Jennifer N. Cha, Ph.D. - US grants

The research objective of this award is to investigate the use of a novel inexpensive printing approach for patterning wafer-level, parallel assemblies of nanoscale electronic materials for biological and chemical sensing from DNA templates. To address features smaller than 20 nm yet still maintain low manufacturing costs, the proposed research will combine a novel soft lithography stamping technique with patterned silicon surfaces to build ordered, spatially defined arrays of DNA and peptide-DNA based scaffolds on flat polydimethylsiloxane. These patterns can then be easily transferred to any receiving flat substrate, such as thermal oxide, metals, or polymers. Limiting the number of nanostructured oxide or silicon substrates needed in the fabrication process will dramatically lower manufacturing costs, while biomolecular scaffolds, such as DNA templates, can address features below 20 nm. The four main research objectives of the proposal are to 1) integrate low-cost, high-throughput inking and stamping methods to generate large area patterned arrays of DNA scaffolds, 2) engineer larger, well-controlled assemblies of DNA scaffolds, 3) incorporate specific molecular recognition motifs to bind nanoscale electronic materials in high yields and with minimal defects, and 4) fabricate fabricating multiplexed parallel, addressable biosensing electrical arrays.

An inexpensive, high throughput method to generate wafer-level arrays of nanoelectronic devices and sensors via benign chemistry and engineering would have an enormous impact on both the electronics and healthcare industries. Despite the wealth of nanoscale materials available, the current limitations of photolithography have prevented the realization of their potential in these applications. The proposed education and research plan will also draw and encourage active participation from teachers and students to help young students learn more about nanoscience and its impact upon society and their everyday lives. In addition, various seminars and interactive demonstrations related to the proposed research will be integrated into existing and new curricula, and open laboratory sessions will be held during university open-house days and on-campus science festivals.

DESCRIPTION (provided by applicant): This proposal describes the synthesis and validation of the first example of a biochemical- responsive ultrasound contrast agent, a potentially groundbreaking advance for deep-tissue imaging of intravascular disease. By improving the power of ultrasound, completion of this multidisciplinary proposal would add value and power to an inexpensive, widespread technique and thus has excellent potential for clinical translation. In addition, this mechanism of activation may be translated to other applications like direct enhancement of sonolytic therapy or site- specific release of therapeutic agents. In this proposal, This proposal describes the synthesis and validation of the first example of a biochemical- responsive ultrasound contrast agent, a potentially groundbreaking advance for deep-tissue imaging of intravascular disease. concern for this country. Acute thrombi may form quickly and develop into worse conditions, potentially leading to patient death if left untreated. Detection is preferably performed using compression ultrasound, a painless, non-invasive outpatient procedure that has good predictive value for symptomatic, proximal vein thrombosis. However, ultrasound cannot differentiate between older clots that should not be treated and acute thrombi that must be treated immediately. The team of PIs has developed a new type of microbubble that remains invisible to ultrasound under normal conditions but becomes detectable only after exposure to certain chemical stimuli. Prior to activation, the microbubble is coated by a rigid, crosslinked polymer shell that dampens the microbubble's response to ultrasound. Treatment with a specific stimulus removes the crosslinks, leaving a flexible, freely oscillating microbubble. The resultant microbubble can be detected via its harmonic oscillations with excellent specificity. Microbubbles will be designed to exhibit sensitivity to thrombin, a protease involved in the coagulation cascade. The microbubbles will be coated with polymer shells crosslinked by DNA aptmaters sensitive to thrombin. Next, their behavior in ultrasound will be examined in real-time on the single bubble level through special equipment previously designed by the PI to create ultrasound pulse programs for optimized signal-to-noise imaging. These thrombin-sensitive ultrasound contrast agents will then be validated in an ex vivo flow model and an in vivo rabbit thrombosis model to detect actual acute thrombi. Thus, a new type of microbubble will be tuned to a medically-relevant system and validated in vivo with excellent chance for translation.

? DESCRIPTION (provided by applicant): The goal of the proposed research is to control the assembly and fabrication of discrete clusters of anisotropic gold nanostructures, UCNPs, and responsive polymer coatings to obtain ideal theranostics for the imaging and treatment of solid tumors. Theranostics represent an exciting area of cancer research because they allow noninvasive tracking of therapeutics into the tumor environment followed by tumor-specific release. Light in the tissue transparency window of 650-1000 nm can serve as an energy source to perform both of these functions. However, most IR-induced imaging and therapy requires light flux only available at superficial sites and thus cannot be applied to many other cancers. The proposed research will utilize the PIs' knowledge of photophysics, nanoscale assembly, and macromolecules for the rational design of NIR-utilizing nanostructures. The proposed theranostic will produce either visible image contrast or drug release, depending on irradiation intensity. The proposed nanostructures will consist of an anisotropic gold nanostructure for NIR maximum absorption, an upconverting nanoparticle (UCNP) for conversion to visible light, and a stimulus-responsive polymer coating to release drug molecules in response to irradiation. A nanostructure able to perform these functions at non-superficial depths requires both a fundamental understanding of the energy transfer processes occurring at the gold surface and the mechanisms by which this energy can be harvested. This will be accomplished through: 1. Synthesis of precise Au-UCNP nanostructures and characterization of photoluminescence. Recent theoretical studies have shown that local field enhancement can enhance upconverted luminescence output by orders of magnitude if the particles are assembled correctly. Biomolecular assembly techniques will be used to specifically position UCNPs at the tips of anisotropic Au nanorods (AuNRs) and nanostars (AuNSs) to maximize energy transfer. These structures will be validated using both single particle and ensemble luminescence measurements. 2. Synthesis of thermally-responsive and photodegradable polymers and evaluation of their responsiveness to NIR irradiation of Au-UCNP clusters. The deposition of IR energy at the surface of the Au nanostructures will be employed to facilitate drug delivery by surface-grafted polymers via either Au surface heating or generation of singlet oxygen. These studies will be performed using polymers capable of conformational switching or oxidative depolymerization, respectively. 3. Synthesis of optimized Au-UCNP-polymer theranostics and validation in in vitro models. The imaging and therapeutic capabilities of the optimized theranostics will be tested against 4T1 cells grown in a 3D Matrigel substrate to mimic both the structure and heterogeneity of the tumor environment.

This research will focus on designing new interfacial coatings for gold nanorods with unprecedented sensitivity, imaging depth, and resolution for photoacoustic imaging (PAI). In typical contrast-enhanced PAI, pulsed laser light is absorbed by a NIR-absorbing nanostructure, leading to local heating, thermal expansion, and the generation of acoustic waves that are detected on the surface of the patient. As compared to all-optical imaging approaches such as fluorescence microscopy or optical coherence tomography, PAI allows for high resolution imaging of optical contrast deep below the tissue surface. Nevertheless, PAI of targeted contrast agents is ultimately limited by the amount of light that reaches the agents, photoacoustic conversion efficiency, and the need to distinguish the agents from background absorbers. The photoacoustic response of contrast agents is often linear with laser fluence, making it difficult to isolate the agents from the background. The proposed contrast agents will utilize novel porous coatings that entrap air, thereby greatly increasing the acoustic response of encapsulated gold nanorods (AuNRs). Hydrophobically-modified porous silica coatings will add two benefits beyond standard photoacoustic contrast agents. First, air has a thermal expansion coefficient two orders of magnitude higher than gold or silica, which should produce a larger acoustic signal at low laser fluences. More importantly, the formation of hydrophobic surfaces with amphiphilic coatings will stabilize the formation of nuclei for cavitation. Because cavitation has a much stronger acoustic response than thermal expansion, it is anticipated that the total acoustic response will be nonlinear with laser fluence. By specifically tuning the surface chemistry of the nanostructures, the amount of energy required to generate cavitation is reduced to the point that nonlinear signal generation can be induced at clinically-relevant laser intensities. This highly multidisciplinary research will be carried out through the PIs? complementary areas of expertise. PI Cha?s group will synthesize the AuNRs coated with nano- and mesoporous silica coatings with the intended NIR absorption profile. PI Goodwin?s group will tune the properties of the silica coatings to provide optimal nucleation of gas bubbles on the contrast agent surface and also design in vitro models for imaging. Finally, PI Murray?s lab will test the photoacoustic response at NIR wavelengths and conduct imaging studies. The ultimate goal will be to build from these initial proof-of-principle studies towards future work in clinical validation and translation via the following aims: (1) Build AuNR-hMSN Nanostructures and Optimize Their Surface Chemistry for Facilitating Cavitation; (2) Characterize Optical Properties and Nonlinear Photoacoustic Response of AuNR- hMSN Nanostructures; (3) Validate PAI Contrast of AuNR-hMSNs in a Scattering In Vitro Spheroid Model.

The goal of the proposed research is to develop Affinity-Mediated Covalent Conjugation (AMCC) reactions that directly modify specific cell types while leaving other cells untouched. The AMCC will target specific receptors on the cell surface and create a permanent covalent link. The ability to directly modify cell membranes with designer chemical functionalities would enable a host of new technologies, such as addressing questions of basic cell biology, increasing therapeutic efficacy, capturing single cells for analysis, to building entirely new self-assembled 3D cell systems from differentiated stem cells. The AMCC reaction starts with an initial, specific non-covalent association, followed by a permanent covalent bond formation induced by irradiation. To promote the non-covalent interaction, the PIs will utilize affibodies that show modest target affinity but are highly robust to modification and incorporation into fusion proteins. In Prior Work (see Research Strategy), the PIs showed that if a specific amino acid on an EGFR-binding affibody was replaced with a benzophenone group, photocrosslinking to EGFR was obtained. This result was validated in both 2D culture and 3D spheroid models, using both UV light and near infrared (NIR) light with upconverting nanoparticles. In this research, the PIs propose to study how the AMCC approach could be utilized to attach specific protein subunits to existing cell receptors using a light-activated, single-step modification. Because affibodies are robust to modification, fusion proteins could be designed with both the photocrosslinkable affibody and an active species (e.g. enzyme, green fluorescent protein (GFP), or streptavidin). Photoconjugation to the cell would create a permanent bond to a cell receptor, followed by long-term expression of this active species on the surface of the cell. As a result, specific cells may be modified at multiple locations simultaneously and orthogonally, leading to numerous advances in areas of model tissue design, in vivo tissue modification, and high throughput cell isolation from complex mixtures. A current barrier to success in this approach is ensuring that the ligated functionalities (e.g., GFP or enzymes) will remain active and intact on the cell membrane over necessary time scales. We hypothesize that covalently binding the ligand to the cell receptor will prolong this lifetime by disguising the conjugated portion as part of the receptor, thereby preventing or hindering receptor-mediated endocytosis and subsequent proteolysis. However, it is not currently known what the long-term fate of the modified receptor will be. Therefore, it is critical to study the effect of photocrosslinking affibodies to cell receptors on the aforementioned biochemical and cellular responses. These questions will be addressed by examining the effects of this approach on cell health, dynamics of the EGFR-affibody complex, and activity of the conjugated segment on the cell surface.

The proposed research plan will develop innovative bioconjugation and DNA-mediated cell assembly strategies for rapid creation of self-assembled multicellular scaffolds with programmable shapes, sizes, and dimensions. Over the past several decades, enormous strides have been made in developing cultured epithelial autografts (CEA) from patient-derived keratinocytes and fibroblasts (i.e. autografts) because they have the smallest chance of immune response and host rejection. However, the new skin tissue must be grown and formed into layers in a lengthy process and the weak mechanical properties of the transplanted skin may result in poor integration with the underlying wound area. In addition, the cells natural adhesion molecules that promote 2D structure are poorly optimized for flexibility and compliance, making it difficult to manipulate onto an underlying substrate or a wound. Patient movement is often inevitable for wounds and burns at certain locations and/or for patients with high burn percentages, which in turn can lead to skin transplant delamination and failure. As a result, the wound sites can become infected and form scar tissue, and in more extreme cases they may lead to intense suffering and even death. The proposed research will develop a DNA mediated bottom-up approach to rapidly generate large-area, close-packed skin cell arrays with predetermined final cell sheet thickness and controllable cell-cell spacing, joined together by reversible, programmable bonds. These cell sheets will boast significantly improved mechanical properties over current state-of-the-art, including robustness, compliance, resistance to tearing, and even self-healing. By combining DNA bound to the cells with complementary DNA freely mobile on the surface, the complementary DNA will act as ?linker? strands to bridge neighboring cells and drive interactions in both 2- and 3D to form close packed cell arrays. Having DNA linkers act as a ?glue? between cells should increase the mechanical stability of the formed tissues and also allow for self-healing. The DNA expressed on the cell membranes can also be used to engineer cell sheets with tunable adhesion forces to an underlying substrate to improve the overall mechanical strength of the final engineered tissue. To conjugate DNA to cell membranes while retaining long-term expression, the PIs have developed a new Affinity-Mediated Covalent Photoconjugation (AMCP) cell functionalization method where the PIs discovered that photocrosslinking protein tags to epidermal growth factor receptor (EGFR) allowed the attached proteins to bypass typical proteolytic pathways and return to the cell membrane. In the proposed research, the PIs will take advantage of the abundance of EGFR on skin cells to attach photocrosslinkable affibody-streptavidin fusion proteins, which in turn will be coupled with biotin-DNA, using the strong biotin-streptavidin interactions to increase ultimate tensile strength of the formed cell sheets. This method will allow tuning of both the number of fusion protein tags per cell and DNA strand density to preserve healthy intracellular signaling and proliferation.