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1.  Synthesis, Characterization, and Evaluation of Pluronic-Based β-Cyclodextrin Polyrotaxanes for Mobilization of Accumulated Cholesterol from Niemann-Pick Type C Fibroblasts 
Biochemistry  2013;52(19):3242-3253.
Several lines of evidence suggest that β-cyclodextrin (β-CD) derivatives initiate the efflux of accumulated, unesterified cholesterol from the late endosomal/lysosomal compartment in Niemann Pick C (NPC) disease models. Unfortunately, repeated injections or continuous infusions of current β-CD therapies are required to sustain suppression of symptoms and prolong life. In an effort to make CD treatment a more viable option by boosting efficacy and improving pharmacokinetics, a library of Pluronic surfactant-based β-CD polyrotaxanes has been developed using biocompatible poly(ethylene glycol) (PEG)–polypropylene glycol (PPG)–PEG triblock copolymers. These compounds carry multiple copies of β-CD as shown by 1H NMR, 2D nuclear Overhouser effect spectroscopy, gel permeation chromatography/multiangle light scattering, analytical ultracentrifugation analysis, matrix assisted laser desorption/ionization mass spectrometry, and diffusion-ordered spectroscopy. Analyses of free β-cyclodextrin contamination in the compounds were made by reverse phase high pressure liquid chromatography and hydrophilic interaction liquid chromatography. Dethreading kinetics were studied by reverse phase high pressure liquid chromatography, UV/vis, and 1H NMR analysis. Filipin staining studies using npc2−/− fibroblasts show significant reversal of cholesterol accumulation after treatment with polyrotaxane compounds. The rate and efficacy of reversal is similar to that achieved by equivalent amounts of monomeric β-CD alone.
PMCID: PMC4319568  PMID: 23560535
2.  Synthesis of 2-Hydroxypropyl-β-cyclodextrin/Pluronic-Based Polyrotaxanes via Heterogeneous Reaction as Potential Niemann-Pick Type C Therapeutics 
Biomacromolecules  2013;14(12):4189-4197.
Five polyrotaxanes were synthesized by threading 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) onto a variety of α,ω-ditriethylenediamino-N-carbamoyl-poly-(ethylene oxide)-block-poly(propylene oxide)-block-poly-(ethylene oxide) (Pluronic) triblock copolymers using a two-pot strategy under heterogeneous, nonaqueous conditions. The threaded HP-β-CD units were retained on the pseudopolyrotaxane precursors by end-capping the branched diamine termini with sodium 2,4,6-trinitrobenzene sulfonate. Inclusion of the Pluronic copolymers within the HP-β-CD cavities was more favorable in nonpolar solvents, such as diethyl ether and n-hexane, both of which gave better coverage ratios than polar solvents. 1H NMR and MALDI-TOF were used to estimate the average molecular weights of the purified polyrotaxane products. A globular morphology of aggregated polyrotaxanes was observed by tapping-mode AFM imaging of dried samples. Treatment of Niemann-Pick C (NPC) type 2-deficient fibroblasts with the polyrotaxane derivatives produced substantial reductions in sterol accumulation, as seen by diminished filipin staining in these cells, suggesting that Pluronic-based polyrotaxanes may be promising vehicles for delivery of HP-β-CD to cells with abnormal cholesterol accumulation.
PMCID: PMC4314287  PMID: 24180231
3.  Effect of Pendant Group on pDNA Delivery by Cationic-β-Cyclodextrin:Alkyl-PVA-PEG Pendant Polymer Complexes 
Biomacromolecules  2013;15(1):12-19.
We have previously shown that cationic-β-CD:R-poly(vinyl alcohol)-poly(ethylene glycol) pendant polymer host:guest complexes are safe and efficient vehicles for nucleic acid delivery, where R = benzylidene-linked adamantyl or cholesteryl esters. Herein, we report the synthesis and biological performance of a family of PVA-PEG pendant polymers whose pendant groups have a wide range of different affinities for the β-CD cavity. Cytotoxicity studies revealed that all of the cationic-β-CD:pendant polymer host:guest complexes have 100 – 1000-fold lower toxicity than bPEI, with pDNA transfection efficiencies that are comparable to branched polyethylenimine (bPEI) and Lipofectamine 2000 (L2K). Complexes formed with pDNA at N/P ratios greater than 5 produced particles with diameters in the 100 – 170 nm range and ζ-potentials of 15 – 35 mV. Gel shift and heparin challenge experiments showed that the complexes are most stable at N/P ≥ 10, with adamantyl- and noradamantyl-modified complexes displaying the best resistance toward heparin-induced decomplexation. Disassembly rates of fluoresceinated-pDNA:CD+:R-PVA-PEG-rhodamine complexes within HeLa cells showed a modest dependence on host:guest binding constant, with adamantyl-, noradamantyl-, and dodecyl-based complexes showing the highest FRET efficiency 9 h after cellular exposure. These findings suggest that the host:guest binding constant has a significant impact on the colloidal stability in the presence of serum, cellular uptake efficiency, endosomal disassembly, and transfection performance of cationic-β-CD:R-poly(vinyl alcohol)-poly(ethylene glycol) pendant polymer complexes.
PMCID: PMC3899244  PMID: 24295406
Gene delivery; Cyclodextrins; Pendant Polymers; Transfection; pDNA
4.  Microfluidic Assembly of Cationic-β-Cyclodextrin:Hyaluronic Acid-Adamantane Host:Guest pDNA Nanoparticles 
Biomaterials science  2013;1(10):10.1039/C3BM00189J.
Traditionally, transfection complexes are typically formed by bulk mixing, producing particles with high polydispersity and limited control over vector size. Herein, we demonstrate the use of a commercial micro-reactor to assemble pDNA:cationic cyclodextrin:pendant polymer nanoparticles using a layer-by-layer approach. Our studies reveal that the particles formulated via microfluidic assembly have much smaller sizes, lower polydispersity, lower ζ-potentials, and comparable cell viability and transfection profiles in HeLa cells than bulk mixed particles. The complexes also show a flow rate-dependent stability, with particles formed at slower flow rates giving rise to more stable complexes as determined by heparin challenge. Our findings suggest that microfluidic reactors offer an attractive method for assembling reproducible, size-controlled complexes from multi-component transfection complex assemblies.
PMCID: PMC3859440  PMID: 24349706
5.  Cationic α-Cyclodextrin:Poly(ethylene glycol) Polyrotaxanes for siRNA Delivery 
Molecular pharmaceutics  2013;10(4):1299-1305.
RNA interference has broad therapeutic potential due to its high specificity and ability to potentially evade drug resistance. Three cationic α-cyclodextrin:poly(ethylene glycol) polyrotaxanes derived from polymer axle different sizes (MW 2000, 3400 and 10000) have been synthesized for delivering siRNA. These polyrotaxanes are able to condense siRNA into positively charged particles that are < 200 nm in diameter, enabling their facile internalization into mammalian cells. The cationic polyrotaxanes display cytotoxicity profiles that are >102-fold lower than the commercial standard bPEI and gene silencing efficiencies that are comparable to both Lipofectamine 2000 and bPEI. Our findings suggest that the cationic polyrotaxanes display a size-activity relationship, wherein, the higher molecular weight polyrotaxanes (PEG3400 and 10000) are able to condense and deliver siRNA better than the lower molecular weight material (PEG2000).
PMCID: PMC4083495  PMID: 23398604
gene delivery; siRNA; cyclodextrins
6.  Multi-armed cationic cyclodextrin:poly(ethylene glycol) polyrotaxanes as efficient gene silencing vectors† 
A family of branched polyrotaxanes (bPRTx+), threaded with multiple cationic α-cyclodextrins (α-CDs) onto a multi-armed poly(ethylene glycol) (PEG) core, were synthesized and studied as gene silencing vectors. These bPRTx+ formed stable, positively charged complexes with diameters of 150–250 nm at N/P ratios as low as 2.5. The bPRTx+ materials were shown to have gene-silencing efficiencies comparable to those of Lipofectamine 2000 (L2k) and bPEI, while displaying similar toxicity profiles. The unique structure of these polyrotaxanes allows them to effectively condense and complex siRNA into nanoparticles at much lower N/P ratios than L2k or bPEI. These findings suggest that bPRTx+ may be useful materials for gene therapy applications.
PMCID: PMC3524380  PMID: 23042106
7.  Development of a Low Toxicity, Effective pDNA Vector Based on Non-covalent Assembly of Bioresponsive Amino-β-Cyclodextrin:Adamantane-Poly(vinyl alcohol)-Poly(ethylene glycol) Transfection Complexes 
Bioconjugate chemistry  2012;23(5):933-940.
A host:guest-derived gene delivery vector has been developed, based on the self-assembly of cationic β-CD derivatives with a poly(vinyl alcohol)MW27kD (PVA) main chain polymer bearing poly(ethylene glycol)MW750 (PEG) or MW2000 PEG and acid-labile adamantane-modified (Ad) grafts through an acid-sensitive benzylidene acetal linkage. These components were investigated for their ability to promote supramolecular complex formation with pDNA using two different assembly schemes, involving either pre-complexation of the pendant Ad-PVA-PEG polymer with the cationic β-CD derivatives before pDNA condensation (Method A) or pDNA condensation with the cationic β-CD derivatives prior to addition of Ad-PVA-PEG to engage host:guest complexation (Method B). The pendant polymers were observed to degrade under acidic conditions, while remaining intact for more than 5 d at pH 7. HeLa cell culture data shows that these materials have 103-fold lower cytotoxicities than 25 kD bPEI, while maintaining transfection efficiencies that are superior to those observed for this benchmark cationic polymer transfection reagent when the Method A assembly scheme is employed. These findings suggest that degradable cationic polymer constructs employing multivalent host:guest interactions may be an effective and low-toxicity vehicle for delivering nucleic acid cargo to target cells.
PMCID: PMC3417082  PMID: 22551467
8.  Dynamin-SNARE interactions control trans-SNARE formation in intracellular membrane fusion 
Nature communications  2013;4:1704.
The fundamental processes of membrane fission and fusion determine size and copy numbers of intracellular organelles. While SNARE proteins and tethering complexes mediate intracellular membrane fusion, fission requires the presence of dynamin or dynamin-related proteins. Here we study these reactions in native yeast vacuoles and find that the yeast dynamin homolog Vps1 is not only an essential part of the fission machinery, but also controls membrane fusion by generating an active Qa SNARE- tethering complex pool, which is essential for trans-SNARE formation. Our findings provide new insight into the role of dynamins in membrane fusion by directly acting on SNARE proteins.
PMCID: PMC3630463  PMID: 23591871
9.  Biosynthesis of albomycin δ2 provides a template for assembling siderophore and aminoacyl-tRNA synthetase inhibitor conjugates 
ACS chemical biology  2012;7(9):1565-1575.
“Trojan horse” antibiotic albomycins are peptidyl nucleosides consisting of a highly modified 4′-thiofuranosyl cytosine moiety and a ferrichrome siderophore that are linked by a peptide bond via a serine residue. While the latter component serves to sequester iron from the environment, the seryl nucleoside portion is a potent inhibitor of bacterial seryl-tRNA synthetases, resulting in broad-spectrum antimicrobial activities of albomycin δ2. The isolation of albomycins has revealed this biological activity is only optimized following two unusual cytosine modifications, N4-carbamoylation and N3-methylation. We identified a genetic locus (named abm) for albomycin production in Streptomyces sp. ATCC 700974. Gene deletion and complementation experiments along with bioinformatic analysis suggested 18 genes are responsible for albomycin biosynthesis and resistance, allowing us to propose a potential biosynthetic pathway for installing the novel chemical features. The gene abmI, encoding a putative methyltransferase, was functionally assigned in vitro and shown to modify the N3 of a variety of cytosine-containing nucleosides and antibiotics such as blasticidin S. Furthermore, a ΔabmI mutant was shown to produce the descarbamoyl-desmethyl albomycin analog, supporting that the N3-methylation occurs before the N4-carbamoylation in the biosynthesis of albomycin δ2. The combined genetic information was utilized to identify an abm-related locus (named ctj) from the draft genome of Streptomyces sp. C. Cross-complementation experiments and in vitro studies with CtjF, the AbmI homolog, suggest the production of a similar 4′-thiofuranosyl cytosine in this organism. In total, the genetic and biochemical data provide a biosynthetic template for assembling siderophore-inhibitor conjugates, and modifying the albomycin scaffold to generate new derivatives.
PMCID: PMC3448783  PMID: 22704654
10.  Effective Targeted Gene Delivery to Dendritic Cells via Synergetic Interaction of Mannosylated Lipid with DOPE and BCAT 
Biomacromolecules  2012;13(3):636-644.
The efficient delivery of plasmids encoding antigenic determinants into dendritic cells (DCs) that control immune response is a promising strategy for rapid development of new vaccines. In this study, we prepared a series of targeted cationic lipoplex based on two synthetic lipid components, mannose-poly(ethylene glycol, MW3000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (Mannose-PEG3000-DSPE) and O-(2R-1,2-di-O-(1'Z,9'Z-octadecadienyl)-glycerol)-3-N-(bis-2-aminoethyl)-carbamate (BCAT), that were formulated with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) for evaluation as non-viral vectors for transgene expression in DCs. First, we optimized the N:P ratio for maximum transfection and then screened the effects of mannose targeting for further enhancement of transfection levels. Our results indicate that efficient delivery of gWIZ GFP plasmid into DCs was observed for mannose compositions of ~10%, whereas low transfection efficiencies were observed with non-targeted formulations. Mannose-targeted lipofectamine complexes also showed high GFP expression levels in DCs relative to non-targeted lipofectamine controls. The best transfection performance was observed using 10 mol % Mannose-PEG3000-DSPE, 60 mol% BCAT, and 30 mol % DOPE, indicating that the most efficient delivery into DCs occurs via synergistic interaction between mannose targeting and acid-labile, fusogenic BCAT:DOPE formulations. Our data suggest that mannose-PEG3000-DSPE:BCAT:DOPE formulations may be effective gene delivery vehicles for the development of DC-based vaccines.
PMCID: PMC3299835  PMID: 22229467
11.  Heterogeneous catalytic hydrogenation of unprotected indoles in water: A green solution to a long-standing challenge 
Organic letters  2011;13(19):5124-5127.
An environmentally benign procedure for the hydrogenation of unprotected indoles is described. The hydrogenation reaction is catalyzed by Pt/C and activated by p-toluenesulfonic acid in water as a solvent. The efficacy of the method is illustrated by the hydrogenation of a variety of substituted indoles to their corresponding indolines which were obtained in excellent yields.
PMCID: PMC3184324  PMID: 21902212
12.  A tethering complex dimer catalyzes trans-SNARE complex formation in intracellular membrane fusion 
Bioarchitecture  2012;2(2):59-69.
SNARE complexes mediate membrane fusion in the endomembrane system. They consist of coiled-coil bundles of four helices designated as Qa, Qb, Qc and R. A critical intermediate in the fusion pathway is the trans-SNARE complex generated by the assembly of SNAREs residing in opposing membranes. Mechanistic details of trans-SNARE complex formation and topology in a physiological system remain largely unresolved. Our studies on native yeast vacuoles revealed that SNAREs alone are insufficient to form trans-SNARE complexes and that additional factors, potentially tethering complexes and Rab GTPases, are required for the process. Here we report a novel finding that a HOPS tethering complex dimer catalyzes Rab GTPase-dependent formation of a topologically preferred QbQcR-Qa trans-SNARE complex.
PMCID: PMC3383723  PMID: 22754631
HOPS tethering complex dimer; QbQcR-Qa trans-SNARE complex; Rab GTPase
13.  Sequential Analysis of Trans-SNARE Formation in Intracellular Membrane Fusion 
PLoS Biology  2012;10(1):e1001243.
SM proteins stabilize cis-SNARE complexes leading to a specific preferred topology for trans-SNARE formation.
SNARE complexes are required for membrane fusion in the endomembrane system. They contain coiled-coil bundles of four helices, three (Qa, Qb, and Qc) from target (t)-SNAREs and one (R) from the vesicular (v)-SNARE. NSF/Sec18 disrupts these cis-SNARE complexes, allowing reassembly of their subunits into trans-SNARE complexes and subsequent fusion. Studying these reactions in native yeast vacuoles, we found that NSF/Sec18 activates the vacuolar cis-SNARE complex by selectively displacing the vacuolar Qa SNARE, leaving behind a QbcR subcomplex. This subcomplex serves as an acceptor for a Qa SNARE from the opposite membrane, leading to Qa-QbcR trans-complexes. Activity tests of vacuoles with diagnostic distributions of inactivating mutations over the two fusion partners confirm that this distribution accounts for a major share of the fusion activity. The persistence of the QbcR cis-complex and the formation of the Qa-QbcR trans-complex are both sensitive to the Rab-GTPase inhibitor, GDI, and to mutations in the vacuolar tether complex, HOPS (HOmotypic fusion and vacuolar Protein Sorting complex). This suggests that the vacuolar Rab-GTPase, Ypt7, and HOPS restrict cis-SNARE disassembly and thereby bias trans-SNARE assembly into a preferred topology.
Author Summary
Cellular components often travel between organelles in vesicular entities. This intracellular traffic usually involves production of a vesicle containing cargo from one organelle membrane, movement of the vesicle to its destination, and then fusion of the vesicle with the target organelle. Thus, membrane fusion is a fundamental process required for these intracellular trafficking events. SNARE proteins and SM proteins mediate this fusion process. SNAREs form complexes that are either located on the same membrane or vesicle (called cis-SNARE complexes) or bridge two membrane compartments or vesicles (trans-SNARE complexes). The cis-SNARE complexes must be activated before trans-SNARE complexes can form and allow the membranes to fuse. We investigated the mechanism of cis-SNARE activation and trans-SNARE formation by studying the fusion of highly purified yeast vacuoles. We found that cis-SNARE activation involves the selective removal of a single SNARE protein from a pre-existing cis-SNARE complex, which is replaced by a similar SNARE originating from the other fusion partner. The activated cis-SNARE complexes depended on SM proteins for their stability. Thus, we have shown that the preferred topology of trans-SNARE formation is determined by cis-SNARE–SM protein interactions.
PMCID: PMC3260307  PMID: 22272185

Results 1-13 (13)