Selective Targeting to Glioma with Nucleic Acid Aptamers

Abstract

Malignant glioma is characterised by a rapid growth rate and high capacity for invasive infiltration to surrounding brain tissue; hence, diagnosis and treatment is difficult and patient survival is poor. Aptamers contribute a promising and unique technology for the in vitro imaging of live cells and tissues, with a potentially bright future in clinical diagnostics and therapeutics for malignant glioma. The binding selectivity, uptake capacity and binding target of two DNA aptamers, SA43 and SA44, were investigated in glioma cells and patient tissues. The binding assay showed that SA43 and SA44 bound with strong affinity (Kd, 21.56 ± 4.60 nM and Kd, 21.11 ± 3.30 nM respectively) to the target U87MG cells. Quantitative analysis by flow cytometry showed that the aptamers were able to actively internalise in U87MG and 1321N1 glioma cells compared to the non-cancerous and non-glioma cell types. Confocal microscopy confirmed staining in the cytoplasm, and co-localisation studies with endoplasmic reticulum, Golgi apparatus and lysosomal markers suggested internalisation and compartmentalisation within the endomembrane system. Both aptamers selectively bound to Ku 70 and Ku 80 DNA repair proteins as determined by aptoprecipitation (AP) followed by mass spectrometry analysis and confirmation by Western blot. In addition, aptohistochemical (AHC) staining on paraffin embedded, formalin fixed patient tissues revealed that the binding selectivity was significantly higher for SA43 aptamer in glioma tissues (grade I, II, III and IV) compared to the non-cancerous tissues, whereas SA44 did not show selectivity towards glioma tissues. The results indicate that SA43 aptamer can differentiate between glioma and non-cancerous cells and tissues and therefore, shows promise for histological diagnosis of glioma.

Aptamers: Uptake mechanisms and intracellular applications

“…The colocalization of cell type specific-DNA aptamers with LAMP-1, a late endosomal and lysosomal marker, was confirmed in renal proximal tubule RPTEC/TERT1 cells by in situ fluorescence hybridization [13]. Glioma-specific DNA aptamers bound Ku 70 and Ku 80 were found to colocalize in lysosomes, endoplasmic reticulum (ER), and Golgi apparatus with organelle-specific fluorescent dyes on live cell images by confocal microscopy [14]. The PrP c DNA aptamers were conjugated with fluorescent quantum dots (QDs), which are fluorescent nanoparticles to track the dynamics of intracellular imaging.…”

“…But, chemically synthesized RNApt16 showed inhibitory effects of 85 ± 5%, suggesting that improving the half-life is pivotal. By following studies, the full length anti-HIV UTR aptamers were cloned into a human U6 snRNA cassette, pU6 14 , for stable hairpin-loop domains. HEK293T cells were transfected with the in vitro transcribed anti-HIV UTR aptamers from the U6 plasmid, which inhibited HIV particle production upto 80 ± 7%.…”

Bifunctional Aptamer–Doxorubicin Conjugate Crosses the Blood–Brain Barrier and Selectively Delivers Its Payload to EpCAM-Positive Tumor Cells

“…The development of aptamers for treatment of brain cancers and neurological conditions is not a new concept. There have been numerous reports of aptamers for development of treatments for glioblastoma and brain disease [49][50][51]. However, these aptamers have mainly been suggested for use in diagnostic applications as they lack therapeutic effect.…”

“…Previous methods reported for increasing DOX uptake in the brain have entailed either pre-treatment with agents that interact with multidrug resistance proteins or the use of peptide vectors [52,53]. While these methods resulted in enhanced uptake of DOX, they are limited by the fact that blocking multidrug resistance proteins may lead to uptake of other toxic substances within the bloodstream [51,52]. More importantly, both methods still lack specific delivery upon entering the brain, thus leading to the issue of neurotoxic side effects.…”

Circular RNA CircHIPK3 Elevates CCND2 Expression and Promotes Cell Proliferation and Invasion Through miR-124 in Glioma

“…The growth and metastasis of glioma cells depend on angiogenesis, and a continuous increase in blood vessels has been considered a key feature of gliomas (Lund et al, 1998). This characteristic of rapid growth and high infiltration causes most patients already at stage IV when they are diagnosed with glioma (Shraddha et al, 2015). Pathological diagnosis is the basis for treatment.…”