The Rocky Mountain Medical Technologies Conference is pleased to have the following individuals present their medical technology research at the conference.
Present your research
Thiol-ene-based poly(ethylene glycol) (PEG) hydrogels provide a unique functional platform for the sustained and localized delivery of bioactive small molecules like glucocorticoids. As a proof of concept, the synthetic glucocorticoid Dexamethasone (Dex) was conjugated to the N-terminus of a matrix metalloproteinase(MMP)-degradable peptide, which was then easily co-polymerized into PEG gel scaffolds by a thiol-ene polymerization mechanism. The conjugated Dex was locally sequestered until released by cleavage of the MMP-degradable peptide tether triggered by cell-secreted MMPs, and was only available for uptake by local co-encapsulated cells.
A biomaterial that could allow investigation of dynamically changing cell culture substrate elasticity would help elucidate valvular interstitial cell (VIC) activation in aortic stenosis. To this end, our study focuses on culturing VICs on a soft thiol-ene poly(ethylene glycol) (PEG) substrate, stiffening it by adding a PEG diacrylate photodegradable crosslinker, and subsequently softening it by photodegradation. A high degree of control is given by the material, as the stiffening is controlled by altering the addition of crosslinkers and the photodegradable moiety allows control over softening both spatially and temporally. This control will mimic heart valve elasticity in vivo.
Breast cancer is highly heterogeneous, comprised of multiple discrete histological and pathological tumor entities, each associated with different biological features and clinical behaviors. The triple-negative breast cancer (TNBC) has emerged as a particularly malignant subtype, affecting predominantly younger women of color, displaying a rapid progression to metastasis, early relapse, and a higher mortality, and we have very limited effective therapeutic options targeting this subtype. Multiple lines of evidence have established a compelling model supporting a direct role of ETS proteins in mammary tumorigenesis. The epithelial-specific-ETS protein, ESE-1, is of particular relevance to breast cancer. Prior results indicate that TNBCs rely on ESE-1 for transformation, and we hypothesize that delivering short interfering RNA (siRNA) to silence ESE-1 may provide a therapeutic benefit. In order to test this hypothesis, we need a safe, effective and translatable method to target and transfect TNBC cells. In this study, we develop and characterize microbubble-assisted sonoporation for in vitro transfection of suspended TNBC cells and demonstrate the potential promise for future in vivo research. The microbubble delivery vehicle is comprised of cationic, lipid-encapsulated microbubbles coated with short-hairpin RNA (shRNA), shielded from immunologic influence by a lipid-anchored polyethylene-glycol (PEG) brush layer. Transfection occurs through the destructive oscillation of microbubbles triggered by ultrasonic waves (1 MHz), leading to poration of the cell membrane and insertion of nucleic acids into the cell cytoplasm. Results from flow cytometry will be presented that show the effects of microbubble concentration, treatment volume and ultrasound parameters on (1) transient cellular poration, (2) cell viability, (3) transfection of reporter genes, GFP and luciferase, and (4) ESE-1 knockdown. Discussion will include the potential future application of microbubble-assisted sonoporation with RNAi for treating breast cancer.
Most biological cells operate within a small range of temperatures which occur in physiological systems. Large changes in temperature produce irreversible thermal damage in cells. We have developed an exposure system which provides low frequency temperature oscillations to cultured tumor cells. A thermoelectric heater is used to administer sinusoidal temperature variations to monolayers of cells cultured in the wells of specially designed microscopy slides. In our experiments, we have observed a decrease in cell proliferation due to exposure to low frequency temperature pulses. We observed decreases of 20% due to exposure to temperature pulses of +/- 1.25C at 0.05Hz. Variation of frequency on either side of this value led to further decreases in cell proliferation. Fluctuations of temperature within the human body have been seen to be +/-0.5C. Experiments conducted at static values of elevated (38C) or depressed (36C) temperatures did not show changes in cell proliferation. This leads us to believe that the observed effects are due to oscillating temperatures and their effects on biochemical oscillations which result from the interplay of competing rate processes having different time constants or non-linearity in the reaction rates.
Over the last decade, microbubble contrast agents (MCA’s) have frequently been cited as promising vehicles for targeted gene delivery applications. MCA’s are small micron-sized particles comprised of a gas core surrounded by a thin lipid, protein, or polymer shell. Due to the compressible nature of the gas core, MCA’s respond to applied ultrasound energy by volumetrically changing in size, corresponding to the compression and rarefaction of the pressure waves. The response of MCA’s under ultrasound makes them unique vascular agents capable of ultrasound mediated drug release in vivo. By tailoring the surface properties of MCA’s, therapeutic genes or nanoparticle carriers can be loaded onto the microbubble surface to improve nucleic acid payload and systemic circulation of nucleic acid vectors. This technique can be utilized to improve circulation and targeting of highly potent in vitro transfection agents that typically demonstrate poor circulation in vivo, such as the non-viral vector polyethelyneimine (PEI). In this study, we present a simple method of combining PEI, with lipid-microbubbles to create polyplex-microbubble hybrids for ultrasound-guided gene therapy applications. Novel polyplex-microbubbles were synthesized, characterized and evaluated for systemic circulation and tumor transfection. Branched PEI (25 kDa) was modified with polyethylene glycol (PEG; 5 kDa), thiolated and covalently attached to maleimide groups present on lipid-coated microbubbles. The PEI-microbubbles demonstrated increasingly positive surface charge and DNA loading capacity with increasing maleimide content, achieving DNA loading capacities up to 0.005±0.001 pg/µm2, which is ~2.5 fold higher than the commonly used cationic lipid microbubbles used for DNA loading. The circulation persistence of the polyplex-loaded microbubbles was monitored in vivo following bolus injections of microbubbles into the tail vein of CD-1 mice using a high-frequency ultrasound imaging probe (30 MHz) placed over the mouse kidney. The ultrasound contrast time-intensity curves were fit to a novel two-compartment pharmacokinetic model designed to distinguish freely circulating microbubbles from non-circulating adherent ones. The model suggested that PEI loading (without DNA) dramatically reduced free circulation and increased nonspecific adhesion to the vasculature. However, DNA loading to form polyplex-microbubbles increased circulation in the bloodstream and decreased nonspecific adhesion. PEI-microbubbles coupled to a luciferase bioluminescence reporter plasmid DNA were shown to transfect tumors implanted in the mouse kidney. Site-specific delivery was achieved using ultrasound applied over the tumor area following bolus injection of the DNA/PEI-microbubbles. In vivo imaging showed over 10-fold higher bioluminescence from the tumor region compared to untreated tissue. Ex vivo analysis of excised tumors showed greater than 40-fold higher expression in tumor tissue than non-sonicated control (heart) tissue. These results suggest that the polyplex-microbubble platform offers improved control of DNA loading and packaging suitable for ultrasound-guided tissue transfection.
β-cells within pancreatic islets sense glucose levels and secrete insulin via a coordinated cascade of metabolic and electrical events to maintain healthy blood glucose levels. The release of insulin involves membrane depolarization that results in changes in the concentration of intracellular free calcium [Ca2+]i. Here, we investigate the effect of cell aggregate dimensionality on β-cell signaling and electrical coupling by monitoring changes is calcium concentration. Three-dimensional (3D) β-cell aggregates showed higher levels of coordinated calcium activity in response to changes in glucose concentration than did two-dimensional (2D) aggregates. 2D β-cell aggregates were found to be coordinated along lengths of approximately 65 ± 2 µm, which was, in general, much smaller than that diameter of these aggregates, while 3D aggregates were well-correlated at sizes up to 230 µm in diameter. Additionally, in smaller 2D aggregates, a high percentage of cells exhibited synchronous behavior but this coordination percentage decreased with increasing aggregate size. Conversely, 3D β-cell aggregates remained highly coordinated over the same size range. Increased cell coupling between β-cells in the 3D aggregates also contributed to a suppression of spontaneous calcium fluctuations and insulin secretion at low glucose concentrations, mimicking a more native-like state for this configuration of the cells. These findings indicate that cells within 3D β-cell aggregates communicate effectively and, in general, 3D aggregates exhibit higher levels of coordination than cells aggregated in 2D.
Islets of Langerhans (islets) are cell clusters found in the pancreas that consist primarily of β-cells that secrete insulin in response to glucose. Like many tissues, proper islet function is supported by interaction with extra cellular matrix (ECM) proteins. In the native islet environment, cells interact with ECM proteins through contact with both the basement membrane enveloping the islet and a network of capillaries dispersed throughout the cell clusters. While islet cell transplantation can be used to treat individuals with severe cases of type I diabetes, this procedure requires the removal of the vascular network supplying the cell aggregates. As a result, interaction between interior aggregate cells and ECM proteins is eliminated, and islet function declines shortly after transplantation. Previous research has demonstrated the benefit of re-introducing ECM proteins to the surface of cell aggregates through various encapsulation methods. In this work, we hypothesize that incorporating ECM proteins throughout three-dimensional (3-D) β-cell aggregates may further increase the amount of insulin secretion, and the viability of cells within these clusters. Using a hydrogel microarray technique, uniform 3D clusters are formed that contain two distinct ECM proteins, fibronectin and laminin, bound to resin- based micro-particles. By varying the number of micro-particles seeded within the cell clusters, the total amount of protein presented to the aggregates can be systematically adjusted. These uniform, 3D cell-micro-particle aggregates may provide a platform to investigate the effects of local protein presentation on β-cell viability and function.
More details coming soon...
More details coming soon...
More details coming soon...