Development of a novel DNA aptamer ligand targeting to primary cultured tumor endothelial cells by a cell-based SELEX method.

The present study used a spontaneous cell-based SELEX method (Systemic Evolution of Ligands by EXponential Enrichment) to produce DNA aptamers that specifically bind to cell surface proteins or biomarkers produced by primary cultured mouse tumor endothelial cells (mTECs). In solid tumors, new blood vessels are formed through an angiogenesis process, and this plays a critical role in cancer development as well as metastasis. To combat angiogenesis, an appropriate diagnosis and a molecular-level understanding of the different cancer types are now a high priority. The novel DNA aptamer AraHH001, developed in this study, binds specifically to mTECs with high affinity in the nano-molar range, but does not bind to normal skin endothelial cells (skin-ECs). The selected DNA aptamer was also found to bind to cultured human tumor endothelial cells (hTECs), isolated from a clinical patient with a renal carcinoma. The aptamer AraHH001 showed significant anti-angiogenesis activity by inhibiting tube formation by mTECs on matrigel. Interestingly, a confocal laser scanning microscopy examination of in vitro cellular uptake revealed that AraHH001 was assimilated by mTECs, and became co-localized in acidic compartments, as detected by labeling with Lysotracker Red. Therefore, the development of a specific DNA aptamer that binds to mTECs, as reported here for the first time, holds great promise not only as a therapeutic aptamer but also as a targeted molecular probe that appears to play a major role in angiogenesis, and for the development of a targeted new drug delivery system

Construction of an Aptamer Modified Liposomal System Targeted to Tumor Endothelial Cells

We describe herein the development of a high affinity and specific DNA aptamer as a new ligand for use in liposomal nanoparticles to target cultured mouse tumor endothelial cells (mTECs). Active targeted nanotechnology based drug delivery systems are currently of great interest, due to their potential for reducing side effects and facilitating the delivery of cytotoxic drugs or genes in a site specific manner. In this study, we report on a promising aptamer candidate AraHH036 that shows selective binding towards mTECs. The aptamer does not bind to normal cells, normal endothelial cells or tumor cells. Therefore, we synthesized an aptamer-polyethylene glycol (PEG) lipid conjugate and prepared aptamer based liposomes (ALPs) by the standard lipid hydration method. First, we quantified the higher capacity of ALPs to internalize into mTECs by incubating ALPs containing 1 mol%, 5 mol% and 10 mol% aptamer of total lipids and compared the results to those for unmodified PEGylated liposomes (PLPs). A confocal laser scanning microscope (CLSM) uptake study indicated that the ALPs were taken up more efficiently than PLPs. The measured K-d value of the ALPs was 142 nM. An intracellular trafficking study confirmed that most of the rhodamine labeled ALPs were taken up and co-localized with the green lysotracker, thus confirming that they were located in lysosomes. Finally, using an aptamer based proteomics approach, the molecular target protein of the aptamer was identified as heat shock protein 70 (HSP70). The results suggest that these ALPs offer promise as a new carrier molecule for delivering anti-angiogenesis drugs to tumor vasculature

An aptamer ligand based liposomal nanocarrier system that targets tumor endothelial cells

The objective of this study was to construct our recently developed aptamer-modified targeted liposome nano-carrier (Apt-PEG-LPs) system to target primary cultured mouse tumor endothelial cells (mTEC), both in vitro and in vivo. We first synthesized an aptamer-polyethylene glycol 2000-distearoyl phosphoethanolamine (Apt-PEG(2000)-DSPE). The conjugation of the Apt-PEG(2000)-DSPE was confirmed by MALDI-TOF mass spectroscopy. A lipid hydration method was used to prepare Apt-PEG-LPs, in which the outer surface of the PEG-spacer was decorated with the aptamer. Apt-PEG-LPs were significantly taken up by mTECs. Cellular uptake capacity was observed both quantitatively and qualitatively using spectrofluorometry, and confocal laser scanning microscopy (CLSM), respectively. In examining the extent of localization of aptamer-modified liposomes that entered the cells, approximately 39% of the Apt-PEG-LPs were not co-localized with lysotracker, indicating that they had escaped from endosomes. The uptake route involved a receptor mediated pathway, followed by clathrin mediated endocytosis. This Apt-PEG-LP was also applied for in vivo research whether this system could target tumor endothelial cells. Apt-PEG-LP and PEG(5000)-DSPE modified Apt-PEG-LP (Apt/PEG(5000)-LP) were investigated by human renal cell carcinoma (OS-RC-2 cells) inoculating mice using CLSM. Apt-PEG-LP and Apt/PEG(5000)-LP showed higher accumulation on tumor vasculature compared to PEG-LP and the co-localization efficacy of Apt-PEG-LP and Apt/PEG(5000)-LP on TEC were quantified 16% and 25% respectively, which was also better than PEG-LP (3%). The findings suggest that this system is considerable promise for targeting tumor endothelial cells to deliver drugs or genes in vitro and in vivo. (C) 2014 Elsevier Ltd. All rights reserved

Binding assay of selected FITC-labeled DNA aptamer AraHH001 against a series of cells, and cell lines by a flow cytometry assay.

<p>A flow cytometry binding assay of AraHH001 with A. Normal skin-ECs, B. OS-RC-2, C. RFP-SM, D. HUVEC, E. HMVEC, F. m OS-RC-EC, and G. hTEC. In all cases, the results show non-treated, treated with 200 pmol FITC labeled zero cycle ssDNA pools, treated with 200 pmol and 1000 pmol FITC-labeled DNA aptamer AraHH001.</p

Determination of binding affinity, Kd value of the DNA aptamer AraHH001 by a flow cytometry.

<p>The Binding affinity of AraHH001 was determined by a flow cytometry using FITC-AraHH001 to mTECs and to skin-ECs. The average mean fluorescence intensity of varying concentration of FITC-AraHH001obtained was plotted to determine dissociation constant Kd. The experiment was repeated three times and a Error bar represents the standard deviation of means.</p

Identification of selected FITC labeled DNA aptamers on mTECs by a flow cytometry assay.

<p>Flow cytometry data represents binding assay of five FITC-labeled DNA aptamers with mTECs. In each DNA aptamer binding assay has shown the result of non treated mTECs, treatment mTECs with 200 pmol FITC labeled zero cycle ssDNA pools, treatment mTECs with 200 pmol DNA aptamer and 1000 pmol DNA aptamer independently.</p

<i>In vitro</i> cellular uptake of the AraHH001 aptamer and control zero cycle pools in acidic compartments of mTECs.

<p>mTECs was incubated with FITC-tagged aptamer AraHH001 and zero cycle pools at 37°C for 30 minutes to check internalization, and were stained live nuclei with Hoechst 33342 and acidic compartments with LysoTracker Red and analyses with confocal laser scanning microscopy. <b>A.</b> (FITC-tagged aptamer AraHH001 and zero cycle pools). <b>B. (</b>Uptake of Lysotracker red into the acidic compartments of mTECs). <b>C.</b> (merge of images, Hoechst, A, and B). <b>D.</b> (merge of image Hoechst, A and B+DIC).</p

Flow cytometry binding assay of selected FITC labeled ssDNA pools with mTECs.

<p>Enrichment in binding random ssDNAs in the selection was observed by flow cytometry analysis. Here the curve represents non-treated cells and treated cells with a zero cycle ssDNA 10 and 12 cycle ssDNA pools respectively. 400 pmol of ssDNA library was used in each case. A zero-cycle ssDNA pool was used to observe the differentiation of enrichment from the starting point of selection with increasing cycle of selection.</p