RESUMEN
Background: Hepatocellular carcinoma (HCC) is the predominant subtype of liver cancer with an extraordinary high mortality. Resistance to systemic therapy is a major cause of inferior clinical outcome in most patients with HCC. CD44 is a transmembrane cell-surface glycoprotein that is characterized by its variants displaying differential overexpression in human cancers. Aptamers, also known as chemical antibodies, can target cell-surface molecules with high affinity and specificity via structural recognition. Aptamer-mediated drug delivery hence is of high potentials in guiding therapy to improve efficacy. Methods: Variants CD44E and CD44s were studied for HCC relevance by investigating their expressions in primary HCC tumors, adjacent cirrhotic/fibrotic livers and normal livers using junction specific primers in qPCR assay. CD44E/s dual-targeted aptamers were uncovered by integrating loss-gain cell-SELEX and next generation sequencing. Selected aptamers were characterized for binding affinity and specificity, biostability, in vivo and in vitro cytotoxicity, in vivo homing and biodistribution, and ability to deliver 5-FU into targeted cells in vitro. Results: Both CD44E and CD44s isoforms showed significant upregulations in HCC tumors with CD44E/s activities promoting cell proliferation and migration. Loss-gain cell-SELEX uncover a CD44E/s dual-targeting aptamer, termed CD44-Apt1. Strong binding of CD44-Apt1 to cell-surface CD44 positive cells but not CD44-negative cells was demonstrated by flow-cytometry. CD44-Apt1 displayed strong affinity to CD44E and CD44s with KD as low as 1 nM but not the hyaluronic acid binding domain of CD44. Confocal imaging of CD44-positive cells stained with fluorescent-labeled CD44-Apt1 showed profound cytoplasmic localization, suggesting efficient cell-penetrating ability. Meanwhile, no apparent staining was observed in CD44-negative cells. CD44-Apt1 when conjugated with inhibitor 5-FU showed efficient guidance of 5-FU into HCC cells that significantly enhanced drug toxicity by more than thousands-fold. Both in vitro cell treatment and in vivo animal biodistribution indicated that CD44-Apt1 is non-toxic. In HCC xenograft model, CD44-Apt1 efficiently homed to tumor xenografts in a CD44 expression-dependent manner. Conclusion: Novel discovery of aptamer CD44-Apt1 that can bind both CD44E and CD44s illustrates high potential as nanoprobe to deliver anti-cancer therapeutics.