Optical fiber-based in vivo quantification of growth factor receptors.

BACKGROUND: Growth factor receptors such as epidermal growth factor receptor 1 and human epidermal growth receptor 2 (HER2) are overexpressed in certain cancer cells. Antibodies against these receptors (eg. cetuximab and transtuzumab [Herceptin]) have shown therapeutic value in cancer treatment. The existing methods for the quantification of these receptors in tumors involve immunohistochemistry or DNA quantification, both in extracted tissue samples. The goal of the study was to evaluate whether an optical fiber-based technique can be used to quantify the expression of multiple growth factor receptors simultaneously. METHODS: The authors examined HER2 expression using the monoclonal antibody trastuzumab as a targeting ligand to test their system. They conjugated trastuzumab to 2 different Alexa Fluor dyes with different excitation and emission wavelengths. Two of the dye conjugates were subsequently injected intravenously into mice bearing HER2-expressing subcutaneous tumors. An optical fiber was then inserted into the tumor through a 30-gauge needle, and using a single laser beam as the excitation source, the fluorescence emitted by the 2 conjugates was identified and quantified by 2-photon optical fiber fluorescence. RESULTS: The 2 conjugates bound to the HER2-expressing tumor competitively in a receptor-specific fashion, but they failed to bind to a similar cell tumor that did not express HER2. The concentration of the conjugate present in the tumor as determined by 2-photon optical fiber fluorescence was shown to serve as an index of the HER2 expression levels. CONCLUSIONS: These studies offer a minimally invasive technique for the quantification of tumor receptors simultaneously.

Dendrimer-based tumor cell targeting of fibroblast growth factor-1.

Fibroblast Growth Factor Receptor (FGFR) is overexpressed in a wide variety of tumors, and therefore is an attractive target for drug delivery. Recombinant FGF-1 was purified and attached to a fifth-generation (G5) polyamidoamine dendrimer. The specific binding and internalization of this conjugate labeled with FITC was demonstrated by flow cytometry as well as by confocal microscopic analysis in cell lines expressing FGFR. The binding and uptake of FGF-conjugated dendrimers was completely blocked by excess nonconjugated FGF-1. Confocal microscopic analysis showed cytosolic as well as nuclear localization. Multivalent G5-FGF nanoparticles may serve as a platform for cytosolic as well as nuclear drug delivery in tumor cells, and as an FGF delivery agent for angiogenesis and wound healing. Our study shows for the first time the applicability of a dendrimer nanodevice for tumor cell targeting through FGFR.

RGD dendron bodies; synthetic avidity agents with defined and potentially interchangeable effector sites that can substitute for antibodies.

Poly(amidoamine) (PAMAM) dendrons were synthesized with c(RGDyK) peptide on the surface to create a scaffold for cellular targeting and multivalent binding. Binary dendron-RGD conjugates were synthesized with a single Alexa Fluor 488, biotin, methotrexate drug molecule, or additional functionalized dendron at the focal point. The targeted dendron platform was shown to specifically target αvß3 integrin expressing human umbilical vein endothelial cells (HUVEC) and human glioblastoma cells (U87MG) in Vitro via flow cytometry. Specific targeting of the dendron-RGD platform was further confirmed by confocal microscopy. Biological activity of the targeted drug conjugate was confirmed via XTT assay. The orthogonal reaction chemistry used at the dendron focal point gives a precise 1:1 ratio of the attachment of multiple functionalities to a small-molecular-weight, chemically stable, high avidity molecule. These studies serve as a framework to selectively combine biologically relevant functions with enhanced specific binding activity to substitute for antibodies in many diagnostic and therapeutic applications.

Dendrimer-epidermal growth factor conjugate displays superagonist activity.

Binding of ligands on to epidermal growth factor receptor (EGFR) can stimulate cell growth; therefore, any application employing EGF as a targeting ligand for a "drug carrier" must evaluate the effect of the conjugate on cell growth. We report the synthesis and in vitro biological activity of EGF molecules coupled to a fluorescein-labeled polyamidoamine dendrimer. The conjugate bound and internalized into several EGFR-expressing cell lines in a receptor-specific fashion. The conjugate effectively induced EGFR phosphorylation and acted as a superagonist by stimulating cell growth to a greater degree than free EGF. Concomitant administration of the chemotherapeutic drug methotrexate completely inhibited cell growth to a degree similar to its effect in the absence of the conjugate. Thus, dendrimer-EGF conjugates serve as EGFR superagonists, but this activity can be overcome by chemotherapeutic drugs. The agonist activity of these materials must be taken into consideration when using EGF conjugates for imaging applications.

HER2 specific delivery of methotrexate by dendrimer conjugated anti-HER2 mAb.

Herceptin, a humanized monoclonal antibody that binds to human growth factor receptor-2 (HER2), was covalently attached to a fifth-generation (G5) polyamidoamine dendrimer containing the cytotoxic drug methotrexate. The specific binding and internalization of this conjugate labeled with FITC was clearly demonstrated in cell lines overexpressing HER2 by flow cytometry as well as confocal microscopic analysis. In addition, binding and uptake of antibody conjugated dendrimers was completely blocked by excess non-conjugated herceptin. The dendrimer conjugate was also shown to inhibit the dihydrofolate reductase with similar activity to methotrexate. Co-localization experiments with lysotracker red indicate that antibody conjugate, although internalized efficiently into cells, has an unusually long residence time in the lysosome. Somewhat lower cytotoxicity of the conjugate in comparison to free methotrexate was attributed to the slow release of methotrexate from the conjugate and its long retention in the lysosomal pocket.

Tumor microvasculature targeting with dendrimer-entrapped gold nanoparticles.

Monodisperse dendrimer-entrapped gold nanoparticles (diameter = 3.0 nm) were prepared using G5 poly(amidoamine) (PAMAM) dendrimer functionalized with fluorescein isothiocyanate (FI) and Arg-Gly-Asp (RGD) peptide as template; in vitro targeting efficacy to integrin receptor expressing cells was confirmed by flow cytometry, confocal microscopy, and ICP-MS.

Synthetic PAMAM-RGD conjugates target and bind to odontoblast-like MDPC 23 cells and the predentin in tooth organ cultures.

Screening techniques now allow for the identification of small peptides that bind specifically to molecules like cells. However, despite the enthusiasm for this approach, single peptides often lack the binding affinity to target in vivo and regulate cell function. We took peptides containing the Arg-Gly Asp(RGD) motif that bind to the alpha Vbeta 3 integrin and have shown potential as therapeutics. To improve their binding affinity, we synthesized polyamidoamine (PAMAM) dendrimer-RGD conjugates that that contain 12-13 copies of the peptide. When cultured with human dermal microvessel endothelial cells (HDMEC), human vascular endothelial cells (HUVEC), or odontoblast-like MDPC-23 cells, the PAMAM dendrimer conjugate targets this receptor in a manner that is both time- and dose-dependent. Finally, this conjugate selectively targets RGD binding sites in the predentin of human tooth organ cultures. Taken together, these studies provide proof of principle that synthetic PAMAM-RGD conjugates could prove useful as carriers for the tissue-specific delivery of integrin-targeted therapeutics or imaging agents and could be used to engineer tissue regeneration.

HER2 specific tumor targeting with dendrimer conjugated anti-HER2 mAb.

In the present study, we report the synthesis and human growth factor receptor-2 (HER2) specific tumor targeting properties of a dendrimer conjugated to anti-HER2 mAb (monoclonal antibody) conjugate. The polyamidoamine (PAMAM) dendrimer generation five (G5) was labeled with alexaFluor 488 and conjugated to anti-HER2 mAb. The binding and internalization of the antibody-conjugated dendrimer to HER2-expressing cells was evaluated by flow cytometry and confocal microscopy. Uniquely, the conjugate demonstrated cellular uptake and internalization in HER2-expressing cells as compared to free antibody. The time course of internalization and blocking experiments with free antibody suggest that the rapid and efficient cellular internalization of the dendrimer-antibody conjugate was achieved without alterations in specificity of targeting. Animal studies demonstrated that the conjugate targets HER2-expressing tumors.

Tumor angiogenic vasculature targeting with PAMAM dendrimer-RGD conjugates.

PAMAM dendrimer-RGD-4C peptide conjugate was synthesized and in vitro targeting efficacy to integrin receptor expressing cells was studied by flow cytometry and confocal microscopy.

Substrate discrimination by cholapod anion receptors: geometric effects and the "affinity-selectivity principle".

Cholapod anion receptors can achieve high affinities while maintaining compatibility with nonpolar media. Previously they have been shown to transport anions across cell and vesicle membranes. In the present work, the scope of the architecture is expanded and structure-selectivity relationships are investigated. Eight new receptors have been synthesized, with up to six H-bond donor centers. Using Cram's extraction method, these compounds plus five known examples have been tested for binding to seven monovalent anions (tetraethylammonium salts, wet chloroform as solvent). Association constants in excess of 10(10) M(-1) have been measured for several pairings. Selectivities vary with receptor geometry, as expected. More remarkably, they also depend on receptor strength: more powerful receptors show a wider range of binding free energies, and therefore a greater spread of Ka(X-)/Ka(Y-). This "affinity-selectivity" effect can be derived from empirical relationships for H-bond strengths, and could prove widely operative in supramolecular chemistry.

Identification of synthetic phosphatidylserine translocases from a combinatorial library prepared by directed split-and-pool synthesis.

Simple sulfonamide and amide derivatives of tris(2-aminoethyl)amine (Tren) are known to promote the translocation or flip-flop of phosphatidylcholine, but not phosphatidylserine, across bilayer membranes. This paper describes the synthesis of a 300-member, spatially encoded library of Tren derivatives with appended peptide--sulfonamide and peptide--urea arms. The library was synthesized using the Encore method with SynPhase lanterns as the solid support. A high-throughput assay was developed to screen individual members of the library for an ability to translocate a fluorescent NBD derivative of phosphatidylserine across vesicle membranes. Several lead compounds were identified, and one was synthesized independently to confirm its high phosphatidylserine translocation activity.

Facilitated phosphatidylserine flip-flop across vesicle and cell membranes using urea-derived synthetic translocases.

Tris(2-aminoethyl)amine derivatives with appended urea and sulfonamide groups are shown to facilitate the translocation of fluorescent phospholipid probes and endogenous phosphatidylserine across vesicle and erythrocyte cell membranes. The synthetic translocases appear to operate by binding to the phospholipid head groups and forming lipophilic supramolecular complexes which diffuse through the non-polar interior of the bilayer membrane.

Selective phosphatidylethanolamine translocation across vesicle membranes using synthetic translocases.

Two sulfonamide derivatives of tris(aminoethyl)amine selectively facilitate the translocation of a fluorescent phospholipid probe containing the phosphoethanolamine head-group across vesicle membranes.

Templated conversion of a crown ether-containing macrobicycle into [2]rotaxanes.

A crown ether-containing macrobicycle was used as the wheel component in a templated synthesis of a [2]rotaxane with an acetal-containing axle. The molecular structures of the macrobicycle and the [2]rotaxane were characterized by NMR spectroscopy and X-ray crystallography. The chloride-binding ability of the macrobicycle, either free in solution or when it is part of a [2]rotaxane, is quite weak as determined by NMR titration experiments. A second analogous [2]rotaxane, with a longer axle, was synthesized, and its solvent-dependent co-conformation was characterized by 2D NMR spectroscopy. The position of the wheel along the axle can be controlled by the solvent polarity, however, attempts to use metal cations such as Na(+), K(+), Ba(2+), and Ag(+) to switch the wheel position in polar solvents were unsuccessful.