Chemical studies of viral entry mechanisms: I. Hydrophobic protein-lipid interactions during sendai virus membrane fusion. II. Kinetics of bacteriophage λ DNA injection.

Viruses possess very specific methods of targeting and entering cells. These methods would be extremely useful if they could also be applied to drug delivery, but little is known about the molecular mechanisms of the viral entry process. In order to gain further insight into mechanisms of viral entry, chemical and spectroscopic studies in two systems were conducted, examining hydrophobic protein-lipid interactions during Sendai virus membrane fusion, and the kinetics of bacteriophage λ DNA injection.

Sendai virus glycoprotein interactions with target membranes during the early stages of fusion were examined using time-resolved hydrophobic photoaffinity labeling with the lipid-soluble carbene generator3-(trifluoromethyl)-3-(m-^(125 )I] iodophenyl)diazirine (TID). The probe was incorporated in target membranes prior to virus addition and photolysis. During Sendai virus fusion with liposomes composed of cardiolipin (CL) or phosphatidylserine (PS), the viral fusion (F) protein is preferentially labeled at early time points, supporting the hypothesis that hydrophobic interaction of the fusion peptide at the N-terminus of the F_1 subunit with the target membrane is an initiating event in fusion. Correlation of the hydrophobic interactions with independently monitored fusion kinetics further supports this conclusion. Separation of proteins after labeling shows that the F_1 subunit, containing the putative hydrophobic fusion sequence, is exclusively labeled, and that the F_2 subunit does not participate in fusion. Labeling shows temperature and pH dependence consistent with a need for protein conformational mobility and fusion at neutral pH. Higher amounts of labeling during fusion with CL vesicles than during virus-PS vesicle fusion reflects membrane packing regulation of peptide insertion into target membranes. Labeling of the viral hemagglutinin/neuraminidase (HN) at low pH indicates that HN-mediated fusion is triggered by hydrophobic interactions, after titration of acidic amino acids. HN labeling under nonfusogenic conditions reveals that viral binding may involve hydrophobic as well as electrostatic interactions. Controls for diffusional labeling exclude a major contribution from this source. Labeling during reconstituted Sendai virus envelope-liposome fusion shows that functional reconstitution involves protein retention of the ability to undergo hydrophobic interactions.

Examination of Sendai virus fusion with erythrocyte membranes indicates that hydrophobic interactions also trigger fusion between biological membranes, and that HN binding may involve hydrophobic interactions as well. Labeling of the erythrocyte membranes revealed close membrane association of spectrin, which may play a role in regulating membrane fusion. The data show that hydrophobic fusion protein interaction with both artificial and biological membranes is a triggering event in fusion. Correlation of these results with earlier studies of membrane hydration and fusion kinetics provides a more detailed view of the mechanism of fusion.

The kinetics of DNA injection by bacteriophage λ. into liposomes bearing reconstituted receptors were measured using fluorescence spectroscopy. LamB, the bacteriophage receptor, was extracted from bacteria and reconstituted into liposomes by detergent removal dialysis. The DNA binding fluorophore ethidium bromide was encapsulated in the liposomes during dialysis. Enhanced fluorescence of ethidium bromide upon binding to injected DNA was monitored, and showed that injection is a rapid, one-step process. The bimolecular rate law, determined by the method of initial rates, revealed that injection occurs several times faster than indicated by earlier studies employing indirect assays.

It is hoped that these studies will increase the understanding of the mechanisms of virus entry into cells, and to facilitate the development of virus-mimetic drug delivery strategies.

Development of a Cationic Mucic Acid Polymer-Based Nanoparticle siRNA Delivery System

Cancer chemotherapy has advanced from highly toxic drugs to more targeted treatments in the last 70 years. Chapter 1 opens with an introduction to targeted therapy for cancer. The benefits of using a nanoparticle to deliver therapeutics are discussed. We move on to siRNA in particular, and why it would be advantageous as a therapy. Specific to siRNA delivery are some challenges, such as nuclease degradation, quick clearance from circulation, needing to enter cells, and getting to the cytosol. We propose the development of a nanoparticle delivery system to tackle these challenges so that siRNA can be effective.

Chapter 2 of this thesis discusses the synthesis and analysis of a cationic mucic acid polymer (cMAP) which condenses siRNA to form a nanoparticle. Various methods to add polyethylene glycol (PEG) for stabilizing the nanoparticle in physiologic solutions, including using a boronic acid binding to diols on mucic acid, forming a copolymer of cMAP with PEG, and creating a triblock with mPEG on both ends of cMAP. The goal of these various pegylation strategies was to increase the circulation time of the siRNA nanoparticle in the bloodstream to allow more of the nanoparticle to reach tumor tissue by the enhanced permeation and retention effect. We found that the triblock mPEG-cMAP-PEGm polymer condensed siRNA to form very stable 30-40 nm particles that circulated for the longest time – almost 10% of the formulation remained in the bloodstream of mice 1 h after intravenous injection.

Chapter 3 explores the use of an antibody as a targeting agent for nanoparticles. Some antibodies of the IgG1 subtype are able to recruit natural killer cells that effect antibody dependent cellular cytotoxicity (ADCC) to kill the targeted cell to which the antibody is bound. There is evidence that the ADCC effect remains in antibody-drug conjugates, so we wanted to know whether the ADCC effect is preserved when the antibody is bound to a nanoparticle, which is a much larger and complex entity. We utilized antibodies against epidermal growth factor receptor with similar binding and pharmacokinetics, cetuximab and panitumumab, which differ in that cetuximab is an IgG1 and panitumumab is an IgG2 (which does not cause ADCC). Although a natural killer cell culture model showed that gold nanoparticles with a full antibody targeting agent can elicit target cell lysis, we found that this effect was not preserved in vivo. Whether this is due to the antibody not being accessible to immune cells or whether the natural killer cells are inactivated in a tumor xenograft remains unknown. It is possible that using a full antibody still has value if there are immune functions which are altered in a complex in vivo environment that are intact in an in vitro system, so the value of using a full antibody as a targeting agent versus using an antibody fragment or a protein such as transferrin is still open to further exploration.

In chapter 4, nanoparticle targeting and endosomal escape are further discussed with respect to the cMAP nanoparticle system. A diboronic acid entity, which gives an order of magnitude greater binding (than boronic acid) to cMAP due to the vicinal diols in mucic acid, was synthesized, attached to 5kD or 10kD PEG, and conjugated to either transferrin or cetuximab. A histidine was incorporated into the triblock polymer between cMAP and the PEG blocks to allow for siRNA endosomal escape. Nanoparticle size remained 30-40 nm with a slightly negative ca. -3 mV zeta potential with the triblock polymer containing histidine and when targeting agents were added. Greater mRNA knockdown was seen with the endosomal escape mechanism than without. The nanoparticle formulations were able to knock down the targeted mRNA in vitro. Mixed effects suggesting function were seen in vivo.

Chapter 5 summarizes the project and provides an outlook on siRNA delivery as well as targeted combination therapies for the future of personalized medicine in cancer treatment.

Quantum error correction with biased noise

Quantum computing offers powerful new techniques for speeding up the calculation of many classically intractable problems. Quantum algorithms can allow for the efficient simulation of physical systems, with applications to basic research, chemical modeling, and drug discovery; other algorithms have important implications for cryptography and internet security.

At the same time, building a quantum computer is a daunting task, requiring the coherent manipulation of systems with many quantum degrees of freedom while preventing environmental noise from interacting too strongly with the system. Fortunately, we know that, under reasonable assumptions, we can use the techniques of quantum error correction and fault tolerance to achieve an arbitrary reduction in the noise level.

In this thesis, we look at how additional information about the structure of noise, or "noise bias," can improve or alter the performance of techniques in quantum error correction and fault tolerance. In Chapter 2, we explore the possibility of designing certain quantum gates to be extremely robust with respect to errors in their operation. This naturally leads to structured noise where certain gates can be implemented in a protected manner, allowing the user to focus their protection on the noisier unprotected operations.

In Chapter 3, we examine how to tailor error-correcting codes and fault-tolerant quantum circuits in the presence of dephasing biased noise, where dephasing errors are far more common than bit-flip errors. By using an appropriately asymmetric code, we demonstrate the ability to improve the amount of error reduction and decrease the physical resources required for error correction.

In Chapter 4, we analyze a variety of protocols for distilling magic states, which enable universal quantum computation, in the presence of faulty Clifford operations. Here again there is a hierarchy of noise levels, with a fixed error rate for faulty gates, and a second rate for errors in the distilled states which decreases as the states are distilled to better quality. The interplay of of these different rates sets limits on the achievable distillation and how quickly states converge to that limit.

A multi-scale approach to shaping carbon nanotube structures for hollow microneedles

The concept of a carbon nanotube microneedle array is explored in this thesis from multiple perspectives including microneedle fabrication, physical aspects of transdermal delivery, and in vivo transdermal drug delivery experiments. Starting with standard techniques in carbon nanotube (CNT) fabrication, including catalyst patterning and chemical vapor deposition, vertically-aligned carbon nanotubes are utilized as a scaffold to define the shape of the hollow microneedle. Passive, scalable techniques based on capillary action and unique photolithographic methods are utilized to produce a CNT-polymer composite microneedle. Specific examples of CNT-polyimide and CNT-epoxy microneedles are investigated. Further analysis of the transport properties of polymer resins reveals general requirements for applying arbitrary polymers to the fabrication process.

The bottom-up fabrication approach embodied by vertically-aligned carbon nanotubes allows for more direct construction of complex high-aspect ratio features than standard top-down fabrication approaches, making microneedles an ideal application for CNTs. However, current vertically-aligned CNT fabrication techniques only allow for the production of extruded geometries with a constant cross-sectional area, such as cylinders. To rectify this limitation, isotropic oxygen etching is introduced as a novel fabrication technique to create true 3D CNT geometry. Oxygen etching is utilized to create a conical geometry from a cylindrical CNT structure as well as create complex shape transformations in other CNT geometries.

CNT-polymer composite microneedles are anchored onto a common polymer base less than 50 µm thick, which allows for the microneedles to be incorporated into multiple drug delivery platforms, including modified hypodermic syringes and silicone skin patches. Cylindrical microneedles are fabricated with 100 µm outer diameter and height of 200-250 µm with a central cavity, or lumen, diameter of 30 µm to facilitate liquid drug flow. In vitro delivery experiments in swine skin demonstrate the ability of the microneedles to successfully penetrate the skin and deliver aqueous solutions.

An in vivo study was performed to assess the ability of the CNT-polymer microneedles to deliver drugs transdermally. CNT-polymer microneedles are attached to a hand actuated silicone skin patch that holds a liquid reservoir of drugs. Fentanyl, a potent analgesic, was administered to New Zealand White Rabbits through 3 routes of delivery: topical patch, CNT-polymer microneedles, and subcutaneous hypodermic injection. Results demonstrate that the CNT-polymer microneedles have a similar onset of action as the topical patch. CNT-polymer microneedles were also vetted as a painless delivery approach compared to hypodermic injection. Comparative analysis with contemporary microneedle designs demonstrates that the delivery achieved through CNT-polymer microneedles is akin to current hollow microneedle architectures. The inherent advantage of applying a bottom-up fabrication approach alongside similar delivery performance to contemporary microneedle designs demonstrates that the CNT-polymer composite microneedle is a viable architecture in the emerging field of painless transdermal delivery.