This study was designed to investigate the intratumoral uptake of hollow gold nanospheres (HAuNS) after hepatic intra-arterial (IA) and intravenous (IV) injection in a liver tumor model.
Materials and Methods
Fifteen VX2 tumor-bearing rabbits were randomized into five groups (N=3 in each group) that received either IV 64Cu-labeled PEG-HAuNS (IV-PEG-HAuNS), IA 64Cu-labeled PEG-HAuNS (IA-PEG-HAuNS), IV cyclic peptide (RGD)-conjugated 64Cu-labeled PEG-HAuNS (IV-RGD-PEG-HAuNS), IA RGD-conjugated 64Cu-labeled PEG-HAuNS (IA-RGD-PEG-HAuNS), or IA 64Cu-labeled PEG-HAuNS with lipiodol (IA-PEG-HAuNS-lipiodol). The animals underwent PET/CT 1 hour after injection, and uptake expressed as percentage of injected dose per gram of tissue (%ID/g) was measured in tumor and major organs. The animals were euthanized 24 hours after injection, and tissues were evaluated for radioactivity.
At 1 hour after injection, animals in the IA-PEG-HAuNS-lipiodol group showed significantly higher tumor uptake (P < 0.001) and higher ratios of tumor-to-normal liver uptake (P < 0.001) than those in all other groups. The biodistribution of radioactivity 24 hours after injection showed that IA delivery of PEG-HAuNS with lipiodol resulted in the highest tumor uptake (0.33 %ID/g; P < 0.001) and tumor-to-normal liver ratio (P < 0.001) among all delivery methods. At 24 hours, the IA-RGD-PEG-HAuNS group showed higher tumor uptake than the IA-PEG-HAuNS group (0.20 %ID/g vs. 0.099 %ID/g; P < 0.001).
Adding iodized oil to IA-PEG-HAuNS maximizes nanoparticle delivery to hepatic tumors and therefore may be useful in targeted chemotherapy and photoablative therapy. PET/CT can be used to noninvasively monitor the biodistribution of radiolabeled HAuNS after IV or IA injection.
Hollow gold nanospheres; liver tumor; intraarterial injection; PET/CT; copper-64; lipiodol
Apoptosis; Caspase 3; Dual Modality; DEVD; Technetium99m; Gamma imaging; Optical Imaging
Photothermal ablation (PTA) is an emerging technique that uses near-infrared laser light-generated heat to destroy tumor cells. However, complete tumor eradication by PTA therapy alone is difficult because heterogeneous heat distribution can lead to sub-lethal thermal dose in some areas of the tumor. Successful PTA therapy requires selective delivery of photothermal conducting nanoparticles to mediate effective PTA of tumor cells, and the ability to combine PTA with other therapy modalities. Here, we synthesized multifunctional doxorubicin (DOX)-loaded hollow gold nanospheres (DOX@HAuNS) that target EphB4, a member of the Eph family of receptor tyrosine kinases overexpressed on the cell membrane of multiple tumors and angiogenic blood vessels. Increased uptake of targeted nanoparticles T-DOX@HAuNS was observed in three EphB4-positive tumors both in vitro and in vivo. In vivo release of DOX from DOX@HAuNS, triggered by near-infrared laser, was confirmed by dual radiotracer technique. Treatment with T-DOX@HAuNS followed by near-infrared laser irradiation resulted in significantly decreased tumor growth when compared to treatments with non-targeted DOX@HAuNS plus laser or HAuNS plus laser. The tumors in six of the eight mice treated with T-DOX@HAuNS plus laser regressed completely with only residual scar tissue by 22 days following injection, and none of the treatment groups experienced a loss in body weight. Together, our findings demonstrate that concerted chemo-photothermal therapy with a single nanodevice capable of mediating simultaneous PTA and local drug release may have promise as a new anticancer therapy.
Hollow Gold Nanospheres; EphB4 receptors; Targeting; Doxorubicin; Multimodal Therapy
Targeted nanoparticle-based delivery systems have been used extensively to develop effective cancer theranostics. However, how targeting ligands affect extravascular transport of nanoparticles in solid tumors remains unclear. Here, we show, using B16/F10 melanoma cells expressing melanocortin type-1 receptor (MC1R), that the nature of targeting ligands, i.e., whether they are agonists or antagonists, directs tumor uptake and intratumoral distribution after extravasation of nanoparticles from tumor vessels into the extravascular fluid space. Pegylated hollow gold nanospheres (HAuNS, diameter≈40 nm) coated with MC1R agonist are internalized upon ligand-receptor binding, whereas MC1R antagonist-conjugated HAuNS remain attached on the cell surface. Transcellular transport of agonist-conjugated HAuNS was confirmed by a multilayer tumor cell model and by transmission electron microscopy. MC1R agonist- but not MC1R antagonist-conjugated nanoparticles exhibit significantly higher tumor uptake than nontargeted HAuNS and are quickly dispersed from tumor vessels via receptor-mediated endocytosis and subsequent transcytosis. These results confirm an active transport mechanism that can be used to overcome one of the major biological barriers for efficient nanoparticle delivery to solid tumors.
Nanoparticles; Melanocortin type-1 receptor (MC1R); Transcytosis; Agonists; Antagonists
The purposes of this study were to develop an efficient method of labeling D-glucosamine hydrochloride with gallium 68 (68Ga) and investigate the imaging properties of the resulting radiotracer in a human tumor xenograft model using micro-positron emission tomography (μPET). The precursor compound 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-2-deoxy-D-glucosamine (DOTA-DG) was synthesized from D-glucosamine hydrochloride and 2-(4-isothiocyanatobenzyl)-DOTA. Radiolabeling of DOTA-DG with 68Ga was achieved in 10 minutes using microwave heating. The labeling efficiency a nd radiochemical purity after purification of 68Ga-DOTA-DG were ~85% and greater than 98%, respectively. In A431 cells, the percentages of 68Ga-DOTA-DG and 18F-FDG uptakes after 60 min incubation were 15.7% and 16.2%, respectively. In vivo, the mean ± standard deviation of 68Ga-DOTADG uptake values in A431 tumors were 2.38±0.30, 0.75±0.13, and 0.39±0.04 percent of the injected dose per gram of tissue at 10, 30, and 60 minutes after intravenous injection, respectively. μPET imaging of A431-bearing mice clearly delineated tumors at 60 minutes after injection of 68Ga-DOTA-DG at a dose of 3.7 MBq. 68Ga-DOTA-DG displayed significantly higher tumor-to-heart, tumor-to-brain, and tumor-to-muscle ratios than 18F-FDG did. Further studies are needed to identify the mechanism of tumor uptake of this new glucosamine-based PET imaging tracer.
Gallium 68; 2-deoxy-D-glucose; μPET imaging; microwave heating-assisted synthesis
Advancements in nanotechnology have made it possible to create multifunctional nanostructures that can be used simultaneously to image and treat cancers. For example, hollow gold nanospheres (HAuNS) have been shown to generate intense photoacoustic signals and induce efficient photothermal ablation (PTA) therapy. In this study, we used photoacoustic tomography (PAT), a hybrid imaging modality, to assess the intravenous delivery of HAuNS targeted to integrins that are overexpressed in both glioma and angiogenic blood vessels in a mouse model of glioma. Mice were then treated with near-infrared laser, which elevated tumor temperature by 20.7 °C. We found that PTA treatment significantly prolonged the survival of tumor-bearing mice. Taken together, these results demonstrate the feasibility of using a single nanostructure for image-guided local tumor PTA therapy using photoacoustic molecular imaging.
EphB4, a member of the largest family of receptor tyrosine kinases, is overexpressed in numerous tumors. In this study, we developed a new class of multimodal nanoplatform for dual single photon emission computed tomography (SPECT) and near-infrared fluorescence imaging of EphB4. EphB4-binding peptide TNYL-FSPNGPIARAW (TNYL-RAW) was conjugated to polyethylene glycol-coated, core-crosslinked polymeric micelles (CCPM) dually labeled with near-infrared fluorescence fluorophores (Cy7) and a radioisotope (indium 111). In vitro, TNYL-RAW-CCPM selectively bound to EphB4-positive PC-3M prostate cancer cells, but not to EphB4-negative A549 lung cancer cells. In vivo, PC-3M tumors were clearly visualized by both SPECT and near-infrared fluorescence tomography after intravenous administration of 111In-labeled TNYL-RAW-CCPM. In contrast, there was little signal in A549 tumors of mice injected with 111In-labeled TNYL-RAW-CCPM or in PC-3M tumors of mice injected with 111In-labeled CCPM. The high accumulation of 111In-labeled TNYL-RAW-CCPM in PC-3M tumor could be significantly reduced after co-injection with an excess amount of TNYL-RAW peptide. Immunohistochemical analysis showed that fluorescence signal from the nanoparticles correlated with their radioactivity count, and co-localized with the EphB4 expressing region. 111In-labeled TNYL-RAW-CCPM allowed visualization of cancer cells overexpressing EphB4 by both nuclear and optical techniques. The complementary information acquired with multiple imaging techniques should be advantageous in early detection of cancer.
Micelle; Nanoparticle; Fluorescence; Molecular Imaging
Efforts to develop adenovirus vectors suitable for genetic interventions in humans have identified three major limitations of the most frequently used vector prototype, human adenovirus serotype 5 (Ad5). These limitations—widespread preexisting anti-Ad5 immunity in humans, the high rate of transduction of normal nontarget tissues, and the lack of target-specific gene delivery—justify the exploration of other Ad serotypes as vector prototypes. In this paper, we describe the development of an alternative vector platform using simian Ad serotype 24 (sAd24). We found that sAd24 virions formed unstable complexes with blood coagulation factor X and, because of that, transduced the liver and other organs at low levels when administered intravenously. The overall pattern of biodistribution of sAd24 particles was similar, however, to that of Ad5, and the intravenously injected sAd24 was cleared by Kupffer cells, leading to their depletion. We modified the virus's fiber protein to design a Her2-specific derivative of sAd24 capable of infecting target human tumor cells in vitro. In the presence of neutralizing anti-Ad5 antibodies, Her2-mediated infection with targeted sAd24 compared favorably to that with the Ad5-derived vector. When used to target Her2-expressing tumors in animals, this fiber-modified vector achieved a higher level of gene transfer to metastasis-containing murine lungs than to tumor-free lungs. In aggregate, these studies provide important insights into sAd24 biology, identify its advantages and limitations as a vector prototype, and are thus essential for further development of an sAd24-based gene delivery platform.
In cancer cells, signal transducer and activator of transcription 3 (Stat3) participates in aberrant growth, survival, angiogenesis, and invasion signals and is a validated target for anti-cancer drug design. We are targeting its SH2 domain to prevent docking to cytokine and growth factor receptors and subsequent signaling. One of the important elements of the recognition sequence, pTyr-Xxx-Xxx-Gln, is glutamine. We incorporated novel Gln mimics into a lead peptide, pCinn-Leu-Pro-Gln-NHBn, and found that a linear, unconstrained side chain and carboxamide are necessary for high affinity, and the benzamide can be eliminated. Replacement of Gln-NHBn with (R)-4-aminopentanamide or 2-aminoethylurea produced inhibitors with equal or greater potency than that of the lead, as judged by fluorescence polarization (IC50 values were 110 and 130 nM, respectively). When Pro was replaced with cis-3,4-methanoproline, the glutamine mimic, (4R,5S)-4-amino-5-benzyloxyhexanamide resulted in an IC50 of 69 nM, the highest affinity Stat3 inhibitor reported to date.
Signal transducer and activator of transcription 3 (Stat3) is involved in aberrant growth and survival signals in malignant tumor cells and is a validated target for anti-cancer drug design. We are targeting its SH2 domain to prevent docking to cytokine and growth factor receptors and subsequent signaling. The amino acids of our lead phosphopeptide, Ac-pTyr-Leu-Pro-Gln-Thr-Val-NH2, were replaced with conformationally constrained mimics. Structure-affinity studies led to the peptidomimetic, pCinn-Haic-Gln-NHBn (21) which had an IC50 of 162 nM (fluorescence polarization), as compared to 290 nM for the lead phosphopeptide (pCinn = 4-phosphoryloxycinnamate, Haic = (2S,5S)-5-amino-1,2,4,5,6,7-hexahydro-4-oxo-azepino[3,2,1-hi]indole-2-carboxylic acid). pCinn-Haic-Gln-OH was docked to the SH2 domain (AUTODOCK) and the two highest populated clusters were subjected to molecular dynamics simulations. Both converged to a common peptide conformation. The complex exhibits unique hydrogen bonding between Haic and Gln and Stat3 as well as hydrophobic interactions between the protein and pCinn and Haic.
To develop melanoma-targeted hollow gold nanospheres (HAuNS) and evaluate their potential utility for selective photothermal ablation (PTA) in melanoma.
A new class of photothermal coupling agents based on HAuNS was synthesized. HAuNS were stabilized with poly(ethylene-glycol) coating and attached with α-melanocyte-stimulating hormone analog, [Nle4,D-Phe7]α-MSH (NDP-MSH), which is a potent agonist of melanocortin type-1 receptor overexpressed in melanoma. The intracellular uptake of the NDP-MSH-conjugated PEGylated HAuNS (NDP-MSH-PEG-HAuNS) and the distribution of β-arrestin were examined in murine B16/F10 melanoma cells. The biodistribution of NDP-MSH-PEG-HAuNs was assessed at 4 h post intravenous injection in tumor-bearing nude mice. PTA effect of the nanoparticles was evaluated both histologically using excised tissue and functionally by [18F]fluorodeoxyglucose positron emission tomography ([18F]FDG-PET).
NDP-MSH-PEG-HAuNS consist only of a thin gold wall with hollow interior (outer diameter, 43.5±2.3 nm; shell thickness, 3–4 nm), which display strong and tunable resonance absorption in near-infrared region (NIR, peak 808 nm). The nanoparticles were specifically taken up by melanoma cells, which initiated the recruitment of β-arrestins, the adapters to link the activated G-protein-coupled receptors to clathrin, indicating the involvement of receptor-mediated endocytosis. This resulted in enhanced extravasation of NDP-MSH-PEG-HAuNS from tumor blood vessels and their dispersion into tumor matrix compared with non-specific PEGylated HAuNS. Successful selective PTA of B16/F10 melanoma with targeted HAuNS was confirmed by histological and [18F]FDG-PET evaluation at 24 h post NIR laser irradiation at a low dose energy of 30 J/cm2.
NDP-MSH-PEG-HAuNS have the potentials to mediate targeted photothermal ablation of melanoma.
hollow gold nanospheres; melanocyte-stimulating hormone; photothermal ablation; melanoma; targeting