Various glutamate urea ligands have displayed high affinities to prostate specific membrane antigen (PSMA), which is highly overexpressed in prostate and other cancer sites. The multivalent versions of small PSMA-targeted molecules are known to be even more efficiently bound to the receptor. Here, we employ a well-known urea-based ligand, 2-[3-(1,3-dicarboxypropyl)-ureido] pentanedioic acid (DUPA) and triazine dendrimers in order to study the effect of molecular size on multivalent targeting in prostate cancer. The synthetic route starts with the preparation of a dichlorotriazine bearing DUPA in 67% overall yield over five steps. This dichlorotriazine reacts with G1, G3, and G5 triazine dendrimers bearing a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) group for 64Cu-labeling at the core to afford poly(monochlorotriazine) intermediates. Addition of 4-aminomethylpiperidine (4-AMP) and the following deprotection produce the target compounds, G1-(DUPA)4, G3-(DUPA)16, and G5-(DUPA)64. These targets include 4/16/64 DUPA groups on the surface and a DOTA group at the core, respectively. In vitro cell assay using PC3-PIP (PSMA positive) and PC3-FLU (PSMA negative) cells reveals that G1-(DUPA)4 has the highest PC3-PIP to PC3-FLU uptake ratio (10-fold) through the PSMA-mediated specific uptake. While G5-(DUPA)64 displayed approximately 12 times higher binding affinity (IC50 23.6 nM) to PC3-PIP cells than G1-(DUPA)4 (IC50 282.3 nM) as evaluated in a competitive binding assay, the G5 dendrimer also showed high non-specific binding to PC3-FLU cells. In vivo uptake of the 64Cu-labeled dendrimers was also evaluated in severe combined inmmunodeficient (SCID) mice bearing PC3-PIP and PC3-FLU xenografts on each shoulder, respectively. Interestingly, quantitative imaging analysis of positron emission tomograph (PET) displayed the lowest tumor uptake in PC3-PIP cells for the midsize dendrimer G3-(DUPA)16 (19.4 kDa) (0.66 ± 0.15%ID/g at 1 h. p.i., 0.64 ± 0.11%ID/g at 4 h. p.i., and 0.67 ± 0.08%ID/g at 24 h. p.i.). Through the specific binding of G1-(DUPA)4 to PSMA, the smallest dendrimer (5.1 kDa) demonstrated the highest PC3-PIP to muscle and PC3-PIP to PC3-FLU uptake ratios (17.7 ± 5.5 and 6.7 ± 3.0 at 4 h p.i., respectively). In addition, the enhanced permeability and retention (EPR) effect appeared to be an overwhelming factor for tumor uptake of the largest dendrimer G5-(DUPA)64 as the uptake was at a similar level irrelevant to the PSMA expression.

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.

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.

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.

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.

Background: 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.Methods: We investigate the mechanisms of cell death that accompanies photochemical internalization of the fluorescent peptide TMR-TAT.Results: TMR-TAT kills cells after endocytosis and light irradiation. The lysis of endocytic organelles by TMR-TAT causes a rapid increase in the concentration of calcium in the cytosol. TMR-TAT co-localizes with endocytic organelles containing calcium prior to irradiation and photochemical internalization leads to the release of the lumenal content of these organelles. Ruthenium red and cyclosporin A, inhibitors of calcium import in mitochondria and of the mitochondria permeability transition pore, inhibit cell death.Conclusions: TMR-TAT mediated photochemical internalization leads to a disruption of calcium homeostasis. The subsequent import of calcium in mitochondria is a causative factor of the cell death that accompanies photochemical intemalization.General significanceUnderstanding how the lysis of endocytic organelles affects cellular physiology and causes cell death is crucial to the development of optimal delivery methodologies. (C) 2012 Elsevier B.V. All rights reserved.

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.

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.

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.

This chapter reviews work from our laboratory over the last 10 years as it pertains to efforts focused on the exploration of triazine dendrimers as vehicles for drug delivery. The discussion is restricted to our efforts in paclitaxel-laden constructs, an area that we have had the most success and have made the greatest investment in time and energy. Areas of emphasis include the evolution of synthetic strategies, characterization, biodistribution, biocompatibility, safety and toxicity, efficacy, and therapeutic advantage. Throughout, limitations and challenges to these efforts are delineated. This chapter is not intended to exhaustively recapitulate the published work. Interested readers are referred to the original research publications and the supporting information provided with each. Instead, the intent is to provide insight into the evolution of the design and to realize this opportunity to share intuitions currently guiding these efforts which, accordingly, are less (or as yet un-) substantiated with experiment.

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 15.6–19.4 nm. Mass spectrometry and light scattering suggest radically different molecular weights with the former ~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 non-linear, ranging from 7–20% cumulative release over a 48 hour 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 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 Abraxane. Both formulations were found to be non-toxic at the administered doses, and the dendrimer had an acute MTD greater than the highest dose administered. In a prostate tumor model (PC-3-luc), efficacy was observed over 70 days with an arrest of tumor growth and lack of luciferase activity observed in the twice treated cohort. washed with media, and fresh media added. Cytotoxicity was evaluated 6 days later by the MTT cell viability. IC 50 values were approximated from the MTT dose-response curves.

Proper management of diabetes requires the frequent measurement of a patient's 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.

BACKGROUND: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).METHODOLOGY/PRINCIPAL FINDINGS:We report that TMR-TAT acts as a photosensitizer that can destroy membranes. TMR-TAT escapes from endosomes after exposure to moderate light doses. However, this is also accompanied by loss of plasma membrane integrity, membrane blebbing, and cell-death. In addition, the peptide causes the destruction of cells when applied extracellularly and also triggers the photohemolysis of red blood cells. These photolytic and photocytotoxic effects were inhibited by hydrophobic singlet oxygen quenchers but not by hydrophilic quenchers.CONCLUSIONS/SIGNIFICANCE:Together, these results suggest that TAT can convert an innocuous fluorophore such as TMR into a potent photolytic agent. This effect involves the targeting of the fluorophore to cellular membranes and the production of singlet oxygen within the hydrophobic environment of the membranes. Our findings may be relevant for the design of reagents with photo-induced endosomolytic activity. The photocytotoxicity exhibited by TMR-TAT also suggests that CPP-chromophore conjugates could aid the development of novel Photodynamic Therapy agents.

Our lab group is currently developing a fluorescent competitive binding assay between the Alexa fluor 647 labeled lectin, Concanavalin A, and highly structured glycosylated dendrimers to be sensitive to varying levels of glucose. Previously, this chemistry has elicited a high sensitivity to additions of physiological concentrations of glucose. However, the exact mechanism behind the sensing has not yet been well understood. This work presents a conceptual model of the response in which competitive binding results in different distributions of aggregates size to varying amounts of glucose. Preliminary experiments were performed by using Numerical Tracking Analysis (NTA) which correlates the movement of particles, positioned by light scattering, to the equivalent Brownian motion associated with particles of a certain spherical diameter. Using this method, the sensing chemistry was exposed to two different glucose concentrations and histograms of the size distribution for glucose concentrations were obtained. Herein the aggregation profile, mean aggregate size, and the number of aggregates (aggregates per mL) for two glucose concentrations are displayed, showing a correlation between the aggregation and glucose concentration.

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.

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.

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.

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.

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.

This manuscript focuses on the routes, methods and reagents used to synthesize triazine-based dendrimers. Our pursuit of macromolecular architectures for drug delivery—dendrimers based on triazines—has been an ongoing effort for 8 years. To date, we have produced complex dendrimers with diverse peripheries as proof-of-concept, less complex molecules tailored for specific applications including DNA and RNA delivery and drug-decorated dendrimers for potential therapeutic applications including infectious disease and cancer. These syntheses have been executed at scales that range from high milligrams to over a kilogram. The routes, reagents and diversity displayed by a target anchors it in time. Early targets derive from convergent synthetic routes while later targets are prepared using divergent syntheses. The core of early dendrimers was a simple diamine, including piperazine, yielding the so-called bow-tie structures, middle period targets boast either a trispiperazinyltriazine core or a ‘super-core’ with six piperazine groups. Later targets return to the trispiperazinyltriazine core. The choice of linking diamine has also changed. Over time, p-aminobenzylamine was replaced by piperazine and then by aminomethylpiperidine with more exotic diamines sprinkled in throughout. Peripheral group choice has undergone similar variations: from AB2 to AB4 to, more recently, AB3. The diversity communicated by these groups yields dendrimers ranging from those with a common surface to examples where two groups were presented to those where four orthogonally reactive groups appear. Over time, these groups have grown in complexity from protected amines to tags for biodistribution and drugs like paclitaxel. Herein, strategies adopted and lessons learned are reviewed, intuitions relayed and future directions forecast.