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Drug delivery vehicle efficacy is directly influenced by cellular internalization. Therefore, determination and optimization of calcium carbonate nanoparticle (CaCO3) fabrication and administration was investigated. Influencing factors of size distribution, morphology, and surface stabilization were found to be dependent on synthesis temperature regulation, amount of water used in washes post-synthesis, surface functionalization for aggregation control with sulfonyl dodecyl sulfate (SDS), and the duration of time particles in aqueous suspension. It was hypothesized that particles of 60 to 100 nm or less in size are optimal carriers capable of facilitating the use of innate cellular internalization mechanisms. Furthermore, it is hypothesized that nanoparticle uptake and biocompatibility may be cell-cycle dependent. Chemically synthesized CaCO3 nanoparticles were determined to have a positive surface charge and size of 188 nm for calcium chloride dihydrate and 596 nm for calcium chloride hexahydrate derivatives using photon correlation spectroscopy with a dual 30 mW laser. Transmission electron microscopy (TEM) was used to confirm size distribution, and chemical composition was analyzed with x-ray diffraction (XRD). Human fetal osteoblasts (hFOBs) were used in stepped serum deprivation studies to evaluate nanoparticle uptake and to assess potential cytotoxicity at each stage of the cell cycles.
OPTIMIZATION OF THE THERMODYNAMICS AND KINETICS OF BIOPOLYMER-BASED SMART MATERIALSLuke Walls-Smith, Anna Simon, Kevin Plaxco.
University of California, Santa Barbara, Santa Barbara, CA.
The availability of materials that change their properties, such as shape or permeability, in response to specific physical and/or chemical cues would revolutionize technologies ranging from targeted drug delivery to medical diagnostics. An emerging strategy for developing such “smart” materials parallels the gated ion channels used to
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control transport into or out of cells. Specifically, biopolymer networks can be engineered to form and dissociate in response to small molecule or protein cues, allowing for specific gating within nanoscale pores. Significant tradeoffs, however, likely exist for such materials between their thermodynamic stability and their kinetic responsiveness (i.e., stable materials respond only very slowly to their cues), which could limit their utility. Here we explore this tradeoff in a simple, tunable biopolymer network consisting of alternating aptamer (i.e., a molecular cue-binding DNA sequence) and structural support strands that hybridize to form a functional, concatenated chain (a concatamer), which breaks-down in the presence of its molecular target. Specifically, we will explore the extent to which changes in the overlapping base-pairing regions of the concatamer, such as the overlap length and the inclusion of mismatches, affects concatamer stability and opening kinetics. Our experiments take advantage of fluorescence resonance energy transfer, stop-flow, and equilibrium fluorimetry to monitor these effects. Our goal is to discern the design parameters that will lead to optimal material stability and responsiveness.
CELLULAR RESPONSES TO NANOPATTERNED BULK METALLIC GLASSESRebecca Betances1, Themis Kyriakides2, Jagannath Padmanabhan2 University of Puerto Rico at Mayagüez, Mayagüez, PR, 2Yale University, New Haven, CT.
1 Bulk metallic glasses (BMGs) are metallic alloys with an amorphous atomic structure. In addition to metallic properties like high strength and exceptional mechanical durability, they have good wear and high corrosion and resistance, which make them attractive for use as biomaterials for short- and long-term implants. Processing BMGs to fabricate microscale and nanoscale features has also been an active area of research. We have recently reported a novel molding technology to fabricate BMGs with precise surface nanostructures. Although there have been no studies exploring cellular responses to BMGs nanopatterns, nanopatterns on other substrates like polysterene, polydimethylsiloxane, and other polymers have been shown to influence cell behavior. In this study, we fabricated platinum BMGs disks with nanorods of multiple dimensions. Our overall goal was to use nanopatterns to influence cell response and ultimately inhibit biomaterial rejection. Specifically, we investigated the impact of various nanorod aspect ratios on fibroblast cell shape and function. Cells were exposed to nanopatterns for 24 hr and then fixed and stained with rhodamine phalloidin and DAPI to visualize the actin cytoskeleton and nuclei, respectively. Images were collected with a fluorescence microscope. Preliminary image analysis of fibroblasts indicated differences in area, perimeter, circularity, and elongation factor when exposed to different aspect ratios. Statistical analysis was performed by ANOVA (analysis of variance). In ongoing studies, we are investigating the effects of nanorod aspect ratios on inflammatory cells such as neutrophils and macrophages, which will allow us to address the foreign body response.
INVESTIGATING THE USE OF GOLD NANOPARTICLES AS CONTRAST AGENTS FOR CELLULAR TRACKINGElda A. Trevino, Laura Ricles, Laura Suggs.
University of Texas at Austin, Austin, TX.
Heart disease is the leading cause of death in the United States.Current therapies for heart disease, including angioplasty and bypass grafting, are not effective and cannot repair irreversible damage following an ischemic attack. The use of bone marrow derived mesenchymal stem cells (MSCs) has been widely investigated for cellbased therapies to promote vascular regeneration following an ischemic event. Prior research has shown that MSCs can undergo tubulogenesis and exhibit phenotypic characteristics of endothelial cells when implanted in a PEGylated-fibrin gel.Thus, MSCs delivered via a PEGylated-fibrin gel have the ability to reestablish blood flow to ischemic areas through the process of neovascularization. However, the current understanding of MSC participation in neovascularization is limited by the inability to monitor and track the cells following implantation. Using a nanoparticle (NP)-based system and noninvasive, photoacoustic imaging we will be able to simultaneously track MSCs and detect the transfer of contrast agents to nonstem cells in vivo following delivery. Nevertheless, introducing a foreign component into a healthy MSC could compromise its function. Thus, this project is focused on investigating the use of nanoparticles as contrast agents. MSC viability and retention of function (including proliferation, differentiation capability, and tubular network formation) following NP-labeling will be examined using various assays and qualitative analysis. Should the data analysis of the various assays and observations prove statistically insignificant between the control and experimental groups, we could conclude that our nanoparticle system is sufficient to determine the mechanism of MSC neovascularization.
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MODIFYING MECHANICAL AND BIOACTIVE PROPERTIES OF HYDROXYAPATITE SCAFFOLDS VIA
COLLAGEN COATINGSClaudio Macias, Joo Ong.
University of Texas at San Antonio, San Antonio, TX.
Currently, many biomaterials are being explored as components for tissue engineering scaffolds. In the case of bone tissue engineering, these materials must provide a matrix that allows for osteoconduction, biocompatibility, biodegradability, mechanical stability, and load-bearing once implanted in vivo. Much success has been had with scaffolds made of hydroxyapatite (HA), which is a calcium phosphate mineral similar to the biological apatite that is found naturally in bone. Although HA has had success at producing reasonable osteoconductivity and high biocompatibility, these scaffolds are unable to carry high loads. In this study, porous HA scaffolds were created by Engineering replicating a polymer template. Briefly, an HA slurry was applied to a polyutherane template, which was then sintered to a maximum temperature of 1,250 oC. Afterward, the scaffolds will be treated with a 25 µg/ml collagen solution for time periods of 15 min, 30 min, and 1 h after which a brief air current will be applied. Collagen, a structural protein naturally found in bone, will be applied in order to improve the scaffold’s structural integrity and enhance biological activity. Helium pycnometry measurements have been taken before collagen coating and will be taken after coating to confirm the porosity of the collagen-coated scaffolds. Further characterization could be obtained by scanning electron microscopy and Fourier-transformed infrared spectroscopy. Mechanical testing will also be carried out to further determine the mechanical integrity of these scaffolds. Currently, we have successfully produced scaffolds using sponges with 40 pores per inch (ppi) and 60 ppi with final volumes of 42.3 mm3 ± 0.00081 mm3 and 61.2 mm3 ±
0.00067 mm3 respectively.
SPATIAL COCULTURES OF ENDOTHELIAL AND OSTEOPROGENITORS ON HYDROXYAPATITE-COLLAGEN
COMPOSITE SCAFFOLDSRebekah Rodriguez, Laura Gaviria, Teja Guda.
University of Texas at San Antonio, San Antonio, TX Large bone injuries such as those caused by trauma resulting from military or automobile accidents often do not heal of their own accord and require a bone/tissue graft to stimulate regeneration. Vessel in-growth is a crucial factor in determining the success of bone regeneration because it provides the nutrient supply and waste removal pathways from the injured area. The focus of this study will be to understand the importance of the spatial distribution of progenitor cells on the simultaneous development of bone and vessels in a coculture model. The scaffold system consists of a hydroxyapatite (HA) substrate topped with a collagen hydrogel, the 2, natural extracellular matrix components of native bone. Human umbilical vein endothelial cells (HUVEC) and bone marrow stem cells (BMSC) will be used in a coculture. The BMSCs will be seeded on the surface of the HA disk or within the collagen hydrogel and the HVECs will be seeded within the hydrogel or above the hydrogel respectively. Different ratios of BMSC to HUVEC (1:0, 5:1, 1:1, 1:5, and 1:1) will be used to identify the most favorable ratio for the simultaneous promotion of osteogenesis and angiogenesis. Matrix protein analysis and cell staining for bone tissue and blood vessel formation will be performed on days 3, 7, 10, 12, and 14. We hypothesize that osteogenesis and angiogenesis will be more prevalent in the cocultures with more BMSCs and with the BMSCs on the HA substrate with the HUVECs within the collagen gel. (Partially supported by NIGMS MARC-U*STAR GM007717.) FRI-259
PRIME-BOOST WITH CELL SURFACE EXPRESSED CLINICAL ISOLATES OF HIV ENVGerardo Perez, Javier Morales, Sarah M. O’Rourke, Phillip W. Berman.
University of California, Santa Cruz, Santa Cruz, CA.
The goal of HIV research is to produce a vaccine that offers complete protection from the virus.To date, no vaccine has shown this. The RV144 clinical trial was the first successful clinical trial in HIV vaccine research; however the vaccine showed only partial efficacy. The recent identification of broadly neutralizing antibodies in HIV infected individuals has given hope that a vaccine may be possible.Broadly neutralizing antibodies are antibodies capable of blocking infection from a wide variety of clinical isolates.Swarm analysis is a method developed in our lab that identifies pairs of mutation(s) that confer resistance or sensitivity to broadly neutralizing antibodies.It examines the quasispecies (swarm) from an HIV infected individual and examines the changes that the envelope protein undergoes.The changes can sometimes alter sensitivity or resistance to broadly neutralizing antibodies.Our lab has
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identified several pairs of mutation(s) from clinical isolates that confer a change in sensitivity. Our lab will use the clinical isolates identified by swarm analysis as part of a prime-boost immunization protocol. Our project involved increasing the cell surface expression of the envelope protein from the clinical isolates.This involved shortening the cytoplasmic tail of Env by placing a stop codon shortly after the transmembrane domain.This modification has been shown to dramatically alter the level of cell surface expression.The hope is that the higher cell surface expression of Env will lead to a better immune response in the DNA prime-boost protocol.
FUNCTIONALIZATION OF GOLD-NANOROD-BASED IMMUNOSENSORS FOR LABEL FREE BIO-DETECTIONFrancisco Gomez-Rivera1, Yanyan Wang2, Liang Tang2.
University of Puerto Rico at Ponce, Santa Isabel, PR, 2University of Texas at San Antonio, San Antonio, TX.