Synthesis, Internalization and Visualization of N-(4-Carbomethoxy) Pyrrolidone Terminated PAMAM [G5:G3-TREN] Tecto(dendrimers) in Mammalian Cells.

Tecto(dendrimers) are well-defined, dendrimer cluster type covalent structures. In this article, we present the synthesis of such a PAMAM [G5:G3-(TREN)]-N-(4-carbomethoxy) pyrrolidone terminated tecto(dendrimer). This tecto(dendrimer) exhibits nontraditional intrinsic luminescence (NTIL; excitation 376 nm; emission 455 nm) that has been attributed to three fluorescent components characterized by different fluorescence lifetimes. Furthermore, it has been shown that this PAMAM [G5:G3-(TREN)]-N-(4-carbomethoxy) pyrrolidone terminated tecto(dendrimer) is able to form a polyplex with double stranded DNA, and is nontoxic for HeLa and HMEC-1 cells up to a concentration of 10 mg/mL, even though it accumulates in endosomal compartments as demonstrated by its unique NTIL emission properties. Many of the above features would portend the proposed use of this tecto(dendrimer) as an efficient transfection agent. Quite surprisingly, transfection activity could not be demonstrated in HeLa cells, and the possible reasons are discussed in the article.

A Systematic Framework and Nanoperiodic Concept for Unifying Nanoscience: Hard/Soft Nanoelements, Superatoms, Meta-Atoms, New Emerging Properties, Periodic Property Patterns, and Predictive Mendeleev-like Nanoperiodic Tables.

Development of a central paradigm is undoubtedly the single most influential force responsible for advancing Dalton's 19th century atomic/molecular chemistry concepts to the current maturity enjoyed by traditional chemistry. A similar central dogma for guiding and unifying nanoscience has been missing. This review traces the origins, evolution, and current status of such a critical nanoperiodic concept/framework for defining and unifying nanoscience. Based on parallel efforts and a mutual consensus now shared by both chemists and physicists, a nanoperiodic/systematic framework concept has emerged. This concept is based on the well-documented existence of discrete, nanoscale collections of traditional inorganic/organic atoms referred to as hard and soft superatoms (i.e., nanoelement categories). These nanometric entities are widely recognized to exhibit nanoscale atom mimicry features reminiscent of traditional picoscale atoms. All unique superatom/nanoelement physicochemical features are derived from quantized structural control defined by six critical nanoscale design parameters (CNDPs), namely, size, shape, surface chemistry, flexibility/rigidity, architecture, and elemental composition. These CNDPs determine all intrinsic superatom properties, their combining behavior to form stoichiometric nanocompounds/assemblies as well as to exhibit nanoperiodic properties leading to new nanoperiodic rules and predictive Mendeleev-like nanoperiodic tables, and they portend possible extension of these principles to larger quantized building blocks including meta-atoms.

A Serendipitous Journey Leading to My Love of Dendritic Patterns and Chemistry.

As the oldest of four Midwestern boys who were offsprings of an accountant and a housewife, each with less than a formal high school degree, we were blessed to have such parents.[...].

Special Issue: "Functional Dendrimers".

This special issue entitled "Functional Dendrimers" focuses on the manipulation of at least six "critical nanoscale design parameters" (CNDPs) of dendrimers including: size, shape, surface chemistry, flexibility/rigidity, architecture and elemental composition. These CNDPs collectively define properties of all "functional dendrimers". This special issue contains many interesting examples describing the manipulation of certain dendrimer CNDPs to create new emerging properties and, in some cases, predictive nanoperiodic property patterns (i.e., dendritic effects). The systematic engineering of CNDPs provides a valuable strategy for optimizing functional dendrimer properties for use in specific applications.

Novel RNA oligonucleotide improves liver function and inhibits liver carcinogenesis in vivo.

UNLABELLED: Hepatocellular carcinoma (HCC) occurs predominantly in patients with liver cirrhosis. Here we show an innovative RNA-based targeted approach to enhance endogenous albumin production while reducing liver tumor burden. We designed short-activating RNAs (saRNA) to enhance expression of C/EBPα (CCAAT/enhancer-binding protein-α), a transcriptional regulator and activator of albumin gene expression. Increased levels of both C/EBPα and albumin mRNA in addition to a 3-fold increase in albumin secretion and 50% decrease in cell proliferation was observed in C/EBPα-saRNA transfected HepG2 cells. Intravenous injection of C/EBPα-saRNA in a cirrhotic rat model with multifocal liver tumors increased circulating serum albumin by over 30%, showing evidence of improved liver function. Tumor burden decreased by 80% (P = 0.003) with a 40% reduction in a marker of preneoplastic transformation. Since C/EBPα has known antiproliferative activities by way of retinoblastoma, p21, and cyclins, we used messenger RNA (mRNA) expression liver cancer-specific microarray in C/EBPα-saRNA-transfected HepG2 cells to confirm down-regulation of genes strongly enriched for negative regulation of apoptosis, angiogenesis, and metastasis. Up-regulated genes were enriched for tumor suppressors and positive regulators of cell differentiation. A quantitative polymerase chain reaction (PCR) and western blot analysis of C/EBPα-saRNA-transfected cells suggested that in addition to the known antiproliferative targets of C/EBPα, we also observed suppression of interleukin (IL)6R, c-Myc, and reduced STAT3 phosphorylation. CONCLUSION: A novel injectable saRNA-oligonucleotide that enhances C/EBPα expression successfully reduces tumor burden and simultaneously improves liver function in a clinically relevant liver cirrhosis/HCC model.

Analysis of polyamidoamine dendrimers by isoelectric focusing.

Polyamidoamine dendrimers have been studied extensively for their potential applications in nanomedicine. Their uses as imaging, drug, and nucleic acid delivery agents are nearing clinical trials. As such, characterization of polyamidoamine dendrimers and their nano-devices is of immense importance for monitoring the efficiency of their synthesis, purity, and quality control of manufactured products as well as their in vivo behavior. We report here the analysis of polyamidoamine dendrimers possessing various cores and surface groups with a simple and inexpensive isoelectric focusing method. The isoelectric points of the dendrimers were readily determined from a calibration plot generated by running proteins with known pI values. The isoelectric points for various surface-modified polyamidoamine dendrimers ranged from 4 to 9. Polyamidoamine dendrimers possessing terminal hydroxyl groups gave a pI > 7, while those with terminal carboxyl groups exhibit a pI < 7. Generation number and cores of the dendrimers did not significantly affect their isoelectric points. Isoelectric focusing thus offers another important tool for characterizing these nanomolecules.

The Role of Branch Cell Symmetry and Other Critical Nanoscale Design Parameters in the Determination of Dendrimer Encapsulation Properties.

This article reviews progress over the past three decades related to the role of dendrimer-based, branch cell symmetry in the development of advanced drug delivery systems, aqueous based compatibilizers/solubilizers/excipients and nano-metal cluster catalysts. Historically, it begins with early unreported work by the Tomalia Group (i.e., The Dow Chemical Co.) revealing that all known dendrimer family types may be divided into two major symmetry categories; namely: Category I: symmetrical branch cell dendrimers (e.g., Tomalia, Vögtle, Newkome-type dendrimers) possessing interior hollowness/porosity and Category II: asymmetrical branch cell dendrimers (e.g., Denkewalter-type) possessing no interior void space. These two branch cell symmetry features were shown to be pivotal in directing internal packing modes; thereby, differentiating key dendrimer properties such as densities, refractive indices and interior porosities. Furthermore, this discovery provided an explanation for unimolecular micelle encapsulation (UME) behavior observed exclusively for Category I, but not for Category II. This account surveys early experiments confirming the inextricable influence of dendrimer branch cell symmetry on interior packing properties, first examples of Category (I) based UME behavior, nuclear magnetic resonance (NMR) protocols for systematic encapsulation characterization, application of these principles to the solubilization of active approved drugs, engineering dendrimer critical nanoscale design parameters (CNDPs) for optimized properties and concluding with high optimism for the anticipated role of dendrimer-based solubilization principles in emerging new life science, drug delivery and nanomedical applications.

Liver Activation of Hepatocellular Nuclear Factor-4α by Small Activating RNA Rescues Dyslipidemia and Improves Metabolic Profile.

Non-alcoholic fatty liver disease (NAFLD) culminates in insulin resistance and metabolic syndrome. Because there are no approved pharmacological treatment agents for non-alcoholic steatohepatitis (NASH) and NAFLD, different signaling pathways are under investigation for drug development with the focus on metabolic pathways. Hepatocyte nuclear factor 4-alpha (HNF4A) is at the center of a complex transcriptional network where its disruption is directly linked to glucose and lipid metabolism. Resetting HNF4A expression in NAFLD is therefore crucial for re-establishing normal liver function. Here, small activating RNA (saRNA) specific for upregulating HNF4A was injected into rats fed a high-fat diet for 16 weeks. Intravenous delivery was carried out using 5-(G5)-triethanolamine-core polyamidoamine (PAMAM) dendrimers. We observed a significant reduction in liver triglyceride, increased high-density lipoprotein/low-density lipoprotein (HDL/LDL) ratio, and decreased white adipose tissue/body weight ratio, all parameters to suggest that HNF4A-saRNA treatment induced a favorable metabolic profile. Proteomic analysis showed significant regulation of genes involved in sphingolipid metabolism, fatty acid ß-oxidation, ketogenesis, detoxification of reactive oxygen species, and lipid transport. We demonstrate that HNF4A activation by oligonucleotide therapy may represent a novel single agent for the treatment of NAFLD and insulin resistance.

Unexpected in vivo anti-inflammatory activity observed for simple, surface functionalized poly(amidoamine) dendrimers.

We report unexpected anti-inflammatory properties for naked, unmodified poly(amidoamine) (PAMAM) dendrimers bearing simple surface functionality (e.g., -NH(2), -OH, etc.). This property was discovered serendipitously while studying the drug delivery features of PAMAM dendrimer-indomethacin complexes. Activity was quantitated by using three independently recognized in vivo anti-inflammatory assay methods, namely, (1) the carrageenan-induced paw edema model (acute activity), (2) the cotton pellet test, and (3) the adjuvant-induced arthritis assay in rats (chronic activities). Those dendrimers bearing amine or hydroxyl surface groups exhibited significant anti-inflammatory activity in the carrageenan-induced paw edema model. For example, [core: 1,2-diaminoethane]; (G = 4.0); {dendri-poly(amidoamine)-(NH(2))(64)} (i.e., G4-NH(2)) exhibited a mean percent inhibition of 35.50 +/- 1.6% 3 h after administration and [core: 1,2-diaminoethane] (G = 4.0); {dendri-poly(amidoamine)-(OH)(64)} (i.e., G4-OH) gave a mean percent inhibition of 31.22 +/- 1.58% 3 h after administration. On the other hand, [core: 1,2-diaminoethane] (G = 4.5); {dendri-poly(amidoamine)-(CO(2)H)(128)} (i.e., G4.5-CO(2)H) exhibited mild anti-inflammatory activity with a mean percent inhibition of 14.00 +/- 2.5% 3 h after administration. Unexpectedly, G4-NH(2) showed significantly higher activity compared to naked indomethacin (i.e., 50 +/- 3.1% vs 22 +/- 1.2%) using the cotton pellet granuloma model. Similarly, in the adjuvant-induced arthritis model, G4-NH(2) compared to naked indomethacin gave a mean percent inhibition of 30 +/- 1.9% versus 11 +/- 0.9% 14 days after administration.

EPR characterization of gadolinium(III)-containing-PAMAM-dendrimers in the absence and in the presence of paramagnetic probes.

Gd(III)-containing dendrimers are promising contrast agents for magnetic resonance imaging (MRI). An important issue in the effectiveness and toxicity of a Gd(III) based MRI contrast agent is knowledge of the relative locations and concentrations of Gd(III) in dendrimer drug delivery hosts. In order to provide experimental information on this issue, we have investigated the electron paramagnetic resonance (EPR) of a stable Gd(III) complex with diethylenetriaminepentaacetic acid (DTPA) in various polyammidoamine (PAMAM) dendrimers as a function of dendrimer generation (G2, G4, and G6), dendrimer core (ethylenediamine = EDA, and cystamine = cys), and dendrimer surface functionality (NH(2), 5-oxo-3-pyrrolidinecarboxylic acid methyl ester = pyr, and tris(hydroxymethyl) methylamine = tris). The dendrimer systems were investigated in the presence and absence of paramagnetic probes, that is, Cu(II) and nitroxide radicals (4-(trimethylammonium and dodecyl-dimethylammonium) 2,2,6,6-tetramethylpiperidine 1-oxyl bromide = CAT1 and CAT12, respectively). The analysis of the EPR spectra revealed anisotropic locations of Gd-DTPA inside the dendrimer. Computer analysis of the EPR spectra of the probes identified the interactions of the Gd-dendrimers with ions and organic molecules. The interaction between the probes and the dendrimer internal and external surface depends on the type of core, the composition of the external surface and the generation of the dendrimer. The negatively charged Gd-DTPA complex attracts the positively charged species and this provokes spin-spin interactions between Gd and the probes, which increases with a decrease in generation, mainly from G6 to G4, and with an increase in both the Gd-dendrimer concentration and the probe concentration. The cys core increases the internal volume and decreases the packing of the branches.

Nanomedicine and drug delivery.

This article discusses the use of nanotechnology in drug delivery approaches. Magnetic nanotechnology is finding wide applications in medicine, most notably in MRI and magnetic separation. The impedance biosensor is expected to find applications in monitoring cytokines in cancer, bone turnover markers in osteoporosis, and understanding neural-degenerative diseases.

AFM analysis of C60 and a poly(amido amine) dendrimer-C60 nanoconjugate.

Atomic force microscopy (AFM) was used to study the nanoscopic structure and topography of buckminsterfullerene (C60) and a conjugate of C60 with generation four, amine-terminated, poly(amido amine) dendrimer (PAMAM-G4). The conjugate contains a PAMAM-G4 core and C60 shell formed by reacting PAMAM-G4 with an excess of C60. Fractal patterns of C60 were observed in nanoscopic AFM images when solutions of different concentrations of C60 in pyridine or toluene were dried at room temperature. In contrast, no fractal patterns were detected in the AFM images of the dendrimer-C60 nanoconjugate, prepared from pyridine solution in a similar manner. Thus, the C60-shell alone is not sufficient to impart the same fractal patterns on the conjugate.

From bench to bedside: successful translational nanomedicine: highlights of the Third Annual Meeting of the American Academy of Nanomedicine.

The Third Annual Meeting of the American Academy of Nanomedicine (AANM) was held at the University of California San Diego, in San Diego, California during September 7-8, 2007. The meeting was focused on successful translational nanomedicine: from bench to bedside. There were four keynote lectures and eight scientific symposiums in this meeting. The researchers and investigators reported the results and process of current nanomedicine research and approaches to clinical applications. The meeting provided exciting information for nanomedicine clinical-related researches and strategy for further development of nanomedicine research which will be benefits to clinical practice.

Engineering dendrimers to produce dendrimer dipole excitation based terahertz radiation sources suitable for spectrometry, molecular and biomedical imaging.

Two critical nanoscale design parameters (CNDPs); namely, surface chemistry and interior compositions of poly(amidoamine) (PAMAM) dendrimers were systematically engineered to produce unique hyperpolarizable, electro-optical substrates. These electro-optically active dendritic films were demonstrated to produce high quality, continuous wave terahertz radiation when exposed to a suitable pump laser that could be used for spectrometry and molecular imaging. These dendrimer based dipole excitation (DDE) terahertz sources were used to construct a working spectrometer suitable for many practical applications including THz imaging and analysis of encapsulated hydrogen species in fullerenes.

Modified PAMAM dendrimer with 4-carbomethoxypyrrolidone surface groups-its uptake, efflux, and location in a cell.

Traditional amine terminated PAMAM dendrimers may be readily surface engineered by a facile one-pot conversion with dialkyl itaconate esters into 4-carbomethoxypyrrolidone terminated PAMAM (G=0-4) dendrimers. These terminated dendrimers are uniquely characterized by exhibiting blue fluorescence emissions (λexc=370nm, λmaxem=440nm). Thanks to this property they can be directly analyzed by confocal microscopy and flow cytometry without additional fluorescence labeling, treatment of dendrimers with chemicals or adjusting pH. These intrinsically fluorescent dendrimers were shown to be very effective for assessing important biological cell features such as cellular entry, intracellular trafficking/localization and efflux properties. For example, all tested cell lines (e.g., B14, BRL-3A, and mHippoE-18) rapidly accumulated PAMAM-pyrrolidone dendrimer. The BRL-3A cell line exhibited both cytoplasmic and nuclear localization patterns; whereas in B14 cells and mHippoE-18 cells, the blue dendrimer fluorescence could only be detected in intracellular endosome-like structures. The dendrimer was observed to be released from all three cell lines during the first 24h; however, efflux was substantially slower from the B-14 cell line. The highest efflux rate was observed for the mHippoE-18 cells. This first successful biological experiment opens a broad spectrum of using these dendrimers as new bioimaging agents for extensive biological cell characterizations.

New technology and clinical applications of nanomedicine: highlights of the second annual meeting of the American Academy of Nanomedicine (Part I).

The Second Annual Meeting of the American Academy of Nanomedicine (AANM) was held at the National Academy of Science Building in Washington, DC, September 9-10, 2006. The program included two Nobel Prize Laureate Lectures, two Keynote Lectures, and 123 invited outstanding State-in-Art lectures presenting in 23 special concurrent symposia. In addition, there were 22 poster presentations in the meeting addressing different areas in nanomedicine research. All of the presenters at the meeting are outstanding investigators and researchers in the field. The Second Annual Meeting of the AANM was a great success. The meeting provides investigators from different world areas a forum and an opportunity for discussion. We believe that nanomedicine research will develop rapidly in the future. The AANM invites basic and clinical researchers from the world to join this exciting research.

Dendrimers in biomedical applications--reflections on the field.

The formation of particulate systems with well-defined sizes and shapes is of eminent interest in certain medical applications such as drug delivery, gene transfection, and imaging. The high level of control possible over the architectural design of dendrimers; their size, shape, branching length/density, and their surface functionality, clearly distinguishes these structures as unique and optimum carriers in those applications. The bioactive agents may be encapsulated into the interior of the dendrimers or chemically attached/physically adsorbed onto the dendrimer surface, with the option of tailoring the carrier to the specific needs of the active material and its therapeutic applications. In this regard, the high density of exo-presented surface groups allows attachment of targeting groups or functionality that may modify the solution behavior or toxicity of dendrimers. Quite remarkably, modified dendrimers have been shown to act as nano-drugs against tumors, bacteria, and viruses. Recent successes in simplifying and optimizing the synthesis of dendrimers such as the 'lego' and 'click' approaches, provide a large variety of structures while at the same time reducing the cost of their production. The reflections on biomedical applications of dendrimers given in this review clearly demonstrate the potential of this new fourth major class of polymer architecture and indeed substantiate the high hopes for the future of dendrimers.

Structure/function relationships of polyamidoamine/DNA dendrimers as gene delivery vehicles.

PAMAM dendrimers are members of a class of polyamine polymers that demonstrate significant gene delivery ability. In this study, a selection of PAMAM dendrimers, spanning a range of sizes (generations 2, 4, 7, and 9) and transfection efficiencies, are characterized by various biophysical methods to search for structural properties that correlate with transfection. Measurements of colloidal properties (size and zeta potential) as a function of charge ratio reveal that highly transfecting dendrimer/DNA complexes have size/zeta potential values between 4 and 8. Circular dichroism (CD) and FTIR spectroscopy of complexes confirm the DNA component remains in B form when associated with all dendrimer generations up to a 5:1 charge ratio (+/-). Isothermal titration calorimetry and differential scanning calorimetry detect changes that are related to polymer structure and charge ratio but do not directly correlate with transfection efficiency. Despite DNA structural and stability changes detected by CD, FTIR, DSC, and ITC that are similar to those seen with other cationic delivery vehicles [e.g., cationic lipids, peptoids/lipitoids, peptides, polyethyleneimines (PEIs), etc.], clear correlations with transfection activity are not readily apparent. This may be due, at least in part, to the heterogeneity of the complexes.

Interview: An architectural journey: from trees, dendrons/dendrimers to nanomedicine. Interview by Hannah Stanwix.

Donald Tomalia received his Bachelor of Arts degree in Chemistry from the University of Michigan (MI, USA). He received his PhD in physical-organic Chemistry from Michigan State University (MI, USA) in 1968 while working at The Dow Chemical Company (MI, USA). In 1990 he moved to Michigan Molecular Institute (MI, USA) as Professor and Director of Nanoscale Chemistry and Architecture. He has subsequently founded three dendrimer based-nanotechnology companies, Dendritech, Inc. (MI, USA), Dendritic Nanotechnologies, Inc. (MI, USA) and NanoSynthons LLC (MI, USA). Donald Tomalia is currently Director of the National Dendrimer & Nanotechnology Center (MI, USA), CEO/founder of NanoSynthons LLC (MI, USA), distinguished visiting Professor, Columbia University (NY, USA) and affiliate Professor, Department of Physics, Virginia Commonwealth University (VA, USA). He is best known for his discovery of dendrimers and has received several awards for his accomplishments and contributions to science, including the 2012 Wallace H Carothers Award. He has authored over 250 publications, as well as over 128 patents.

Dendrimer-based drug and imaging conjugates: design considerations for nanomedical applications.

Dendrimers are members of a versatile, fourth new class of polymer architecture (i.e. dendritic polymers after traditional linear, crosslinked and branched types). Typically, dendrimers are used as well-defined scaffolding or nanocontainers to conjugate, complex or encapsulate therapeutic drugs or imaging moieties. As a delivery vector, the dendrimer conjugate linker or spacer chemistry plays a crucial part in determining optimum drug delivery to disease sites by conserving active drug efficacy while influencing appropriate release patterns. This review focuses on several crucial issues related to those dendrimer features, namely the role of dendrimers as nanoscaffolding and nanocontainers, crucial principles that might be invoked for improving dendrimer cytotoxicity properties, understanding dendrimer cellular transport mechanisms and the exciting role of dendrimers as high-contrast MRI imaging agents. The review concludes with a brief survey of translational efforts from research and development phases to clinical trials that are actively emerging.