The Interaction of Targeted and Non-Targeted Nanoparticles with Cells and Model Membranes.

The first component of the thesis examines the binding of a multivalent, folic acid (FA) receptor targeted, generation 5 (G5) PAMAM dendrimer using force pulling spectroscopy. This targeted nanoparticle, when conjugated to the therapeutic methotrexate and then acetylated to form a neutral complex, has been shown to be effective at reducing FA-receptor expressing KB cell tumors. Surface plasmon resonance (SPR) showed a 100,000-fold decrease in the dissociation rate of the G5-FAn from a model FA receptor surface, folate binding protein (FBP), as the number of FAs (n) increased from 1-12. Force pulling spectroscopy was then used to compare the force required to rupture the interaction between the FBP and a single G5-FAn taken from a solution of G5-FAn where n = 0, 4.7, 2.7, and 7.2. A difference in rupture force was measured but due to the heterogeneity of the number of FAs per G5-FAn within a given solution, it was not possible to assign the measured forces to a specific number of FA-FBP interactions. The second component of this thesis examines the interaction of a variety of non-targeted, charged nanoparticles with cells and model membranes. Five polycationic polymers (G5 and G7 PAMAM dendrimer, branched polyethylenimine (PEI), poly-L-lysine (PLL) and diethylaminoethyl-dextran (DEAE-DEX)) were shown to induce nanoscale hole formation in cells as measured by enzyme and dye diffusion assays, as well as in dimyristoylphosphatidylcholine (DMPC) supported lipid bilayers (SLB) as measured by atomic force microscopy (AFM). In contrast, neutral polymers polyethylene glycol and polyvinyl alcohol did not induce nanoscale hole formation in cells or DMPC SLBs. This suggests that a possible mechanism for polycationic polymer internalization and/or nanoparticle-induced cytotoxicity of cells is through nanoscale hole formation. The interaction between SLBs and a variety of other charged nanoparticles (MSI-78, Au-NH2, G3-NH2 PAMAM dendron, and silica-NH2 were also investigated using the AFM-SLB assay. The general trend taken from the AFM-SLB studies is that surface area of the polycationic nanoparticles is the largest contributing factor to membrane disruption. In addition, micelles of charged detergents cetyl trimethylammonium bromide bromide (CTAB) and sodium dodecyl sulfate (SDS) were also shown to induce hole formation in SLBs.Ph.D.ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/58463/1/lerouepr_1.pd

A Rapid Cost Modeling Tool for Evaluating and Improving Public Health Supply Chain Designs.

Effective and efficient health supply chains play a vital role in achieving health outcomes by ensuring supplies are available for people to access quality health services. However, supplying health commodities to service delivery points is complex and costly in many low- and middle-income countries. Thus, governments and partner organizations are often interested in understanding how to design their health supply chains more cost efficiently.Several modeling tools exist in the public and private market that can help assess supply chain efficiency and identify supply chain design improvements. These tools are generally capable of providing users with very precise cost estimates, but they often use proprietary software and require detailed data inputs. This can result in a somewhat lengthy and expensive analysis process, which may be prohibitive for many decision makers, especially in the early stages of a supply chain design process. For many use cases, such as advocacy, informing workshop and technical meetings, and narrowing down initial design options, decision makers may often be willing to trade some detail and accuracy in exchange for quicker and lower-cost analysis results. To our knowledge, there are no publicly available tools focused on generating quick, high-level estimates of the cost and efficiency of different supply chain designs.To address this gap, we designed and tested an Excel-based Rapid Supply Chain Modeling (RSCM) Tool. Our assessment indicated that, despite requiring significantly less data, the RSCM Tool can generate cost estimates that are similar to other common analysis and modeling methods. Furthermore, to better understand how the RSCM Tool aligns with real-world processes and decision-making timelines, we used it to inform an ongoing immunization supply chain redesign in Angola. For the use cases described above we believe that the RSCM Tool addresses an important need for quicker and less expensive ways to identify more cost-efficient supply chain designs

A model for estimating costs and benefits of new vaccine technologies from the perspective of both buyers and sellers.

Although vaccination is widely considered one of the most cost-effective health interventions available, global coverage rates for many vaccines remain lower than necessary for disease elimination and eradication. New vaccine technologies can play an important role in addressing barriers to vaccination and increasing coverage rates. To identify and prioritize vaccine technology investments, decision makers must be able to compare the overall costs and benefits of each investment option. While these data points may exist, they are often confined to silos. Decision makers would benefit from a model that synthesizes this broad range of data and provides clear and actionable information. To facilitate vaccine investment, purchasing and deployment decisions, we developed a systematic and transparent cost-benefit model that estimates the value and risk of a given investment scenario from the perspective of both "buyers" (e.g., global donors, country governments) and "sellers" (e.g., developers, manufacturers) of vaccines. This model, which can be used to evaluate scenarios related to a single vaccine presentation or a portfolio of vaccine presentations, leverages our published approach for estimating the impact of improved vaccine technologies on vaccination coverage rates. This article presents a description of the model and provides an illustrative example application to a portfolio of measles-rubella vaccine technologies currently under development. Although the model is generally applicable to organizations involved in vaccine investment, manufacturing or purchasing, we believe it may be particularly useful to those engaged in vaccine markets that rely strongly on funding from institutional donors

Evaluating Binding Avidities of Populations of Heterogeneous Multivalent Ligand-Functionalized Nanoparticles

Ligand-functionalized, multivalent nanoparticles have been extensively studied as targeted carriers in biomedical applications for drug delivery and imaging. The chemical synthesis method used, however, generates nanoparticles that are heterogeneous with respect to the number of ligands on each nanoparticle. This article examines the role this heterogeneity in ligand number plays in multivalent interactions between nanoparticle ligands and targeted receptors. We designed and synthesized a model heterogeneous multivalent nanoparticle system and developed a unique kinetic analysis to quantify the avidity interactions. This system used mono-dispersed poly(amidoamine) (PAMAM) dendrimers that were then chemically functionalized with ssDNA oligonucleotides as to yield the heterogeneous nanoparticle platform (ligand valencies <i>n</i> = 1.7, 3.1, 6), and employed complementary oligonucleotides as targeted receptors on a surface plasmon resonance (SPR) biosensor to evaluate the multivalent binding of the nanoparticle population. Kinetic analysis of both parallel initial rate and dual-Langmuir analyses of SPR binding curves was performed to assess avidity distributions. We found that batches of multivalent nanoparticles contain both fast- and slow-dissociation subpopulations, which can be characterized as having “weak” and “strong” surface interactions (“binding”), respectively. Furthermore, we found that the proportion of “strong” binders increased as a function of the mean oligonucleotide valence of the nanoparticle population. These analyses allowed an assessment of how avidity distributions are modulated by the number of functionalized ligands and suggested that there are threshold valences that differentiated fast- and slow-dissociation nanoparticles

Ligand Characteristics Important to Avidity Interactions of Multivalent Nanoparticles

Multivalent interactions involve the engagement of multiple ligand–receptor pairs and are important in synthetic biology as design paradigms for targeted nanoparticles (NPs). However, little is known about the specific ligand parameters important to multivalent interactions. We employed a series of oligonucleotides as ligands conjugated to dendrimers as nanoparticles, and used complementary oligonucleotides on a functionalized SPR surface to measure binding. We compared the effect of ligand affinity to ligand number on the avidity characteristics of functionalized NPs. Changing the ligand affinity, either by changing the temperature of the system or by substitution noncomplementary base pairs into the oligonucleotides, had little effect on multivalent interaction; the overall avidity, number of ligands required for avidity per particle, and the number of particles showing avidity did not significantly change. We then made NP conjugates with the same oligonucleotide using an efficient copper-free click chemistry that resulted in essentially all of the NPs in the population exceeding the threshold ligand value. The particles exceeding the threshold ligand number again demonstrated high avidity interactions. This work validates the concept of a threshold ligand valence and suggests that the number of ligands per nanoparticle is the defining factor in achieving high avidity interactions

Specific and Cooperative Interactions between Oximes and PAMAM Dendrimers As Demonstrated by ¹H NMR Study

Oximes are important in the treatment of organophosphate (OP) poisoning, but have limited biological half-lives. Complexing these drugs with a macromolecule, such as a dendrimer, could improve their pharmacokinetics. The present study investigates the intermolecular interactions that drive the complexation of oxime-based drug molecules with fifth generation poly­(amidoamine) (PAMAM) dendrimers. We performed steady-state binding studies of two molecules, pralidoxime and obidoxime, employing multiple NMR methods, including 1D titration, ¹H–¹H 2D spectroscopy (COSY, NOESY), and ¹H diffusion-ordered spectroscopy (DOSY). Several important insights were gained in understanding the host–guest interactions occurring between the drug molecules and the polymer. First, the guest molecules bind to the dendrimer macromolecule through a specific interaction rather than through random, hydrophobic encapsulation. Second, this specificity is driven primarily by the electrostatic or H-bond interaction of the oxime at a dendrimer amine site. Also, the average strength for each drug and dendrimer interaction is affected by the surface modification of the polymer. Third, individual binding events between oximes and a dendrimer have a negative cooperative effect on subsequent oxime binding. In summary, this report provides a novel perspective important for designing host systems for drug delivery

Characterization of Folic Acid and Poly(amidoamine) Dendrimer Interactions with Folate Binding Protein: A Force-Pulling Study

Atomic force microscopy force-pulling experiments have been used to measure the binding forces between folic acid (FA) conjugated poly­(amidoamine) (PAMAM) dendrimers and folate binding protein (FBP). The generation 5 (G5) PAMAM conjugates contained an average of 2.7, 4.7, and 7.2 FA per dendrimer. The most probable rupture force was measured to be 83, 201, and 189 pN for G5-FA_2.7, G5-FA_4.7, and G5-FA_7.2, respectively. Folic acid blocking experiments for G5-FA_7.2 reduced the frequency of successful binding events and increased the magnitude of the average rupture force to 274 pN. The force data are interpreted as arising from a network of van der Waals and electrostatic interactions that form between FBP and G5 PAMAM dendrimer, resulting in a binding strength far greater than that expected for an interaction between FA and FBP alone