Yu-Fen Huang

Cancer Cell Targeting Using Multiple Aptamers Conjugated on Nanorods

Molecular recognition toward specific cells is a key issue for effective disease, such as cancer, diagnosis and therapy. Although many molecular probes such as aptamers and antibodies can recognize the unique molecular signatures of cancer cells, some of these probes only have relatively weak binding affinities. This results in poor signaling and hinders cell targeting. Here, we use Au-Ag nanorods (NRs) as a nanoplatform for multivalent binding by multiple aptamers on the rod to increase both the signal and binding strengths of these aptamers in cancer cell recognition. Up to 80 fluorophore-labeled aptamers can be attached on a 12 nm x 56 nm NR, resulting in a much stronger fluorescence signal than that of an individual dye-labeled aptamer probe. The molecular assembly of aptamers on the NR surfaces also significantly improves the binding affinity with cancer cells through simultaneous multivalent interactions with the cell membrane receptors. This leads to an affinity at least 26-fold higher than the intrinsic affinity of the original aptamer probes. As determined by flow cytometric measurements, an enhancement in fluorescence signal in excess of 300-fold is obtained for the NR-aptamer-labeled cells compared with those labeled by individual aptamer probes. Therefore, the molecular assembly of aptamers clearly shows potential applications for the elucidation of cells with low density of binding sites, or with relatively weak binding probes, and can thus greatly improve our ability to perform cellular imaging and targeting. This is an excellent example of using nanomaterials to develop advanced molecular binders with greatly improved properties for cellular studies.

Molecular Assembly of an Aptamer–Drug Conjugate for Targeted Drug Delivery to Tumor Cells

Special delivery! An aptamer‐directed anticancer drug was molecularly engineered to be delivered to target cells for efficient therapeutic application. The covalent conjugation of drug and aptamer creates alternative opportunities for targeted therapy, as multiple yet specific aptamers can be “generated” relatively easily by cell‐SELEX for any target cells; this demonstrates the full potential of cell‐SELEX as a molecular discovery tool for biomedical studies and drug development.

Selective photothermal therapy for mixed cancer cells using aptamer-conjugated nanorods.

Safe and effective photothermal therapy depends on efficient delivery of heat for killing cells and molecular specificity for targeting cells. To address these requirements, we have designed an aptamer-based nanostructure which combines the high absorption efficiency of Au-Ag nanorods with the target specificity of molecular aptamers, a combination resulting in the development of an efficient and selective therapeutic agent for targeted cancer cell photothermal destruction. Most nanomaterials, such as gold nanoshells or nanorods (NRs), require a relatively high power of laser irradiation (1 x 10 (5)-1 x 10 (10) W/m (2)). In contrast, the high absorption characteristic of our Au-Ag NRs requires only 8.5 x 10 (4) W/m (2) laser exposure to induce 93 (+/-11)% cell death of NR-aptamer-labeled cells. Aptamers, the second component of the nanostructure, are generated from a cell-SELEX (systematic evolution of ligands by exponential enrichment) process and can be easily selected for specific recognition of individual tumor cell types without prior knowledge of the biomarkers for the cell. When tested with both cell suspensions and artificial solid tumor samples, these aptamer conjugates were shown to have excellent hyperthermia efficiency and selectivity. Under a specific laser intensity and duration of laser exposure, about 50 (+/-1)% of target (CEM) cells were severely damaged, while more than 87 (+/-1)% of control (NB-4) cells remained intact in a suspension cell mixture. These results indicate that the Au-Ag nanorod combination offers selective and efficient photothermal killing of targeted tumor cells, thus satisfying the two key challenges noted above. Consequently, for future in vivo application, it is fully anticipated that the tumor tissue will be selectively destroyed at laser energies which will not harm the surrounding normal tissue.

Release of photoactivatable drugs from plasmonic nanoparticles for targeted cancer therapy.

Chemotherapy is an important modality in cancer treatment. The major challenges of recent works are to improve drug loading, increase selectivity to target cells, and control the precise release of drugs. In the present study, we devised a smart drug carrier, an aptamer/hairpin DNA-gold nanoparticle (apt/hp-Au NP) conjugate for targeted delivery of drugs. The DNA aptamer sgc8c, which possesses strong affinity for protein tyrosine kinase 7 (PTK7), abundantly expressed on the surface of CCRF-CEM (T-cell acute lymphoblastic leukemia) cells, was assembled onto the surface of Au NPs. The repeated d(CGATCG) sequence within the hpDNA on the Au NP surface was used for the loading of the anticancer drug doxorubicin (Dox). After optimization, 25 (±3) sgc8c and 305 (±9) Dox molecules were successfully loaded onto the AuNP (13 nm) surface. The binding capability of apt/hp-Au NP conjugates toward targeted cells was investigated by flow cytometry and atomic absorption spectroscopy, which showed that the aptamer-functionalized nanoconjugates were selective for targeting of cancer cells. A cell toxicity (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, MTT) assay also demonstrated that these drug-loaded nanoconjugates could kill targeted cancer cells more effectively than nontargeted (control) cells. Most importantly, when illuminated with plasmon-resonant light (532 nm), Dox:nanoconjugates displayed enhanced antitumor efficacy with few side effects. The marked release of Dox from these nanoconjugates in living cells was monitored by increasing fluorescence signals upon light exposure. In vitro studies confirmed that aptamer-functionalized hp-Au NPs can be used as carriers for targeted delivery of drugs with remote control capability by laser irradiation with high spatial/temporal resolution.

Near-infrared light-responsive core-shell nanogels for targeted drug delivery.

A near-infrared light-responsive drug delivery platform based on Au-Ag nanorods (Au-Ag NRs) coated with DNA cross-linked polymeric shells was constructed. DNA complementarity has been applied to develop a polyacrylamide-based sol-gel transition system to encapsulate anticancer drugs into the gel scaffold. The Au-Ag NR-based nanogels can also be readily functionalized with targeting moieties, such as aptamers, for specific recognition of tumor cells. When exposed to NIR irradiation, the photothermal effect of the Au-Ag NRs leads to a rapid rise in the temperature of the surrounding gel, resulting in the fast release of the encapsulated payload with high controllability. In vitro study confirmed that aptamer-functionalized nanogels can be used as drug carriers for targeted drug delivery with remote control capability by NIR light with high spatial/temporal resolution.

Using Aptamer-Conjugated Fluorescence Resonance Energy Transfer Nanoparticles for Multiplexed Cancer Cell Monitoring

To facilitate the selection of effective therapeutic pathways and improve clinical outcomes, sensitive and simultaneous diagnosis of multiple trace biomarkers or cancer cells from complex living samples is particularly critical in the early stages of tumor development. To achieve this, we have combined the selectivity and affinity of aptamers with the spectroscopic advantages of fluorescence resonance energy transfer (FRET) nanoparticles (NPs). This has produced an aptamer-conjugated FRET NP assay that performs simultaneous multiplexed monitoring of cancer cells with the desired degree of sensitivity and selectivity. First, by changing the doping ratio of three different dyes, the FRET-mediated emission signatures could be tuned such that the nanoparticles would exhibit multiple colors upon excitation with a single wavelength. These FRET nanoparticles were then modified by a few aptamers specific for different cancer cell lines, in this case, T-cell leukemia and B-cell lymphoma. As a result, simultaneous and sensitive detection of multiple cancer cell targets was achieved. Therefore, our aptamer-conjugated FRET NPs are highly promising for potential applications in the sensitive monitoring of multiple cancer cells for biomedical research and medical diagnostics.

Colorimetric determination of urinary adenosine using aptamer-modified gold nanoparticles

This paper describes a colorimetric sensing approach for the determination of adenosine triphosphate (ATP) using aptamer-modified gold nanoparticles (Apt-Au NPs). In the absence of the analytes, the color of the Apt-Au NPs solution changed from wine-red to purple as a result of salt-induced aggregation. Binding of the analytes to the Apt-Au NPs induced folding of the aptamers on the Au NP surfaces into four-stranded tetraplex structures (G-quartet) and/or an increase in charge density. As a result, the Apt-Au NPs solution was wine-red in color in the presence of the analytes under high salt conditions. For mixtures of ATP (20.0-100.0nM), Apt-Au NPs (3.0nM), 10.0% poly(ethylene glycol), 0.2microM TOTO-3, 150.0mM NaCl, 15.0mM KCl, and 16.0mM Tris-HCl (pH 7.4), a linear correlation (R(2)=0.99) existed between the ratio of the extinctions of the Apt-Au NPs at 650 and 520nm (Ex(650/520)) and the concentration of ATP. The limit of detection for ATP was 10.0nM. The practicality of this simple, sensitive, specific, and cost-effective approach was demonstrated through the determination of the concentration of adenosine in urine samples.

Aptamer-conjugated and drug-loaded acoustic droplets for ultrasound theranosis

Tumor therapy requires multi-functional treatment strategies with specific targeting of therapeutics to reduce general toxicity and increase efficacy. In this study we fabricated and functionally tested aptamer-conjugated and doxorubicin (DOX)-loaded acoustic droplets comprising cores of liquid perfluoropentane compound and lipid-based shell materials. Conjugation of sgc8c aptamers provided the ability to specifically target CCRF-CEM cells for both imaging and therapy. High-intensity focused ultrasound (HIFU) was introduced to trigger targeted acoustic droplet vaporization (ADV) which resulted in both mechanical cancer cell destruction by inertial cavitation and chemical treatment through localized drug release. HIFU insonation showed a 56.8% decrease in cell viability with aptamer-conjugated droplets, representing a 4.5-fold increase in comparison to non-conjugated droplets. In addition, the fully-vaporized droplets resulted in the highest DOX uptake by cancer cells, compared to non-vaporized or partially vaporized droplets. Optical studies clearly illustrated the transient changes that occurred upon ADV of droplet-targeted CEM cells, and B-mode ultrasound imaging revealed contrast enhancement by ADV in ultrasound images. In conclusion, our fabricated droplets functioned as a hybrid chemical and mechanical strategy for the specific destruction of cancer cells upon ultrasound-mediated ADV, while simultaneously providing ultrasound imaging capability.

A Surface Energy Transfer Nanoruler for Measuring Binding Site Distances on Live Cell Surfaces

Measuring distances at molecular length scales in living systems is a significant challenge. Methods like Förster resonance energy transfer (FRET) have limitations due to short detection distances and strict orientations. Recently, surface energy transfer (SET) has been used in bulk solutions; however, it cannot be applied to living systems. Here, we have developed an SET nanoruler, using aptamer-gold nanoparticle conjugates with different diameters, to monitor the distance between binding sites of a receptor on living cells. The nanoruler can measure separation distances well beyond the detection limit of FRET. Thus, for the first time, we have developed an effective SET nanoruler for live cells with long distance, easy construction, fast detection, and low background. This is also the first time that the distance between the aptamer and antibody binding sites in the membrane protein PTK7 was measured accurately. The SET nanoruler represents the next leap forward to monitor structural components within living cell membranes.

Photo-assisted synthesis of highly fluorescent ZnSe(S) quantum dots in aqueous solution

This paper describes the synthesis of highly water-soluble and fluorescent ZnSe(S)-alloyed quantum dots (QDs). We used zinc perchlorate hexahydrate, sodium hydrogen selenide as precursors and mercaptopropionic acid as stabilizer to synthesize ZnSe QDs in aqueous solution at 160 °C for 9 h. The as-prepared ZnSe QDs possess a quantum yield (QY) of 8.1% and high trapped emission. After UV irradiation using a 100 W Hg–Xe lamp for 0.5 h, ZnSe(S) QDs having a QY of 19.0% are formed from ZnSe QDs. However, aggregation of ZnSe(S) QDs under longer UV irradiation (> 0.5 h) takes place, leading to instability and irreproducibility. To overcome this, additional thiol compounds (mercaptopropionic acid, mercaptosuccinic acid, 11-mercaptoundecanoic acid, and thioglycolic acid) were separately added to ZnSe QD solutions during UV irradiation. UV irradiation and oxygen accelerate the release of S2− from the thiol compounds, leading to the formation of ZnSe(S) QDs. Among the thiol compounds, mercaptosuccinic acid is the most suitable in terms of stability and photoluminescence intensity. We suggest that the size and functional group of the thiol compounds play an important role in determining the optical properties and stability of ZnSe(S) QDs. The as-prepared ZnSe(S) QDs fluoresce strongly (QY up to 44.0%) at 407 nm with a narrow bandwidth (W1/2 < 25 nm) when excited at 325 nm.