Since the discovery of liposomes in the 1960s, lipid-based materials have played a significant role in the development of nanotechnology. Present applications of liposomes range from cosmetics, food, drug formulation, adjuvant carriers, to gene therapy. However, specifically within drug delivery applications, there are major deficiencies concerning the stability of agent carriers with respect to drug loading, controlled release, overcoming biological barriers, and efficient active targeting. Consequently, there is a serious need for the development of stable materials towards improving the delivery of therapeutics. Herein, we present a strategy for the preparation of robust and stable lipid materials capable of encapsulating small molecules and assembling into well-defined structures as a potential therapeutic delivery system. These systems aim to address the key issues of high permeability of membranes and the subsequent release of encapsulated cargo. Taking inspiration from Archaea organisms, in their ability to withstand extreme environments, archaea-type lipids were synthesized towards generating archaeosomes as an alternative and improved approach for the encapsulation and release of small molecule cargo. These biomimetic materials may potentially serve as stabilizing agents, drug storage, and drug carriers in therapeutics.

Abstract

Recognition and cellular uptake by macrophages and ensuing clearance by the mononuclear phagocyte system (MPS) stands as a significant biological barrier for nanoparticle (NP) therapeutics. Here, we report a readily deployable method to prepare peptide brush polymers (PBPs) by ROMP, which are used to examine the effects of charge, size, and composition on macrophage uptake using RAW 264.7 cells. In our long-standing interest of developing enzyme-responsive systems, the polymers were encoded with multiple copies of a peptide substrate (GPLGLAG) for proteolytic enzymes specific to diseased or inflamed tissues. The incorporation of a permanent cation (quaternary amine) into the peptide sequence further permitted water-solubility for homopolymers and facilitated formulation of discrete, uniform NPs formulated from block copolymers, which were also investigated. An enzyme-activatable cell uptake strategy is also reported by way of a zwitterionic-to-cationic charge switch using a model system.

Similarly, enzyme hydrolysis of N-terminus conjugated peptide brush polymers (NPBPs) was shown to promote the assembly of discrete, nanoscale cylindrical micelles, upon exposure of carboxylates on the polymer after proteolytic cleavage. In contrast, C-terminus conjugated PBPs (CPBPs), which are structural isomers of NPBPs and expose protonated amines when subjected to proteolysis, remain as fully dispersed polymers, as indicated by TEM. As an initial assessment of their behavior in vivo, intravenous (IV) and intratumoral (IT) injections of N- and CPBP analogues were performed in a metastatic and solid tumor model known to have elevated levels of matrix metalloproteases capable of cleaving peptide substrates on NPBP and CPBP. These systems incorporated a Gd-DOTA label for tracking as well as assessment of relaxivity properties by MRI.

In a separate application, peptide polymer amphiphiles (PPAs) were used to decorate liquid crystal (LC) microdroplets comprised of the nematic 5CB, in order to provide the basis for multi-length scale response systems. The PPAs were designed with biphenyl, mesogen-like side-chains to promote assembly at the LC microdroplet interfaces and peptidic moieties (GPLGLAG) capable of enzymatic processing by thermolysin. Enzymatic cleavage of the PPAs was shown to trigger changes in PPA-surfactant complexes formed at the interface of the LC, thus giving rise to an easily measured optical response. To improve the design of PPAs for triggering LC interfaces, a library of norbornyl mesogens was synthesized to study the nature of homopolymer and copolymer anchoring at the LC/aqueous interface. From these collective experiments, a set of design principles emerged that was then used to design an optimized PPA copolymer.

Ring opening metathesis polymerization (ROMP) was used to generate a variety of self-assembled nanostructures, including purely synthetic and bio-hybrid materials. The properties of polynorbornene amphiphilic block copolymer structures and their relationship to resulting morphology was explored, paving the way for nanoparticle design at the structural and processing level. In the context of bio-synthetic polymer amphiphiles, hydrophobic ROMP polymers were attached to hydrophilic DNA strands to produce self-assembly micelles on the order of 20 nm in diameter. Herein, we explored the self-assembly properties, stability, and applications of these assemblies in pristine conditions and cellular environments.

Morphology plays an important yet poorly understood role in dictating how nanomaterials interact with cells and tissues. Uncovering this relationship relies on working out how to control particle morphology in the first place. We prepared purely synthetic amphiphiles using ROMP to prepare aqueous phase diagrams of block copolymer assemblies. By preparing polymers with varying properties, such as block lengths, block identity, and block ratios, the relationship between polymer structure and the resulting self-assembly nanostructure could be observed under certain conditions. Furthermore, by manipulating the assembly conditions of these polymers, we have shown that multiple stable morphologies can be generated from the same block copolymer starting material. This represents the first study of its kind for ROMP-derived amphiphilic assemblies, which exhibit variations in self-assembly dynamics compared to more traditional block copolymers.

Adding a level of complexity to our block copolymer system, we next explored more therapeutically relevant systems by conjugating DNA to a hydrophobic ROMP homopolymer and assembling them into DNA-displaying micelles. We determined the stability of the DNA on the micelle surface by treating the structures with various nucleases and human serum. The stability of the DNA on the micelle corona resisted degradation by nucleases in some circumstances, but not all, relative to the free DNA control, highlighting the importance of careful design of the amphiphile for a given application.

After determining the stability of DNA polymer assemblies (DPAs), antisense DPAs were designed against a known therapeutic target in cancer cells, MDR1. Importantly, these materials were designed with sequences not containing chemical modifications, such as locked nucleic acids or other backbone alterations. After treating MDR1-dependent doxorubicin resistant cells with the antisense micelles, sensitivity to the chemotherapeutic could be restored to near-parental cell line IC50 values.

Despite many desirable properties nanoparticles have in therapeutic applications, a major bottleneck in their development is the fact that very little is known about how they interact with cells and tissues. Next, a high throughput, whole-genome approach to elucidate the pathways responsible for nanomaterial uptake by cells was developed and tested using DPAs as a proof-of-concept. Using Genome-wide CRISPR Knock Out (GeCKO), a population of cells representing knockouts across the entire genetic spectrum was tested against uptake of cyanine 5 labeled DPAs. Using this approach, we have identified the transmembrane protein SLC18B1, among a handful of other proteins, as candidates for mediating uptake; a previously unknown interaction by DNA-displaying nanomaterials with cell surfaces. By expanding this technique to other categories of nanoparticle medicines with different structures and surface modifications the generation of new design rules for nanomaterial therapeutics may be prepared to help researchers avoid off-target accumulation and advance many more nanotherapies to the clinic.

In recent history, application of macromolecular biomaterials for therapeutic tissue engineering after severe injury has presented a promising strategy to for positive remodeling. Herein, the design of structurally dynamic vehicles for noninvasive delivery and stimuli-responsive self-assembly is investigated. These proof-of-concept platforms were explored for the purpose of expanding the utility of insert biomaterial platforms for practical clinical applications. Ultimately, we envision formulation with biotherapeutics will provide a smart mechanism for treating inflammatory diseases.

First, nanomaterials were designed to respond to matrix metalloproteinases (MMPs) and acutely upregulated at the site of myocardial infarction (MI). Ring opening metathesis polymerization (ROMP) of a brush peptide-polymer amphiphile was explored to generate intravenous (IV) injectable 20 nm nanoparticles that respond to MMP-2/9 through self-assembly into micron-scale aggregate scaffolds. The in vivo targeting efficiency, biodistribution, and toxicity of such constructs was investigated. These materials demonstrate targeted accumulation, nonimmunogenicity, and prolonged retention up to 30 days at the site of MI.

Cyclic peptide progelators were designed as structurally dynamic and biodegradable materials for targeted hydrogelation at the site of myocardial infarction. Self-assembling peptides (SAPs), which stack as β-sheets via intermolecular electrostatic, hydrophobic, and in some cases, π-π interactions were sterically constrained through macrocyclization. This modified construct was sufficient to prevent gelation and lower sample viscosity, enabling delivery via minimally invasive catheter delivery. These peptides respond to MMP-2/9 and elastase upregulated at the MI, as well as to the robust nonspecific protease, thermolysin, through self-assembly into viscoelastic hydrogels. Hemocompatibility and in vivo response of one such construct demonstrated the utility of this platform design.

Next, the strategy for generation of catheter-injectable SAPs was expanded into pH-sensitive low viscosity progelators. The pH-responsiveness of the KLD-12 SAP was tuned via temporary modification with substituted maleamic acids to afford polyanionic peptide solutions. The deprotection of these “caps” recapitulates the zwitterionic KLD-12 SAP under physiologically relevant levels of tissue acidosis (pH 6.8). Progelators were catheter injectable and hemocompatible, whereas acid-treated samples produce solid gels that resist material spreading and are reheable. Finally, this platform exhibited acid-induced drug encapsulation, a useful property for targeted therapeutics without complex synthetic manipulation.

Finally, we turned back to nondegradable ROMP co-polymers for studying architectural influence on biomimetic hydrogel assembly. Three amino acid-functionalized monomers were designed to mimic the alternating KLD-12 SAP cationic, neutral hydrophobic, and anionic charges, respectively, that imbue self-assembling behavior. Modification to the degree of polymerization (DP), number of blocks, block lengths, and hydrophobicicity, were explored to investigate their propensity for hydrogel self-assembly and stability. This investigation provides general insight into physical hydrogel polymer design as well as proteolytic-resistant biomaterials for potential applications as long-term implantable hydrogels.

A strategy for the synthesis of peptides displayed as high density brush polymers is detailed. With this strategy, numerous high density brush polymers displaying peptides were prepared and studied for their ability to protect the peptides from proteolysis. Furthermore, strategies to enable cellular internalization of peptide high density brush polymers were investigated. Finally, peptides were incorporated into polymeric nanomaterials and used for tumor and ischemic tissue targeting.

Peptide high density brush polymers were prepared using graft through ring opening metathesis polymerization (ROMP) to investigate which amino acids could be incorporated into the peptide and polymerized successfully using a ruthenium-based initiator. It was found that only cysteine was unable to be polymerized, whereas lysine and methionine showed difficulty. Strategies to incorporate these problematic amino acids include maintaining protecting groups on the side chains, and incorporating a longer linker between the peptide and the polymerizable moiety.

With this strategy in hand, a variety of high density brush polymers bearing peptides that are enzyme substrates were prepared. These peptides were investigated for their ability to be proteolytically degraded by thrombin, trypsin and pepsin. It was found that in general, peptides displayed as high density brush polymers were protected from proteolysis compared to their monomeric counterparts. When the recognition sequence was spaced closer to the norbornene backbone, the peptide was more protected from proteolysis. Furthermore, peptides were more protected from proteolysis when polymerized to higher molecular weights. With these data, it was believed that polymerizing peptides into high density brushes could protect peptides from proteolysis, a major problem in peptide therapeutics.

A second problem in peptide therapeutics is the inability to internalize into cells. Using high density brushed polymers displaying cationic peptides, the polymeric material is able to internalize into cells in a concentration and degree of polymerization dependent process. Furthermore, a therapeutic peptide was displayed as a high density brush polymer and internalized into cells, and maintained its bioactivity of inducing mitochondrial dependent apoptosis.

Matrix metalloproteinases (MMPs) are enzymes that are upregulated in many diseased states such as hind limb ischemia and cancer. A peptide polymer amphiphile that displayed an MMP substrate as the hydrophilic block, and was labeled with a near infrared dye was prepared. Peptide polymer amphiphiles are able to self-assemble into micellar nanoparticles. It was previously shown that when the nanomaterial is incubated with MMPs, the enzyme cleaves off the peptide, resulting in a change in polymer amphiphilicity inducing an aggregation event. Near infrared labeled polymeric materials were intravenously administered to animal models of cancer and peripheral artery disease, two diseases where MMPs are upregulated, and the particles were able to accumulate into the diseased tissue, albeit not significantly over the non-responsive control. It was found, however, that when incorporating a zwitterionic dye to the polymeric material, there was a significant decrease in off target accumulation.

Nanoparticles are the hallmark of nanomedicine, proving clinically relevant for the delivery of therapeutic and diagnostic drugs. Nanomaterials are highly versatile, with the ability for scientists to tailor composition and function to suit the desired application. Herein, two types of nanocarriers are investigated in the context of magnetic resonance imaging contrast agents: polymeric nanoparticles and protein carriers.

A key research focus in applying nanoparticles for drug delivery is in understanding physicochemical properties affecting in vivo fate. Shape or morphology of a particle is a vastly underutilized property in the design of nanoparticles, and is difficult to predictably control in the context of polymeric nanoparticles. Investigations of block copolymer nanoassemblies evaluate effects of the polymer structure and the self-assembly process to form interesting and diverse structures.

In this work, ring-opening metathesis polymerization is employed to prepare and study block copolymer amphiphile self-assembly. First, a small library of amphiphiles are prepared in which the hydrophobic block functionality is varied. In varying the polymer structure and the solvent for assembly, different morphologies are produced, including small and large spheres, cylinders, y-junctions, and rods. In several cases, one polymer can take on different morphologies depending on the organic cosolvent used during micellization, highlighting the importance of assembly conditions and dynamics in forming kinetically trapped structures versus thermodynamically stable structures. These are important considerations when designing, synthesizing, and formulating polymeric nanoparticles for in vivo applications.

Next, direct incorporation of a gadolinium based contrast agent for magnetic resonance imaging is studied. A novel monomer and chain transfer agent of a gadolinium-chelate are used to directly incorporate the contrast agent in to a polynorbornene polymer backbone. The resulting spherical and fibrillar nanoparticles exhibited enhanced relaxivity and are studied as MRI contrast agents using live imaging in murine models.

Finally, a fatty acid ligand is conjugated to a gadolinium-based contrast agent is prepared and formulated with human serum albumin. In formulation with HSA, the agent exhibits high relaxivity and prolonged blood circulation. In addition, therapeutic conjugates formulated with HSA are evaluated for in vitro cytotoxicity and found to be effective in tumor growth suppression in vivo.

Targeted therapeutics should accumulate at disease sites in higher quantities relative to other locations in the body. In cancer therapy, one strategy towards this is to exploit features of the tumor microenvironment (TME) for drug delivery. Herein, drug delivery strategies focusing on the TME are investigated.

First, nanomaterials designed to respond to matrix metalloproteinases (MMPs) are explored for delivery of cytotoxins to tumors. Ring opening metathesis polymerization (ROMP) is employed to generate amphiphilic diblock copolymers containing paclitaxel (PTX) prodrugs and MMP-responsive peptides. These polymers form 20 nm nanoparticles that rearrange to microscale aggregates upon MMP exposure. This process is observed and efficacy evaluated in a fibrosarcoma xenograft model. Critically, these materials have equivalent efficacy to PTX at equivalent doses, but with a 16-fold higher maximum tolerated dose (MTD). Further, nanoparticles containing an MMP-responsive peptide together with platinum (Pt) drugs, 15N-labelled moieties, and near infrared (NIR) fluorophores are developed. These materials are evaluated for their therapeutic efficacy in vivo and the simultaneous, yet independent, tracking of carrier and drug is completed ex vivo using correlative microscopy techniques.

This platform is then extended to the delivery of immunotherapeutics. MMP-responsive nanoparticles containing an immunotherapeutic compound (1V209) are developed and assessed in vitro for immunogenicity and in vivo for efficacy. In a syngeneic orthotopic breast cancer model, 1V209-containing nanoparticles do not cause nonspecific cytokine upregulation and significantly inhibit lung metastasis formation relative to 1V209, non-responsive nanoparticles, and saline.

Dual-responsive nanomaterials are also explored that are designed to respond to two separate features of the TME. Polymers and nanoparticles are designed to release cargo only when both MMPs and reactive oxygen species (ROS) are present.

Finally, a biomaterial-based delivery strategy is explored using human serum albumin (HSA) as a drug carrier of long chain fatty acids (LCFAs) to the TME. Mono-functionalizing octadecanedioic acid (ODDA) with PTX affords a fatty acid-PTX ester prodrug that retains one carboxylic acid moiety to form stable electrostatic interactions with HSA in its natural binding sites for LCFAs. This prodrug is capable of binding to HSA and shows differentiated pharmacokinetics, as well as remarkable tolerability and efficacy in vivo, relative to clinical formulations in multiple xenograft models.