Liposomes featuring a lipid bilayer surrounding an aqueous core, have been utilized as carriers for drugs and macromolecules since the 1970s. Liposomal drug encapsulation improves the pharmacokinetics, biodistribution, and pharmacodynamics of drugs. This research has culminated in over ten approved liposomal therapies, including Doxil® for the treatment of Kaposi’s sarcoma.

Despite the longevity, further clinical adoption of liposomes and nanomaterials has been limited due to a lack of effective targeting, undesired drug release kinetics, or inadequate drug penetration. In this dissertation, we present strategies to improve nanoparticle drug delivery through synthetic modifications of the particle or alterations of the target tissue. Our initial efforts centered on synthesizing novel lipids to focus the release of drugs or augment nanoparticle stability. Specifically, we detail the synthesis and characterization of sulfolipids with the potential for triggered-release of drug cargo in the tumor microenvironment through the use of sulfatases. These sulfolipids formed highly stable aggregates that were unable to form vesicles. Next, we describe the synthesis and biophysical characteristics of a modified polyethylene glycol (PEG), a key component for extending the circulation of nanomaterials. We anchored PEG to cholesterol to improve the interactions with neighboring lipids. These sterol-anchored PEG molecules exhibited an array of canonical liposome behaviors including the formation of vesicles, encapsulation of drugs, and limited non-specific protein adhesion.

Subsequently, we turned our focus to approaches to make the target tissue more receptive to drug delivery. We communicate procedures to recombinantly purify matrix metalloproteinase-8 (MMP-8), a key enzyme for tissue remodeling. We demonstrate methods to utilize MMPs in drug delivery through the attachment to the surface of liposomes. These procedures enabled the use of MMP-8 to improve the permeability of cartilage endplate tissue through removal of inhibitory extracellular matrix components. Altogether, the work presented provides several strategies to enhance the utility of liposomes through improvements in the release of drug cargo, particle stability and tissue penetration.

The large molecular weight, poly-anionic, and labile nature of small interfering RNA (siRNA) necessitate that these macromolecules are associated with carriers for cellular delivery. Lipid-based particles (LNPs) are very efficient at both cellular internalization and contents delivery; however, the cell has evolved mechanisms to efficiently degrade exogenous macromolecules and prevent the alteration of cellular function by foreign nucleic acids. An important aspect to understand is how cells differentially traffic LNPs so that more efficacious formulations can be designed for therapeutic purposes.

The majority of this dissertations presents a series of investigations into how cells endocytosis and sort LNPs encapsulating siRNA. First, I developed a set of fluorescently tagged endosomal marker proteins for clathrin-, caveolin-, and flotillin-dependent internalization pathways. In addition, I prepared markers for early and late endosomes (Rab5a and Rab7a), recycling endosomes (Rab11a), multivesicular bodies (CD82), and lysosomes (LAMP1). Second, I utilized the technique of ratiometric fluorescence microscopy to measure endosomal pH. Third, I developed an image-processing program built into ImageJ/Fiji to accurately colocalize the lipid, siRNA, and endosomal marker or pH probe signals. I then used these techniques to determine the pathway cells employ to internalize three different LNP formulations comprised of cholesteryl hemisuccinate (CHEMS), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA).

I present evidence that DOTAP LNPs are more potent at delivering siRNA compared to CHEMS or DLinDMA LNPs. I also demonstrate that cells endocytose the LNPs in measurably different ways, specifically, DOTAP exhibits at 0.62 pH unit increase in endosomal pH, pH dependent delivery, 30% colocalization with Rab11a and CD82 during the first 30 minutes of transfection, and a slow accumulation into LAMP1 positive lysosomes over a 4 hour transfection ending at 25% colocalization. In contrast, CHEMS is the least efficient LNP at delivering siRNA and colocalizes with LAMP1 at 25% during a 4 hour transfection. DLinDMA shows a 0.4 pH unit increase in endosomal pH and low colocalization (10 - 15%) with the Rab11a, CD82, and LAMP1 markers tested, despite being present at similar levels as the other formulations, and is presumably trafficked into compartments I did not label. Correlations between cellular trafficking and siRNA delivery efficiency are discussed that may aid in the design of potent LNP formulations.

Finally, I also investigated the human dsRNA transporter SIDT-1, and present evidence that high expression levels are required for cellular siRNA uptake, and the that extracellular domain does not bind directly to dsRNA. In the appendix, I present efforts to use site directed mutagenesis to convert human cytidine deaminase into an enzyme with cytosine deaminase activity.

Non-viral gene therapy vectors have not yet achieved the gene transfer efficiency of viral vectors but continue to be an attractive alternative to viruses due to reduced toxicity and ease of preparation. Successful improvements to non-viral vectors will likely require rationally designed strategies that consider the many barriers that nucleic acid drugs must overcome, including distribution to target organs, internalization into cells, endosomal escape, transport in the cytoplasm, and nuclear uptake. Studies that increase our understanding of these barriers will therefore also represent a significant contribution to the development of new vectors.

This dissertation presents a series of studies related to the intracellular delivery of plasmid DNA to the nucleus. First, a new strategy was introduced to increase the cytoplasmic mobility of plasmid DNA: Biomolecular Adaptor for Retrograde Transport (BART) peptides designed to hijack endogenous molecular motors. Proof-of-concept experiments showed that a BART peptide can bind to a dynein light chain and that BART-bound cargo can be actively transported along microtubules by dynein in a reconstituted system. Second, an assay was developed to quantify the amount of plasmid DNA that is delivered to cell nuclei using various delivery conditions. Application of this assay revealed that cells transfected with Lipofectamine^TM lipoplexes or polyethylenimine polyplexes contained between 75 and 50,000 plasmids/nucleus and that lipoplex-delivered plasmids were more efficiently expressed, based on protein expression per plasmids in the nucleus, than polyplex-delivered plasmids. Third, optimized conditions were established for a robust high throughput assay to screen for small molecule enhancers of transfection. Enhancer molecules discovered with this screen could help to elucidate rate-limiting steps in the transfection process and could also be used as adjuvants in gene therapy formulations. These studies summarize my efforts to design new tools and gauges to improve both the design of and our understanding of gene vectors.

In addition, the final report of a field study in the Dominican Republic to assess the feasibility of needle-free injection technology for that country's immunization program is included at the end of this dissertation. This report presents recommendations on how current devices could be improved and how the technology could be implemented.

In the last decade, the promise of cell-based therapy has come closer to fruition. Although macrophages (MΦ) play important roles in immunity, development and homeostatic tissue maintenance, they have not yet been incorporated into this cell therapy revolution. This reality may change due to a better understanding of MΦ development, plasticity and diverse phenotypical behaviors. As such, there is now the information needed to devise and develop MΦ cell-based therapies.

In this dissertation, I describe the engineering approaches I employed to improve and develop MΦ for cell-based therapies. I first focused on developing a means of generating large numbers of functional MΦ in vitro by the reversible expression of the Hoxb8 transcription factor. I next investigated the biodistribution and survival of transplanted Hoxb8 dependent progenitors (HDP) and macrophages arising from them (HDP-MΦ) in immunocompetent BALB/c and immunodeficient NCG mice. This was enabled by incorporating a number of genetic modifications into the HDP including: constitutively active GMCSFR, luciferase and an inducible IRF8, and by pretreating animals with liposomal clodronate. This combination of factors led to improving the post-transplantation survival of HDP from <1 day to 7 days in BALB/c mice. In addition, I engineered HDP to overexpress iduronidase (IDUA) as a potential cell vector to deliver therapeutic doses of IDUA in the mouse for the treatment of Hurlers Syndrome. These techniques and results described here can guide new approaches for developing novel MΦ-based therapeutics.

Synergistic anti-cancer drug combinations have superior tumor-killing activity, the potential to reduce drug toxicity to healthy tissues, and the ability to minimize the development of drug resistance. Since drug synergism is dependent on the ratio of the combined drugs, synergistic agents must be maintained at fixed ratios to achieve the maximum therapeutic effect in vivo. We hypothesize that targeted liposomes can enhance the efficacy of synergistic anti-cancer drug combinations in vivo by facilitating the intracellular delivery of both drugs at their synergistic ratio and dose. To test this hypothesis, select combinations of anti-cancer drugs were screened in vitro for synergism in KB folate receptor over-expressing cancer cells. The combination activity of drug pairs was evaluated with the median effect method. Irinotecan (IRN) and 5-fluoroorotic acid (FOA) emerged as the most synergistic pair in the screen since they exhibited synergism at a wide range of concentrations and molar ratios. Investigating my hypothesis required that I devise and validate new encapsulation techniques for liposome formulations of IRN, FOA, and the combination of IRN + FOA. Safety studies with the single agents in non-targeted liposomes (NTLs) were conducted in normal mice to identify the maximum tolerated dose. Therapeutic studies in HT29 and C26 tumor mouse models confirmed that the new NTL formulations had anti-cancer potency. We designed folate-targeted liposomes (FTLs) that target the folate receptors on KB tumor cells in vitro and in vivo when as little as 0.03 mol% of the synthesized folate ligand was displayed on the liposome surface. Biodistribution and anti-tumor studies in the KB model with liposome encapsulated doxorubicin confirmed that FTLs deliver chemotherapeutics to the tumor and have anti-tumor activity. The anti-tumor efficacy of IRN + FOA co-delivered in the same FTLs or in a mixture of FTLs was compared to their NTL counterparts in the KB model. Liposomes significantly enhanced the in vivo efficacy of the synergistic combinations. However, folate-targeted liposomes with IRN + FOA did not provide a statistically significant therapeutic advantage over co-delivery of this synergistic pair in non-targeted liposomes.

Lipid-based nanoparticles have long been used to deliver biologically active molecules such as drugs, proteins, peptides, DNA, and siRNA in vivo. Liposomes and lipoplexes alter the biodistribution, pharmacokinetics, and cellular uptake of their encapsulated or associated cargo. This can increase drug efficacy while reducing toxicity, resulting in an increased therapeutic index and better clinical outcomes. Unlike small molecule drugs, which passively diffuse through lipid membranes, nucleic acids and proteins require an active, carrier mediated escape mechanism to reach their site of action. As such, the therapeutic application and drug properties dictate the required biophysical characteristics of the lipid nanoparticle. These carrier properties depend on the structure and biophysical characteristics of the lipids and other components used to formulate them.

This dissertation presents a series of studies related to the development of novel synthetic lipids for use in drug delivery systems. First, we developed a novel class of zwitterionic lipids with head groups containing a cationic amine and anionic carboxylate and ester-linked oleic acid tails. These lipids exhibit structure-dependent, pH-responsive biophysical properties, and may be useful components for next-generation drug delivery systems. Second, we extended the idea of amine/carboxylate containing zwitterionic head groups and synthesized a series of acetate terminated diacyl lipids containing a quaternary amine. These lipids have an inverted headgroup orientation compared to naturally occurring zwitterionic lipids, and show interesting salt-dependent biophysical properties. Third, we synthesized and characterized a focused library of ionizable lysine-based lipids, which contain a lysine head group linked to a long-chain dialkylamine. A focused library was synthesized to determine the impact of hydrophobic fluidity, lipid net charge, and lipid pKa on the biophysical and siRNA transfection characteristics of these lipids. Our results indicate that structural variations significantly impact the biophysical and transfection behavior of this class of lipids.

In summary, we have synthesized several new classes of lipids with biophysical characteristics that may be useful for drug delivery applications. Our results show that slight modifications to lipid structure impacts their biophysical behavior, which in turn dictates their potential utility in drug delivery systems. Further understanding lipid structure-activity relationships will allow for the rational design and engineering of lipids with appropriate properties for specific delivery applications.

Lipids have been known to self-assemble into vesicles for over four decades; a process that has been exploited in fields ranging from drug delivery to the study of cellular mimics. The supramolecular properties of the liposome are primarily determined by the individual lipids which comprise it. Small changes to the architecture or chemistry of the lipid can result in significant alterations to both liposomal properties and the ease of liposome formation, and sometimes result in the formation of an entirely different aggregation phase (micelle, fiber, tube, etc...). Both experimental and theoretical methods have been employed to devise a framework for predicting how a given lipid will assemble in water and how the resulting liposomal will behave. To date, a number of basic principles exist that focus primarily on traditional phospholipids systems. My thesis work has added a number of new lipids that might be used to further test the properties of bilayer formation.

In the first chapter of this thesis, I discuss how lipid structure can affect liposomal properties relevant to drug delivery. In the second and third chapters, I report on novel lipids that explore how alterations to the charge-orientation and type of anion in the lipid headgroup affect the liposome's biophysical characteristics (i.e. transition temperature, permeability, surface potential, interaction with divalent cations) and in one case, even eliminates the lipid's ability to form vesicles under standard conditions. In the fourth and fifth chapters of this thesis, I describe a class of synthetic zwitterionic bolaamphiphiles that is capable of forming small diameter vesicles and discuss the possible configurations of the compounds within the vesicle wall as well as modifications to the bolaamphiphile that may enable them to be used in drug delivery. Each of these four classes of new lipids produced vesicles with unpredicted and interesting properties. They should provide biophysical chemists additional tools to study the physical and chemical properties of membranes and could contribute to the development of new liposome therapeutics.

The membrane proximal region (MPR) of HIV-1 gp41 is a desirable candidate for development of a vaccine that elicits neutralizing antibodies since it is targeted by three of the most potent broadly neutralizing monoclonal antibodies isolated from infected patients. Structural studies of these antibodies and their epitopes suggest that MPR immunogens may be presented in a lipid membrane environment. In this thesis, I report the synthesis and evaluation of MPR lipid-peptide conjugates for membrane presentation of the MPR and elicitation of neutralizing antibodies to HIV-1.

In Chapter 2, I hypothesized that covalent attachment of lipid anchors would enhance the humoral immune response to MPR-derived peptides presented in liposomes. In a comparison of eight lipids conjugated to MPR peptides, cholesterol hemisuccinate promoted the strongest anti-peptide titers in mice. Lipid conjugation was employed to manipulate the biophysical properties and antibody inducing capability of MPR peptides, and this lipopeptide toolkit will be useful for interrogating the role of structure in the immune response to the MPR.

In Chapter 3, MPR lipopeptide immunogens were further utilized to study the cause of weak immune responses to the MPR. We initially hypothesized that the antibody response against the MPR is restricted by immunologic tolerance, but a comparative assessment of antibody responses to MPR lipopeptides in two mouse models of defective immune tolerance indicated that tolerance mechanisms are insufficient to explain the poor antibody responses to the MPR.

I also hypothesized that MPR immunogenicity could be increased through phosphorylation of amino acid side chains. Evaluation of modified MPR immunogens revealed a modification that increased anti-MPR antibodies by an order of magnitude. Importantly, the sera of rabbits immunized with these modified constructs neutralized a laboratory-adapted HIV-1 strain in vitro, providing a foundation for further work on this strategy.

Finally, supplementary studies were conducted involving delivery systems and novel molecular adjuvants for MPR peptide immunogens. In Chapter 4, metal chelation through lipid-anchored multivalent nitrilotriacetic acid was investigated as a non-covalent strategy to attach peptide and protein antigens to liposomal vaccine carriers. Also, in Chapter 5 retinoids were explored as candidate liposomal adjuvants for enhancing the antibody response to MPR immunogens. These studies may lead to improved formulations for delivery of MPR peptide antigens to elicit neutralizing antibodies against HIV-1.

Nature challenges healthy mammals with constant risk of infection by a wide variety of pathogens and with degradation of healthy tissue and control systems. I have been interested in drug delivery and improved delivery of therapeutics primarily to attack pathogens but with incidental value in assessing vital functions of a healthy mammal. My recent work uses protein design to approach such problems. Delivering biomacromolecules remains important both for therapeutics and in discerning and shaping functions of cells.My first thesis project focused on designing a better glomerular filtration rate (GFR) marker to facilitate assessment of renal function and seeking a marker reflecting water distribution in the body, which is relevant to distribution of highly soluble drugs [1]. Tritiated polyethylene glycol (PEG) was known to clear the kidneys effectively and correlated well with a GFR standard assay. A better radioactive label for PEG would allow for easy detection, including imaging. At the time of the project, only one biological conjugation with PEG was reported. Attaching an iodinatable moiety to polyethylene glycol (PEG) polymers of different sizes enabled tracking the compound using radioactive iodine. I made a series of related compounds and studied the pharmacokinetics (PK) and pharmacodynamics (PD) of these in rodents and ultimately in a dog. Using relatively long PEG polymers of molecular weight (MW) 5,000 to 6,000 daltons, the PEG dominated the behavior of the compounds, clearing rapidly through the kidneys. With shorter PEG polymers, the chemistry of the iodinatable group was more significant and the compounds were more likely to clear through the bile, to a degree making them unsuitable for a GFR marker but possibly useful to study liver function. Chapter 1, the published manuscript from my first thesis project, is cited in 20 scientific publications and 51 issued US patents. Variations on the design principles of my project have been used widely in the pharmaceutical industry. During a break in my PhD studies, I improved the formulation of a human Phase-2-ready antifungal drug and designed and organized extensive testing in mice and dogs to show that a sustained-release formulation would overcome PK limitations and made the drug much more potent. This work is discussed briefly in Chapter 2. My second thesis project studied brilacidin activity against 40 fungal isolates from 20 different species, showing useful activity against several important human pathogens [2]. The human and many other innate immune systems includes a variety of peptides known as defensins that weaken or kill a variety of pathogens, including bacteria, fungi, and viruses. Brilacidin is a synthetic defensin-mimic, designed to exhibit the physicochemical properties of defensins as a class. Brilacidin is in human Phase 2 trials. Despite its potential, Brilacidin's efficacy against fungi had not been comprehensively explored until my studies, which showcased its viability as a therapeutic agent against challenging-to-treat fungal infections, thereby offering a beacon of hope for future clinical interventions. Chapter 3, the published manuscript based on this second thesis project, has recently been submitted for review, available online in Preprints.