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1.  PTD-Modified ATTEMPTS for Enhanced Toxin-based Cancer Therapy: An In Vivo Proof-of-Concept Study 
Pharmaceutical research  2015;32(8):2690-2703.
Purpose
To investigate the feasibility of applying PTD-modified ATTEMPTS (Antibody Targeted Triggered Electrically Modified Prodrug-Type Strategy) for enhanced toxin therapy for the treatment of cancer.
Methods
A heparin-functionalized murine anti-CEA monoclonal antibody (mAb), T84.66-heparin (T84.66-Hep), was chemically synthesized and characterized for specific binding to CEA overexpressed cells. The T84.66-Hep was then applied to the PTD-modified ATTEMPTS approach and the crucial features of the drug delivery system (DDS), ‘antibody targeting’ and ‘heparin/protamine-based prodrug’, were evaluated in vitro to examine whether it could selective delivery a PTD-modified toxin, recombinant TAT-gelonin chimera (TAT-Gel), to CEA high expression cancer cells (LS174T). Furthermore, the feasibility of the drug delivery system (DDS) was assessed in vivo by biodistribution and efficacy studies using LS174T s.c. xenograft tumor bearing mice.
Results
T84.66-Hep displayed specific binding, but limited internalization (35% after 48 h incubation) to CEA high expression LS174T cells over low expression HCT116 cells. When mixed together with TAT-Gel, the T84.66-Hep formed a strong yet reversible complex. This complex formation provided an effective means of active tumor targeting of TAT-Gel, by 1) directing the TAT-Gel to CEA overexpressed tumor cells and 2) preventing nonspecific cell transduction to non-targeted normal cells. The cell transduction of TAT-Gel could, however, be efficiently reversed by addition of protamine. Feasibility of in vivo tumor targeting and “protamine-induced release” of TAT-Gel from the T84.66-Hep counterpart was confirmed by biodistribution and preliminary efficacy studies.
Conclusions
This study successfully demonstrated in vitro and in vivo the applicability of PTD-modified ATTEMPTS for toxin-based cancer therapy.
doi:10.1007/s11095-015-1653-y
PMCID: PMC4490053  PMID: 25701313
Toxin; Protein transduction domains; Anti-CEA monoclonal antibody; Heparin; Cancer
2.  Recombinant TAT-Gelonin Fusion Toxin: Synthesis and Characterization of Heparin/Protamine-Regulated Cell Transduction 
Protein toxins, such as gelonin, are highly desirable anti-cancer drug candidates due to their unparalleled potency and repetitive reaction mechanism in inhibiting protein translation. However, for its potential application in cancer therapy, there remains the cell membrane barrier that allows permeation of only small molecules, which must be overcome. To address this challenge, we conjugated gelonin with a protein transduction domain (PTD), the TAT peptide, via genetic recombination. The chimeric TAT-gelonin fusion protein (TAT-Gel) retained equipotent N-glycosidase activity yet displayed greater cell uptake than unmodified recombinant gelonin (rGel), thereby yielding a significantly augmented cytotoxic activity. Remarkably, TATGel displayed up to 177-fold lower IC50 (avg. 54.3 nM) than rGel (avg. IC50: 3640 nM) in tested cell lines. This enhanced cytotoxicity, however, also raised potential toxicity concerns due to the non-selectivity of PTD in its mediated cell transduction. To solve this problem, we investigated the plausibility of regulating the cell transduction of TAT-Gel via a reversible masking using heparin and protamine. Here, we demonstrated, both in vitro and in vivo, that the cell transduction of TAT-Gel can be completely curbed with heparin and yet this heparin block can be efficiently reversed by the addition of protamine. This reversible tight regulation of the cell transduction of TAT-Gel by heparin and protamine sheds light of possible application of TATGel in achieving a highly effective yet safe drug therapy for the treatment of tumors.
doi:10.1002/jbm.a.35188
PMCID: PMC4198515  PMID: 24733757
Gelonin; TAT; Heparin; Protamine; Tumor
3.  Massive Bioaccumulation and Self-Assembly of Phenazine Compounds in Live Cells 
Advanced science  2015;2(8):1500025.
Clofazimine is an orally administered, FDA-approved drug that massively bioaccumulates in macrophages, forming membrane-bound intracellular structures possessing nanoscale supramolecular features. Here, a library of phenazine compounds derived from clofazimine was synthesized and tested for their ability to accumulate and form ordered molecular aggregates inside cells. Regardless of chemical structure or physicochemical properties, bioaccumulation was consistently greater in macrophages than in epithelial cells. Microscopically, some self-assembled structures exhibited 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 was 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 led 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.
doi:10.1002/advs.201500025
PMCID: PMC4569013  PMID: 26380168
Medicinal chemistry; clofazimine; imaging; aggregation; chromophores; biocrystals; polarization
4.  Chemically and Biologically Synthesized CPP-Modified Gelonin for Enhanced Anti-tumor Activity 
The ineffectiveness of small molecule drugs against cancer has generated significant interest in more potent macromolecular agents. Gelonin, a plant-derived toxin that inhibits protein translation, has attracted much attention in this regard. Due to its inability to internalize into cells, however, gelonin exerts only limited tumoricidal effect. To overcome this cell membrane barrier, we modified gelonin, via both chemical conjugation and genetic recombination methods, with low molecular weight protamine (LMWP), a cell-penetrating peptide (CPP) which was shown to efficiently ferry various cargos into cells. Results confirmed that gelonin-LMWP chemical conjugate (cG-L) and recombinant gelonin-LMWP chimera (rG-L) possessed N-glycosidase activity equivalent to that of unmodified recombinant gelonin (rGel); however, unlike rGel, both gelonin-LMWPs were able to internalize into cells. Cytotoxicity studies further demonstrated that cG-L and rG-L exhibited significantly improved tumoricidal effects, with IC50 values being 120-fold lower than that of rGel. Moreover, when tested against a CT26 s.c. xenograft tumor mouse model, significant inhibition of tumor growth was observed with rG-L doses as low as 2 μg/tumor, while no detectable therapeutic effects were seen with rGel at 10-fold higher doses. Overall, this study demonstrated the potential of utilizing CPP-modified gelonin as a highly potent anticancer drug to overcome limitations of current chemotherapeutic agents.
doi:10.1016/j.jconrel.2013.08.016
PMCID: PMC3849409  PMID: 23973813
Gelonin; LMWP; Cell penetrating peptide; Cancer; Ribosome-inactivating protein; Toxin
5.  The Extracellular Microenvironment Explains Variations in Passive Drug Transport across Different Airway Epithelial Cell Types 
Pharmaceutical research  2013;30(8):2118-2132.
Purpose
We sought to identify key variables in cellular architecture and physiology that might explain observed differences in the passive transport properties of small molecule drugs across different airway epithelial cell types.
Methods
Propranolol (PR) was selected as a weakly basic, model compound to compare the transport properties of primary (NHBE) vs. tumor-derived (Calu-3) cells. Differentiated on Transwell™ inserts, the architecture of pure vs. mixed cell co-cultures was studied with confocal microscopy followed by quantitative morphometric analysis. Cellular pharmacokinetic modeling was used to identify parameters that differentially affect PR uptake and transport across these two cell types.
Results
Pure Calu-3 and NHBE cells possessed different structural and functional properties. Nevertheless, mixed Calu-3 and NHBE cell co-cultures differentiated as stable cell monolayers. After measuring the total mass of PR, the fractional areas covered by Calu-3 and NHBE cells allowed deconvoluting the transport properties of each cell type. Based on the apparent thickness of the unstirred, cell surface aqueous layer, local differences in extracellular microenvironment explained the measured variations in passive PR uptake and permeation between Calu-3 and NHBE cells.
Conclusion
Mixed cell co-cultures can be used to compare the local effects of the extracellular microenvironment on drug uptake and transport across two epithelial cell types.
doi:10.1007/s11095-013-1069-5
PMCID: PMC3706189  PMID: 23708857
cellular pharmacokinetics; Calu-3 cells; local drug absorption; inhaled drug delivery; computational modeling
6.  Pulsed Magnetic Field Improves the Transport of Iron Oxide Nanoparticles through Cell Barriers 
ACS nano  2013;7(3):2161-2171.
Understanding how a magnetic field affects the interaction of magnetic nanoparticles (MNPs) with cells is fundamental to any potential downstream applications of MNPs as gene and drug delivery vehicles. Here, we present a quantitative analysis of how a pulsed magnetic field influences the manner in which MNPs interact with, and penetrate across a cell monolayer. Relative to a constant magnetic field, the rate of MNP uptake and transport across cell monolayers was enhanced by a pulsed magnetic field. MNP transport across cells was significantly inhibited at low temperature under both constant and pulsed magnetic field conditions, consistent with an active mechanism (i.e. endocytosis) mediating MNP transport. Microscopic observations and biochemical analysis indicated that, in a constant magnetic field, transport of MNPs across the cells was inhibited due to the formation of large (>2 μm) magnetically-induced MNP aggregates, which exceeded the size of endocytic vesicles. Thus, a pulsed magnetic field enhances the cellular uptake and transport of MNPs across cell barriers relative to a constant magnetic field by promoting accumulation while minimizing magnetically-induced MNP aggregates at the cell surface.
doi:10.1021/nn3057565
PMCID: PMC3609927  PMID: 23373613
Magnetic field; Superparamagnetic iron oxide nanoparticles; Magnetic targeting; Drug delivery; Bioimaging; Magnetically-guided therapy; Cell-based assays
7.  Pulmonary Administration of a Water-Soluble Curcumin Complex Reduces Severity of Acute Lung Injury 
Local or systemic inflammation can result in acute lung injury (ALI), and is associated with capillary leakage, reduced lung compliance, and hypoxemia. Curcumin, a plant-derived polyphenolic compound, exhibits potent anti-inflammatory properties, but its poor solubility and limited oral bioavailability reduce its therapeutic potential. A novel curcumin formulation (CDC) was developed by complexing the compound with hydroxypropyl-γ-cyclodextrin (CD). This results in greatly enhanced water solubility and stability that facilitate direct pulmonary delivery. In vitro studies demonstrated that CDC increased curcumin’s association with and transport across Calu-3 human airway epithelial cell monolayers, compared with uncomplexed curcumin solubilized using DMSO or ethanol. Importantly, Calu-3 cell monolayer integrity was preserved after CDC exposure, whereas it was disrupted by equivalent uncomplexed curcumin solutions. We then tested whether direct delivery of CDC to the lung would reduce severity of ALI in a murine model. Fluorescence microscopic examination revealed an association of curcumin with cells throughout the lung. The administration of CDC after LPS attenuated multiple markers of inflammation and injury, including pulmonary edema and neutrophils in bronchoalveolar lavage fluid and lung tissue. CDC also reduced oxidant stress in the lungs and activation of the proinflammatory transcription factor NF-κB. These results demonstrate the efficacy of CDC in a murine model of lung inflammation and injury, and support the feasibility of developing a lung-targeted, curcumin-based therapy for the treatment of patients with ALI.
doi:10.1165/rcmb.2011-0175OC
PMCID: PMC3488693  PMID: 22312018
cyclodextrin; LPS; turmeric; Calu-3; oxidative stress; TEER
8.  A Cell-based Computational Modeling Approach for Developing Site-Directed Molecular Probes 
PLoS Computational Biology  2012;8(2):e1002378.
Modeling the local absorption and retention patterns of membrane-permeant small molecules in a cellular context could facilitate development of site-directed chemical agents for bioimaging or therapeutic applications. Here, we present an integrative approach to this problem, combining in silico computational models, in vitro cell based assays and in vivo biodistribution studies. To target small molecule probes to the epithelial cells of the upper airways, a multiscale computational model of the lung was first used as a screening tool, in silico. Following virtual screening, cell monolayers differentiated on microfabricated pore arrays and multilayer cultures of primary human bronchial epithelial cells differentiated in an air-liquid interface were used to test the local absorption and intracellular retention patterns of selected probes, in vitro. Lastly, experiments involving visualization of bioimaging probe distribution in the lungs after local and systemic administration were used to test the relevance of computational models and cell-based assays, in vivo. The results of in vivo experiments were consistent with the results of in silico simulations, indicating that mitochondrial accumulation of membrane permeant, hydrophilic cations can be used to maximize local exposure and retention, specifically in the upper airways after intratracheal administration.
Author Summary
We have developed an integrative, cell-based modeling approach to facilitate the design and discovery of chemical agents directed to specific sites of action within a living organism. Here, a computational, multiscale transport model of the lung was adapted to enable virtual screening of small molecules targeting the epithelial cells of the upper airways. In turn, the transport behaviors of selected candidate probes were evaluated to establish their degree of retention at a site of absorption, using computational simulations as well as two in vitro cell-based assay systems. Lastly, bioimaging experiments were performed to examine candidate molecules' distribution in the lungs of mice after local and systemic administration. Based on computational simulations, the higher mitochondrial density per unit absorption surface area is the key parameter determining the higher retention of small molecule hydrophilic cations in the upper airways, relative to lipophilic weak bases, specifically after intratracheal administration.
doi:10.1371/journal.pcbi.1002378
PMCID: PMC3285574  PMID: 22383866
9.  Transcellular Transport of Heparin-coated Magnetic Iron Oxide Nanoparticles (Hep-MION) Under the Influence of an Applied Magnetic Field 
Pharmaceutics  2010;2(2):119-135.
In this study, magnetic iron oxide nanoparticles coated with heparin (Hep-MION) were synthesized and the transcellular transport of the nanoparticles across epithelial cell monolayers on porous polyester membranes was investigated. An externally applied magnetic field facilitated the transport of the Hep-MION across cell monolayers. However, high Hep-MION concentrations led to an increased aggregation of nanoparticles on the cell monolayer after application of the magnetic field. Our results indicate that magnetic guidance of Hep-MION most effectively promotes transcellular transport under conditions that minimize formation of magnetically-induced nanoparticle aggregates. Across cell monolayers, the magnet’s attraction led to the greatest increase in mass transport rate in dilute dispersions and in high serum concentrations, suggesting that magnetic guidance may be useful for in vivo targeting of Hep-MION.
doi:10.3390/pharmaceutics2020119
PMCID: PMC2997712  PMID: 21152371
magnetic iron oxide nanoparticles (MION); magnetic field; transcellular transport; MDCK cell monolayer; drug targeting
10.  Transcellular Transport of Heparin-coated Magnetic Iron Oxide Nanoparticles (Hep-MION) Under the Influence of an Applied Magnetic Field 
Pharmaceutics  2010;2(2):119-135.
In this study, magnetic iron oxide nanoparticles coated with heparin (Hep-MION) were synthesized and the transcellular transport of the nanoparticles across epithelial cell monolayers on porous polyester membranes was investigated. An externally applied magnetic field facilitated the transport of the Hep-MION across cell monolayers. However, high Hep-MION concentrations led to an increased aggregation of nanoparticles on the cell monolayer after application of the magnetic field. Our results indicate that magnetic guidance of Hep-MION most effectively promotes transcellular transport under conditions that minimize formation of magnetically-induced nanoparticle aggregates. Across cell monolayers, the magnet’s attraction led to the greatest increase in mass transport rate in dilute dispersions and in high serum concentrations, suggesting that magnetic guidance may be useful for in vivo targeting of Hep-MION.
doi:10.3390/pharmaceutics2020119
PMCID: PMC2997712  PMID: 21152371
magnetic iron oxide nanoparticles (MION); magnetic field; transcellular transport; MDCK cell monolayer; drug targeting

Results 1-10 (10)