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Find video protocols related to scientific articles indexed in Pubmed.
Light-fuelled transport of large dendrimers and proteins.
J. Am. Chem. Soc.
PUBLISHED: 05-06-2014
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This work presents a facile water-based supramolecular approach for light-induced surface patterning. The method is based upon azobenzene-functionalized high-molecular weight triazine dendrimers up to generation 9, demonstrating that even very large globular supramolecular complexes can be made to move in response to light. We also demonstrate light-fuelled macroscopic movements in native biomolecules, showing that complexes of apoferritin protein and azobenzene can effectively form light-induced surface patterns. Fundamentally, the results establish that thin films comprising both flexible and rigid globular particles of large diameter can be moved with light, whereas the presented material concepts offer new possibilities for the yet marginally explored biological applications of azobenzene surface patterning.
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Design, synthesis and biological assessment of a triazine dendrimer with approximately 16 Paclitaxel groups and 8 PEG groups.
Mol. Pharm.
PUBLISHED: 11-15-2013
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The synthesis and characterization of a generation three triazine dendrimer that displays a phenolic group at the core for labeling, up to eight 5 kDa PEG chains for solubility, and 16 paclitaxel groups is described. Three different diamine linkers-dipiperidine trismethylene, piperazine, and aminomethylpiperidine-were used within the dendrimer. To generate the desired stoichiometric ratio of 8 PEG chains to 16 paclitaxel groups, a monochlorotriazine was prepared with two paclitaxel groups attached through their 2-hydroxyls using a linker containing a labile disulfide. This monochlorotriazine was linked to a dichlorotriazine with aminomethylpiperidine. The resulting dichlorotriazine bearing two paclitaxel groups could be reacted with the eight amines of the dendrimer. NMR and MALDI-TOF confirm successful reaction. The eight monochlorotriazines of the resulting material are used as the site for PEGylation affording the desired 2:1 stoichiometry. The target and intermediates were amenable to characterization by (1)H and (13)C NMR, and mass spectrometry. Analysis revealed that 16 paclitaxel groups were installed along with 5-8 PEG chains. The final construct is 63% PEG, 22% paclitaxel, and 15% triazine dendrimer. Consistent with previous efforts and computational models, 5 kDa PEG groups were essential for making the target water-soluble. Molecular dynamics simulations showed a high degree of hydration of the core, and a radius of gyration of 2.8 ± 0.2 nm. The hydrodynamic radius of the target was found to be 15.8 nm by dynamic light scattering, an observation indicative of aggregation. Drug release studies performed in plasma showed slow and identical release in mouse and rat plasma (8%, respectively). SPECT/CT imaging was used to follow biodistribution and tumor uptake. Using a two component model, the elimination and distribution half-lives were 2.65 h and 38.2 h, respectively. Compared with previous constructs, this dendrimer persists in the vasculature longer (17.33 ± 0.88% ID/g at 48 h postinjection), and showed higher tumor uptake. Low levels of dendrimer were observed in lung, liver, and spleen (?6% ID/g). Tumor saturation studies of small prostate cancer tumors (PC3) suggest that saturation occurs at a dose between 23.2 mg/kg and 70.9 mg/kg.
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Dendrimers terminated with dichlorotriazine groups provide a route to compositional diversity.
Org. Lett.
PUBLISHED: 07-19-2013
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Triazine dendrimers terminated with either four or eight dichlorotriazines can be prepared in high yields by reacting an amine-terminated dendrimer with cyanuric chloride. These materials exist as white powders and are stable to storage at room temperature. Sequential nucleophilic aromatic substitution with two different amine nucleophiles yields compounds that display the desired compositional diversity. Reaction conditions for the substitution were developed using a model dichlorotriazine with amine nucleophiles at -20, 0, and 25 °C. Selective substitution is favored at lower temperatures and with more nucleophilic amine groups.
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Computational design principles for the discovery of bioactive dendrimers: [s]-triazines and other examples.
Expert Opin Drug Discov
PUBLISHED: 07-05-2013
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Chemistry yields dendrimers of many classes and compositions. Translating this synthetic success to bioactivity is significantly aided by the use of computational modeling and our knowledge of the three-dimensional shapes of these macromolecules.
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Synthesis of large dendrimers with the dimensions of small viruses.
J. Am. Chem. Soc.
PUBLISHED: 03-13-2013
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The dendrimer chemistry reported offers a route to synthetic target molecules with spherical shape, well-defined surface chemistries, and dimensions that match the size of virus particles. The largest target, a generation-13 dendrimer comprising triazines linked by diamines, is stable across ranges of concentration, pH, temperature, solvent polarity and in the presence of additives. This dendrimer theoretically presents 16,384 surface groups and has a molecular weight exceeding 8.4 MDa. Transmission electron and atomic force microscopies, dynamic light scattering, and computations reveal a diameter of ~30 nm. The target was synthesized through an iterative divergent approach using a monochlorotriazine macromonomer providing two generations of growth per synthetic cycle. Fidelity in the synthesis is supported by evidence from NMR spectroscopy, mass spectrometry, and high-pressure liquid chromatography.
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Computational design principles for bioactive dendrimer based constructs as antagonists of the TLR4-MD-2-LPS complex.
Biomaterials
PUBLISHED: 07-19-2011
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The cell surface interaction between bacterial lipopolysaccharide (LPS), Toll-like receptor 4 (TLR4) and MD-2 is central to bacterial sepsis syndromes and wound healing. We have shown that a generation (G) 3.5 polyamidoamine (PAMAM) dendrimer that was partially glycosylated with glucosamine inhibits TLR4-MD-2-LPS induced inflammation in a rabbit model of tissue scaring. However, it was a mixture of closely related chemical species because of the polydispersity of the starting PAMAM dendrimer. Generation 2 triazine dendrimers with single chemical entity material status are available at low cost and at the kilogram scale. PAMAM dendrimer can be synthetically grafted onto this triazine core dendrimer to make new triazine-PAMAM hybrid dendrimers. This led us to examine whether molecular modelling methods could be used to identify the key structural design principles for a bioactive lead molecule that could be synthesized and biologically evaluated. We describe our computer aided molecular studies of several dendrimer based constructs and the key design principles identified. Our approach should be more broadly applicable to the biologically focused, rational and accelerated design of molecules for other TLR receptors. They could be useful for treating infectious, inflammatory and malignant diseases.
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Encapsulation of a Concanavalin A/dendrimer glucose sensing assay within microporated poly (ethylene glycol) microspheres.
Biomed Opt Express
PUBLISHED: 02-23-2011
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Proper management of diabetes requires the frequent measurement of a patients blood glucose level. To create a long-term, minimally-invasive sensor that is sensitive to physiological concentrations of glucose a fluorescent glucose sensing assay using a competitive binding approach between fluorescently tagged Concanavalin-A (Con-A) and glycodendrimer is being developed. Until now, the essential step of effectively encapsulating this aggregative sensing assay while allowing a reversible response has yet to be reported. In this paper, a microporation technique is described in which microspheres are synthesized in a manner that creates fluid-filled pores within a poly (ethylene glycol) hydrogel. This dual-nature technique creates hydrophilic, biocompatible microcapsules in which the aggregative binding kinetics of the sensing assay within the pores are not constrained by spatial fixation in the hydrogel matrix. Confocal images displaying the localization of pockets filled with the assay within the polymeric matrix are presented in this paper. In addition, fluorescent responses to varying glucose concentrations, leaching studies, and long-term functionality of the encapsulated assay are demonstrated. To our knowledge, this is the first time that the Con-A/glycodendrimer assay has been shown to be reversible and repeatable within hydrogel spheres, including the display of functionality up to fourteen days under ambient conditions.
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Conjugation to the cell-penetrating peptide TAT potentiates the photodynamic effect of carboxytetramethylrhodamine.
PLoS ONE
PUBLISHED: 02-11-2011
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Cell-penetrating peptides (CPPs) can transport macromolecular cargos into live cells. However, the cellular delivery efficiency of these reagents is often suboptimal because CPP-cargo conjugates typically remain trapped inside endosomes. Interestingly, irradiation of fluorescently labeled CPPs with light increases the release of the peptide and its cargos into the cytosol. However, the mechanism of this phenomenon is not clear. Here we investigate the molecular basis of the photo-induced endosomolytic activity of the prototypical CPPs TAT labeled to the fluorophore 5(6)-carboxytetramethylrhodamine (TMR).
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Molecular modeling and in vivo imaging can identify successful flexible triazine dendrimer-based siRNA delivery systems.
J Control Release
PUBLISHED: 01-28-2011
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This study aimed to identify suitable siRNA delivery systems based on flexible generation 2-4 triazine dendrimers by correlating physico-chemical and biological in vitro and in vivo properties of the complexes with thermodynamic parameters calculated using molecular modeling. The siRNA binding properties of the dendrimers and PEI 25 kDa were simulated, binding and stability were measured in SYBR Gold assays, and hydrodynamic diameters, zeta potentials, and cytotoxicity were quantified. These parameters were compared with cellular uptake of the complexes and their ability to mediate RNAi. Radiolabeled complexes were administered intravenously, and pharmacokinetic profiles and biodistribution of these polyplexes were assessed both invasively and non-invasively. All flexible triazine dendrimers formed thermodynamically more stable complexes than PEI. While PEI and the generation 4 dendrimer interacted more superficially with siRNA, generation 2 and 3 virtually coalesced with siRNA, forming a tightly intertwined structure. These dendriplexes were therefore more efficiently charge-neutralized than PEI complexes, reducing agglomeration. This behavior was confirmed by results of hydrodynamic diameters (72.0 nm-153.5 nm) and zeta potentials (4.9 mV-21.8 mV in 10 mM HEPES) of the dendriplexes in comparison to PEI complexes (312.8 nm-480.0 nm and 13.7 mV-17.4 mV in 10 mM HEPES). All dendrimers, even generation 3 and 4, were less toxic than PEI. All dendriplexes were efficiently endocytosed and showed significant and specific luciferase knockdown in HeLa/Luc cells. Scintillation counting confirmed that the generation 2 triazine complexes showed more than twofold prolonged circulation times as a result of their good thermodynamic stability. Conversely, generation 3 complexes dissociated in vivo, and generation 4 complexes were captured by the reticulo-endothelial system due to their increased surface charge. Molecular modeling proves very valuable for rationalizing experimental parameters based on the dendrimers structural properties. Non-invasive molecular imaging predicted the in vivo fate of the complexes. Therefore, both techniques effectively promote the rapid development of safe and efficient siRNA formulations that are stable in vivo.
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Triazine dendrimers as nonviral vectors for in vitro and in vivo RNAi: the effects of peripheral groups and core structure on biological activity.
Mol. Pharm.
PUBLISHED: 06-08-2010
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A family of triazine dendrimers, differing in their core flexibility, generation number, and surface functionality, was prepared and evaluated for its ability to accomplish RNAi. The dendriplexes were analyzed with respect to their physicochemical and biological properties, including condensation of siRNA, complex size, surface charge, cellular uptake and subcellular distribution, their potential for reporter gene knockdown in HeLa/Luc cells, and ultimately their stability, biodistribution, pharmacokinetics and intracellular uptake in mice after intravenous (iv) administration. The structure of the backbone was found to significantly influence siRNA transfection efficiency, with rigid, second generation dendrimers displaying higher gene knockdown than the flexible analogues while maintaining less off-target effects than Lipofectamine. Additionally, among the rigid, second generation dendrimers, those with either arginine-like exteriors or peripheries containing hydrophobic functionalities mediated the most effective gene knockdown, thus showing that dendrimer surface groups also affect transfection efficiency. Moreover, these two most effective dendriplexes were stable in circulation upon intravenous administration and showed passive targeting to the lung. Both dendriplex formulations were taken up into the alveolar epithelium, making them promising candidates for RNAi in the lung. The ability to correlate the effects of triazine dendrimer core scaffolds, generation number, and surface functionality with siRNA transfection efficiency yields valuable information for further modifying this nonviral delivery system and stresses the importance of only loosely correlating effective gene delivery vectors with siRNA transfection agents.
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Biological assessment of triazine dendrimer: toxicological profiles, solution behavior, biodistribution, drug release and efficacy in a PEGylated, paclitaxel construct.
Mol. Pharm.
PUBLISHED: 05-21-2010
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The physicochemical characteristics, in vitro properties, and in vivo toxicity and efficacy of a third generation triazine dendrimer bearing approximately nine 2 kDa polyethylene glycol chains and twelve ester linked paclitaxel groups are reported. The hydrodynamic diameter of the neutral construct varies slightly with aqueous solvent ranging from 15.6 to 19.4 nm. Mass spectrometry and light scattering suggest radically different molecular weights with the former approximately 40 kDa mass consistent with expectation, and the latter 400 kDa mass consistent with a decameric structure and the observed hydrodynamic radii. HPLC can be used to assess purity as well as paclitaxel release, which is insignificant in organic solvents or aqueous solutions at neutral and low pH. Paclitaxel release occurs in vitro in human, rat, and mouse plasma and is nonlinear, ranging from 7 to 20% cumulative release over a 48 h incubation period. The construct is 2-3 orders of magnitude less toxic than Taxol by weight in human hepatocarcinoma (Hep G2), porcine renal proximal tubule (LLC-PK1), and human colon carcinoma (LS174T) cells, but shows similar cytotoxicity to Abraxane in LS174T cells. Both Taxol and the construct appear to induce caspase 3-dependent apoptosis. The construct shows a low level of endotoxin, is not hemolytic and does not induce platelet aggregation in vitro, but does appear to reduce collagen-induced platelet aggregation in vitro. Furthermore, the dendrimer formulation slightly activates the complement system in vitro due most likely to the presence of trace amounts (<1%) of free paclitaxel. An animal study provided insight into the maximum tolerated dose (MTD) wherein 10, 25, 50, and 100 mg of paclitaxel/kg of construct or Abraxane were administered once per week for three consecutive weeks to non tumor bearing athymic nude mice. The construct showed in vivo toxicity comparable to that of Abraxane. Both formulations were found to be nontoxic at the administered doses, and the dendrimer had an acute MTD greater than the highest dose administered. In a prostate tumor model (PC-3-h-luc), efficacy was observed over 70 days with an arrest of tumor growth and lack of luciferase activity observed in the twice treated cohort.
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Synthesis and characterization of a triazine dendrimer that sequesters iron(III) using 12 desferrioxamine B groups.
Bioorg. Med. Chem.
PUBLISHED: 04-14-2010
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The synthesis of a third generation triazine dendrimer, 1, containing multiple, iron-sequestering desferrioxamine B (DFO) groups is described. Benzoylation of the hydroxamic acid groups of DFO and formation of a reactive dichlorotriazine provide the intermediate for reaction with the second generation dendrimer displaying twelve amines. This strategy further generalizes the functional monomer approach to generate biologically active triazine dendrimers. Dendrimer 1 is prepared in seven steps in 35% overall yield and displays 12 DFO groups making it 56% drug by weight. Spectrophotometric titrations (UV-vis) show that 1 sequesters iron(III) atoms with neither cooperativity nor significant interference from the dendrimer backbone. Evidence from NMR spectroscopy and mass spectrometry reveals a limitation to this functional monomer approach: trace amounts of O-to-N acyl migration from the protected hydroxamic acids to the amine-terminated dendrimer occurs during the coupling step leading to N-benzoylated dendrimers displaying fewer than 12 DFO groups.
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Divergent synthesis of triazine dendrimers using a trimethylene-dipiperidine linker that increases efficiency, simplifies analysis, and improves product solubility.
Tetrahedron Lett.
PUBLISHED: 03-31-2010
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To combine benefits stemming from the high nucleophilicity of piperidine and the flexibility afforded by aliphatic triamine linkers, a trimethylene-dipiperidine linker has been used to synthesize triazine dendrimers using a divergent route. The cyclic, secondary amine of the linker reacts with monochlorotriazine monomer units, 1, leading to a dendrimer growth strategy that requires two-steps-per-generation. This strategy reduces the number of steps required for synthesis by 50%. The new linker also reduces complexity in the NMR spectra because rotational isomerism observed in linkers with primary amines is not present. In addition, the final products contain no interior hydrogen-bond donating groups. The high solubility observed in organic solvents for protected dendrimers is attributed to this factor and the inherent flexibility provided by the linker. Gas phase simulation suggests that globular structure emerges after generation three, wherein the core of the dendrimer is effectively shielded from solvent.
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A retro-inverso TAT-like peptide designed to deliver cysteamine to cells.
Bioorg. Med. Chem. Lett.
PUBLISHED: 02-25-2010
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A retro-inverso, TAT-like peptide wherein lysine residues are replaced with cysteine residues bearing a disulfide-linked cysteamine group is found to engage in thiol-disulfide exchange with cysteine. These peptides are transported into cells and localize to lysosomes. Cellular uptake is enhanced in peptides bearing two cysteamine groups over those with one or none, by factors of approximately 1.5 and 12, respectively.
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Synthesis of odd generation triazine dendrimers using a divergent, macromonomer approach.
Org. Lett.
PUBLISHED: 02-23-2010
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Using a macromonomer, first, third, and fifth generation triazine dendrimers can be prepared using a divergent approach. The nine-step process to the fifth generation target relies on an iterative two-reactions-per-generation strategy to yield the desired material in approximately 48% overall yield. This target displays 96 surface groups. NMR spectroscopy and mass spectrometry show that exceptionally narrow polydispersity is achieved using this strategy.
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Computational insights into the interactions between DNA and siRNA with "rigid" and "flexible" triazine dendrimers.
Biomacromolecules
PUBLISHED: 02-06-2010
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In this study, simulation challenges intuitive models of "flexible" and "rigid" generation two triazine dendrimers as it pertains to solution conformation and conformation on binding DNA or siRNA sequences. These results derive from structural and energetic analyses of the binding events. Simulations of the rigid structure reinforce the role of the constrained piperazine linker in positioning the peripheral groups at significant distance from each other and the core of the dendrimer. In contrast, the flexible dendrimer, characterized by triethyleneglycol-like linkers, collapses in solution. On binding DNA and siRNA, these conformations are largely retained. The rigid dendrimer undergoes reorganization of peripheral groups to generate a large number of contacts to the nucleic acid. In contrast, the flexible dendrimer, originally conceived to create multivalent interactions with nucleic acids, generates only a few contacts and collapses further. This paper provides unique insight in the role played by molecular flexibility in the binding phenomenon.
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Cancer therapies utilizing the camptothecins: a review of the in vivo literature.
Mol. Pharm.
PUBLISHED: 01-30-2010
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This review summarizes the in vivo assessment-preliminary, preclinical, and clinical-of chemotherapeutics derived from camptothecin or a derivative. Camptothecin is a naturally occurring, pentacyclic quinoline alkaloid that possesses high cytotoxic activity in a variety of cell lines. Major limitations of the drug, including poor solubility and hydrolysis under physiological conditions, prevent full clinical utilization. Camptothecin remains at equilibrium in an active lactone form and inactive hydrolyzed carboxylate form. The active lactone binds to DNA topoisomerase I cleavage complex, believed to be the single site of activity. Binding inhibits DNA religation, resulting in apoptosis. A series of small molecule camptothecin derivatives have been developed that increase solubility, lactone stability and bioavailability to varying levels of success. A number of macromolecular agents have also been described wherein camptothecin(s) are covalently appended or noncovalently associated with the goal of improving solubility and lactone stability, while taking advantage of the tumor physiology to deliver larger doses of drug to the tumor with lower systemic toxicity. With the increasing interest in drug delivery and polymer therapeutics, additional constructs are anticipated. The goal of this review is to summarize the relevant literature for others interested in the field of camptothecin-based therapeutics, specifically in the context of biodistribution, dosing regimens, and pharmacokinetics with the desire of providing a useful source of comparative data. To this end, only constructs where in vivo data is available are reported. The review includes published reports in English through mid-2009.
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Polycationic triazine-based dendrimers: effect of peripheral groups on transfection efficiency.
New J Chem
PUBLISHED: 12-05-2009
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A panel of eight, second generation triazine dendrimers differing in the number of amines, guanidines, hydroxyls and aliphatic groups on the periphery was synthesized and assayed for gene transfer in an attempt to correlate the effects of surface functionality on transfection efficiency. The physicochemical and biological properties of the dendrimers and dendriplexes, such as condensation of DNA, size, surface charge and morphology of dendriplexes, toxicity and ultimately transfection efficiency in MeWo cells, were analyzed. The results from an ethidium bromide exclusion assay showed that the complexation efficiency of the dendrimers with DNA is moderately affected by surface groups. Increasing the number of surface amines, reducing the number of surface hydroxyl groups, or replacing the amine moiety with guanidines all help strengthen the complex formed. Results from dynamic light scattering and zeta potential analyses indicate that the smallest particles correlate with complexes that exhibit the highest zeta potentials. Cytotoxicity was low for all compounds, particularly for the G2-5 dendrimer containing alkyl groups on the periphery, indicating the benefit of incorporating such neutral functionality onto the surface of the triazine dendrimers. Within this panel, the highest transfection efficiency was observed for the dendrimers that formed the smallest complexes, suggesting that this physicochemical property is an accurate predictor for determining which dendrimers will show high transfection efficiency.
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Synthesis and Characterization of Anionic Triazine Dendrimers with a Labile Disulfide Core.
Isr. J. Chem.
PUBLISHED: 11-26-2009
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Anionic dendrimers based on melamine with disulfide bonds at the core were prepared to investigate the solubility of these architectures, the ability of these molecules to solubilize pyrene as a model drug, and the ability of these architectures to undergo thiol-disulfide exchange. The ability to solubilize pyrene is directly correlated with molecular weight of the dendrimer-aggregation of dendrons does not occur. Thiol-disulfide exchange occurs rapidly using dithiothreitol as the reductant to yield dendrimers with thiol cores that can undergo oxidation in air to yield the original dendrimer.
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Design, synthesis, characterization, and biological evaluation of triazine dendrimers bearing paclitaxel using ester and ester/disulfide linkages.
Bioconjug. Chem.
PUBLISHED: 11-03-2009
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The design, synthesis, characterization, and preliminary biological assessment of three dendrimers are reported. All three dendrimers, 1-3, present twelve paclitaxel groups linked by acylation of the 2-hydroxyl group. The linker for dendrimers 2 and 3 also includes a disulfide. Installation of the paclitaxel group relies on reacting twelve primary amines of a second generation triazine dendrimer, a scaffold available on kilogram scale, with a dichlorotriazine bearing the drug. This dichlorotriazine is available in four steps by (i) reacting paclitaxel with glutaric anhydride, (ii) activating with N-hydroxysuccinimide (NHS), (iii) treating the resulting ester with either 1,3-diaminopropane (for 1) or cystamine (for 2 and 3), and (iv), finally, reacting with cyanuric chloride. After reaction with the dendrimer, the resulting monochlorotriazine groups are reacted with 4-aminomethylpiperidine (AMP) and then a poly(ethylene glycol) (PEG) group of molecular weight 2 kDa. Two different PEG-NHS esters are employed that differ in lability. For 1 and 2, the PEG incorporates an ester-linked succinic acid group. For 3, the PEG incorporates an ether-linked acetic acid group. Both mass spectrometry and 1H NMR spectroscopy prove valuable for determining the final ratios of dendrimer:paclitaxel:AMP:PEG. These values are typically 1:12:12:9. Cytotoxicity of these constructs using an MTT-based assay and PC-3 cells reveals IC(50) values in the low nanomolar range with dithiothreitol and glutathione enhancing the toxicity of the disulfide-containing constructs 2 and 3. Preliminary toxicology assessment of 1 suggests that it is well tolerated in vivo with preferential renal clearance. The elimination half-lives of all of the dendrimers appear shorter than predicted from the previous results. Tumor localization is observed for all the three dendrimers.
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Triazine dendrimers as nonviral gene delivery systems: effects of molecular structure on biological activity.
Bioconjug. Chem.
PUBLISHED: 08-28-2009
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A family of generation 1, 2, and 3 triazine dendrimers differing in their core flexibility was prepared and evaluated for their ability to accomplish gene transfection. Dendrimers and dendriplexes were analyzed by their physicochemical and biological properties such as condensation of DNA, size, surface charge, morphology of dendriplexes, toxic and hemolytic effects, and ultimately transfection efficiency in L929 and MeWo cells. Flexibility of the backbone was found to play an important role with generation 2 dendrimer displaying higher transfection efficiencies than 25 kDa poly(ethylene imine) or SuperFect at a lower cytotoxicity level. This result is surprising, as PAMAM dendrimers require generations 4 or 5 to become effective transfection reagents. The ability to delineate effects of molecular structure and generation of triazine dendrimers with biological properties provides valuable clues for further modifying this promising class of nonviral delivery system.
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Intercepting the synthesis of triazine dendrimers with nucleophilic pharmacophores: a general strategy toward drug delivery vehicles.
Chem. Commun. (Camb.)
PUBLISHED: 08-11-2009
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The camptothecin ester of isonipecotic acid is installed on a triazine dendrimer intermediate obtained through an iterative, scalable route to ultimately yield cationic and PEGylated targets with activities in cell culture comparable to free drug.
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Mimicking PAMAM Dendrimers with Ampholytic, Hybrid Triazine Dendrimers: A Comparison of Dispersity and Stability.
Macromolecules
PUBLISHED: 06-29-2009
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Two strategies are applied to mimic the ampholytic nature of the surfaces of half-generation PAMAM dendrimers and yet retain the very narrow dispersity inherent of triazine dendrimers. Both strategies start with a monodisperse, single-chemical entity, generation two triazine dendrimer presenting twelve surface amines that is available at the kilogram scale. The first method relies on reaction with methyl bromoacetate. Complete conversion of the surface primary amines to tertiary amines occurs to provide 24 surface esters. Extended reaction times lead to quarternization of the amines while other unidentified species are also present. The resulting polyester can be quantitatively hydrolyzed using 4M aqueous HCl to yield a dendrimer with 12 tertiary amines and 24 carboxylic acids about a hydrophobic triazine core. The second method utilizes Michael additions of methyl acrylate to yield 24 surface esters. This reaction proceeds more rapidly and more cleanly than the former strategy. Hydrolysis of this material proceeds quantitatively using 4M aqueous HCl to yield desired dendrimer. In both cases, MALDI-TOF mass spectrometry provides compelling evidence of reaction progress. Electrophoretic analysis confirms the ampholytic nature of these materials with the former targets having a pI value in the 1.8 < pI < 3.4 range, and the latter having a pI value in the 4.7 < pI < 5.9. These ranges bookend the pH range within which PAMAM dendrimers become zwitterionic, 3.4 < pI < 4.7. The strategy of using monodisperse amine-terminated dendrimer constructs as core offers significant advantage over PAMAM homopolymers including dispersity, ease of characterization and batch-to-batch reproducibility. These triazine dendrimers could ultimately be adopted into materials with applications wherein the demands of purity have hitherto remained unsatisfied.
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Electrophoretic Behavior of Anionic Triazine and PAMAM Dendrimers: Methods for Improving Resolution and Assessing Purity Using Capillary Electrophoresis.
Macromolecules
PUBLISHED: 04-28-2009
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The synthesis and characterization of second- and third-generation triazine dendrimers bearing carboxylic acid groups on the periphery are reported. These materials were synthesized by exhaustive succinylation of amine-terminated dendrimers. (1)H and (13)C NMR spectra are consistent with the desired products, but these techniques are limited by degeneracy in signals. MALDI-TOF mass spectrometry confirms the presence of the desired material. These materials display pH-dependent solubility in water. Capillary electrophoresis proves to be valuable in multiple elements of this work, and general protocols emerge that appear to be useful for the characterization of lower-generation anionic dendrimers. Specifically, capillary electrophoresis provides a convenient method for monitoring the removal of excess succinic anhydride/succinic acid and offers additional clues to the chemical nature of the impurities in these samples. Optimization of the background electrolyte and instrumental parameters allows for the assessment of the purity of these triazine targets as well as comparison with two sets of commercially available anionic poly(amidoamine) (PAMAM) dendrimers. Corroborative information from the different orthogonal analytical techniques employed supports the hypothesis that triazine dendrimers exist as very narrowly disperse mixtures of macromolecules approaching, in some cases, single chemical entities.
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Gadolinium MRI contrast agents based on triazine dendrimers: relaxivity and in vivo pharmacokinetics.
Bioconjug. Chem.
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Four gadolinium (Gd)-based macromolecular contrast agents, G3-(Gd-DOTA)(24), G5-(Gd-DOTA)(96), G3-(Gd-DTPA)(24), and G5-(Gd-DTPA)(96), were prepared that varied in the size of dendrimer (generation three and five), the type of chelate group (DTPA or DOTA), and the theoretical number of metalated chelates (24 and 96). Synthesis relied on a dichlorotriazine derivatized with a DOTA or DTPA ligand that was incorporated into the dendrimer and ultimately metalated with Gd ions. Paramagnetic characteristics and in vivo pharmacokinetics of all four contrast agents were investigated. The DOTA-containing agents, G3-(Gd-DOTA)(24) and G5-(Gd-DOTA)(96), demonstrated exceptionally high r1 relaxivity values at off-peak magnetic fields. Additionally, G5-(Gd-DOTA)(96) showed increased r1 relaxivity in serum compared to that in PBS, which was consistent with in vivo images. While G3-(Gd-DOTA)(24) and G3-(Gd-DTPA)(24) were rapidly excreted into the urine, G5-(Gd-DOTA)(96) and G5-(Gd-DTPA)(96) did not clear as quickly through the kidneys. Molecular simulation of the DOTA-containing dendrimers suggests that a majority of the metalated ligands are accessible to water. These triazine dendrimer-based MRI contrast agents exhibit several promising features such as high in vivo r1 relaxivity, desirable pharmacokinetics, and well-defined structure.
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TAT-mediated photochemical internalization results in cell killing by causing the release of calcium into the cytosol of cells.
Biochim. Biophys. Acta
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Lysis of endocytic organelles is a necessary step in many cellular delivery methodologies. This is achieved efficiently in the photochemical internalization approach but the cell death that accompanies this process remains a problem.
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Triazine dendrimers as drug delivery systems: from synthesis to therapy.
Adv. Drug Deliv. Rev.
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The use of triazine dendrimers as drug delivery systems benefits from their synthetic versatility and well-defined structure. Triazine dendrimers can be designed and readily synthesized to display orthogonally functional surfaces that facilitate post-synthetic manipulation such as attachment of drug, PEGylation, and/or the installation of ligands or reporting groups. The synthesis is scalable, and large generations can be accessed. To date, triazine dendrimers have been probed for a variety of medicinal applications including drug delivery with an emphasis on cancer, nonviral DNA and RNA delivery systems, in sensing applications, and as bioactive materials. Specifically, triazine adducts with paclitaxel, camptothecin, brefeldin A, and desferrioxamine have been prepared and assessed. Paclitaxel constructs show promising activity in vivo. The use of these materials in fluorescence-based glucose sensors is being pursued. Glycosylated triazine dendrimers interfere with signal transduction in the Toll-4 receptor pathway.
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Antitumor activity and molecular dynamics simulations of paclitaxel-laden triazine dendrimers.
Mol. Pharm.
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The antitumor activities of triazine dendrimers bearing paclitaxel, a well-known mitotic inhibitor, are evaluated in SCID mice bearing human prostate cancer xenografts. To increase the activity of a first generation prodrug 1 that contained twelve paclitaxel molecules tethered via an ester linkage, the new construct described here, prodrug 2, tethers paclitaxel with linkers containing both an ester and disulfide. While PEGylation is necessary for solubility, and may improve biocompatibility and increase plasma half-life, it increases the heterogeneity of the sample with an average of eight to nine PEG chains (2 kDa each) incorporated. The heterogeneous population of PEGylated materials was used without fractionation based on models obtained from molecular dynamics simulations. Three models were examined; hexaPEGylated, nonaPEGylated, and dodecaPEGylated constructs. Intravenous delivery of prodrug 2 was performed by single, double or triple dosing regimes with doses spaced by one week. The doses varied from 50 mg of paclitaxel/kg to 200 mg of paclitaxel/kg. Tumor growth arrest and regression was observed over the 10-week treatment period without mortality for mice treated with the 50 mg of paclitaxel/kg treated three times.
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Experimental and computational evidence for an inversion in guest capacity in high-generation triazine dendrimer hosts.
J. Am. Chem. Soc.
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The synthesis, characterization, and host-guest chemistry of high-generation triazine dendrimers are described. With pyrene and camptothecin as guests, experiments revealed that the guest capacity of odd-generation triazine dendrimers increased until generation 7 but decreased at generation 9. Molecular dynamics simulations conducted in explicit solvent showed a useful fingerprint for this behavior in radial distribution functions of water molecules penetrating the interior of the dendrimers. A linear relationship between the guest capacity of dendrimers measured experimentally and the number of water molecules within the interior determined computationally was observed.
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