani, S Sud, C McHugh, V Vitvitsky, R Banerjee, L Yeomans, Y Sun, GR Rosania & KA Stringer, "Macrophage-mediated Clofazimine Sequestration is Accompanied by the Reprogramming of Host Metabolism”, Journal of Pharmaceutical Sciences, 2017;106(4):1162-1174 (IF:2.59) (link) (‡ - equal contributions).
- Metabolic reprogramming is a hallmark of a newly defined field of study - Immunometabolomics. Here, we show how bioaccumulation of a drug within a work-horse of the immune system - macrophages radically modulates levels of classic metabolites in blood and urine.
ABSTRACT
Prolonged (8 weeks) oral administration of clofazimine results in a profound pharmacodynamic response - bioaccumulation in macrophages (including Kupffer cells) as intracellular crystal-like drug inclusions (CLDIs) with an associated increase in interleukin-1 receptor antagonist production. Notably, CLDI formation in Kupffer cells concomitantly occurs with the formation of macrophage-centric granulomas. Accordingly, we sought to understand the impact of these events on host metabolism using 1H-nuclear magnetic resonance metabolomics. Mice received a clofazimine or vehicle-enriched (sham) diet for at least 8 weeks. At 2 weeks, the antimicrobial activity of clofazimine was evident by changes in urine metabolites. From 2 to 8 weeks, there was a striking change in metabolite levels indicative of a reorientation of host energy metabolism paralleling the onset of CLDI and granuloma formation. This was evidenced by a progressive reduction in urine levels of metabolites involved in one-carbon metabolism with corresponding increases in whole blood, and changes in metabolites associated with lipid, nucleotide and amino acid metabolism, and glycolysis. Although clofazimine-fed mice ate more, they gained less weight than control mice. Together, these results indicate that macrophage sequestration of clofazimine as CLDIs and granuloma formation is accompanied by a profound metabolic disruption in energy homeostasis and one-carbon metabolism.
- P Rzeczycki, GS Yoon, RK Keswani, S Sud, TA Koehn, KA Stringer & GR Rosania, 2017, "Detecting Ordered Small Molecule Drug Aggregates in Live Macrophages: A Multi-Parameter Microscope Image Data Acquisition and Analysis Strategy", Biomedical Optics Express, 8(2):860-872 (link)
- Identification of crystals or particulate matter within living cells can be tough, given the dimensions we typically work with. Here, we define a multi-parametric microscopy setup which is capable of quantifying crystal presence via absorbance, fluorescence and polarized light properties.
ABSTRACT
Following prolonged administration, certain orally bioavailable but poorly soluble small molecule drugs are prone to precipitate out and form crystal-like drug inclusions (CLDIs) within the cells of living organisms. In this research, we present a quantitative multiparameter imaging platform for measuring the fluorescence and polarization diattenuation signals of cells harboring intracellular CLDIs. To validate the imaging system, the FDAapproved drug clofazimine (CFZ) was used as a model compound. Our results demonstrated that a quantitative multi-parameter microscopy image analysis platform can be used to study drug sequestering macrophages, and to detect the formation of ordered molecular aggregates formed by poorly soluble small molecule drugs in animals.
- M Murashov, V LaLone, PM Rzeczycki, RK Keswani, GS Yoon, S Sud, W Rajeswaran, SD Larsen, KA Stringer, GR Rosania, “The Physicochemical Basis of Clofazimine-Induced Skin Pigmentation", Journal of Investigative Dermatology, 2018;138(3):697-703 (link)
- Drugs can bioaccumulate in the skin when administered over a prolonged duration of time. In this study, we explored the physicochemical mechanisms of such accumulation through optical and chemical analysis of drug-fed mice and tissues.
ABSTRACT
Clofazimine is a weakly basic, FDA-approved antibiotic recommended by the World Health Organization to treat leprosy and multi-drug-resistant tuberculosis. Upon prolonged treatment, clofazimine extensively bioaccumulates and precipitates throughout the organism, forming crystal-like drug inclusions (CLDIs). Because of the drug’s red color, it is widely believed that clofazimine bioaccumulation results in skin pigmentation, its most common side effect. To test whether clofazimine-induced skin pigmentation is due to CLDI formation, we synthesized a closely related clofazimine analog that does not precipitate under physiological pH and chloride conditions that are required for CLDI formation. Despite the absence of detectable CLDIs in mice, administration of this analog still led to significant skin pigmentation. In clofazimine treated mice, skin cryosections revealed no evidence of CLDIs when analyzed with a microscopic imaging system specifically designed for detecting clofazimine aggregates. Rather, the reflectance spectra of the skin revealed a signal corresponding to the soluble, free base form of the drug. Consistent with the low concentrations of clofazimine in the skin, these results suggest that clofazimine-induced skin pigmentation is not due to clofazimine precipitation and CLDI formation, but rather to the partitioning of the circulating, free base form of the drug into subcutaneous fat.
- T Woldemichael‡, RK Keswani‡, PM Rzeczycki, MD Murashov, V LaLone, B Gregorka, JA Swanson, KA Stringer, GR Rosania, “Reverse Engineering the Intracellular Self-Assembly of a Functional Mechanopharmaceutical Device”, Scientific Reports, 2018;8(1):2934 (‡ - equal authorship) (link).
- Mechanopharmaceutical intracellular agents can drive cellular homeostasis mechanisms in novel hitherto unknown mechanisms. In this study (in a first of its kind), we describe the self-assembly of such agents driven by ionic gradients and channels via experimental and computational approaches .
ABSTRACT
Weakly basic, poorly soluble chemical agents could be exploited as building blocks for constructing sophisticated molecular devices inside the cells of living organisms. Here, using experimental and computational approaches, we probed the relationship between the biological mechanisms mediating lysosomal ion homeostasis and the self-assembly of a weakly basic small molecule building block (clofazimine) into a functional, mechanopharmaceutical device (intracellular Crystal-Like Drug Inclusions - "CLDIs") in macrophage lysosomes. Physicochemical considerations indicate that the intralysosomal stabilization of the self-assembled mechanopharmaceutical device depends on the pHmax of the weakly basic building block and its affinity for chloride, both of which are consistent with the pH and chloride content of a physiological lysosomal microenvironment. Most importantly, in vitro and in silico studies revealed that high expression levels of the vacuolar ATPase (V-ATPase), irrespective of the expression levels of chloride channels, are necessary and sufficient to explain the cell-type dependent formation, stabilization, and biocompatibility of the self-assembled mechanopharmaceutical device within macrophages.
- PM Rzeczycki, T Woldemichael, Andrew Willmer, MD Murashov, J Baik, RK Keswani, GS Yoon, KA Stringer, N Rodriguez-Hornedo, GR Rosania, “An Expandable Mechanopharmaceutical Device (1): Measuring the Cargo Capacity of Macrophages in a Living Organism”, Pharmaceutical Research, 2018;36(1):12 (link).
- Mechanopharmaceutical intracellular agents can drive cellular homeostasis mechanisms in novel hitherto unknown mechanisms. In an extension of our first study, we show the macrophage's response to the presence of such a dynamic endolysosomal load.
ABSTRACT
PURPOSE:Clofazimine (CFZ) is an FDA-approved, poorly soluble small molecule drug that precipitates as crystal-like drug inclusions (CLDIs) which accumulate in acidic cytoplasmic organelles of macrophages. In this study, we considered CLDIs as an expandable mechanopharmaceutical device, to study how macrophages respond to an increasingly massive load of endophagolysosomal cargo.
METHODS:First, we experimentally tested how the accumulation of CFZ in CLDIs impacted different immune cell subpopulations of different organs. Second, to further investigate the mechanism of CLDI formation, we asked whether specific accumulation of CFZ hydrochloride crystals in lysosomes could be explained as a passive, thermodynamic equilibrium phenomenon. A cellular pharmacokinetic model was constructed, simulating CFZ accumulation driven by pH-dependent ion trapping of the protonated drug in the acidic lysosomes, followed by the precipitation of CFZ hydrochloride salt via a common ion effect caused by high chloride concentrations.
RESULTS:While lower loads of CFZ were mostly accommodated in lung macrophages, increased CFZ loading was accompanied by organ-specific changes in macrophage numbers, size and intracellular membrane architecture, maximizing the cargo storage capabilities. With increasing loads, the total cargo mass and concentrations of CFZ in different organs diverged, while that of individual macrophages converged. The simulation results support the notion that the proton and chloride ion concentrations of macrophage lysosomes are sufficient to drive the massive, cell type-selective accumulation and growth of CFZ hydrochloride biocrystals.
CONCLUSION:CLDIs effectively function as an expandable mechanopharmaceutical device, revealing the coordinated response of the macrophage population to an increasingly massive, whole-organism endophagolysosomal cargo load.
METHODS:First, we experimentally tested how the accumulation of CFZ in CLDIs impacted different immune cell subpopulations of different organs. Second, to further investigate the mechanism of CLDI formation, we asked whether specific accumulation of CFZ hydrochloride crystals in lysosomes could be explained as a passive, thermodynamic equilibrium phenomenon. A cellular pharmacokinetic model was constructed, simulating CFZ accumulation driven by pH-dependent ion trapping of the protonated drug in the acidic lysosomes, followed by the precipitation of CFZ hydrochloride salt via a common ion effect caused by high chloride concentrations.
RESULTS:While lower loads of CFZ were mostly accommodated in lung macrophages, increased CFZ loading was accompanied by organ-specific changes in macrophage numbers, size and intracellular membrane architecture, maximizing the cargo storage capabilities. With increasing loads, the total cargo mass and concentrations of CFZ in different organs diverged, while that of individual macrophages converged. The simulation results support the notion that the proton and chloride ion concentrations of macrophage lysosomes are sufficient to drive the massive, cell type-selective accumulation and growth of CFZ hydrochloride biocrystals.
CONCLUSION:CLDIs effectively function as an expandable mechanopharmaceutical device, revealing the coordinated response of the macrophage population to an increasingly massive, whole-organism endophagolysosomal cargo load.
- PM Rzeczycki, GS Yoon, RK Keswani, S Sud, J Baik, MD Murashov, IL Bergin, KA Stringer, GR Rosania, “An Expandable Mechanopharmaceutical Device (2): Drug Induced Granulomas Maximize the Cargo Sequestering Capacity of Macrophages in the Liver”, Pharmaceutical Research, 2018;36(1):3 (link).
- Mechanopharmaceutical intracellular agents can drive cellular homeostasis mechanisms in novel hitherto unknown mechanisms. In a second extension of our first study, we show that granulomas provide a safe-house toward harboring crystals thereby limiting their damage toward the organ as well.
ABSTRACT
PURPOSE:Drug-induced liver injuries (DILI) comprise a significant proportion of adverse drug reactions leading to hospitalizations and death. One frequent DILI is granulomatous inflammation from exposure to harmful metabolites that activate inflammatory pathways of immune cells of the liver, which may act as a barrier to isolate the irritating stimulus and limit tissue damage.
METHODS:Paralleling the accumulation of CFZ precipitates in the liver, granulomatous inflammation was studied to gain insight into its effect on liver structure and function. A structural analog that does not precipitate within macrophages was also studied using micro-analytical approaches. Depleting macrophages was used to inhibit granuloma formation and assess its effect on drug bioaccumulation and toxicity.
RESULTS:Granuloma-associated macrophages showed a distinct phenotype, differentiating them from non-granuloma macrophages. Granulomas were induced by insoluble CFZ cargo, but not by the more soluble analog, pointing to precipitation being a factor driving granulomatous inflammation. Granuloma-associated macrophages showed increased activation of lysosomal master-regulator transcription factor EB (TFEB). Inhibiting granuloma formation increased hepatic necrosis and systemic toxicity in CFZ-treated animals.
CONCLUSIONS:Granuloma-associated macrophages are a specialized cell population equipped to actively sequester and stabilize cytotoxic chemotherapeutic agents. Thus, drug-induced granulomas may function as drug sequestering "organoids" -an induced, specialized sub-compartment- to limit tissue damage.
METHODS:Paralleling the accumulation of CFZ precipitates in the liver, granulomatous inflammation was studied to gain insight into its effect on liver structure and function. A structural analog that does not precipitate within macrophages was also studied using micro-analytical approaches. Depleting macrophages was used to inhibit granuloma formation and assess its effect on drug bioaccumulation and toxicity.
RESULTS:Granuloma-associated macrophages showed a distinct phenotype, differentiating them from non-granuloma macrophages. Granulomas were induced by insoluble CFZ cargo, but not by the more soluble analog, pointing to precipitation being a factor driving granulomatous inflammation. Granuloma-associated macrophages showed increased activation of lysosomal master-regulator transcription factor EB (TFEB). Inhibiting granuloma formation increased hepatic necrosis and systemic toxicity in CFZ-treated animals.
CONCLUSIONS:Granuloma-associated macrophages are a specialized cell population equipped to actively sequester and stabilize cytotoxic chemotherapeutic agents. Thus, drug-induced granulomas may function as drug sequestering "organoids" -an induced, specialized sub-compartment- to limit tissue damage.
- X Hong, PM Rzeczycki, RK Keswani, MD Murashov, Fan Z, Deng CX, GR Rosania, “Acoustic tweezing cytometry for mechanical phenotyping of macrophages and mechanopharmaceutical cytotripsy”, Scientific Reports, 2019;9(1):5702.
- We have shown that CLDIs themselves are quite elastic. Does that impact the cell in which it resides to modulate its viscoelastic behavior?
ABSTRACT
Macrophages are immune cells responsible for tissue debridement and fighting infection. Clofazimine, an FDA-approved antibiotic, accumulates and precipitates as rod-shaped, crystal-like drug inclusions within macrophage lysosomes. Drug treatment as well as pathophysiological states could induce changes in macrophage mechanical property which in turn impact their phenotype and function. Here we report the use of acoustic tweezing cytometry as a new approach for in situ mechanical phenotyping of macrophages and for targeted macrophage cytotripsy. Acoustic tweezing cytometry applies ultrasound pulses to exert controlled forces to individual cells via integrin-bound microbubbles, enabling a creep test for measuring cellular mechanical property or inducing irreversible changes to the cells. Our results revealed that macrophages with crystal-like drug inclusions became significantly softer with higher cell compliance, and behaved more elastic with faster creep and recovery time constants. On the contrary, phagocytosis of solid polyethylene microbeads or treatment with soluble clofazimine rendered macrophages stiffer. Most notably, application of ultrasound pulses of longer duration and higher amplitude in ATC actuated the integrin-bound microbubbles to mobilize the crystal-like drug inclusions inside macrophages, turning the rod-shaped drug inclusions into intracellular microblender that effectively destructed the cells. This phenomenon of acoustic mechanopharmaceutical cytotripsy may be exploited for ultrasound activated, macrophage-directed drug release and delivery.
INTERNATIONAL/NATIONAL CONFERENCE PROCEEDINGS
LOCAL/REGIONAL MEETINGS/SYMPOSIA
- MD Murashov, V LaLone, PM Rzeczycki, RK Keswani, GS Yoon, S Sud, R Walajapet, SD Larsen, KA Stringer, GR Rosania, “The Physicochemical Basis of Clofazimine’s Skin Pigmentation Side Effect”, American Association of Pharmaceutical Scientists Annual Meeting, San Diego, CA, Nov 12-15, 2017.
- T Woldemichael, RK Keswani, GR Rosania, “Inhibiting the Lysosomal V-ATPase Decreases Intracellular Hydrochloride Crystal Formation by a Weakly Basic Poorly Soluble Drug”, American Association of Pharmaceutical Scientists Annual Meeting, Denver, CO, Nov 13-17, 2016.
- M Arocho, RK Keswani, GR Rosania, "Characterizing the Surface Properties of Biocrystals Based on Their Interactions with Polystyrene Microspheres", Emerging Researchers National Conference in STEM, Washington, DC, February 25-27, 2016.
- C Tian‡, RK Keswani‡, T Peryea, G Gandikota, GR Rosania & X Wang, "Contrast-enhanced photoacoustic tomography of human joints", Proceedings of SPIE (Photonics West), San Francisco, CA, February 13-18, 2016 (‡ - equal authorship).
- RK Keswani, J Baik, A Pawate, PJA Kenis & GR Rosania, "A Three Dimensional Structural Model of Intracellular Crystal-Like Drug Inclusions", American Association of Pharmaceutical Scientists Annual Meeting, Orlando, FL, Oct 25-29, 2015.
- GS Yoon, RK Keswani, S Sud, KA Stringer, GR Rosania, "A Macrophage-Mediated Drug Sequestration and Detoxification Pathway Leads to Inhibition of Inflammation", American Association of Pharmaceutical Scientists Annual Meeting, Orlando, FL, Oct 25-29, 2015.
- GS Yoon, RK Keswani, S Sud, KA Stringer, TJ Standiford, GR Rosania, “Massive Bioaccumulation of Clofazimine Biocrystals in Lung Macrophages Suppress Pro-inflammatory Response Pathways and Alleviate Lung Injury”, American Association of Pharmaceutical Scientists Annual Meeting, Orlando, FL, Oct 25-29, 2015.
- GS Yoon, RK Keswani, S Sud, KA Stringer, TJ Standiford, GR Rosania, "Clofazimine Activates Anti-Inflammatory Signals While Suppressing Pro-Inflammatory Response Pathways Following Massive Bioaccumulation In Alveolar Macrophages", Annual Meeting of the American Thoracic Society - ATS 2015, Denver, CO, May 15-21, 2015.
- KA Stringer, J Trexel, S Sud, RK Keswani, GS Yoon, C McHugh, Y Sun & GR Rosania, "Clofazimine Alters Host Energy Homeostasis that Involves Choline, Glycine and Methionine Metabolism", Metabolomics 2015, Annual Meeting of the Metabolomics Society, San Francisco, CA, Jun 29 - Jul 2, 2015.
- RK Keswani, S Sud, KA Stringer & GR Rosania, "A Novel Coupled Hydrochloride/Chloride-Based Co-precipitation Mechanism for Xenobiotic Sequestration", American Association of Pharmaceutical Scientists Annual Meeting, San Diego, CA, Nov 2-6, 2014.
- RK Keswani, GS Yoon, S Sud, KA Stringer & GR Rosania, "Clofazimine’s Anti-inflammatory Effect is Mediated via Its Macrophage-Targeted Crystallized Forms", American Association of Pharmaceutical Scientists Annual Meeting, San Diego, CA, Nov 2-6, 2014.
- RK Keswani, S Sud, KA Stringer & GR Rosania, "Crystal-Like-Drug-Inclusions (CLDIs) of Clofazimine—A Novel Fluorescent Macrophage Marker", American Association of Pharmaceutical Scientists Annual Meeting, San Diego, CA, Nov 2-6, 2014.
- GS Yoon, RK Keswani, S Sud, KA Stringer, GR Rosania, "Clofazimine crystal-like drug inclusions (CLDIs) downregulate TLR2- and TLR4-mediated inflammatory response in murine macrophages", American Association of Pharmaceutical Scientists Annual Meeting, San Diego, CA, Nov 2-6, 2014.
- RK Keswani, S Sud, KA Stringer & GR Rosania, 2014, "Flow Cytometric Analysis of Specialized Drug-Sequestering Cells of the Immune System", CYTO - Congress of the International Society for Advancement of Cytometry, Fort Lauderdale, FL, May 17-21, 2014.
- NP Gothe, RK Keswani & E McAuley, “Yoga Practice Improves Working Memory Capacity By Attenuating Stress Levels”, The Gerontological Society of America Annual Scientific Meeting, New Orleans, LA, November 20-24, 2013.
- RK Keswani & DW Pack, “Design of Hybrid Retroviral/Synthetic Gene Delivery Vectors”, Annual American Society of Gene and Cell Therapy (ASGCT) Meeting, Philadelphia, PA, May 16-19, 2012.
- RK Keswani, M Lazebnik & DW Pack, “Intracellular Trafficking of Hybrid Retroviral/Synthetic Gene Delivery Vectors”, Annual American Society of Gene and Cell Therapy (ASGCT) Meeting, Philadelphia, PA, May 16-19, 2012.
- RK Keswani & DW Pack, “Recovery of Retroviral Infectivity via Non-Covalent Conjugation with Chitosan”, Annual American Institute of Chemical Engineers (AIChE) Meeting, Minneapolis, MN, October 16-21, 2011. (oral)
- RK Keswani, M Lazebnik & DW Pack, “Reinstatement of Retroviral Infectivity via Non-Covalent Conjugation with lipids”, Annual American Institute of Chemical Engineers (AIChE) Meeting, Minneapolis, MN, October 16-21, 2011. (oral)
- ME Hwang, RK Keswani & DW Pack, “Non-Viral Gene Delivery with 25 kDa PEI and G4 PAMAM are dependent on both clathrin and caveolin trafficking in HeLa cells”, American Society for Gene & Cell Therapy, Seattle, WA, May 19-22, 2011.
- RK Keswani, ME Hwang & DW Pack, “Interplay of clathrin and caveolin trafficking pathways in polymer-based gene delivery”, American Society for Gene & Cell Therapy, Washington, D.C., May 20-22, 2010.
LOCAL/REGIONAL MEETINGS/SYMPOSIA
- TA Koehn, GS Yoon, RK Keswani, GR Rosania & KA Stringer, "Clofazimine (CFZ) Biocrystal Formation in Macrophages", Research Forum - University of Michigan College of Pharmacy, Ann Arbor, MI, Mar 18, 2016.
- RK Keswani, J Baik, A Pawate, PJA Kenis, GR Rosania, "A Three Dimensional Structural Model of Intracellular Crystal-like Drug Inclusions", Great Lakes Drug Metabolism and Disposition Group Annual Meeting, Ann Arbor, MI, May 7-8, 2015
- RK Keswani, GS Yoon, S Sud, P Rzeczycki, KA Stringer, GR Rosania, "Chemical & Fluorescence Analysis of Intracellular Drug Disposition within Macrophages", Great Lakes Drug Metabolism and Disposition Group Annual Meeting, Ann Arbor, MI, May 7-8, 2015
- P Rzeczycki, RK Keswani, GS Yoon, GR Rosania, "Experimental Microanalysis of the Formation of Insoluble Intracellular Drug Complexes", Great Lakes Drug Metabolism and Disposition Group Annual Meeting, Ann Arbor, MI, May 7-8, 2015
- GS Yoon, RK Keswani, S Sud, KA Stringer, GR Rosania, "Clofazimine crystal-like-drug inclusions (CLDIs) down-regulate TLR2- and TLR-4 mediated inflammatory response in murine macrophages", Great Lakes Drug Metabolism and Disposition Group Annual Meeting, Ann Arbor, MI, May 7-8, 2015
- GS Yoon, S Sud, RK Keswani, KA Stringer, GR Rosania, "Clofazimine exhibits profound anti-inflammatory activity in vivo by boosting systemic IL-1RA expression", Great Lakes Drug Metabolism and Disposition Group Annual Meeting, Ann Arbor, MI, May 7-8, 2015
- TA Koehn, RK Keswani, GR Rosania & KA Stringer, "Establishing in vitro Growth Conditions for Clofazimine (CFZ) Crystals using Macrophages", Annual MICHR Symposium, Ann Arbor, MI, Oct 1, 2014.
- RK Keswani, S Sud, KA Stringer & GR Rosania, 2014, "Old is Gold - an in vivo self-assembling crystal of clofazimine", Great Lakes Drug Metabolism and Drug Disposition Meeting Annual Meeting, Indianapolis, IN, May 15-16, 2014.
- RK Keswani, R Banerjee, J Swanson & GR Rosania, “Molecular Engineering of Self-Organizing & Site-Directed Intracellular Crystals in Living Organisms”, 1st Annual M-Cubed Symposium, Ann Arbor, MI, November 15, 2013.
- IM Pozdol, RK Keswani & DW Pack, "Design of Lipid-based Hybrid Retroviral Vectors for Cancer Gene Therapy", Cancer Community at Illinois Symposium, Urbana, IL, April 4-5, 2012.
- M Lazebnik, RK Keswani, M Thompson & DW Pack, "Elucidation of Endocytosis Pathways in siRNA Delivery", Cancer Community at Illinois Symposium, Urbana, IL, April 4-5, 2012.
- RK Keswani & DW Pack, “Development of Hybrid Viral/Non-Viral Vectors: Chitosan-MLV Conjugates”, Chemical & Molecular Biology Training Grant Symposium, Urbana, IL, October 22, 2010. - selected for oral presentation.
- DM Drake, RK Keswani, DW Pack, “Design, Optimization, and Intracellular Trafficking of Polyethylenimine/Virus-Like Particle Hybrid Gene Delivery Vectors”, AIChE Midwest Regional Conference, Chicago, IL, September 30-October 1, 2010.
- RK Keswani & DW Pack, “Design of Hybrid/Viral Gene Therapy Vectors via Association of Murine Leukemia Viruses with Synthetic Agents”, BioEngineering@Illinois 2010, Urbana, IL, April 9, 2010.
- RK Keswani, A Chiu & DW Pack, “Surface Modification of a Murine Leukemia Virus Particle”, Chemical & Molecular Biology Training Grant Symposium, Urbana, IL, October 16, 2009. - selected for oral presentation
- RK Keswani, DM Drake & DW Pack, “Design of hybrid-viral gene therapy vectors via association of murine leukemia viruses with synthetic agents”, ChBE Graduate Research Symposium, Urbana, IL, October 10, 2008.
- RK Keswani, DM Drake & DW Pack, “Design of hybrid-viral gene therapy vectors via association of murine leukemia viruses with synthetic agents”, AIChE Midwest Regional Conference, Chicago, IL, September 22-23, 2008. (oral)
- “On Supramolecular Assembly and Subcellular Biochemistry: Mechanistic Principles for Design and Delivery of Therapeutics”, Integrative Biosciences Center (IBio), Wayne State University, Detroit, MI, February 15, 2017.
- “On Supramolecular Assembly and Subcellular Biochemistry: Mechanistic Principles for Design and Delivery of Therapeutics”, Department of Chemical Engineering, University of Louisville, Louisville, KY, October 31, 2016.
- “Synthesizing Functional Mimics of the Retroviral Envelope Protein: Applications in Gene Therapy”, Eli Lilly and Company, Indianapolis, IN, September 15, 2016.
- “On Supramolecular Assembly and Subcellular Biochemistry: Mechanistic Principles for Design and Delivery of Therapeutics”, Department of Chemical Engineering, University of New Hampshire, Durham, NH, April 11, 2016.
- “On Supramolecular Assembly and Subcellular Biochemistry: Mechanistic Principles for Design and Delivery of Therapeutics”, Department of Chemical Engineering, Auburn University, Auburn, AL, March 7, 2016.
- “On Supramolecular Assembly and Subcellular Biochemistry: Mechanistic Principles for Design and Delivery of Therapeutics”, Department of Chemical and Biomedical Engineering, Florida A&M University-Florida State University, Tallahassee, FL, March 1, 2016.
- “On Supramolecular Assembly and Subcellular Biochemistry: Mechanistic Principles for Design and Delivery of Therapeutics”, Department of Biomedical Engineering, Rowan University, Glassboro, NJ, February 22, 2016.
- GR Rosania*, KA Stringer*, RK Keswani*, GS Yoon*,“Supercharging Macrophages”, 2nd Annual M-Cubed Symposium, Ann Arbor, MI, October 9, 2014 - selected for showcase talk, * - denotes co-presenters.
- “Hybrid Retroviral/Synthetic Gene Delivery Vectors: Design and Mechanisms”, University of Michigan Ann Arbor College of Pharmacy, Ann Arbor, MI, May 6, 2013
- “Design of Hybrid Retroviral/Synthetic Gene Delivery Vectors”, Pfizer, Pearl River, NY, April 16, 2012
- “Design of Hybrid Viral/Non-Viral Gene Delivery Vectors”, Orochem Technologies, Lombard, IL, February 4, 2012
- "Hybrid Viral Vectors", Catherine Connor Dissertation Award Reception, Biotechnology Center, March 1, 2011
- “Preparation for Graduate Studies”, ACS Student Chapter, Urbana, IL, October 2009
- EM Horstman, RK Keswani, T Woldemichael, PM Rzeczycki, MD Murashov, PJA Kenis, GR Rosania, “Macrophage-Stabilized Polymorphs of Weakly Basic Drug Salts”, International Patent Application Number WO2018/081072 A1
- RK Keswani, GS Yoon, KA Stringer, C Tian, XD Wang, GR Rosania, "Compositions and Methods for Delivering Pharmaceutical Agents", US Patent Application No. 15/011047, 2016. (link) (pdf)
- D Sharma, RK Keswani, W Mahmood, AA Oroskar, "SMB Process for the Purification of Ethanol and Butanediol with Integrated Regeneration”, US Patent 8704016 B2. (link) (pdf)