Emerging Technology and Best Practices Seminar Series

Nanotechnology in Medicine: From Diagnostics to Therapeutics

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POSTER PRESENTATION ABSTRACTS

Wynter Duncanson (Wong Lab)

Title: Tethered architecture improves PLA colloid targeting

Abstract: Polymeric colloidal emulsions are an attractive delivery vehicle as they can efficiently encapsulate drugs, or imaging agents. For site-targeted delivery of these particles, the surface must be functionalized with surface ligands to direct the vehicle to specific receptors at the site of disease; however, a major obstacle in the delivery of site targeted colloids is the reticuloendothelial system (RES). In order to optimize a particulate surface for effective targeting, a combination of ligands and a protein blocking agent like polyethylene glycol (PEG) should be on the surface colloid to lead to the desired in vivo particle to surface interactions. Here we form polylactic acid (PLA) microspheres with the common single emulsion technique and incorporate various mixtures of functionalized and nonfunctionalized polyethylene glycol lipids. Particles are characterized for morphology, surface charge, and composition with field-emission scanning electron microscopy (FESEM), zeta potential measurements, and proton nuclear magnetic resonance ((1)H NMR) spectroscopy, respectively. The surface densities of PEG lipids determined by (1)H NMR and particle size distributions are consistent with scaling theory for adsorption of chains onto a surface. We observe significant binding of liganded PEG-lipid tethers when the molecular weight is greater than the unliganded PEG-lipids for significant binding events. This novel technique used to fabricate these liganded particles combined with the lipid bilayer binding studies provides a platform for systematic optimization of particle binding and prevention of protein adsorption.

Aaron P. Griset (Grinstaff Lab)

Title: Polymeric pH-Responsive Nanospheres for Delivery of Anticancer Drugs

Abstract: There is a pressing need for effective drug delivery systems as many biologically active compounds suffer from deficiencies that prohibit their use in the clinic, such as poor water solubility, rapid clearance from circulation, undesirable biodistribution, severe side effects, and degradation in vivo. Nanotechnology holds an enormous amount of potential for applications in the area of drug delivery, with nanoparticle systems being particularly attractive for this application. We have synthesized polymeric nanospheres which swell in response to the drop in pH which occurs after endocytosis, leading to release of encapsulated compounds and potential disruption of the endosome. With the endosome no longer intact, delivery of the drug to the cytoplasm can then occur. Given these properties, delivery of anticancer agents was selected for our initial studies. The pH-based triggering mechanism allows for high intracellular concentrations of drug to be achieved at the site of delivery and reduce systemic exposure, increasing efficacy and reducing the toxicity commonly caused by conventional chemotherapy. The nanospheres were characterized using UV-Vis spectroscopy, dynamic light scattering, electron microscopy, and cellular uptake assays.

Graham Houtchens (Wong Lab)

Title: Microscale Double Emulsions Using Microfluidic Flow Focusing

Abstract: Microparticles serve an increasingly important function in drug delivery and medical imaging. Particles that are a few microns in size are able to pass through the smallest blood vessels and effectively carry drugs through the body. Hollow particles in this size range have shown promise in ultrasound contrast imaging, in which the echogenicity of the hollow spheres enhance ultrasound signals. In practice, microparticles are currently made by combining the constituents in bulk and aggravating the mixture to generate emulsions. However, this creates an inhomogeneous batch of particles with a wide dispersion of sizes, which can lead to particles which are too large to effectively travel through the body in the case of drug delivery, and which produce varying responses to sound waves when used in ultrasound imaging.

We have created a microfluidic device capable of generating monodisperse double emulsions in the 1-20 microns range. The device takes advantage of flow focusing, which has been established in single emulsion systems as being capable of producing equally sized particles at regular intervals. The current design uses the same flow focusing technique, with an added stream for an additional phase and consequently another emulsion. The design is etched in silicon and bonded with glass, allowing the use of a large range of organic solvents in the device . Preliminary trials of the design in PDMS with water and oil have shown the device as being capable of generating double emulsions, and translation into the silicon device with the appropriate solvents is promising. The use of such a device to create double emulsions will lead to homogeneous batches of particles that are better suited for their ultimate function in drug delivery and medical imaging.

M. Dominika Kulinski (Klapperich Lab)

Title: Sample Preparation Module for Bacterial Lysis and Isolation of DNA from Infected Hematuric Human Urine

Abstract: Cell lysis and nucleic acid purification are essential steps in the construction of fully integrated devices for the detection of bacterial infections in real patient samples at the point of care. Urinary tract infections result in millions of physician visits annually with estimated costs due to infection in the billions of dollars. Most infections are provisionally diagnosed using dipstick tests that detect the presence of leukocyte esterase and nitrite. The dipstick test does not give any information about the infecting organism. For this information, time-consuming culture methods are required. As a result, initial treatments are given based on empiric symptoms, which may lead to the inappropriate use of antibiotics or to leaving a sick patient untreated. An integrated device with the capability of detecting specific strains of infecting organisms will enable clinicians to distinguish between those that are antibiotic resistant and those that are not. Here we use Escherichia coli as a test organism for a point of care thermoplastic microfluidic device designed to take in a urine sample, mix it with lysis buffer, and perform a hybrid chemical/mechanical lysis followed by a solid phase extraction of nucleic acids from the sample. To demonstrate proof-of-concept, we doped human hematuric urine samples with E. coli at concentrations ranging from 10¹ – 10⁵ colony-forming units/mL (CFU/mL) to simulate real patient samples. We then successfully performed on-chip lysis and on-chip DNA extraction. The bacterial DNA was then amplified with real-time PCR establishing efficient lysis and isolation down to 10¹ CFU/mL. We had comparable results to the Qiagen kit at higher concentrations and performed better at recovering DNA at lower concentrations. Infection was confirmed by the presence of more than one gene, and the results were compared to commercial kits for lysis and solid phase extraction. With further development, the sample preparation module described here can be extended to sample preparation on-chip for other bodily fluids.

Lynell R. Skewis (Reinhard Lab)

Title: Spermidine modulated ribonuclease activity probed by RNA plasmon rulers

Abstract: The importance of RNA-protein interactions in biology is tremendous. However, the dynamic details of these interactions are still poorly understood for most systems. Single molecule techniques are needed to access the level of detail required to fully understand such processes as protein translation, gene regulation, and retroviral genome replication.
The plasmon ruler is a dynamic molecular probe based on the distance dependent coupling of noble metal nanoparticle plasmons.^1-3 The signal from plasmon rulers is based on scattering, so they neither blink nor bleach.⁴ Their long lifetime and strong signal intensity enables long observation times and high throughput. Plasmon rulers have been used to measure the dynamics of DNA – protein interactions in highly parallel single molecule in vitro studies.^1-3 We developed geometry to extend their applicability to single stranded RNA systems. We used the RNA plasmon rulers to study the modulation of ribonuclease A (RNase A) activity by the endogenous triamine, spermidine. In this work, we demonstrate the robustness and sensitivity of RNA plasmon rulers. Here we present evidence that the kinetics of RNA cleavage by RNase A is regulated by spermidine induced charge and structure stabilization of the RNA. We show that the time to cleavage by RNase A is reduced with increasing spermidine concentration. In addition to an overall deceleration in cleavage kinetics, discreet subpopulations of cleavage times arise with increased spermidine concentration. We attribute these subpopulations to transient stabilization of secondary structures that prevent cleavage by RNase A. We also present evidence for structural shifts found in intensity trajectories for individual RNA plasmon rulers.

(1) Reinhard, B. M.; Sheikholeslami, S.; Mastroianni, A.; Alivisatos, A. P.; Liphardt, J. Proceedings of the National Academy of Sciences of the United States of America 2007, 104, 2667-2672.
(2) Reinhard, B. M.; Siu, M.; Agarwal, H.; Alivisatos, A. P.; Liphardt, J. Nano Letters 2005, 5, 2246-2252.
(3) Sonnichsen, C.; Reinhard, B. M.; Liphardt, J.; Alivisatos, A. P. Nature Biotechnology 2005, 23, 741-745.
(4) Yguerabide, J.; Yguerabide, E. E. Analytical Biochemistry 1998, 262, 157-176.

Chris Sucato and Teresa Eichinger (Hamilton Lab)

Title: Cytoxicity Studies on HAEC and CASMC with Functionalized Magnetite Nanoparticles

Abstract: The inflammatory response to plaque formation along arterial inner walls is a chronic, slowly progressing complication marked by accumulation of lipid-laden macrophage cells, and polymeric deposits including fibrin, proteoglycans, collagen, elastin, and cellular debris. Atherogenesis in humans is largely asymptomatic until acute events such as plaque rupture and thrombosis lead to loss of circulation to vital tissues of the heart or brain. Atheromatous plaque formation is considered the most important underlying cause of heart attack and stroke, and the leading cause of mortality and morbidity in developed countries, yet remains difficult to detect by practical diagnostic means prior to the onset of life-threatening complications. The objective of this work is the development and evaluation of nanoparticle-based imaging contrast agents capable of direct in-vivo visualization of components of atheromatous plaques, and specifically, differentiation between benign and vulnerable plaques at risk for thrombosis. Such direct visualization may be beneficial for both diagnosis and guidance of therapy. Toward advancing development of MRI contrast agents, our specific aims are: (1) Identification of novel affinity targets inherent to vulnerable plaques, and organic synthesis of monodisperse magnetic nanoparticles with functionalization to bind selectively to plaque targets.

Characterization of novel compounds for morphology and composition, and adhesion properties under flow conditions modeling that of blood vessels. (2) Evaluation of novel image contrast agents by toxicology testing in cell culture (macrophage, endothelial cells). An ultimate goal, after completion of the first two aims, is (3) Evaluation of MRI signal enhancement and differentiation between target and non-target tissues in the rabbit model.

Peng Zhang (Porter Lab)

Title: Ultrasound-induced Thermal Lesion Formation With Phase Shift Emulsion

Abstract: Cancer is a group of diseases in which abnormal cells grow out of control and in the end destroy the whole cellular society. In theory, cancer can be cured by removing cancer cells, which is conventionally attempted to be done by surgery, chemotherapy, radiation therapy, etc. However, the effectiveness of these treatments is limited since either completely removal is hard to achieve or side effects will damage healthy tissues, which sometimes are fatal.

Compared with conventional methods, HIFU (high intensity focused ultrasound), has advantage in non-invasive and few side effect. By focusing ultrasound beam into diseased tissue, most acoustic energy is deposited into the targeted site and converted to heat. When hot enough, a volume of tissue around focal spot would be thermally coagulated and form a lesion.

The heating rate of could be dramatically elevated if bubbles present in focal spot since bubbles will be driven by sound field (cavitation) and increase the absorption of acoustic energy. However, the pre-nucleation sites are omnipresent in most tissues in vivo and make the bubble-enhanced heating unpredictable.

To take advantage of bubble-enhanced heating, we propose to deliver Phase-Shift Emulsion (PSE) into tumor as nucleation site. The presence of PSE is expected to provide site-specified cavitation and more controllable thermal efficiency.

Ankur Shah (Bharti Lab)

Title: Enrichment of serum phosphopeptide by nanoparticles to identify cancer biomarkers

Abstract: The role of kinases in transformation and cancer progression is established, though isolation and characterization of low abundance serum phosphoproteins remains a challenge. The existing technology lacks the specificity, efficiency and flexibility necessary to analyze larger sample volumes. To overcome these problems we have used nanoparticles to enrich phosphopeptide from serum and then analyze by mass spectrometry (MS). Nanoparticles provide a very large surface area, specificity and ability to analyze larger serum samples. We have developed and characterized Fe3O4-SiO2-TiO2 core/shell/shell nanoparticles to specifically enrich phosphopeptides. Preliminary data indicates that particles bind specifically to phosphopeptides, and can be analyzed directly by MALDI-MS. The ongoing work will determine the efficiency of phosphopeptide enrichment from serum.

Xiaojuan Khoo (Grinstaff Lab)

Title: Multifunctional Peptide-based Coatings for the Facile Modification of Device Interfaces

Events that direct the integration and subsequent performance of a medical implant take place largely at the tissue-implant interface. Precise control over the interactions between material surfaces and the surrounding biological environment is therefore critical to the success of biomedical devices. Increasingly, peptides capable of recognizing synthetic materials are being explored for this purpose. We have designed and developed novel functional peptide-based coatings or "Interfacial Biomaterials" (IFBMs) for the rapid modification of device surfaces. Unique linear peptide sequences with specific binding affinity for a target biomaterial were conjugated to a functional moiety with distinct biological activity, affording multifunctional molecules capable of directing and modulating the specific biological and chemical responses at a material surface. 

Peptide sequences with high specific affinity for titanium (Ti) were identified using a combinatorial phage display screening process. A variety of surface characterization techniques were employed to investigate the physical characteristics of these peptides and to quantify their affinity, kinetics and overall performance as a surface coating. We have previously demonstrated the ability of RGD-functionalized coatings to guide endothelialization of Ti surfaces for cardiovascular stenting applications. In our latest studies, peptides functionalized with polyethylene glycol (PEG) chains were characterized for their ability to confer protein and cell resistance onto Ti substrates. Surfaces treated with the PEGylated coating demonstrated excellent resistance to fibronectin adsorption, as well as a greatly reduced the in vitro colonization of S. Aureus bacteria cultures. These PEGylated coatings show promise in terms of resolving two major problems in device applications - (i) non-specific protein adsorption and (ii) bacterial infections.