Development and characterization of CD73-siRNA-loaded nanoemulsion: effect on C6 glioma cells and primary astrocytes

Glioblastoma (GB) is the most common malignant brain tumor and is characterized by high invasiveness, poor prognosis, and limited therapeutic options. Silencing gene expression, through the use of small interfering RNA (siRNA), has been proposed as an alternative to conventional cancer therapy. Here, we evaluated the potential of CD73 as a new therapeutic target, since it is overexpressed in solid tumors and has emerged as a promising target to control GB progression. Methods: A cationic nanoemulsion (NE) as an intravenous siRNA-CD73 delivery system was developed and its effect on C6 glioma cell viability was determined. Results: The nanostructured system was effective in complexing oligonucleotides for delivery to target cells. In addition, we observed that the NE-siRNA-CD73 complex was effective in reducing CD73 protein levels and AMPase activity, which were related to decreased C6 glioma cell viability. Conclusions: These findings indicate the potential of siRNA-CD73-loaded cationic NE as a therapeutic alternative for glioma treatment254408415CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DO RIO GRANDE DO SUL - FAPERGS422298/2016-6; 312187/2018-1sem informação12/2011-7; 16/2551-0000265-7; 16/2551-0000473-

Histopathological analysis of human MB implanted tumors.

<p>The implanted tumors were excised, fixed and destined to posterior analysis as described in the Materials and Methods. Representative H&E sections of MB tumors <b>(A, B)</b> Daoy, <b>(C)</b> D283ev and <b>(D)</b> D283hCD73 demonstrate the histopathological characteristics of human MB: the presence of nodularity (Nod), intratumoral vascularization (V), necrotic areas (N) and the presence of mitosis are indicated by arrows. The results of additional analyses are detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140996#pone.0140996.t001" target="_blank">Table 1</a>. The images were obtained using an inverted fluorescence microscope (Nikon Eclipse TE 300).</p

Evaluation of immunolabeling of active Caspase-3.

<p>Caspase-3 immunolabeling was evaluated in MB tumor cells lines by flow cytometry (A) and tumor samples by immunohistochemical staining (B) as described in the Materials and Methods. (***) p < 0.001 indicates a statistically relevant difference in relation to negative cells. The images were obtained using an inverted fluorescence microscope (Carl Zeiss-Imager M2 microscope) at 20× magnification.</p

P1 adenosine receptor expression in human MB lines.

<p>The relative expression levels of A₁, A_2A, A_2B and A₃ in Daoy, D283, D283ev and D283hCD73 MB cell lines were assessed by real-time PCR. Endogenous GAPDH expression was used to normalize the adenosine expression levels. * p < 0.05, ** p < 0.01 and *** p < 0.001 indicate a significant difference between the analyzed samples and the Daoy cell line; # p < 0.05 corresponds to differences between the analyzed samples and the D283 cell line.</p

Immunohistochemical characterization of engrafted D283ev and D283hCD73 human MB tumors.

<p>Immunohistochemical labeling was performed as described in the Materials and Methods. Anti-synaptophysin and enolase immunostaining were used to evaluate whether the tumor samples were representative of human MB. The proliferation profile was determined using the anti-human Ki67 antibody. Tissue vascularization was visualized with an anti-human CD31 antibody. Labeling is indicated by arrows. Additional analyses are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140996#pone.0140996.t002" target="_blank">Table 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140996#pone.0140996.s003" target="_blank">S3 Fig</a>.</p

Ecto-5’-NT expression and activity following transfection of the D283 MB cell line.

<p>Ecto-5’-NT expression was determined by <b>(A)</b> RT-PCR analysis, <b>(B)</b> flow cytometry and <b>(C)</b> enzymatic activity as described in the Materials and Methods. <b>(D)</b> Cell proliferation indices for each cell line were obtained during five days of culture. (*) p < 0.05; (**) p < 0.01; and (***) p < 0.001 indicate significant differences compared to the Daoy cell line, and (#) p < 0.05; (##) p < 0.01; and (###) p < 0.001 indicate significant differences compared to the D283 cell line.</p

Immunohistochemical analysis of implanted MB.

<p>Immunohistochemistry slides were analyzed by a pathologist in a blinded manner. Analysis were performed using ten randomly chosen fields (x200) per tumor (Olympus BH-2 microscope). Ki67 positive cells were quantified by counting labeled cells in each field.</p><p>Immunohistochemical analysis of implanted MB.</p

Effect of APCP on MB cell proliferation.

<p>At 60% confluence, the cells were treated with 5 μM APCP for 24 and 48 h, and cell counting was performed as described in the Materials and Methods. Controls were considered 100%. The data were analyzed by a Student t-test, and (*) p < 0.05 and (***) p < 0.001 indicate significant differences compared to the control of each respective cell line.</p

Effect of APCP on AMPase activity in human MB cell lines.

<p>After reaching confluence, MB cell lines were pre-incubated for 10 min with APCP at the following concentrations: 1, 5, 10, 20 and 50 μM. At sequencing, AMP was added as a substrate at 2 mM for all cell lines. For Daoy and D283hCD73, the cells were incubated for 10 min and for D283 and D283ev, 30 min. The control did not receive APCP at any time. Specific activities were expressed as nmol/Pi/mg of protein. (*) p < 0.05; (**) p < 0.01; and (***) p < 0.001 indicate significant differences compared to the control of each respective cell line.</p

Ecto-5’-NT expression in D283 decreases the growth of tumor cells.

<p>To determine human MB tumor growth in nude mice, equal amounts of Daoy, D283ev and D283hCD73 cells (1 × 10⁶ cells) were implanted by subcutaneous injection into the dorsal region of nude mice. During tumor growth, the following data were obtained: <b>(A)</b> Measurements of tumor mass, which determine tumor growth (mm³). <b>(B)</b> Prior to euthanasia, nude mice were injected with the IRDYE^® 800 CW PEG Contrast Agent, and images were captured in the Odyssey^®CLx Infrared Imaging System plus MousePOD <i>in vivo</i> Imaging Accessory. Thus, the location and size of the tumor could be qualitatively measured <i>in vivo</i>. The white circle highlights the tumor mass in each animal that was examined. <b>(C)</b> Detection of ecto-5’-NT immunoreactivity in D283ev and D283hCD73 tumors. The values represent the mean values ± SD (n = 10) for each analyzed group, where (*) p < 0.05.</p