Darren Yohan

Elucidating the Uptake and Distribution of Nanoparticles in Solid Tumors via a Multilayered Cell Culture Model

Multicellular layers (MCLs) have previously been used to determine the pharmacokinetics of a variety of different cancer drugs including paclitaxel, doxorubicin, methotrexate, and 5-fluorouracil across a number of cell lines. It is not known how nanoparticles (NPs) navigate through the tumor microenvironment once they leave the tumor blood vessel. In this study, we used the MCL model to study the uptake and penetration dynamics of NPs. Gold nanoparticles (GNPs) were used as a model system to map the NP distribution within tissue-like structures. Our results show that NP uptake and transport are dependent on the tumor cell type. MDA-MB-231 tissue showed deeper penetration of GNPs as compared to MCF-7 one. Intracellular and extracellular distributions of NPs were mapped using CytoViva imaging. The ability of MCLs to mimic tumor tissue characteristics makes them a useful tool in assessing the efficacy of particle distribution in solid tumors.

Quantitative monitoring of radiation induced skin toxicities in nude mice using optical biomarkers measured from diffuse optical reflectance spectroscopy.

Monitoring the onset of erythema following external beam radiation therapy has the potential to offer a means of managing skin toxicities via biological targeted agents - prior to full progression. However, current skin toxicity scoring systems are subjective and provide at best a qualitative evaluation. Here, we investigate the potential of diffuse optical spectroscopy (DOS) to provide quantitative metrics for scoring skin toxicity. A DOS fiberoptic reflectance probe was used to collect white light spectra at two probing depths using two short fixed source-collector pairs with optical probing depths sensitive to the skin surface. The acquired spectra were fit to a diffusion theory model of light transport in tissue to extract optical biomarkers (hemoglobin concentration, oxygen saturation, scattering power and slope) from superficial skin layers of nude mice, which were subjected to erythema inducing doses of ionizing radiation. A statistically significant increase in oxygenated hemoglobin (p < 0.0016) was found in the skin post-irradiation - confirming previous reports. More interesting, we observed for the first time that the spectral scattering parameters, A (p = 0.026) and k (p = 0.011), were an indicator of erythema at day 6 and could potentially serve as an early detection optical biomarker of skin toxicity. Our data suggests that reflectance DOS may be employed to provide quantitative assessment of skin toxicities following curative doses of external beam radiation.

Vasculotide, an Angiopoietin-1 mimetic, reduces acute skin ionizing radiation damage in a preclinical mouse model

BackgroundMost cancer patients are treated with radiotherapy, but the treatment can also damage the surrounding normal tissue. Acute skin damage from cancer radiotherapy diminishes patients’ quality of life, yet effective biological interventions for this damage are lacking. Protecting microvascular endothelial cells from irradiation-induced perturbations is emerging as a targeted damage-reduction strategy. Since Angiopoetin-1 signaling through the Tie2 receptor on endothelial cells opposes microvascular perturbations in other disease contexts, we used a preclinical Angiopoietin-1 mimic called Vasculotide to investigate its effect on skin radiation toxicity using a preclinical model.MethodsAthymic mice were treated intraperitoneally with saline or Vasculotide and their flank skin was irradiated with a single large dose of ionizing radiation. Acute cutaneous damage and wound healing were evaluated by clinical skin grading, histology and immunostaining. Diffuse reflectance optical spectroscopy, myeloperoxidase-dependent bioluminescence imaging of neutrophils and a serum cytokine array were used to assess inflammation. Microvascular endothelial cell response to radiation was tested with in vitro clonogenic and Matrigel tubule formation assays. Tumour xenograft growth delay experiments were also performed. Appreciable differences between treatment groups were assessed mainly using parametric and non-parametric statistical tests comparing areas under curves, followed by post-hoc comparisons.ResultsIn vivo, different schedules of Vasculotide treatment reduced the size of the irradiation-induced wound. Although skin damage scores remained similar on individual days, Vasculotide administered post irradiation resulted in less skin damage overall. Vasculotide alleviated irradiation-induced inflammation in the form of reduced levels of oxygenated hemoglobin, myeloperoxidase bioluminescence and chemokine MIP-2. Surprisingly, Vasculotide-treated animals also had higher microvascular endothelial cell density in wound granulation tissue. In vitro, Vasculotide enhanced the survival and function of irradiated endothelial cells.ConclusionsVasculotide administration reduces acute skin radiation damage in mice, and may do so by affecting several biological processes. This radiation protection approach may have clinical impact for cancer radiotherapy patients by reducing the severity of their acute skin radiation damage.

Size dependent gold nanoparticle interaction at nano-micro interface using both monolayer and multilayer (tissue-like) cell models

Gold nanoparticles (GNPs) can be used as a model NP system to improve the interface between nanotechnology and medicine since their size and surface properties can be tailored easily. GNPs are being used as radiation dose enhancers and as drug carriers in cancer research. Hence, it is important to know the optimum NP size for uptake not only at monolayer level but also at tissue level. Once GNPs leave tumor vasculature, they enter the tumor tissue. Success of any therapeutic technique using NPs depends on how well NPs penetrate the tumor tissue and reach individual tumor cells. In this work, multicellular layers (MCLs) were grown to model the post-vascular tumor environment. GNPs of 20 nm and 50 nm diameters were used to elucidate the effects of size on the GNP penetration and distribution dynamics. Larger NPs (50 nm) were better at monolayer level, but smaller NPs (20 nm) were at tissue level. The MCLs exhibited a much more extensive extracellular matrix (ECM) than monolayer cell cultures. This increased ECM created a barrier for NP transport and ECM was also dependent on the tumor cell lines. Smaller NPs penetrated better compared to larger NPs. Transport of NPs was better in MDA-MB231 vs MCF-7. This MCL model tissue structures are better tools to optimize NP transport through tissue before using them in animal models. Based on our study, we believe that smaller NPs are better for improved outcome in future cancer therapeutics.

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model.

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and negatively impact patient quality of life and long term survival. Advances in the understanding of the biological pathways associated with normal tissue toxicities have allowed for the development of interventional drugs, however, current response studies are limited by a lack of quantitative metrics for assessing the severity of skin reactions. Here we present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe the instrumentation design of the DOS system as well as the inversion algorithm for extracting the optical parameters. Finally, to demonstrate clinical utility, we present representative data from a pre-clinical mouse model of radiation induced erythema and compare the results with a commonly employed visual scoring. The described DOS method offers an objective, high through-put evaluation of skin toxicity via functional response that is translatable to the clinical setting.

Early biomarker for radiation-induced wounds: day one post-irradiation assessment using hemoglobin concentration measured from diffuse optical reflectance spectroscopy

Normal tissue radiation toxicities are evaluated subjectively and cannot predict the development of severe side-effects. Using a hand-held diffuse reflectance optical spectroscopy probe, we measured optical parameters in mouse skin 1-4 days after irradiation. Using a radiation toxicity model and a therapeutic mitigator described previously [BMC Cancer14, 614 (2014)], we found that hemoglobin (Hb) levels increased sharply 24 h after irradiation only in the irradiated group without the mitigator. This group also had the largest peak wound areas after 14 days. We conclude that increased Hb one day after skin irradiation predicts the severity of the subsequent irradiation-induced wound.

Increase in uptake of peptide modified gold nanoparticles (GNPs)

The interactions of synthetically produced gold nanoparticles (GNPs) with living organisms are getting attention in the biomedical sciences. While non-viral carriers, like GNPs, are safer and more cost-efficient than viral vectors, the efficiency of cell penetration is lower. Unmodified GNPs enter the cell through a receptor-medicated endocytosis (RME) and is encapsulated inside a double membrane endosome, which leads to exocytosis. Increasing uptake of GNPs into the cell is a challenging task as the GNPs has to escape the regular endo-lyso pathway. In this paper, various modifications using peptide conjugates have been performed to the GNPs to increase the efficiency of cell penetration. Quantitative and qualitative data are obtained from a UV spectrometer, an Atomic Absorption spectrometer, and Transmission Electron Microscope.