Glioblastoma is the deadliest form of brain cancer, yet treatment remains largely palliative. Recurrence and resistance to conventional radiation therapies are largely due to subsets of radioresistant glioma stem cells (GSCs) which survive and can repropagate the tumor. Phage display is a well-established high-throughput screening technique which can identify high-affinity, surface-binding peptides against targets of interest. We used phage display to identify a single peptide sequence, YPHKWHEFKQRV, which was highly enriched in irradiated patient-derived GSC neurospheres compared to both non-tumor populations and unirradiated GSCs. Irradiation significantly increased surface binding of the target peptide in a number of different patient-derived GSC neurospheres in vitro and in vivo. This binding increase was abrogated by dissociation into single-cell suspensions prior to peptide binding, suggesting a possible cell-cell adhesion-related peptide binding partner. We identified four candidate genes which preferentially bind the peptide, and we observed aberrant plasma membrane localization in both Ezrin (EZR) and Metadherin (MTDH) following irradiation. All four candidate genes were essential for GSC proliferation based on shRNA knockdowns, while only EZR knockdown significantly impaired unirradiated proliferation, post-irradiation proliferation, and survival to ionizing radiation. Together, these data suggest a possible radiation-induced translocation mechanism for EZR supporting radioresistance uniquely in GSCs. Additionally, the specificity of the phage display-identified for irradiated GSCs highlight the power of this technique and the potential for this and other tumor-targeting peptides in GBM immunotherapy.

Traumatic brain injury (TBI) is a major public health concern that can result in long-term neurological impairments. Calpain is a calcium-dependent cysteine protease that is activated within minutes after TBI and sustained calpain activation is known to contribute to neurodegeneration and blood-brain barrier dysregulation. Based on its role in disease progression, calpain inhibition has been identified as a promising therapeutic target. Efforts to develop therapeutics for calpain inhibition would benefit from the ability to measure calpain activity with spatial precision within the injured tissue. In this work, I have engineered nanomaterials that can be used to measure and/or inhibit calpain activity after TBI. First, I used a hyaluronic acid-targeted activity-based nanosensor for TBI (HA-targeted TBI-ABN) to measure calpain activity in the cortex, hippocampus, and hindbrain after injury in both female and male mice. In a mouse model of TBI, I show that the HA-targeted TBI-ABN detects high calpain activity in the cortex and hippocampus ipsilateral to injury in both female and male mice. Second, I designed an activity-based nanotheranostic for TBI (TBI-ABNT) that can both sense and inhibit calpain activity. To inhibit calpain activity, I incorporated calpastatin peptide (CAST), an endogenous inhibitor of calpain. In a mouse model of TBI, I show that the TBI-ABNT construct is able to sense and inhibit calpain activity in the injured cortex and hippocampus. In an analysis of cellular calpain activity, I observe ABNT-mediated inhibition of calpain activity in neurons, endothelial cells, and microglia of the cortex. In a comparison of neuronal calpain activity by brain structure, I observe greater ABNT-mediated inhibition of calpain activity in cortical neurons compared to hippocampal neurons. Furthermore, I found that apoptosis was dependent on both calpain inhibition and by brain structure. Lastly, I designed a calpasatin peptide-based nanomaterial (CAST-PEG) to improve the pharmacokinetics of therapeutic calpastatin peptide. I show that a high valency of CAST per polymeric scaffold improves inhibitory activity, blood half-life, and biodistribution compared to free CAST. In a mouse model of TBI, I see a dose-dependent improvement in the accumulation of CAST-PEG in the injured brain compared to off-target organs. Finally, I show that neither CAST nor CAST-PEG delivery reduces calpain-specific α-spectrin breakdown products at 24 hours after injury. I present three platforms that can be used to understand the regional- and cell-specific activity of calpain and its inhibition to inform therapeutic interventions for TBI.

Traumatic brain injury (TBI) is a critical public health concern affecting millions of people globally per year. The primary trauma initiates a complex cascade of secondary injury which can extend for days to weeks post-injury and involves disease hallmarks like ectopic protease activity, vascular dysregulation, and neuroinflammation. Unfortunately, several hurdles constrain clinical translation of TBI therapeutics, including limited passive accumulation into injured brain tissue. As an alternative approach, my thesis leverages the extracellular matrix (ECM) to target the brain after TBI. Native brain ECM is an attractive candidate for active material targeting via affinity ligands or click chemistry to improve selectivity and distribution within the injured brain. Additionally, decellularized ECM derived biomaterials have previously demonstrated injury-specific deposition, retention, and therapeutic repair in preclinical models of disease, and are thus promising candidates for investigation within TBI.

First, the native brain ECM was leveraged as a scaffold to improve delivery and function of a nanosensor for TBI. We performed an in vivo screen of peptide sequences that bind ECM constituents in the injured brain and found that hyaluronic acid (HA) targeting peptides increased nanomaterial distribution within the injury. We then utilized HA targeting to improve delivery and function of a protease-responsive imaging material. The addition of HA targeting peptides to our fluorogenic activity-based nanosensor for TBI improved nanosensor activation and enabled robust 3D visualization of protease activity within a cleared brain. Second, we developed a novel in vivo chemical targeting approach for TBI by leveraging the extracellular fibrin clot as a scaffold for in situ click chemistry. We delivered strained cyclooctyne (SCO) modified fibrinogen which incorporated within the injured brain. The SCO-modified clot was subsequently utilized for intra-injury capture and retention of diverse materials including a small molecule azide-dye, 40kDa azide-PEG, and PEG-catalase antioxidant. Third, we investigated the pharmacokinetics, biodistribution, and therapeutic potential of an infusible extracellular matrix derived biomaterial (iECM) within TBI. iECM accumulated and was retained within the injured brain and a multi-dose regimen was established to optimize delivery. Further, iECM reduced vascular permeability at the injury and modulated pro-inflammatory and neuroprotective genes. Taken together, we demonstrate the utility of ECM as a scaffold for material targeting, and as an injury-homing biomaterial therapeutic, within TBI.

Traumatic brain injury (TBI) affects over 2.8 million people annually in the United States and leads to the hospitalization of ~300,000 patients per year. Current methods of diagnosis for TBI are either subjective and poor at discriminating mild TBI (Glasgow Coma Scale) or take extensive time and resources to run (computed tomography and magnetic resonance imaging). These diagnostic modalities also do not capture information on the biological processes driving the pathology of secondary injury, where there is a window of opportunity to prevent further damage with treatment. As a supplement to these diagnostics, the blood or cerebrospinal fluid can be sampled for biomarkers in the form of breakdown products which are released during degenerative processes after TBI. Many of these biomarkers are produced by ectopic proteases including the calcium-dependent protease calpain-1, which is implicated in cellular death and worsened prognosis after TBI. Thus, the measurement of calpain-1 activity may help to diagnose injury progression and inform patient prognosis after TBI.

To diagnose secondary injury in TBI, we developed an activity-based nanosensor for TBI (TBI-ABN) which can detect activity from the protease calpain-1. The nanosensor is comprised of a FRET peptide conjugated to a 40 kDa 8-arm PEG scaffold, and can produce a fluorescent signal once it is specifically cleaved by active calpain-1. In a mouse model of TBI, systemically administered TBI-ABNs were found to accumulate and activate in the injured brain tissue as assessed by fluorescence in brain tissue slices. Next, we investigated whether adding active targeting to components of the brain extracellular matrix, such as hyaluronic acid, could improve the sensitivity of TBI-ABNs. We conjugated hyaluronic acid-targeting peptides to TBI-ABNs and found that the activation of sensor in injured brain tissue was increased by approximately 2.8- and 6.6-fold when targeting peptides were added at moderate and high valencies, respectively, compared to non-targeted nanosensor within brain tissue homogenates. Finally, we redesigned the TBI-ABN to release a synthetic biomarker into the blood or urine after systemic administration for a minimally-invasive measurement of protease activity after TBI. The synthetic biomarker could be quantified both via fluorescence and immunoassays to detect calpain-1 activity in TBI. These nanosensors are the first demonstrations of protease activity measurement in the context of TBI and have potential both as tools to study protease activation in the injured brain and as diagnostics to identify the biological processes taking place after injury.

Many traumatic brain injury (TBI) survivors suffer cognitive impairments after injury, yet there are currently no FDA approved therapeutics that address long-term patient brain health. Development of vascularly delivered TBI therapeutics is challenging due to their poor pharmacokinetics, which reduces their accumulation and retention in the brain. Nanoparticles are promising technologies that can improve the vascular delivery of TBI therapeutics by protecting the drug payload in circulation, increasing blood half-life, and passively accumulating in injured brain tissue across the transiently permeable blood-brain barrier (BBB). Nanoparticle surfaces can be modified to change their physicochemical properties and implement targeting strategies, thereby tuning their pharmacokinetics to improve delivery. Thus, vascular delivery of nanoparticle-based TBI therapeutics can be improved by engineering the surface properties of nanoparticles.To engineer nanoparticles that accumulate in the injured brain, we modified the surfaces of diverse nanoparticle platforms to tune their physicochemical properties and implement active targeting. First, we engineered the steric forces and hydrophobicity of lipid nanoparticles (LNP) by modifying their polyethylene glycol (PEG) layer with short and long anchor PEG-lipids. We found that vascularly delivered LNPs formulated with long anchored PEG-lipids had longer blood half-lives and extended activity in the injured brain. Next, we engineered the charge and hydrophobicity of model polystyrene nanoparticles via surface modification with peptides. We found that peptide physicochemical properties affected nanoparticle pharmacokinetics, with neutral, zwitterionic, and negatively charged nanoparticles demonstrating greater passive accumulation in the injured brain than positively charged nanoparticles. Finally, we applied these ideas to a porous silicon nanoparticle (pSiNP) drug delivery system for brain derived neurotrophic factor (BDNF) surface modified with PEG and a peptide that targets the injured brain (CAQK). After intravenous administration of pSiNP-BDNF, we observed a ~24% reduction in brain lesion volumes compared to both free BDNF and untreated controls. Understanding how engineered nanoparticle surface properties affect their pharmacokinetics in TBI models informs the design of nanoparticle-based TBI therapeutics and is broadly applicable to the design of vascularly delivered therapeutic nanoparticles.

Traumatic brain injury (TBI) presents a serious challenge for modern medicine due to the poor regenerative capabilities of the brain, complex pathophysiology, and lack of effective treatment for TBI to date. The overall goal of this research is to develop as nanoparticle capable of sustained drug release for the delivery and treatment of TBI so that therapeutic drug can be delivered after the Blood Brain Barrier (BBB) closes 24 hours after injury. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles have shown some experimental success; as of yet, none have yielded improved results of clinical efficacy. Polyethylene Glycol (PEG) has been shown to act as a stealth molecule that can increase the circulation time of particles. N-acetylcysteine has shown neuroprotective and anti-inflammatory potential. This paper presents the development of PEGylated PLGA NPs synthesized using double and single emulsion for potential therapeutic delivery to TBI. Dynamic light scattering (DLS) show PEG-PLGA NPs to have an average diameter of 200 nm. Loading capacity was obtained at 2%, and drug encapsulation efficiency was obtained at 55%. A biphasic drug release pattern was noted in situ and drug release was assessed in vitro over 72 h using FRET nanoparticles, almost complete release of drug was seen during this time period. Finally, in vivo administration of PEG-PLGA NPs demonstrated that PEGylated NPS were able to show reduced accumulation in filtration organs such as the liver and kidneys compared to bare PLGA particles. This evidence supports the potential of PEG-PLGA NPs as a promising carrier to develop TBI therapeutics.

Myocardial infarction (MI) affects nearly 800,000 people every year in the United States. As it stands, treating patients during the acute phase of MI is highly limited due to the fragility of the infarcted heart. Thus, minimally invasive intravenous administration of targeted nanoscale therapeutics has the potential to allow for more effective treatment of the heart during this acute phase of MI, a therapeutic window in which more invasive procedures are not feasible. Using an ischemia-reperfusion injury model in rats, we assessed three different nanoscale platforms: polynorbornene nanoparticles, a degradable copolymer nanoparticle, and a polynorbornene protein-like polymer. All these systems leverage the enhanced permeability and retention effect in the infarcted region of the heart and extravasates into the injured myocardium, undergoing a morphological switch from the nano to the micron-scale when cleaved by endogenous MMPs in the infarct. First, we demonstrated the aggregation and localization of matrix metalloproteinase (MMP) responsive peptide-polymer amphiphile polynorbornene nanoparticles carrying a small molecule MMP-inhibitor to the heart following acute myocardial infarction (MI). In addition, we showed that drug incorporation onto the nanoparticle backbone improved significantly improved it’s the maximum concentration at which it could be tolerated compared to free drug treatment in vitro while also maintain the drug’s bioactivity. Next, we assess the degradable nanoparticle for cytocompatibility, infarct-localization, and biodistribution over time. We found that this material successfully degrades over time while exhibiting distinct and beneficial biodistribution compared to previous work. Lastly, we investigated the biodistribution and mechanism of accumulation using a protein-like polymer. We observed strong colocalization of this material with necrotic cardiomyocytes in the infarcted region and favorable biodistribution that preferentially biased accumulation in the kidneys over other satellite organs. All these material approaches are highly versatile and can be easily adapted to carry various therapeutic payloads, making them a potential platform for targeted drug delivery to areas of inflammation.

With the advent of targeted immunotherapies, survival rates of some cancers have improved tremendously over the years; however, cancer continues to be the second leading cause of death overall in the United States. Most cancer-related deaths can be attributed to the onset of metastatic disease, defined by the spread of disseminated cancer cells from the primary tumor into distal organs following trafficking into the lymphatic and circulatory systems. Once metastasis has occurred, survival rates drop precipitously as metastatic cancers exhibit therapeutic resistance and plasticity. Therefore, it is imperative that newer technologies that can not only treat metastatic cancers, but also prevent them are continuously developed. In this dissertation, I detail many of these efforts in fighting against metastatic cancer using a repurposed plant virus called the cowpea mosaic virus (CPMV). Past studies have demonstrated the potent immunotherapeutic ability of CPMV as a direct in situ vaccine with efficacy established in murine models of colon, ovarian, glioma, breast cancer, and melanoma as well as in canine patients with melanoma and inflammatory mammary cancer. However, metastatic cancers are uninjectable, which required engineering of the CPMV nanoparticles for the treatment and prophylaxis of metastatic cancers. Specifically, I conjugated peptides termed H6 and G3 that target a protein called S100A9, which becomes overexpressed in a myriad of cancer types and is indicated in lung metastasis. Targeting S100A9 led to intrapulmonary accumulation of CPMV in mice allowing for both prophylaxis and treatment of metastatic breast cancer and melanoma to the lungs. The same nanoparticles were also used to target intraperitoneal metastases from colon cancer leading to improved tumor rejection and survival. While efficacious, CPMV-H6/G3 treatment can be limited to S100A9-expressing cancers. I then broadened the applicability of native CPMV by utilizing it as an adjuvant therapy for the prevention of a wide range of cancers demonstrating its efficacy in four different metastatic cancer models in two species of mice. My data highlight that the antitumor efficacy of CPMV is species, tissue, and tumor-type agnostic.The adjuvanticity of CPMV was also repurposed for vaccine applications – concomitant injection of CPMV with antigens causes potent titer production. Given the role of S100A9 in lung metastasis, I created a vaccine utilizing CPMV and another viral adjuvant Q and chemically conjugated a S100A9 epitope to the viral carriers to induce the production of -S100A9 antibodies leading to reduced metastasis within the lungs. I then further developed the CPMV and Q adjuvant and carrier systems by developing a histidine-tag nickel binding chemistry that allows for the rapid production of plug-and-play vaccine candidates. This allows for the generation of vaccine candidates that co-deliver antigen and adjuvant while still demonstrating plug-and-play capabilities – results with ovalbumin showed that titers against ovalbumin were improved compared to bolus injections leading to improved antitumor responses against specialized ovalbumin-expressing cell lines. Overall, my thesis showcases the potent ability of CPMV to both prevent and treat metastatic cancers in prophylactic, therapeutic, and vaccine formats and highlights the development of CPMV and Q in plug-and-play vaccine applications.