Enhanced Targeted Gene Transduction: AAV2 Vectors Conjugated to Multiple Aptamers via Reducible Disulfide Linkages.

Enhanced targeted gene transduction by AAV2 vectors is achieved by linking the vector to multiple sgc8 aptamers, which are selective for cell membrane protein PTK7. Aptamer molecules are conjugated to multiple sites on a DNA dendrimer (G-sgc8), which is then linked to AAV2 via a dithiobis(succinimidyl propionate) cross-linker containing a disulfide group, which can facilitate the release of AAV2 vectors by reaction with the reduced form of intracellular glutathione. The G-sgc8-AAV2 vectors showed a 21-fold enhancement in binding affinity and an enhanced ability to protect sgc8 aptamers against nuclease degradation to cells expressing PTK7 compared to single aptamer-AAV2 conjugates. The transduction efficiency was tested by loading AAV2 with the gene for green fluorescent protein. Therefore, this modified recombinant vector is an attractive and promising tool for targeted biomedical applications.

Recognition-then-Reaction Enables Site-Selective Bioconjugation to Proteins on Live-Cell Surfaces.

Site-selective protein modification is a key step in facilitating protein functionalization and manipulation. To accomplish this, genetically engineered proteins were previously required, but the procedure was laborious, complex, and technically challenging. Herein we report the development of aptamer-based recognition-then-reaction to guide site-selective protein/DNA conjugation in a single step with outstanding selectivity and efficiency. As models, several proteins, including human thrombin, PDGF-BB, Avidin, and His-tagged recombinant protein, were studied, and the results showed excellent selectivity under mild reaction conditions. Taking advantage of aptamers as recognition elements with extraordinary selectivity and affinity, this simple preparation method can tag a protein in a complex milieu. Thus, with the aptamer obtained from cell-SELEX, real-time modification of live-cell membrane proteins can be achieved in one step without any pre-treatment.

Aptasensor with Expanded Nucleotide Using DNA Nanotetrahedra for Electrochemical Detection of Cancerous Exosomes.

Exosomes are extracellular vesicles (50-100 nm) circulating in biofluids as intercellular signal transmitters. Although the potential of cancerous exosomes as tumor biomarkers is promising, sensitive and rapid detection of exosomes remains challenging. Herein, we combined the strengths of advanced aptamer technology, DNA-based nanostructure, and portable electrochemical devices to develop a nanotetrahedron (NTH)-assisted aptasensor for direct capture and detection of hepatocellular exosomes. The oriented immobilization of aptamers significantly improved the accessibility of an artificial nucleobase-containing aptamer to suspended exosomes, and the NTH-assisted aptasensor could detect exosomes with 100-fold higher sensitivity when compared to the single-stranded aptamer-functionalized aptasensor. The present study provides a proof-of-concept for sensitive and efficient quantification of tumor-derived exosomes. We thus expect the NTH-assisted electrochemical aptasensor to become a powerful tool for comprehensive exosome studies.

ICG-conjugated Magnetic Graphene Oxide for Dual Photothermal and Photodynamic Therapy.

Aptamer-functionalized magnetic graphene oxide conjugates loaded with indocyanine green (ICG) dye, or Apt@ICG@mGO, have been successfully developed for dual-targeted photothermal and photodynamic therapy. In general, a drug or its carrier or their dosage can be imprtant important issues in terms of toxicity. However, in this system, each component used is quite safe, biocompatibe and clean. For instance, ICG, a Food and Drug Administration (FDA) approved near-infrared (NIR) dye, serves as both a photothermal and photodynamic agent. It is immobilized on the surface of mGO via a physical interaction called "π-π stacking". The mGO, as a most biocomptible member of the carbo family, is selected for use as a platform for aptamer and ICG dye conjugation, as well as as a photothermal agent. The light in the near-infrared region (NIR) was chosen as a harmless light source for activating the agents for photothermal therapy (PTT) and photodynamic therapy (PDT). The magnetic properties of mGO are also used for separation of Apt@ICG@mGO conjugates from the reaction medium. Aptamer sgc8 acts as a targeting ligand to selectively and specifically bind to a protein on the membrane of cancer cell line CCRF-CEM. After the aptamer- functionalized ICG@mGO conjugates are incubated with target CEM cells at 37 °C for 2 hours, they are bound to cells or they may be internalized into the cell via endocytosis. More significantly, we demonstrated that the Apt@ICG@mGO conjugates produce heat for photothermal therapy (PTT) and singlet oxygen for photodynamic therapy (PDT) upon NIR laser irradiation at 808 nm. Thus, remarkably efficient cancer cell destructions with ~41% and ~60% and ~82% cell killing using 10, 50 and 100 ppm Apt@ICG@mGO, respectively are achieved in 5 min light exposure.

Aptamers against Cells Overexpressing Glypican†3 from Expanded Genetic Systems Combined with Cell Engineering and Laboratory Evolution.

Laboratory in vitro evolution (LIVE) might deliver DNA aptamers that bind proteins expressed on the surface of cells. In this work, we used cell engineering to place glypican 3 (GPC3), a possible marker for liver cancer theranostics, on the surface of a liver cell line. Libraries were then built from a six-letter genetic alphabet containing the standard nucleobases and two added nucleobases (2-amino-8H-imidazo[1,2-a][1,3,5]triazin-4-one and 6-amino-5-nitropyridin-2-one), Watson-Crick complements from an artificially expanded genetic information system (AEGIS). With counterselection against non-engineered cells, eight AEGIS-containing aptamers were recovered. Five bound selectively to GPC3-overexpressing cells. This selection-counterselection scheme had acceptable statistics, notwithstanding the possibility that cells engineered to overexpress GPC3 might also express different off-target proteins. This is the first example of such a combination.

A Facile Process for the Preparation of Three-Dimensional Hollow Zn(OH)2 Nanoflowers at Room Temperature.

A facile strategy has been developed to synthesize double-shelled Zn(OH)2 nanoflowers (DNFs) at room temperature. The nanoflowers were generated via conversion of Cu2 O nanoparticles (NPs) using ZnCl2 and Na2 S2 O3 by a simple process. Outward diffusion of the Cu(2+) , produced by an oxidation process on the surface of NPs, and the inward diffusion of Zn(2+) by coordination and migration, eventually lead to a hollow cavity in the inner NPs with a double-shelled 3D hollow flower shapes. The thickness of the inner and outer shells is estimated to be about 20 nm, and the thickness of nanopetals is about 7 nm. The nanoflowers have large surface areas and excellent adsorption properties. As a proof of potential applications, the DNFs exhibited an excellent ability to remove organic molecules from aqueous solutions.

DNA micelle flares: a study of the basic properties that contribute to enhanced stability and binding affinity in complex biological systems.

DMFs are spherical DNA-diacyllipid nanostructures formed by hydrophobic effects between lipid tails coupled to single-stranded DNAs. Such properties as high cellular permeability, low critical micelle concentration (CMC) and facile fabrication facilitate intracellular imaging and drug delivery. While the basic properties of NFs have been amply described and tested, few studies have characterized the fundamental properties of DMFs with particular respect to aggregation number, dissociation constant and biostability. Therefore, to further explore their conformational features and enhanced stability in complex biological systems, we herein report a series of characterization studies. Static light scattering (SLS) demonstrated that DMFs possess greater DNA loading capacity when compared to other DNA-based nanostructures. Upon binding to complementary DNA (cDNA), DMFs showed excellent dissociation constants (Kd) and increased melting temperatures, as well as constant CMC (10 nM) independent of DNA length. DMFs also present significantly enhanced stability in aqueous solution with nuclease and cell lysate. These properties make DMFs ideal for versatile applications in bioanalysis and theranostics studies.

Development of a panel of DNA Aptamers with High Affinity for Pancreatic Ductal Adenocarcinoma.

Pancreatic cancer costs nearly 40,000 lives in the U.S. each year and has one of the lowest survival rates among cancers. Effective treatment of pancreatic ductal adenocarcinoma is hindered by lack of a reliable biomarker. To address this challenge, aptamers were selected by cell-SELEX (Systematic Evolution of Ligands by EXponential enrichment) targeting human pancreatic ductal adenocarcinoma (PL45). Five promising aptamers presenting low Kd values and good specificity were generated. Among these five aptamers, one was tailored into a nanostructure carrying a high drug payload for specific drug delivery. The results show a viability of almost 80% for negative cells while only 50% of the target cells remained alive after 48 h incubation. These results lead to the conclusion that further research could reveal protein biomarkers specific to pancreatic adenocarcinoma, with probes available for early detection.

Ionic Functionalization of Hydrophobic Colloidal Nanoparticles To Form Ionic Nanoparticles with Enzymelike Properties.

Inorganic colloidal nanoparticles (NPs) stabilized by a layer of hydrophobic surfactant on their surfaces have poor solubility in the aqueous phase, thus limiting their application as biosensors under physiological conditions. Here we report a simple model to ionize various types of hydrophobic colloidal NPs, including FePt, cubic Fe3O4, Pd, CdSe, and NaYF4 (Yb 30%, Er 2%, Nd 1%) NPs, to multicharged (positive and negative) NPs via ligand exchange. Surfaces of neutral hydrophobic NPs were converted to multicharged ions, thus making them soluble in water. Furthermore, peroxidase-like activity was observed for ionic FePt, Fe3O4, Pd, and CdSe NPs, of which FePt and CdSe catalyzed the oxidation of the colorless substrate 3,3',5,5'-tetramethylbenzidine (TMB) to the blue-colored product in the absence of H2O2, while Pd and Fe3O4 catalyzed the oxidization of TMB in the presence of H2O2. With the benefit of the ionic functionalization protocols described herein, colloidal NPs should gain wider use as biomarkers, nanozymes, and biosensors.

DNA Aptamer Based Nanodrugs: Molecular Engineering for Efficiency.

In the past two decades, the study of cancer therapy has gradually advanced to the "nano" era. Numerous novel nanomaterials armed with unique physical properties have been introduced into biomedical research. At the same time, functional nucleic acid molecules, especially aptamers, have aroused broad attention from the biomedical community. Benefiting from the advancement of molecular engineering strategies, it is now feasible to combine the cancer-specific recognition capability of aptamers with various other special functions of nanomaterials to develop cancer-specific drugs at the nanoscale. Nanodrugs are now offering an unprecedented opportunity to achieve the goal of efficient targeted delivery as well as controlled release. This review highlights some achievements made in multiple aptamer-based nanodrug systems that have emerged in recent years, including studies in the infant stage of "proof-of-concept".

Molecular Recognition of Human Liver Cancer Cells Using DNA Aptamers Generated via Cell-SELEX.

Most clinical cases of liver cancer cannot be diagnosed until they have evolved to an advanced stage, thus resulting in high mortality. It is well recognized that the implementation of early detection methods and the development of targeted therapies for liver cancer are essential to reducing the high mortality rates associated with this disease. To achieve these goals, molecular probes capable of recognizing liver cancer cell-specific targets are needed. Here we describe a panel of aptamers able to distinguish hepatocarcinoma from normal liver cells. The aptamers, which were selected by cell-based SELEX (Systematic Evolution of Ligands by Exponential Enrichment), have Kd values in the range of 64-349 nM toward the target human hepatoma cell HepG2, and also recognize ovarian cancer cells and lung adenocarcinoma. The proteinase treatment experiment indicated that all aptamers could recognize target HepG2 cells through surface proteins. This outcome suggested that these aptamers could be used as potential probes for further research in cancer studies, such as developing early detection assays, targeted therapies, and imaging agents, as well as for the investigation of common membrane proteins in these distinguishable cancers.

Evolution of functional six-nucleotide DNA.

Axiomatically, the density of information stored in DNA, with just four nucleotides (GACT), is higher than in a binary code, but less than it might be if synthetic biologists succeed in adding independently replicating nucleotides to genetic systems. Such addition could also add functional groups not found in natural DNA, but useful for molecular performance. Here, we consider two new nucleotides (Z and P, 6-amino-5-nitro-3-(1'-ß-D-2'-deoxyribo-furanosyl)-2(1H)-pyridone and 2-amino-8-(1'-ß-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one). These are designed to pair via complete Watson-Crick geometry. These were added to a library of oligonucleotides used in a laboratory in vitro evolution (LIVE) experiment; the GACTZP library was challenged to deliver molecules that bind selectively to liver cancer cells, but not to untransformed liver cells. Unlike in classical in vitro selection, low levels of mutation allow this system to evolve to create binding molecules not necessarily present in the original library. Over a dozen binding species were recovered. The best had Z and/or P in their sequences. Several had multiple, nearby, and adjacent Zs and Ps. Only the weaker binders contained no Z or P at all. This suggests that this system explored much of the sequence space available to this genetic system and that GACTZP libraries are richer reservoirs of functionality than standard libraries.

A Nonenzymatic Hairpin DNA Cascade Reaction Provides High Signal Gain of mRNA Imaging inside Live Cells.

Enzyme-free signal amplification has enabled sensitive in vitro detection of biomolecules such as proteins and nucleic acids. However, monitoring targets of interest in live cells via enzyme-free amplification is still challenging, especially for analytes with low concentrations. To the best of our knowledge, this paper reports the first attempt to perform mRNA imaging inside live cells, using a nonenzymatic hairpin DNA cascade reaction for high signal gain, termed a hairpin DNA cascade amplifier (HDCA). In conventional nucleic acid probes, such as linear hybridization probes, mRNA target signaling occurs in an equivalent reaction ratio (1:1), whereas, in HDCA, one mRNA target is able to yield multiple signal outputs (1:m), thus achieving the goal of signal amplification for low-expression mRNA targets. Moreover, the recycled mRNA target in the HDCA serves as a catalyst for the assembly of multiple DNA duplexes, generating the fluorescent signal of reduced MnSOD mRNA expression, thus indicating amplified intracellular imaging. This programmable cascade reaction presents a simple and modular amplification mechanism for intracellular biomarkers of interest, providing a significant boost to the search for clues leading to the accurate identification and effective treatment of cancers.

Self-assembly of DNA nanohydrogels with controllable size and stimuli-responsive property for targeted gene regulation therapy.

Here, we report the synthesis and characterization of size-controllable and stimuli-responsive DNA nanohydrogels as effective targeted gene delivery vectors. DNA nanohydrogels were created through a self-assembly process using three kinds of building units, respectively termed Y-shaped monomer A with three sticky ends (YMA), Y-shaped monomer B with one sticky end (YMB), and DNA linker (LK) with two sticky ends. Hybridization at the sticky ends of monomers and LK leads to nanohydrogel formation. DNA nanohydrogels are size-controllable by varying the ratio of YMA to YMB. By incorporating different functional elements, such as aptamers, disulfide linkages, and therapeutic genes into different building units, the synthesized aptamer-based nanohydrogels (Y-gel-Apt) can be used for targeted and stimuli-responsive gene therapy. Y-gel-Apt strongly inhibited cell proliferation and migration in target A549 cells, but not in control cells. By taking advantage of facile modular design and assembly, efficient cellular uptake, and superior biocompatibility, this Y-gel-Apt holds great promise as a candidate for targeted gene or drug delivery and cancer therapy.

Self-assembled Multifunctional DNA Nanoflowers for the Circumvention of Multidrug Resistance in Targeted Anticancer Drug Delivery.

Cancer chemotherapy has been impeded by side effects and multidrug resistance (MDR) partially caused by drug efflux from cancer cells, which call for targeted drug delivery systems additionally able to circumvent MDR. Here we report multifunctional DNA nanoflowers (NFs) for targeted drug delivery to both chemosensitive and MDR cancer cells and circumvent MDR in both leukemia and breast cancer cell models. NFs are self-assembled via liquid crystallization of DNA generated by Rolling Circle Replication, during which NFs are incorporated with aptamers for specific cancer cell recognition, fluorophores for bioimaging, and Doxorubicin (Dox)-binding DNA for drug delivery. NF sizes are tunable (down to ~200 nm in diameter), and the densely packed drug-binding motifs and porous intrastructures endow NFs with high drug loading capacity (71.4%, wt/wt). The Dox-loaded NFs (NF-Dox) are stable at physiological pH, yet drug release is facilitated in acidic or basic conditions. NFs deliver Dox into target chemosensitive and MDR cancer cells, preventing drug efflux and enhancing drug retention in MDR cells. Consequently, NF-Dox induces potent cytotoxicity in both target chemosensitive cells and MDR cells, but not nontarget cells, thus concurrently circumventing MDR and reducing side effects. Overall, these NFs are promising to circumvent MDR in targeted cancer therapy.

Facile surface functionalization of hydrophobic magnetic nanoparticles.

Nonpolar phase synthesized hydrophobic nanocrystals show attractive properties and have demonstrated prominent potential in biomedical applications. However, the preparation of biocompatible nanocrystals is made difficult by the presence of hydrophobic surfactant stabilizer on their surfaces. To address this limitation, we have developed a facile, high efficiency, single-phase and low-cost method to convert hydrophobic magnetic nanoparticles (MNPs) to an aqueous phase using tetrahydrofuran, NaOH and 3,4-dihydroxyhydrocinnamic acid without any complicated organic synthesis. The as-transferred hydrophilic MNPs are water-soluble over a wide pH range (pH = 3-12), and the solubility is pH-controllable. Furthermore, the as-transferred MNPs with carboxylate can be readily adapted with further surface functionalization, varying from small molecule dyes to oligonucleotides and enzymes. Finally, the strategy developed here can easily be extended to other types of hydrophobic nanoparticles to facilitate biomedical applications of nanomaterials.

Engineering of switchable aptamer micelle flares for molecular imaging in living cells.

Simultaneous monitoring of the expression, distribution, and dynamics of biological molecules in living cells is one of the most challenging tasks in the analytical sciences. The key to effective and successful intracellular imaging is the development of delivery platforms with high efficiency and ultrasensitive molecular probes for specific targets of interest. To achieve these goals, many nanomaterials are widely used as carriers to introduce nucleic acid probes into living cells for real-time imaging of biomolecules. However, limitations on their use include issues of cytotoxicity and delivery efficiency. Herein, we propose a switchable aptamer micelle flare (SAMF), formed by self-assembly of an aptamer switch probe-diacyllipid chimera, to monitor ATP molecules inside living cells. Similarity of hydrophobic composition between diacyllipids in the micelle flares and phospholipid bilayers in the dynamic membranes of living cells allows SAMFs to be uptaken by living cells more efficiently than aptamer switch probes without external auxiliary. Switchable aptamers were found to bind target ATP molecules with high selectivity and specificity, resulting in restoration of the fluorescence signal from "OFF" to "ON" state, thus indicating the presence of the analyte. These switchable aptamer micelle flares, which exhibit cell permeability and nanoscale controllability, show exceptional promise for molecular imaging in bioanalysis, disease diagnosis, and drug delivery.

Cancer cell sensing and therapy using affinity tag-conjugated gold nanorods.

Through the developments in controlling the shape of gold nanoparticles, synthesis of gold nanorods (AuNRs) can be considered as a milestone discovery in the area of nanomaterial-based cancer treatments. Besides having tuneable absorption maxima at near infrared (NIR) range, AuNRs have superior absorption cross section at NIR frequencies compared with other gold nanoparticles. When this unique optical property is combined with the specificity against cancer cells used by affinity tag conjugations, AuNRs become one of the most important nanoparticles used in both cancer cell sensing and in therapy. In this review, the impact of size and shape control of nanoparticles, especially AuNRs, on cancer cell treatments and a range of aptamer-conjugated AuNR applications in this regard are reviewed.