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.

Significant effect of size on the in vivo biodistribution of gold composite nanodevices in mouse tumor models.

There is growing interest in developing tissue-specific multifunctional drug delivery systems with the ability to diagnose or treat several diseases. One class of such agents, composite nanodevices (CNDs), is multifunctional nanomaterials with several potential medical uses, including cancer imaging and therapy. Nanosized metal-dendrimer CNDs consist of poly(amidoamine) dendrimers (in various sizes, surface substituents, and net charges) and inorganic nanoparticles, properties of both of which can be individually modified and optimized. In this study we examine effects of size and surface charge on the behavior of Au-dendrimer CNDs in mouse tumor models. Quantitative biodistribution and excretion analyses including 5-nm and 22-nm positive surface, 5-nm and 11-nm negative surface, and a 5-nm neutral surface CNDs were carried out in the B16 mouse melanoma tumor model system. Results seen with the 22-nm CND in the B16 melanoma model were corroborated in a prostate cancer mouse tumor model system. Quantitative in vivo studies confirm the importance of charge and show for the first time the importance of size in affecting CND biodistribution and excretion. Interestingly, CNDs of different size and/or surface charge had high levels of uptake ("selective targeting") to certain organs without specific targeting moieties placed on their surfaces. We conclude that size and charge greatly affect biodistribution of CNDs. These findings have significance for the design of all particle-based nanodevices for medical uses. The observed organ selectivity may make these nanodevices exciting for several targeted medical applications.

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.

Tetrathiomolybdate blocks bFGF- but not VEGF-induced incipient angiogenesis in vitro.

BACKGROUND: Angiogenesis is a multi-step process which involves endothelial cell sprouting from existing blood vessels, followed by migration, proliferation, alignment and tube formation. Tetrathiomolybdate (TM) is a multi-hit antiangiogenic agent with actions against multiple angiogenic pathways. These inhibitory effects of TM are attributed to its potent copper level-reducing property. Copper is needed for activation of various angiogenic pathways at the transcriptional and protein levels. MATERIALS AND METHODS: The direct effects of TM on angiogenesis of endothelial cells were examined using an in vitro sprout-forming system. RESULTS: It was shown that depletion of copper by TM selectively repressed bFGF-induced, but not VEGF-induced sprout formation (an early angiogenic step). CONCLUSION: This model permitted the separation of VEGF- and bFGF- induced early angiogenesis in vitro, and indicated the existence of mechanistic differences between bFGF- and VEGF- induced early angiogenic events.

Combination tetrathiomolybdate and radiation therapy in a mouse model of head and neck squamous cell carcinoma.

OBJECTIVE: To assess the effect of combining tetrathiomolybdate therapy and radiation treatment (RT) on tumor growth in the mouse head and neck squamous cell carcinoma (HNSCC) model. DESIGN: One million HNSCC cells were injected subcutaneously into the flanks of C3H/HeJ mice and the tumors grown to an average of 301 mm3 (day 0). Mice were randomized into 4 groups: (a) no therapy, (b) tetrathiomolybdate alone, (c) RT alone, or (d) tetrathiomolybdate + RT. Data from 3 experiments with these 4 groups were analyzed. A gaussian mixed model was fit to the initialized logarithm of the tumor size counts between days 7 and 16 (linear component), and growth rates were compared. Assays using 3-(4,5-dimethylthiazol-2yl)-2,5 diphenyltetrazolium bromide (MTT) were conducted on HNSCC cells in culture with varying doses of tetrathiomolybdate. INTERVENTIONS: Treated mice were given tetrathiomolybdate in their water and observed for clinical evidence of toxic effects associated with copper depletion as measured by ceruloplasmin assay. When tumor sizes reached an average of 535 mm(3), mice receiving RT were given a single fraction of 750 rad (7.5 Gy), a dose determined in previous experiments to slow but not cure tumor growth, permitting an examination of interaction of radiation with tetrathiomolybdate. RESULTS: Data from 3 separate experiments were analyzed. There were a total of 37 mice in the untreated group, 32 mice in the tetrathiomolybdate alone group, 38 mice in the RT alone group, and 46 mice in the tetrathiomolybdate + RT group. Ceruloplasmin assays showed that we had obtained adequate copper reduction throughout the experiments to inhibit angiogenesis with minimal toxic effects. The tetrathiomolybdate + RT combined therapy group of mice showed a statistically significant decrease in tumor growth compared with both the tetrathiomolybdate alone (P = .001) and RT alone groups (P<.001). CONCLUSION: The combination of the anti-angiogenic copper chelating agent tetrathiomolybdate with RT improved local control of HNSCC in an isogenic mouse model compared with either therapy alone.

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.

Sustained Hedgehog signaling is required for basal cell carcinoma proliferation and survival: conditional skin tumorigenesis recapitulates the hair growth cycle.

Temporally and spatially constrained Hedgehog (Hh) signaling regulates cyclic growth of hair follicle epithelium while constitutive Hh signaling drives the development of basal cell carcinomas (BCCs), the most common cancers in humans. Using mice engineered to conditionally express the Hh effector Gli2, we show that continued Hh signaling is required for growth of established BCCs. Transgene inactivation led to BCC regression accompanied by reduced tumor cell proliferation and increased apoptosis, leaving behind a small subset of nonproliferative cells that could form tumors upon transgene reactivation. Nearly all BCCs arose from hair follicles, which harbor cutaneous epithelial stem cells, and reconstitution of regressing tumor cells with an inductive mesenchyme led to multilineage differentiation and hair follicle formation. Our data reveal that continued Hh signaling is required for proliferation and survival of established BCCs, provide compelling support for the concept that these tumors represent an aberrant form of follicle organogenesis, and uncover potential limitations to treating BCCs using Hh pathway inhibitors.

Protein micelles from lipoxygenase 3.

Heat-induced conformational changes in lipoxygenase 3 were characterized by differential scanning calorimetry. The positions of the observed transitions were sensitive to the composition of the buffer. In particular, lipoxygenase 3 heated in carbonate buffer at pH 8.0 formed large soluble aggregates. Variable-temperature circular dichroism revealed that the formation of the aggregates was not accompanied by the unfolding of the C-terminal domain, which is composed primarily of alpha-helix. The aggregates were investigated using size exclusion chromatography, native polyacrylamide gel electrophoresis, dynamic light scattering, and electron microscopy. The data were consistent with the formation of roughly spherical particles with an average hydrodynamic radius of 26 nm and an approximate composite molecular weight of 10,000,000 Da. To account for the formation of soluble aggregates from lipoxygenase 3, we propose that hydrophobic amino acid residues are exposed by unfolding of the N-terminal beta-barrel domain of the protein resulting in the formation of protein micelles with a hydrophilic surface composed of the C-terminal domains.