Beware of phosphate: evidence of specific dendrimer-phosphate interactions.

Dendrimers are extensively studied for drug delivery and catalysis, most of which are pH dependent. Phosphate buffer solutions (PBSs) are often used to adjust the pH. We have found that phosphate ions become incorporated into poly(amidoamine) (PAMAM) dendrimer molecules by forming H-bonds with tertiary nitrogens. We show that this specific interaction between H2PO4- and HPO42- ions and generation five PAMAM dendrimers causes a decrease in hydrodynamic size, disturbing the outcome of the size exclusion chromatography analysis. We monitored this interaction by 1H and 31P high resolution NMR, NMR-diffusiometry, pH-potentiometry and infrared spectroscopy. Failing to take into account this effect may lead to incorrect conclusions and misinterpreting interactions of PAMAM dendrimers with drug molecules and subsequently incorrect dosing. The phosphate salts of amino terminated generation five PAMAM dendrimers are stable for years when stored in the dark, even in dilute aqueous solutions, which has important implications for the shelf-life of dendrimer-based drug delivery systems.

Diverse Applications of Nanomedicine.

The design and use of materials in the nanoscale size range for addressing medical and health-related issues continues to receive increasing interest. Research in nanomedicine spans a multitude of areas, including drug delivery, vaccine development, antibacterial, diagnosis and imaging tools, wearable devices, implants, high-throughput screening platforms, etc. using biological, nonbiological, biomimetic, or hybrid materials. Many of these developments are starting to be translated into viable clinical products. Here, we provide an overview of recent developments in nanomedicine and highlight the current challenges and upcoming opportunities for the field and translation to the clinic.

Transobturator Bypass Utilizing Fluoroscopic Guidance.

INTRODUCTION: Transobturator bypass is an older and less frequently performed revascularization of the lower extremity that is useful in the setting of multiple failed lower extremity bypasses usually due to groin sepsis and infection(s). It can be a technically challenging operation due to anatomic constraints. REPORT: We describe a successful transobturator bypass utilizing fluoroscopy for passage of bypass tunneler and graft through the obturator foramen. DISCUSSION: Live fluoroscopy is a versatile technique and in conjunction with our described surgical technique of lower extremity bypass should help increase the technical success rates of this procedure.

Caging cancer.

This article summarizes the overall issues surrounding cancer for the general audience. Individual differences between patients include genetic and non-genetic differences manifested in various cellular pathways, tumor heterogeneity and variability, differing contribution of tumor microenvironment and potential toward metastasis. Successful treatment of individuals depends on correctly interpreting all these factors and appropriately addressing the actual features by using customized therapy strategies, simultaneously or sequentially. Currently used chemotherapy agents are cytotoxic and typically target one of the major pathways; therefore they have to be applied in combination regimes. Nanomedicines however, have the potential advantage that more than one feature can be built into a complex nanodevice. This yet untapped potential is illustrated on the example of a gadolinium fullerenol cage molecule. Gd@C82(OH)22 has low toxicity, influences several biologic features simultaneously, displays tumoristatic properties, and is effective against triple-negative breast cancer cells. A deeper understanding of the exact relations between the physicochemical characteristics of this system and the biologic events may lead to a new class of efficient anticancer pharmaceutics. FROM THE CLINICAL EDITOR: The search for the magic bullet in the treatment of cancer has long been the dream of clinicians and researchers worldwide. Inherent cellular characteristics of cancer cells have made this task extremely hard to reach. In this article, the author provided a concise summary on the understanding and challenges in the current battle and also illustrated the potential usefulness of the recently developed gadolinium fullerenol cage molecule by describing experimental data from various research groups.

The 4th Annual Meeting of the American Society for Nanomedicine.

The 4th Conference of the American Society for Nanomedicine is being held March 28-30, 2014 at the Universities at Shady Grove, Rockville, Maryland. The meeting's theme is on defining the role of nanomedicines for nervous system diagnostics and disease but balanced by broad and timely topics for nanotechnology. Nanoneuromedicine, as defined by the development of small drug formulations for the diagnosis and treatment of degenerative, inflammatory, infectious, vascular, addictive, behavioral and metabolic disorders of the nervous system, will provide a focus for each of the scientific sessions. This research is interdisciplinary and it's in its infancy. The hurdles that preclude translation from bench to bedside would include its delivery across the blood brain barrier, limiting nervous system toxicities, and improving drug targeting to diseased brain subregions. These all pose challenges. Multidisciplinary works in neuroscience (neurobiology, neurochemistry, neurophysiology, and neuroinflammation), bioimaging, and polymer chemistry to facilitate outcomes for formulation manufacture will be vigorously discussed. How drugs reach sites of action need include neural cell specific subcellular compartments. The ASNM meeting will showcase nanoneuromedicine research from leading investigators of divergent scientific backgrounds who define this new field. It will also serve as an incubator for developing investigators and broad new field discoveries. Welcome to the conference and enjoy!

In vivo toxicity evaluation of gold-dendrimer composite nanodevices with different surface charges.

Composite nanodevices (CNDs) are multifunctional nanomaterials with potential uses in cancer imaging and therapy. Poly(amidoamine) dendrimer-based composite nanodevices are important members of this group and consist of an organic dendrimer component and an incorporated inorganic component, in this case, gold. This study addresses the short- (14 days) and long-term (78 days) in vivo toxicity of generation-5 (G5; 5 nm) PAMAM dendrimer-based gold-CNDs (Au-CNDs) with varying surface charges (positive, negative and neutral) in C57BL/6J male mice. Detailed toxicological analyses of (1) body weight changes, (2) serum chemistry and (3) histopathological examination of 22 organs showed no evidence of organ injury or organ function compromise. Zeta potential of Au-CNDs showed significant change from their parent dendrimers upon gold incorporation, making the normally lethal positive surface dendrimer biologically safe. Also homeostatic mechanisms in vivo may compensate/repair toxic effects, something not seen with in vitro assays.

Physiologically based pharmacokinetic model for composite nanodevices: effect of charge and size on in vivo disposition.

PURPOSE: To characterize temporal exposure and elimination of 5 gold/dendrimer composite nanodevices (CNDs) (5 nm positive, negative, and neutral, 11 nm negative, 22 nm positive) in mice using a physiologically based mathematical model. METHODS: 400 ug of CNDs is injected intravenously to mice bearing melanoma cell lines. Gold content is determined from plasma and tissue samples using neutron activation analysis. A physiologically based pharmacokinetic (PBPK) model is developed for 5 nm positive, negative, and neutral and 11 nm negative nanoparticles and extrapolated to 22 nm positive particles. A global sensitivity analysis is performed for estimated model parameters. RESULTS: Negative and neutral particles exhibited similar distribution profiles. Unique model parameter estimates and distribution profiles explain similarities and differences relative to positive particles. The model also explains mechanisms of elimination by kidney and reticuloendothelial uptake in liver and spleen, which varies with particle size and charge. CONCLUSION: Since the PBPK model can capture the diverse temporal profiles of non-targeted nanoparticles, we propose that when specific binding ligands are lacking, size and charge of nanodevices govern most of their in vivo interactions.

Fabrication of {198Au0} radioactive composite nanodevices and their use for nanobrachytherapy.

We describe the simple fabrication of poly({198Au}) radioactive gold-dendrimer composite nanodevices in distinct sizes (diameter between 10 nm and 29 nm) for targeted radiopharmaceutical dose delivery to tumors in vivo. Irradiation of aqueous solutions of 197Au containing poly(amidoamine) dendrimer tetrachloroaurate salts or {197Au0} gold-dendrimer nanocomposites in a nuclear reactor resulted in the formation of positively charged and soluble poly{198Au0} radioactive composite nanodevices (CNDs). A mouse melanoma tumor model was used to test whether the poly{198Au0} CNDs can deliver a therapeutic dose. A single intratumoral injection of poly{198Au0}(d=22nm) CNDs in phosphate-buffered saline delivering a dose of 74 muCi resulted after 8 days in a statistically significant 45% reduction in tumor volume, when compared with untreated groups and those injected with the "cold" nanodevice. No clinical toxicity was observed during the experiments. This study provides the first proof of principle that radioactive CNDs can deliver therapeutic doses to tumors.

Self assembly and optical properties of dendrimer nanocomposite multilayers.

Ultrathin multilayers are important for electrical and optical devices, as well as for immunoassays, artificial organs, and for controlling surface properties. The construction of ultrathin multilayer films by electrostatic layer-by-layer deposition proved to be a popular and successful method to create films with a range of electrical, optical, and biological properties. Dendrimer nanocomposites (DNCs) form highly uniform hybrid (inorganic-organic) nanoparticles with controlled composition and architecture. In this work, the fabrication, characterization, and optical properties of ultrathin dendrimer/poly(styrene sulfonate) (PSS) and silver-DNC/PSS nanocomposite multilayers using layer-by-layer (LbL) electrostatic assembly techniques are described. UV-vis spectra of the multilayers were found to be a combination of electronic transitions of the surface plasmon peaks, and the regular frequency modulations attributable to the multilayered film structure. The modulations appeared as the consequence of the highly regular and non-intermixed multilayer growth as a function of the resulting structure. A simple model to explain the experimental data is presented. Use of DNCs in multilayers results in abrupt, flat, and uniform interfaces.

Synthesis and characterization of PAMAM dendrimer-based multifunctional nanodevices for targeting alphavbeta3 integrins.

We have synthesized a stable and clinically relevant nanodevice (cRGD-BT-ND; ND for short) that exhibits superior binding to the biologic target alphavbeta3 integrins, when either compared to the same free cRGD peptide or to the biotinylated nanodevice without covalently attached peptides (BT-ND). Selective targeting of alphavbeta3 integrins was achieved by coupling cyclic cRGD peptides to the nanodevice (ND) surface, while biotin groups (BT) were used for amplified detection of bound cRGD-BT-ND by anti-biotin antibody or avidin linked to horseradish peroxidase after binding. The synthesis involved the following steps: the amino-terminated ethylenediamine core generation 5 poly(amidoamine) (PAMAM_E5.NH2) dendrimer was first partially acetylated and then biotinylated, and residual primary amine termini were converted to succinamic acid groups (SAH), some of which finally were conjugated with cRGD peptide residues through the amino group of the lysine side chain. The starting material and all derivatives were extensively characterized by polyacrylamide gel electrophoresis (PAGE), size exclusion chromatography (SEC), potentiometric acid-base titration, MALDI-TOF, and NMR. Cytotoxicity of all dendrimer derivatives was examined in B16F10 melanoma cell cultures using the XTT colorimetric assay for cellular viability. Binding of nanodevices to the biological target was determined using plates coated with human alphavbeta3 integrin and alphavbeta3 receptor expressing human dermal microvascular endothelial cells (HDMECs). The PAMAM_E5.(NHAc)72(NHBT)8(NHSAH)35(NHSA-cR GD)4 nanodevice is nontoxic within physiologic concentration ranges and specifically binds to the alphavbeta3 integrins, apparently much stronger than the cyclic cRGD peptide itself.

Dendrimer 101.

In this chapter dendrimer basics are reviewed. It is impossible to describe, refer to or even list the related literature (our "dendrimer" database consists of over 7,000 papers and patents), thus selection of references has been made based on personal preference, often choosing clarity over details and overarching principles instead of detailed chemical structures. A large number of excellent reviews are available to those who are interested in more detail in particular areas. References will take the reader to original scientific papers that provide more detail about a particular topic, describe experiments, and draw conclusions reflecting each author's personal views. Even listing of books and reviews must be partial, as they number in the hundreds.

Electrophoretic mobility and molecular distribution studies of poly(amidoamine) dendrimers of defined charges.

Generation 5 ethylenediamine (EDA)-cored poly(amidoamine) (PAMAM) dendrimers (E5, E denotes the EDA core and 5 the generation number) with different degrees of acetylation and carboxylation were synthesized and used as a model system to investigate the effect of charge and the influence of dendrimer surface modifications on electrophoretic mobility (EM) and molecular distribution. The surface-modified dendrimers were characterized by size-exclusion chromatography, 1H NMR, MALDI-TOF-MS, PAGE, and CE. The focus of our study was to determine how EM changes as a function of particle charge and molecular mass, and how the molecular distribution changes due to surface modifications. We demonstrate that partially modified dendrimers have much broader migration peaks than those of fully surface functionalized or unmodified E5 dendrimers due to variations in the substitution of individual dendrimer surfaces. EM decreased nonlinearly with increases in surface acetylation for both PAMAM acetamides and PAMAM succinamic acids, indicating a complex migration activity in CE separations that is not solely due to charge/mass ratio changes. These studies provide new insights into dendrimer properties under an electric field, as well as into the characterization of dendrimer-based materials being developed for medical applications.

Silver/dendrimer nanocomposites as biomarkers: fabrication, characterization, in vitro toxicity, and intracellular detection.

We have synthesized water-soluble, biocompatible, fluorescent, and stable silver/dendrimer nanocomposites that exhibit a potential for in vitro cell labeling. Amino-, hydroxyl-, and carboxyl-terminated ethylenediamine core generation 5 poly(amidoamine) dendrimers were utilized to prepare aqueous silver(I)-dendrimer complexes (with the molar ratio of 25 Ag+ per dendrimer) at the biologic pH of 7.4. Conversion of silver(I)-dendrimer complexes into dendrimer nanocomposites was achieved by irradiating the solutions with UV light to reduce the bound Ag+ cations to zerovalent Ag0 atoms, which were simultaneously trapped in the dendrimer network, resulting in the formation of {(Ag0)25-PAMAM_E5.NH2}, {(Ag0)25-PAMAM_E5.NGly}, and {(Ag0)25-PAMAM_E5.NSAH} dendrimer nanocomposites (DNC), respectively. The silver-DNCs were characterized by means of UV-vis, fluorescence spectroscopy, dynamic light-scattering, zeta potential measurements, high-resolution transmission electron microscopy, X-ray energy dispersive spectroscopy, and selected area electron diffraction. The cytotoxicity of dendrimers and related silver nanocomposites was evaluated using an XTT colorimetric assay of cellular viability. The cellular uptake of nanoparticles was examined by transmission electron and confocal microscopy. Results indicate that {(Ag0)25-PAMAM_E5.NH2}, {(Ag0-)25-PAMAM_E5.NGly}, and {(Ag0)25-PAMAM_E5.NSAH} form primarily single particles with diameters between 3 and 7 nm. The dendrimer nanocomposites are fluorescent, and their surface charge, cellular internalization, toxicity, and cell labeling capabilities are determined by the surface functionalities of dendrimer templates. The {(Ag0)25-PAMAM_E5.NH2} and {(Ag0)25-PAMAM_E5.NSAH} nanocomposites exhibit potential application as cell biomarkers.

In vivo biodistribution of dendrimers and dendrimer nanocomposites -- implications for cancer imaging and therapy.

Our results indicate that the surface chemistry, composition, and 3-D structure of nanoparticles are critical in determining their in vivo biodistribution, and therefore the efficacy of nanodevice imaging and therapies. We demonstrate that gold/dendrimer nanocomposites in vivo, present biodistribution characteristics different from PAMAM dendrimers in a B16 mouse tumor model system. We review important chemical and biologic uses of these nanodevices and discuss the potential of nanocomposite devices to greatly improve cancer imaging and therapy, in particular radiation therapy. We also discuss major issues confronting the use of nanoparticles in the near future, with consideration of toxicity analysis and whether biodegradable devices are needed or even desirable.

Analysis of poly(amidoamine)-succinamic acid dendrimers by slab-gel electrophoresis and capillary zone electrophoresis.

Ethylenediamine (EDA)-core poly(amidoamine) (PAMAM) succinamic acid dendrimers (Ex.SAH, where x refers to the generation) were synthesized and analyzed by polyacrylamide gel electrophoresis (PAGE), size-exclusion chromatography (SEC), potentiometric acid-base titration, and capillary zone electrophoresis (CZE). Various generations (E1.SAH-E7.SAH) PAMAMs and a succinamic acid terminated core-shell tecto(dendrimer) (E5(E3.SAH)(n)) were first analyzed by PAGE. PAGE results show that the relative mobilities of generation 2 to generation 7 dendrimers decreased with the increasing number of generations. The molecular mass of a generation 5 core generation 3 shell tecto(dendrimer) (denoted as E5(E3.SAH)(n)) was determined to be between the Mw of E6.SAH and E7.SAH. CZE analysis allowed the evaluation of electrophoretic properties of given-generation dendrimers. The electrophoretic mobilities of individual generations PAMAM polyanions are similar, indicating that the separation mainly depends on their approximately identical charge/mass ratio. The E5(E3.SAH)(n) tectodendrimer had a lower electrophoretic mobility, which was consistent with its lower charge/mass ratio. The combination of PAGE and CZE analysis provides an alternative and effective way to characterize this group of PAMAM-succinamic acid dendrimers.

Capillary electrophoresis of polycationic poly(amidoamine) dendrimers.

Generation 2 to generation 5 poly(amidoamine) (PAMAM) dendrimers having different terminal functionalities were analyzed by capillary electrophoresis (CE). Polyacrylamide gel electrophoresis was also used to assess the composition of the individual generations for comparison with the CE results. Separation of PAMAMs can be accomplished by either using uncoated silica or silanized silica capillaries, although reproducibility is poor using the uncoated silica capillary. To improve run-to-run reproducibility, silanized capillary was used and various internal standards were also tested. Relative and normalized migration times of primary amine terminated PAMAM dendrimers were then determined using 2,3-diaminopyridine (2,3-DAP) as an internal standard. Using silanized capillaries and internal standards, the relative and normalized migration times are fully reproducible and comparable between runs. Apparent dimensionless electrophoretic mobilities were determined and the results were compared to theoretical calculations. It is concluded that for PAMAMs a complex separation mechanism has to be considered in CE, where the movement of the ions is due to the electric field, but the separation is rather the consequence of the adsorption/desorption equilibria on the capillary wall ("electrokinetic capillary chromatography"). The described method may be used for quality control and may serve as an effective technique to analyze polycationic PAMAM dendrimers and their derivatives with different surface modifications.

Nanoparticle targeting of anticancer drug improves therapeutic response in animal model of human epithelial cancer.

Prior studies suggested that nanoparticle drug delivery might improve the therapeutic response to anticancer drugs and allow the simultaneous monitoring of drug uptake by tumors. We employed modified PAMAM dendritic polymers <5 nm in diameter as carriers. Acetylated dendrimers were conjugated to folic acid as a targeting agent and then coupled to either methotrexate or tritium and either fluorescein or 6-carboxytetramethylrhodamine. These conjugates were injected i.v. into immunodeficient mice bearing human KB tumors that overexpress the folic acid receptor. In contrast to nontargeted polymer, folate-conjugated nanoparticles concentrated in the tumor and liver tissue over 4 days after administration. The tumor tissue localization of the folate-targeted polymer could be attenuated by prior i.v. injection of free folic acid. Confocal microscopy confirmed the internalization of the drug conjugates into the tumor cells. Targeting methotrexate increased its antitumor activity and markedly decreased its toxicity, allowing therapeutic responses not possible with a free drug.

Acoustic detection of controlled laser-induced microbubble creation in gelatin.

A high-frequency (85 MHz) acoustic technique is used to identify system parameters for controlled laser-induced microbubble creation inside tissue-mimicking, gelatin phantoms. Microbubbles are generated at the focus of an ultrafast 793-nm laser source and simultaneously monitored through ultrasonic pulse-echo recordings. Displayed in wavefield form, these recordings illustrate microbubble creation, and integrated backscatter plots provide specifics about microbubble characteristics and dissolution behavior. By varying laser parameters, including pulse fluence (or pulse energy flux, J/cm2), total number of pulses delivered, and the period between pulses, the size, lifetime, and dissolution dynamics of laser-induced microbubbles may be independently controlled. Pulse fluence is the main size-controlling parameter, whereas both increases in pulse fluence and pulse number can lengthen microbubble lifetime from tens to hundreds of milliseconds. In short, a microbubble of particular lifetime does not necessarily have to be of a particular size. Microbubble behavior, furthermore, is independent of pulse periods below a fluence-dependent threshold value, but it exhibits stochastic behavior if pulse repetition is too slow. These results demonstrate that laser pulse fluence, number, and period may be varied to deposit energy in a specific temporal manner, creating and stabilizing microbubbles with particular characteristics and, therefore, potential uses in sensitive acoustic detection and manipulation schemes.