Recommendations for Nanomedicine Human Subjects Research Oversight: An Evolutionary Approach for an Emerging Field

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/96363/1/j.1748-720X.2012.00703.x.pd

Mapping Interactions Between Micropatterned Hydrophobic and Hydrophilic Surfaces by Atomic Force Microscopy

115 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1998.Hydrophobic and hydrophilic interactions are not additive and the net interactions between heterogeneous surfaces depend on the relative size of hydrophobic and hydrophilic sites on the surface. Independent on whether the hydrophobic sphere is positioned on top of a hydrophobic or hydrophilic area, repulsions are measured, with the same strength and decay length of the electrostatic interaction. Jump into contact distances and adhesion data, between a 20 $\mu$m hydrophobic sphere and a heterogeneous surface that has equal hydrophobic and hydrophilic areas will average, whereas in all other cases the hydrophobic sphere will clearly differentiate between the different areas, accurately mapping high and low adhesion areas on the surface.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Mapping Interactions Between Micropatterned Hydrophobic and Hydrophilic Surfaces by Atomic Force Microscopy

115 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1998.Hydrophobic and hydrophilic interactions are not additive and the net interactions between heterogeneous surfaces depend on the relative size of hydrophobic and hydrophilic sites on the surface. Independent on whether the hydrophobic sphere is positioned on top of a hydrophobic or hydrophilic area, repulsions are measured, with the same strength and decay length of the electrostatic interaction. Jump into contact distances and adhesion data, between a 20 $\mu$m hydrophobic sphere and a heterogeneous surface that has equal hydrophobic and hydrophilic areas will average, whereas in all other cases the hydrophobic sphere will clearly differentiate between the different areas, accurately mapping high and low adhesion areas on the surface.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Transfection Mechanisms of Polyplexes, Lipoplexes, and Stealth Liposomes in α₅β₁ Integrin Bearing DLD‑1 Colorectal Cancer Cells

Receptor targeted, PEGylated transfection agents can improve stability and delivery specificity of current cationic lipid and polymer based nonviral gene delivery vehicles, but their mode of transfection is poorly understood. We therefore investigated the transfection mechanisms of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/1,2-dioleoyl-<i>sn</i>-glycero-3-phosphoethanolamine (DOPE) lipoplexes, branched polyethylenimine (bPEI) polyplexes, and bPEI encapsulated in either PEGylated (stealth) nontargeted liposomes or PR_b peptide (targeted to α₅β₁ integrin) functionalized stealth liposomes in DLD-1 colorectal cancer cells in vitro with gene expression assays, flow cytometry and confocal microscopy. DOTAP/DOPE and PR_b functionalized stealth liposomes mediated higher gene expression compared to nontargeted stealth liposomes and bPEI. However DOTAP/DOPE was internalized slowly leading to lower levels of DNA uptake. In contrast, despite high internalization of bPEI polyplexes, gene expression levels were low as DNA was unable to escape from the endosomes. Nontargeted stealth liposomes also mediated low gene expression due to low amounts of DNA internalized and slow internalization kinetics. PR_b functionalized stealth liposomes struck an optimal balance among these transfection agents with efficient transfection arising from fast integrin mediated internalization kinetics, high amounts of DNA uptake, and endosomal escape. We found α₅β₁ integrin to be a valuable target for gene delivery and that the caveolar endocytic pathway may offer an advantage to receptor targeted PEGylated transfection agents in DLD-1 cells

Increasing Cancer-Specific Gene Expression by Targeting Overexpressed α₅β₁ Integrin and Upregulated Transcriptional Activity of NF-κB

We developed a modular multifunctional nonviral gene delivery system by targeting the overexpressed cancer surface receptor α₅β₁ integrin and the upregulated transcriptional activity of the cancer resistance mediating transcription factor NF-κB, thereby introducing a new form of transcriptional targeting. NF-κB regulated therapy can improve specificity of gene expression in cancer tissue and also may offset NF-κB mediated cancer resistance. We delivered a luciferase gene under the control of an NF-κB responsive element (pNF-κB-Luc) encapsulated in a PR_b peptide functionalized stealth liposome that specifically targets the α₅β₁ integrin and achieved increased gene expression in DLD-1 colorectal cancer cells compared to BJ-fibroblast healthy cells <i>in vitro</i>. The multitargeted system was also able to differentiate between cancer cells and healthy cells better than either of the individually targeted systems. In addition, we constructed a novel cancer therapeutic plasmid by cloning a highly potent diphtheria toxin fragment A (DTA) expressing gene under the control of an NF-κB responsive element (pNF-κB-DTA). A dose-dependent reduction of cellular protein expression and increased cytotoxicity in cancer cells was seen when transfected with PR_b functionalized stealth liposomes encapsulating the condensed pNF-κB-DTA plasmid. Our therapeutic delivery system specifically eradicated close to 70% of a variety of cancer cells while minimally affecting healthy cells <i>in vitro</i>. Furthermore, the modular nature of the nonviral design allows targeting novel pairs of extracellular receptors and upregulated transcription factors for applications beyond cancer gene therapy

Bioresorbable Polymersomes for Targeted Delivery of Cisplatin

Nontoxic bioresorbable polymersomes have been developed that efficiently and site-selectively tether targeting peptides under mild conditions with no toxic catalysts. The binding and release properties of these polymersomes have been evaluated when targeting DLD-1 human colon cancer cells overexpressing the α₅β₁ integrin. The delivery efficacy to these cells is markedly improved over commonly used RGD targeting peptides by use of an α₅β₁-specific targeting peptide, PR_b. Release profiles in buffered solution from pH 7.4 to 4.5 were evaluated and compared to release after binding to cells, and enzymatic degradation was identified as a major cause of rapid payload release in the cell. Intracellular trafficking and release were imaged <i>via</i> confocal microscopy in live cells and colocalization with organelles was evaluated quantitatively over time. Finally, the anticancer drug cisplatin was encapsulated in the PR_b functionalized polymersomes and the presence of PR_b greatly improved delivery efficacy, with increased cisplatin-induced losses to targeted DLD-1 colon cancer cell viability. When delivered to CACO-2 model human epithelial cells expressing low levels of α₅β₁ integrin, low toxicity was maintained, suggesting that targeting was specific to α₅β₁ overexpressing cells. These results demonstrate that PR_b-functionalized bioresorbable polymersomes may be an attractive route to minimizing the dose-limiting side effects associated with existing approaches to cisplatin chemotherapy

Three-Dimensional Cell Entrapment as a Function of the Weight Percent of Peptide-Amphiphile Hydrogels

The design of scaffolds which mimic the stiffness, nanofiber structure, and biochemistry of the native extracellular matrix (ECM) has been a major objective for the tissue engineering field. Furthermore, mimicking the innate three-dimensional (3D) environment of the ECM has been shown to significantly altered cellular response compared to that of traditional two-dimensional (2D) culture. We report the development of a self-assembling, fibronectin-mimetic, peptide-amphiphile nanofiber scaffold for 3D cell culture. To form such a scaffold, 5 mol % of a bioactive PR_g fibronectin-mimetic peptide-amphiphile was mixed with 95 mol % of a diluent peptide-amphiphile (E2) whose purpose was to neutralize electrostatic interactions, increase the gelation kinetics, and promote cell survival. Atomic force microscopy verified the fibrilar structure of the gels, and the mechanical properties were characterized for various weight percent (wt %) formulations of the 5 mol % PR_g–95 mol % E2 peptide-amphiphile mixture. The 0.5 wt % formulations had an elastic modulus of 429.0 ± 21.3 Pa whereas the 1.0 wt % peptide-amphiphile hydrogels had an elastic modulus of 808.6 ± 38.1 Pa. The presence of entrapped cells in the gels decreased the elastic modulus, and the decrease was a function of cell loading. Although both formulations supported cell proliferation, the 0.5 wt % gels supported significantly greater NIH3T3/GFP fibroblast cell proliferation throughout the gels than the 1.0 wt % gels. However, compared to the 0.5 wt % formulations, the 1.0 wt % hydrogels promoted greater increases in mRNA expression and the production of fibronectin and type IV collagen ECM proteins. This study suggests that this fibronectin-mimetic scaffold holds great promise in the advancement of 3D culture applications and cell therapies