Magnetic Hyperthermia in Y79 Retinoblastoma and ARPE19 Retinal Epithelial Cells: Tumor Selective Apoptotic Activity of Iron Oxide Nanoparticle

Purpose: To evaluate selective apoptosis of Y79 retinoblastoma versus ARPE-19 retinal pigment epithelial cells by using different doses of dextran-coated iron oxide nanoparticles (DCIONs) in a magnetic hyperthermia paradigm. Methods: Y79 and ARPE-19 cells were exposed to different concentrations of DCIONs, namely, 0.25, 0.5, 0.75, and 1 mg/ml. After 2 hours of incubation, cells were exposed to a magnetic field with a frequency of 250 kHz and an amplitude of 4 kA/m for 30 minutes to raise the cellular temperature between 42 and 46°C. Y79 and ARPE-19 cells incubated with DCION without magnetic field exposure were used as controls. Cell viability and apoptosis were assessed at 4, 24, and 72 hours after hyperthermia treatment. Results: At 4 hours following magnetic hyperthermia, cell death for Y79 cells was 1%, 8%, 17%, and 17% for 0.25, 0.5, 0.75 and 1 mg/ml of DCION, respectively. Cell death increased to 47%, 59%, 70%, and 75% at 24 hours and 16%, 45%, 50%, and 56% at 72 hours for 0.25, 0.5, 0.75, and 1 mg/ml of DCIONs, respectively. Magnetic hyperthermia did not have any significant toxic effects on ARPE-19 cells at all DCION concentrations, and minimal baseline cytotoxicity of DCIONs on Y79 and ARPE-19 cells was observed without magnetic field activation. Gene expression profiling showed that genes involved in FAS and tumor necrosis factor alpha signaling pathways were activated in Y79 cells following hyperthermia. Caspase 3/7 activity in Y79 cells increased following treatment, consistent with the activation of caspase-mediated apoptosis and loss of cell viability by magnetic hyperthermia. Conclusion: Magnetic hyperthermia using DCIONs selectively kills Y79 cells at 0.5 mg/ml or higher concentrations via the activation of apoptotic pathways. Translational Relevance: Magnetic hyperthermia using DCIONs might play a role in targeted management of retinoblastoma

Selective Death of Y79 Retinoblastoma Cells by Magnetic Hyperthermia via Caspase Dependent Apoptotic Pathway

Purpose : To evaluate the selective death of Y79 retinoblastoma cells using dextran-coated iron oxide nanoparticles in a magnetic hyperthermia paradigm and assess the molecular pathways that play a role in cell death. Methods : Y79 and ARPE-19 cells were exposed to different concentrations of dextran-coated iron oxide magnetic nanoparticles: 250, 500, 750 and 1000 µg/ml. After 2 hours of incubation, cells were exposed to a magnetic field with frequency and intensity values of 250 kHz and 4 kA/m at temperature of 43-44 degreeC for 30 minutes. Hyperthermia induced apoptosis was assessed at 4, 24 and 72 hours after treatment. Transmission electron microscopy was performed to elucidate the cellular uptake and distribution of nanoparticles in Y79 and ARPE-19 cells. Gene expression profiling for apoptosis was performed using RT2 Profiler PCR arrays. Results : At 4 hours, cell death for Y79 cells was estimated to be 16, 16, 18 and 24% for 250, 500, 750 and 1000 µg/ml, respectively. At 24 hours, cell death in Y79 cells was estimated to be 46, 57, 67, and 73% at respective four concentrations. Y79 retinoblastoma cells underwent apoptosis at 72 hours with cell death estimated at 16, 48, 52 and 57% respectively with increasing nanoparticle concentrations. Magnetic hyperthermia did not have a significant toxic effect on ARPE-19 cells for all four concentrations of nanoparticles. Minimal baseline cytotoxicity of nanoparticles was observed without magnetic field activation. Transmission Electron Microscopy images showed that dextran-coated iron oxide nanoparticles were engulfed into the cytoplasm of both Y79 and ARPE-19 cells and specifically located in endosomes. Gene expression profiling showed that genes in the FAS signaling pathway and TNF alpha signaling were activated in Y79 retinoblastoma cells at 24 hours after magnetic hyperthermia. qRT-PCR results showed an increased gene expression of caspase8, caspase10, caspase14, caspase9 and cytochromeC. 48 hours after magnetic hyperthermia, we observed significantly increased caspase 3/7 activity in Y79 cells, confirming that apoptosis through intrinsic and extrinsic pathways were activated in these cells. Conclusions : Magnetic hyperthermia using dextran-coated iron oxide nanoparticles were engulfed by both Y79 and ARPE-19 cells. They satisfactorily and selectively killed Y79 cells at 750 and 1000 µg/ml concentrations via activation of two apoptotic pathways

Photoacoustic imaging features of intraocular tumors: Retinoblastoma and uveal melanoma.

The purpose of this study is to examine the capability of photoacoustic (PA) imaging (PAI) in assessing the unique molecular and architectural features in ocular tumors. A real-time PA and ultrasonography (US) parallel imaging system based on a research US platform was developed to examine retinoblastoma in mice in vivo and human retinoblastoma and uveal melanoma ex vivo. PA signals were generated by optical illumination at 720, 750, 800, 850, 900 and 950 nm delivered through a fiber optical bundle. The optical absorption spectra of the tumors were derived from the PA images. The optical absorption spectrum of each tumor was quantified by fitting to a polynomial model. The microscopic architectures of the tumors were quantified by frequency domain analysis of the PA signals. Both the optical spectral and architectural features agree with the histological findings of the tumors. The mouse and human retinoblastoma showed comparable total optical absorption spectra at a correlation of 0.95 (p<0.005). The quantitative PAI features of human retinoblastoma and uveal melanoma have shown statistically significant difference in two tailed t-tests (p<0.05). Fully compatible with the concurrent procedures, PAI could be a potential tool complementary to other diagnostic modalities for characterizing intraocular tumors

Illustration of experiment systems.

<p>(A) mouse experiment <i>in vivo</i>. (B) human eye globe imaging <i>ex vivo</i>.</p

Histology photographs of retinoblastoma and uveal melanoma.

<p>(A) Retinoblastoma. (B) uveal melanoma. The arrows in (A) mark the calcification spots forming the heterogeneous tissue architecture in retinoblastoma. The slides were prepared by H&E staining. Images were taken at 4x magnification.</p

Statistics of the optical absorption spectral parameters derived with linear model, i.e. 1^st order polynomial.

<p>Statistics of the optical absorption spectral parameters derived with linear model, i.e. 1^st order polynomial.</p

PASA of the human intraocular tumors.

<p>(A) Representative averaged PA signals spectra at 720 nm derived from the tumor samples. The solid lines are the linear fits to the signal power spectra within the probe frequency bandwidth (7-15MHz). Frequency range of 6.8–14.8 MHz was actually observed due to the limited sampling resolution. The PASA slope is tan(<i>θ</i>). Midband-fit is the magnitude of the linear model at the center of the observed frequency range (10.8 MHz for this case). (B) Statistics of the PASA slopes of retinoblastomas and uveal melanomas. The slopes derived from uveal melanomas have an average of -4.5 and standard deviation of 0.56. The slopes derived from retinoblastomas have an average of -5.6 and standard deviation of 0.60. Two-tailed t-test between the two groups has shown a p-value of 0.025.</p

B-scan PA (acquired at 720 nm) and US images of retinoblastoma tumor in mice.

<p>The yellow arrows mark the contour of the eye globe. The cyan arrows mark the skull of the mouse. The top of the PA images show bright artifacts due to the absorption of backscattered optical energy by the US probe surface. Scale bar: 1 cm.</p

Relative optical absorption spectra complied by combining data acquired animal <i>in vivo</i> and human tissues <i>ex vivo</i>.

<p>(A) retinoblastoma in mice <i>in vivo</i>. (B) Retinoblastoma in human <i>ex vivo</i>. (C) Uveal melanoma in human <i>ex vivo</i>.</p

Statistics of the optical absorption spectral parameters derived with 2^nd order polynomial.

<p>Statistics of the optical absorption spectral parameters derived with 2^nd order polynomial.</p