mRNA-engineered mesenchymal stem cells for targeted delivery of interleukin-10 to sites of inflammation.

Mesenchymal stem cells (MSCs) are promising candidates for cell-based therapy to treat several diseases and are compelling to consider as vehicles for delivery of biological agents. However, MSCs appear to act through a seemingly limited "hit-and-run" mode to quickly exert their therapeutic impact, mediated by several mechanisms, including a potent immunomodulatory secretome. Furthermore, MSC immunomodulatory properties are highly variable and the secretome composition following infusion is uncertain. To determine whether a transiently controlled antiinflammatory MSC secretome could be achieved at target sites of inflammation, we harnessed mRNA transfection to generate MSCs that simultaneously express functional rolling machinery (P-selectin glycoprotein ligand-1 [PSGL-1] and Sialyl-Lewis(x) [SLeX]) to rapidly target inflamed tissues and that express the potent immunosuppressive cytokine interleukin-10 (IL-10), which is not inherently produced by MSCs. Indeed, triple-transfected PSGL-1/SLeX/IL-10 MSCs transiently increased levels of IL-10 in the inflamed ear and showed a superior antiinflammatory effect in vivo, significantly reducing local inflammation following systemic administration. This was dependent on rapid localization of MSCs to the inflamed site. Overall, this study demonstrates that despite the rapid clearance of MSCs in vivo, engineered MSCs can be harnessed via a "hit-and-run" action for the targeted delivery of potent immunomodulatory factors to treat distant sites of inflammation.

Cell-based selection provides novel molecular probes for cancer stem cells.

Cancer stem cells (CSC) represent a malignant subpopulation of cells in hierarchically organized tumors. They constitute a subpopulation of malignant cells within a tumor mass and possess the ability to self-renew giving rise to heterogeneous tumor cell populations with a complex set of differentiated tumor cells. CSC may be the cause of metastasis and therapeutic refractory disease. Because few markers exist to identify and isolate pure CSC, we used cell-based Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX) to create DNA aptamers that can identify novel molecular targets on the surfaces of live CSC. Out of 22 putative DNA sequences, 3 bound to ~90% and 5 bound to ~15% of DU145 prostate cancer cells. The 15% of cells that were positive for the second panel of aptamers expressed high levels of E-cadherin and CD44, had high aldehyde dehydrogenase 1 activity, grew as spheroids under nonadherent culture conditions, and initiated tumors in immune-compromised mice. The discovery of the molecular targets of these aptamers could reveal novel CSC biomarkers.

Bioinspired multivalent DNA network for capture and release of cells.

Capture and isolation of flowing cells and particulates from body fluids has enormous implications in diagnosis, monitoring, and drug testing, yet monovalent adhesion molecules used for this purpose result in inefficient cell capture and difficulty in retrieving the captured cells. Inspired by marine creatures that present long tentacles containing multiple adhesive domains to effectively capture flowing food particulates, we developed a platform approach to capture and isolate cells using a 3D DNA network comprising repeating adhesive aptamer domains that extend over tens of micrometers into the solution. The DNA network was synthesized from a microfluidic surface by rolling circle amplification where critical parameters, including DNA graft density, length, and sequence, could readily be tailored. Using an aptamer that binds to protein tyrosine kinase-7 (PTK7) that is overexpressed on many human cancer cells, we demonstrate that the 3D DNA network significantly enhances the capture efficiency of lymphoblast CCRF-CEM cells over monovalent aptamers and antibodies, yet maintains a high purity of the captured cells. When incorporated in a herringbone microfluidic device, the 3D DNA network not only possessed significantly higher capture efficiency than monovalent aptamers and antibodies, but also outperformed previously reported cell-capture microfluidic devices at high flow rates. This work suggests that 3D DNA networks may have broad implications for detection and isolation of cells and other bioparticles.

Tracking mesenchymal stem cells with iron oxide nanoparticle loaded poly(lactide-co-glycolide) microparticles.

Monitoring the location, distribution and long-term engraftment of administered cells is critical for demonstrating the success of a cell therapy. Among available imaging-based cell tracking tools, magnetic resonance imaging (MRI) is advantageous due to its noninvasiveness, deep penetration, and high spatial resolution. While tracking cells in preclinical models via internalized MRI contrast agents (iron oxide nanoparticles, IO-NPs) is a widely used method, IO-NPs suffer from low iron content per particle, low uptake in nonphagocytotic cell types (e.g., mesenchymal stem cells, MSCs), weak negative contrast, and decreased MRI signal due to cell proliferation and cellular exocytosis. Herein, we demonstrate that internalization of IO-NP (10 nm) loaded biodegradable poly(lactide-co-glycolide) microparticles (IO/PLGA-MPs, 0.4-3 µm) in MSCs enhances MR parameters such as the r(2) relaxivity (5-fold), residence time inside the cells (3-fold) and R(2) signal (2-fold) compared to IO-NPs alone. Intriguingly, in vitro and in vivo experiments demonstrate that internalization of IO/PLGA-MPs in MSCs does not compromise inherent cell properties such as viability, proliferation, migration and their ability to home to sites of inflammation.

Engineered cell homing.

One of the greatest challenges in cell therapy is to minimally invasively deliver a large quantity of viable cells to a tissue of interest with high engraftment efficiency. Low and inefficient homing of systemically delivered mesenchymal stem cells (MSCs), for example, is thought to be a major limitation of existing MSC-based therapeutic approaches, caused predominantly by inadequate expression of cell surface adhesion receptors. Using a platform approach that preserves the MSC phenotype and does not require genetic manipulation, we modified the surface of MSCs with a nanometer-scale polymer construct containing sialyl Lewis(x) (sLe(x)) that is found on the surface of leukocytes and mediates cell rolling within inflamed tissue. The sLe(x) engineered MSCs exhibited a robust rolling response on inflamed endothelium in vivo and homed to inflamed tissue with higher efficiency compared with native MSCs. The modular approach described herein offers a simple method to potentially target any cell type to specific tissues via the circulation.

Cell-surface sensors for real-time probing of cellular environments.

The ability to explore cell signalling and cell-to-cell communication is essential for understanding cell biology and developing effective therapeutics. However, it is not yet possible to monitor the interaction of cells with their environments in real time. Here, we show that a fluorescent sensor attached to a cell membrane can detect signalling molecules in the cellular environment. The sensor is an aptamer (a short length of single-stranded DNA) that binds to platelet-derived growth factor (PDGF) and contains a pair of fluorescent dyes. When bound to PDGF, the aptamer changes conformation and the dyes come closer to each other, producing a signal. The sensor, which is covalently attached to the membranes of mesenchymal stem cells, can quantitatively detect with high spatial and temporal resolution PDGF that is added in cell culture medium or secreted by neighbouring cells. The engineered stem cells retain their ability to find their way to the bone marrow and can be monitored in vivo at the single-cell level using intravital microscopy.

Capturing cancer cells using aptamer-immobilized square capillary channels.

We report a simple square capillary-based cell affinity chromatography device that utilizes a coating of aptamers for selective capture of target cancer cells from a flowing suspension. The device consists of a square capillary with an inner diameter of roughly five cell diameters, connected via Teflon tubing to a syringe. Aptamers are immobilized on the inner surface of the capillary through biotin-avidin chemistry, the extent of which can be controlled by adjusting the aptamer concentration. Introduction of different cell types into separate devices, as well as mixtures of target and non-target cells, demonstrated that aptamer-target cells can be captured in significantly higher concentrations compared to non-target cells. Once optimized, 91.1 ± 3.5% capture efficiency of target leukemia cells was reported, as well as 97.2 ± 2.8% and 83.6 ± 5.8% for two different colon cancer cell lines. In addition, cells captured in the device were imaged, and the square capillary exhibited better optical properties than standard cylindrical capillaries, leading to the detection of leukemia cells in blood samples. Compared to current microfluidic cell affinity devices, this capture device requires no complicated design or fabrication steps. By providing a simple means of detecting and imaging cancer cells in the blood, this work has potential to directly assist clinicians in determining disease prognosis and measuring therapeutic response.

Using azobenzene incorporated DNA aptamers to probe molecular binding interactions.

The rational design of DNA/RNA aptamers for use as molecular probes depends on a clear understanding of their structural elements in relation to target-aptamer binding interactions. We present a simple method to create aptamer probes that can occupy two different structural states. Then, based on the difference in binding affinity between these states, target-aptamer binding interactions can be elucidated. The basis of our two-state system comes from the incorporation of azobenzene within the DNA strand. Azobenzene can be used to photoregulate the melting of DNA-duplex structures. When incorporated into aptamers, the light-regulated conformational change of azobenzene can be used to analyze how aptamer secondary structure is involved in target binding. Azobenzene-modified aptamers showed no change in target selectivity, but showed differences in binding affinity as a function of the number, position, and conformation of azobenzene modifications. Aptamer probes that can change binding affinity on demand may have future uses in targeted drug delivery and photodynamic therapy.

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.

Aptamer-nanoparticle strip biosensor for sensitive detection of cancer cells.

We report an aptamer-nanoparticle strip biosensor (ANSB) for the rapid, specific, sensitive, and low-cost detection of circulating cancer cells. Known for their high specificity and affinity, aptamers were first selected from live cells by the cell-SELEX (systematic evolution of ligands by exponential enrichment) process. When next combined with the unique optical properties of gold nanoparticles (Au-NPs), ANSBs were prepared on a lateral flow device. Ramos cells were used as a model target cell to demonstrate proof of principle. Under optimal conditions, the ANSB was capable of detecting a minimum of 4000 Ramos cells without instrumentation (visual judgment) and 800 Ramos cells with a portable strip reader within 15 min. Importantly, ANSB has successfully detected Ramos cells in human blood, thus providing a rapid, sensitive, and low-cost quantitative tool for the detection of circulating cancer cells. ANSB therefore shows great promise for in-field and point-of-care cancer diagnosis and therapy.

Aptamer-based microfluidic device for enrichment, sorting, and detection of multiple cancer cells.

The ability to diagnose cancer based on the detection of rare cancer cells in blood or other bodily fluids is a significant challenge. To address this challenge, we have developed a microfluidic device that can simultaneously sort, enrich, and then detect multiple types of cancer cells from a complex sample. The device, which is made from poly(dimethylsiloxane) (PDMS), implements cell-affinity chromatography based on the selective cell-capture of immobilized DNA-aptamers and yields a 135-fold enrichment of rare cells in a single run. This enrichment is achieved because the height of the channel is on the order of a cell diameter. The sorted cells grow at the comparable rate as cultured cells and are 96% pure based on flow cytometry determination. Thus, by using our aptamer based device, cell capture is achieved simply and inexpensively, with no sample pretreatment before cell analysis. Enrichment and detection of multiple rare cancer cells can be used to detect cancers at the early stages, diagnose metastatic relapse, stratify patients for therapeutic purposes, monitor response to drugs and therapies, track tumor progression, and gain a deeper understanding of the biology of circulating tumor cells (CTCs).

Nucleic acid aptamers for biosensors and bio-analytical applications.

Oligonucleotides were once considered only functional as molecules for the storage of genetic information. However, the discovery of RNAzymes, and later, DNAzymes, unravelled the innate potential of oligonucleotides in many other biological applications. In the last two decades, these applications have been further expanded through the introduction of Systematic Evolution of Ligands by EXponential enrichment (SELEX) which has generated, by repeated rounds of in vitro selection, a type of molecular probe termed aptamers. Aptamers are oligonucleic acid (or peptide) molecules that can bind to various molecular targets and are viewed as complements to antibodies. Aptamers have found applications in many areas, such as bio-technology, medicine, pharmacology, microbiology, and analytical chemistry, including chromatographic separation and biosensors. In this review, we focus on the use of aptamers in the development of biosensors. Coupled with their ability to bind a variety of targets, the robust nature of oligonucleotides, in terms of synthesis, storage, and wide range of temperature stability and chemical manipulation, makes them highly suitable for biosensor design and engineering. Among the many design strategies, we discuss three general paradigms that have appeared most frequently in the literature: structure-switching, enzyme-based, and aptazyme-based designs.

Single-DNA molecule nanomotor regulated by photons.

We report the design of a single-molecule nanomotor driven by photons. The nanomotor is a DNA hairpin-structured molecule incorporated with azobenzene moieties to facilitate reversible photocontrollable switching. Upon repeated UV-vis irradiation, this nanomotor displayed 40-50% open-close conversion efficiency. This type of nanomotor displays well-regulated responses and can be operated under mild conditions with no output of waste. In contrast to multiple-component DNA nanomachines, the intramolecular interaction in this single-molecule system offers unique concentration-independent motor functionality. Moreover, the hairpin structure of the motor backbone can significantly improve the efficiency of light-to-movement energy conversion. These results suggest that azobenzene-incorporated, hairpin-structured single-molecule DNA nanomotors have promising potential for applications which require highly efficient light-driven molecular motors.

Locked nucleic acid based beacons for surface interaction studies and biosensor development.

DNA sensors and microarrays permit fast, simple, and real-time detection of nucleic acids through the design and use of increasingly sensitive, selective, and robust molecular probes. Specifically, molecular beacons (MBs) have been employed for this purpose; however, their potential in the development of solid-surface-based biosensors has not been fully realized. This is mainly a consequence of the beacon's poor stability because of the hairpin structure once immobilized onto a solid surface, commonly resulting in a low signal enhancement. Here, we report the design of a new MB that overcomes some of the limitations of MBs for surface immobilization. Essentially, this new design adds locked nucleic acid bases (LNAs) to the beacon structure, resulting in a LNA molecular beacon (LMB) with robust stability after surface immobilization. To test the efficacy of LMBs against that of regular molecular beacons (RMBs), the properties of selectivity, sensitivity, thermal stability, hybridization kinetics, and robustness for the detection of target sequences were compared and evaluated. A 25-fold enhancement was achieved for the LMB on surface with detection limits reaching the low nanomolar range. In addition, the LMB-based biosensor was shown to possess better stability, reproducibility, selectivity, and robustness when compared to the RMB. Therefore, as an alternative to conventional DNA and as a prospective tool for use in both DNA microarrays and biosensors, these results demonstrate the potential of the locked nucleic acid bases for nucleic acid design for surface immobilization.

Using photons to manipulate enzyme inhibition by an azobenzene-modified nucleic acid probe.

The ability to inhibit an enzyme in a specific tissue with high spatial resolution combined with a readily available antidote should find many biomedical applications. We have accomplished this by taking advantage of the cis-trans photoisomerization of azobenzene molecules. Specifically, we positioned azobenzene moieties within the DNA sequence complementary to a 15-base-long thrombin aptamer and then linked the azobenzene-modified cDNA to the aptamer by a polyethylene glycol (PEG) linker to make a unimolecular conjugate. During the photoisomerization of azobenzene by visible light, the inhibition of thrombin is disabled because the probe hybridizes with the cDNA in the trans-azobenzene conformation so that the aptamer cannot bind its target thrombin. However, when UV light is applied, melting of the hairpin structure (duplex) is induced via trans-to-cis conversion, thereby changing conformation of the aptamer and making the aptamer free to bind to and inhibit its target thrombin. By using standard clotting assays, we measured the IC(200) of various probe designs in both states and concluded the feasibility of using photon energy to temporally and spatially regulate these enzymatic reactions. Thus, we can report the development of DNA probes in the form of photon-controllable (thrombin) inhibitors, termed PCIs, and we expect that this approach will be highly beneficial in future biomedical and pharmaceutical applications.

Molecular assembly of an aptamer-drug conjugate for targeted drug delivery to tumor cells.

The conjugation of antitumor drugs to targeting reagents such as antibodies is a promising method that can increase the efficacy of chemotherapy and reduce the overall toxicity of the drugs. In this study, we covalently link the antitumor agent doxorubicin (Dox) to the DNA aptamer sgc8c, which was selected by the cell-SELEX method. In doing so, we expected that this sgc8c-Dox conjugate would specifically kill the target CCRF-CEM (T-cell acute lymphoblastic leukemia, T-cell ALL) cells, but with minimal toxicity towards nontarget cells. The results demonstrated that the sgc8c-Dox conjugate possesses many of the properties of the sgc8c aptamer, including high binding affinity (K(d)=2.0+/-0.2 nM) and the capability to be efficiently internalized by target cells. Moreover, due to the specific conjugation method, the acid-labile linkage connecting the sgc8c-Dox conjugate can be cleaved inside the acidic endosomal environment. Cell viability tests demonstrate that the sgc8c-Dox conjugates not only possess potency similar to unconjugated Dox, but also have the required molecular specificity that is lacking in most current targeted drug delivery strategies. Furthermore, we found that nonspecific uptake of membrane-permeable Dox to nontarget cell lines could also be inhibited by linking the drug with the aptamer; thus, the conjugates are selective for cells that express higher amounts of target proteins. Compared to the less effective Dox-immunoconjugates, these sgc8c-Dox conjugates make targeted chemotherapy more feasible with drugs having various potencies. When combined with the large number of recently created DNA aptamers that specifically target a wide variety of cancer cells, this drug-aptoconjugation method will have broad implications for targeted drug delivery.

Enrichment of cancer cells using aptamers immobilized on a microfluidic channel.

This work describes the development and investigation of an aptamer modified microfluidic device that captures rare cells to achieve a rapid assay without pretreatment of cells. To accomplish this, aptamers are first immobilized on the surface of a poly(dimethylsiloxane) microchannel, followed by pumping a mixture of cells through the device. This process permits the use of optical microscopy to measure the cell-surface density from which we calculate the percentage of cells captured as a function of cell and aptamer concentration, flow velocity, and incubation time. This aptamer-based device was demonstrated to capture target cells with >97% purity and >80% efficiency. Since the cell capture assay is completed within minutes and requires no pretreatment of cells, the device promises to play a key role in the early detection and diagnosis of cancer where rare diseased cells can first be enriched and then captured for detection.

Regulation of singlet oxygen generation using single-walled carbon nanotubes.

We have designed a novel photodynamic therapy (PDT) agent using protein binding aptamer, photosensitizer, and single-walled carbon nanotube (SWNT). The PDT is based on covalently linking a photosensitizer with an aptamer then wrapping onto the surface of SWNTs, such that the photosensitizer can only be activated by light upon target binding. We have chosen the human alpha-thrombin aptamer and covalently linked it with Chlorin e6 (Ce6), which is a second generation photosensitizer. Our results showed that SWNTs are great quenchers to singlet oxygen generation (SOG). In the presence of its target, the binding of target thrombin will disturb the DNA interaction with the SWNTs and cause the DNA aptamer to fall off the SWNT surface, resulting in the restoration of SOG. This study validated the potential of our design as a novel PDT agent with regulation by target molecules, enhanced specificity, and efficacy of therapeutic function, which directs the development of photodynamic therapy to be safer and more selective.