Biodegradable Polydisulfide Dendrimer Nanoclusters as MRI Contrast Agents

Gadolinium-conjugated dendrimer nanoclusters (DNCs) are a promising platform for the early detection of disease; however, their clinical utility is potentially limited due to safety concerns related to nephrogenic systemic fibrosis (NSF). In this paper, biodegradable DNCs were prepared with polydisulfide linkages between the individual dendrimers to facilitate excretion. Further, DNCs were labeled with premetalated Gd chelates to eliminate the risk of free Gd becoming entrapped in dendrimer cavities. The biodegradable polydisulfide DNCs possessed a circulation half-life of >1.6 h in mice and produced significant contrast enhancement in the abdominal aorta and kidneys for as long as 4 h. The DNCs were reduced in circulation as a result of thiol–disulfide exchange, and the degradation products were rapidly excreted <i>via</i> renal filtration. These agents demonstrated effective and prolonged <i>in vivo</i> contrast enhancement and yet minimized Gd tissue retention. Biodegradable polydisulfide DNCs represent a promising biodegradable macromolecular MRI contrast agent for magnetic resonance angiography and can potentially be further developed into target-specific MRI contrast agents

Gold-Loaded Polymeric Micelles for Computed Tomography-Guided Radiation Therapy Treatment and Radiosensitization

Gold nanoparticles (AuNPs) have generated interest as both imaging and therapeutic agents. AuNPs are attractive for imaging applications since they are nontoxic and provide nearly three times greater X-ray attenuation per unit weight than iodine. As therapeutic agents, AuNPs can sensitize tumor cells to ionizing radiation. To create a nanoplatform that could simultaneously exhibit long circulation times, achieve appreciable tumor accumulation, generate computed tomography (CT) image contrast, and serve as a radiosensitizer, gold-loaded polymeric micelles (GPMs) were prepared. Specifically, 1.9 nm AuNPs were encapsulated within the hydrophobic core of micelles formed with the amphiphilic diblock copolymer poly(ethylene glycol)-<i>b</i>-poly(ε-capralactone). GPMs were produced with low polydispersity and mean hydrodynamic diameters ranging from 25 to 150 nm. Following intravenous injection, GPMs provided blood pool contrast for up to 24 h and improved the delineation of tumor margins <i>via</i> CT. Thus, GPM-enhanced CT imaging was used to guide radiation therapy delivered <i>via</i> a small animal radiation research platform. In combination with the radiosensitizing capabilities of gold, tumor-bearing mice exhibited a 1.7-fold improvement in the median survival time, compared with mice receiving radiation alone. It is envisioned that translation of these capabilities to human cancer patients could guide and enhance the efficacy of radiation therapy

GNP administration in combination with RT improves survival in mice with advanced GBM tumors.

<p><b>A.</b> BLI of a representative mouse with advanced orthotopic GBM xenografts (radiance ∼10⁸ p/sec/cm²/sr) used for the survival study. <b>B.</b> Photograph of a brain and resected tumor 48 hours after intravenous injection of GNPs. Tumor shows darkened appearance due to extravasation due to EPR into the tumor. <b>C.</b> Survival data in mice with advanced orthotopic GBM treated with or without GNPs followed by mock-irradiation or given stereotactic RT (20 Gy). The right cerebral hemispheres of nude mice were initially implanted with 350,000 U251 cells, and tumors were allowed to grow until the measured radiance reached ∼10⁸p/sec/cm²/sr (approximately 3–5 weeks post-implantation), at which point the mice were given their respective treatments (<i>n</i> = 5 for GNP+RT and <i>n</i> = 4 for control, GNP, and RT groups). Median and mean survival analysis were obtained with Kaplan-Meier analysis, and comparison between RT versus GNP+RT survival curves showed <i>p</i> = 0.011. Mean survival times are shown with 95% confidence intervals. N.S. in the figure indicates lack of statistical significance, while the asterisk (*) denotes that significance was reached (α = 0.05).</p

Assessing GNP enhancement with <i>in vitro</i> assays of radiosensitivity.

<p><b>A.</b> Deconvolution imaging of γh2ax foci in U251 cells that were mock-irradiated (upper) or irradiated with 4 Gy (lower). Cells irradiated with 1 mM GNPs display a 1.7-fold higher density of persistent γh2ax foci 24 hours after RT. <b>B.</b> Quantitative analysis of γh2ax foci for N >100 viable nuclei. Error bars, 95% confidence interval. Statistical significance was determined using a two-tailed <i>t</i>-test (α = 0.05), with <i>p</i><0.05 being considered significant. <b>C.</b> Clonogenic assay of U251 cells treated with (red circles) and without (hollow squares) 1 mM GNPs and given radiation doses of 0, 2, 4 and 6 Gy. Error bars represent the mean survival ± s.d. of at least four replicates.</p

Visualization of vascular dose painting effects via immunofluorescent labeling of DNA DSBs and vascular endothelial markers.

<p><b>A.</b> Mouse brain endothelial cells co-cultured with human GBM cells (1∶7, respectively) <i>in vitro</i> show enhanced RT damage when irradiated (4 Gy) with 1 mM GNPs. Upper : Immunofluorescence imaging of γh2ax foci and DAPI in normal mouse brain endothelial cells with the indicated treatments. Lower: Quantitative analysis of γh2ax foci (yellow) for N >10 viable nuclei (blue) of normal murine brain endothelial cells co-cultured with human GBM cells <i>in vitro</i>. Error bars, 95% confidence interval. Statistical significance was determined using a one-tailed <i>t</i>-test (α = 0.05), with <i>p</i><0.05 considered significant. <b>B.</b> Brains irradiated immediately following GNP injection leads to considerable colocalization of DNA DSB and blood vessels compared to those receiving RT alone. Healthy brains were mock-irradiated or irradiated with 20 Gy (whole-brain) immediately (<5 min) after i.v. administration of 1.25 g Au/kg GNPs or saline. Mice were sacrificed 24 hours later, and their brains were fixed/stained for γh2ax, CD31, and DAPI. C. γh2ax colocalization with CD31-positive cells was performed by calculating Mander’s coefficient (M2) in binary projections of CD31 and γh2ax channels.</p

Radiation-induced modulation of the blood-brain barrier leads to increased uptake of GNPs in orthotopic GBM xenografts.

<p><b>A.</b> Schematic of BBB-disruption with targeted RT, which leads to endothelial cell death and loosening of tight junctions. <b>B.</b> Representative BLI image of a smaller, less disruptive GBM tumor (max BLI ∼10⁶) used in this experiment. <b>C.</b> T2-weighted MRI image of a stereotactically implanted intracranial GBM tumor (approximated by dashed orange line). <b>D.</b> ICP-MS analysis of gold uptake in the right hemispheres of healthy brains and those with orthotopic GBM excised from mice 48 hours after i.v. injection of saline or 0.4 g Au/kg GNPs administered 7–14 days after 20 Gy RT or mock-irradiation. The right cerebral hemispheres of mice were orthotopically inoculated with 350,000 U251 cells. Tumors were allowed to grow until the measured BLI irradiance reached ∼10⁶ p/sec/cm²/sr (approximately 2 weeks post-implantation), at which point the mice were given their respective treatments. Brains with tumors receiving RT (<i>n</i> = 4) prior to GNP administration show significant increase in EPR-driven gold accumulation compared to mock-irradiated controls (<i>n</i> = 4). Mock-irradiated (<i>n</i> = 3) and irradiated (<i>n</i> = 3) healthy brains, however, show no significant difference in gold uptake, suggesting that normal tissue may recover more quickly than tumor. <b>E.</b> Representative H/E staining of sections from orthotopic tumors with (+) and without (−) GNP injection.</p

Gold nanoparticle characterization.

<p><b>A.</b> Transmission electron micrograph of GNPs having approximately 12 nm cores. <b>B.</b> Representative dynamic light scattering measurement of GNPs. Data was fit to a Gaussian function to determine the peak ± s.d. of nanoparticle hydrodynamic diameter (d_H). <b>C.</b> UV-vis absorption spectrum of GNPs showing characteristic surface plasmon resonance at λ ≈ 522 nm. <b>D.</b> MTT viability assay of U251 cells treated with increasing concentrations of GNPs for 24 hours. Error bars, mean viability ± s.d. of three replicates.</p