Cell-Specific Internalization Study of an Aptamer from Whole Cell Selection

Center for Research at the Bio/Nano Interface, Department of Chemistry, Shands Cancer Center and UF Genetics Institute, McKnight Brain Institute, University of Florida, Gainesville, FL 32611-7200, USA.
Chemistry (Impact Factor: 5.73). 02/2008; 14(6):1769-75. DOI: 10.1002/chem.200701330
Source: PubMed


Nucleic acid aptamers have been shown many unique applications as excellent probes in molecular recognition. However, few examples are reported which show that aptamers can be internalized inside living cells for aptamer functional studies and for targeted intracellular delivery. This is mainly due to the limited number of aptamers available for cell-specific recognition, and the lack of research on their extra- and intracellular functions. One of the major difficulties in aptamers' in vivo application is that most of aptamers, unlike small molecules, cannot be directly taken up by cells without external assistance. In this work, we have studied a newly developed and cell-specific DNA aptamer, sgc8. This aptamer has been selected through a novel cell selection process (cell-SELEX), in which whole intact cells are used as targets while another related cell line is used as a negative control. The cell-SELEX enables generation of multiple aptamers for molecular recognition of the target cells and has significant advantages in discovering cell surface binding molecules for the selected aptamers. We have studied the cellular internalization of one of the selected aptamers. Our results show that sgc8 is internalized efficiently and specifically to the lymphoblastic leukemia cells. The internalized sgc8 aptamers are located inside the endosome. Comparison studies are done with the antibody for the binding protein of sgc8, PTK7 (Human protein tyrosine kinase-7) on cell surface. We also studied the internalization kinetics of both the aptamer and the antibody for the same protein on the living cell surface. We have further evaluated the effects of sgc8 on cell viability, and no cytotoxicity is observed. This study indicates that sgc8 is a promising agent for cell-type specific intracellular delivery.

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Available from: Dihua Shangguan, Jun 23, 2014
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    • "For example, an antitumor agent (Doxorubicin; Dox) has been covalently cross-linked with a thiol-modified, cell-internalizing DNA aptamer sgs8c through an acid–labile acylhydrazone linkage (Figure 3a), which facilitates cleavage of the chemical bond that releases the Dox molecule inside the acidic endosomal environment.80 The DNA aptamer that was selected by the cell-SELEX method can specifically recognize and be efficiently internalized by CCRF-CEM cells that express its target protein tyrosine kinase 7 (PTK7).37,38,81 The sgs8c aptamer-Dox conjugate not only showed antitumor activity similar to free Dox, but also preserved the high binding affinity of the parental DNA aptamer. "
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    ABSTRACT: One hundred years ago, Dr. Paul Ehrlich popularized the "magic bullet" concept for cancer therapy in which an ideal therapeutic agent would only kill the specific tumor cells it targeted. Since then, "targeted therapy" that specifically targets the molecular defects responsible for a patient's condition has become a long-standing goal for treating human disease. However, safe and efficient drug delivery during the treatment of cancer and infectious disease remains a major challenge for clinical translation and the development of new therapies. The advent of SELEX technology has inspired many groundbreaking studies that successfully adapted cell-specific aptamers for targeted delivery of active drug substances in both in vitro and in vivo models. By covalently linking or physically functionalizing the cell-specific aptamers with therapeutic agents, such as siRNA, microRNA, chemotherapeutics or toxins, or delivery vehicles, such as organic or inorganic nanocarriers, the targeted cells and tissues can be specifically recognized and the therapeutic compounds internalized, thereby improving the local concentration of the drug and its therapeutic efficacy. Currently, many cell-type-specific aptamers have been developed that can target distinct diseases or tissues in a cell-type-specific manner. In this review, we discuss recent advances in the use of cell-specific aptamers for targeted disease therapy, as well as conjugation strategies and challenges.
    Full-text · Article · Jun 2014 · Molecular Therapy - Nucleic Acids
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    • "In fact, mice with fat-specific disruption of the insulin receptor gene have low fat mass levels, experience a loss of body weight, and undergo obesity-related glucose intolerance [54]. (2) Our study indicates that specific delivery of anti-obesity drug(s) to mature white adipocytes by targeted vehicles via the internalization characteristics of aptamer(s) is possible [55]. (3) This approach can also be used to obtain homogeneous adipocytes for both basic research and for clinical applications [56]. "
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    ABSTRACT: Background Adipose tissue, mainly composed of adipocytes, plays an important role in metabolism by regulating energy homeostasis. Obesity is primarily caused by an abundance of adipose tissue. Therefore, specific targeting of adipose tissue is critical during the treatment of obesity, and plays a major role in overcoming it. However, the knowledge of cell-surface markers specific to adipocytes is limited. Methods and Results We applied the CELL SELEX (Systematic Evolution of Ligands by EXponential enrichment) method using flow cytometry to isolate molecular probes for specific recognition of adipocytes. The aptamer library, a mixture of FITC-tagged single-stranded random DNAs, is used as a source for acquiring molecular probes. With the increasing number of selection cycles, there was a steady increase in the fluorescence intensity toward mature adipocytes. Through 12 rounds of SELEX, enriched aptamers showing specific recognition toward mature 3T3-L1 adipocyte cells were isolated. Among these, two aptamers (MA-33 and 91) were able to selectively bind to mature adipocytes with an equilibrium dissociation constant (Kd) in the nanomolar range. These aptamers did not bind to preadipocytes or other cell lines (such as HeLa, HEK-293, or C2C12 cells). Additionally, it was confirmed that MA-33 and 91 can distinguish between mature primary white and primary brown adipocytes. Conclusions These selected aptamers have the potential to be applied as markers for detecting mature white adipocytes and monitoring adipogenesis, and could emerge as an important tool in the treatment of obesity.
    Full-text · Article · May 2014 · PLoS ONE
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    • "Aptamers generated from whole living cells are excellent molecular recognition probes to characterize target cells on a molecular level. These aptamers are effective for recognizing the target molecules such as cell-membrane receptors in their native conformations and physiological environments in a living cell, thus, it makes them an effective tool for biomarker discovery and molecular medicine [15]. "
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    ABSTRACT: In this paper, a panel of single-stranded DNA aptamers with high affinity and specificity against Salmonella Paratyphi A was selected from an enriched oligonucleotide pool by a whole-cell-Systematic Evolution of Ligands by Exponential Enrichment (SELEX) procedure, during which four other Salmonella serovars were used as counter-selection targets. It was determined through a fluorescence assay that the selected aptamers had high binding ability and specificity to this pathogen. The dissociation constant of these aptamers were up to nanomolar range, and aptamer Apt22 with the lowest Kd (47 ± 3 nM) was used in cell imaging experiments. To detect this bacteria with high specificity and cost-efficiently, a novel useful detection method was also constructed based on the noncovalent self-assembly of single-walled carbon nanotubes (SWNTs) and DNAzyme-labeled aptamer detection probes. The amounts of target bacteria could be quantified by exploiting chemoluminescence intensity changes at 420 nm and the detection limit of the method was 103 cfu/mL. This study demonstrated the applicability of Salmonella specific aptamers and their potential for use in the detection of Salmonella in food, clinical and environmental samples.
    Full-text · Article · May 2013 · Sensors
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