Bioconjugates for Cancer Prevention: Opportunities for Impact.

Cancer prevention encompasses both screening strategies to find cancers early when they are likely to be most treatable and prevention and interception strategies to reduce the risk of developing cancers. Bioconjugates, here defined broadly as materials and molecules that have synthetic and biological components, have roles to play across the cancer-prevention spectrum. In particular, bioconjugates may be developed as affordable, accessible, and effective screening strategies or as novel vaccines and drugs to reduce one's risk of developing cancers. Developmental programs are available for taking novel technologies and evaluating them for clinical use in cancer screening and prevention. While a variety of different challenges exist in implementing cancer-prevention interventions, a thoughtful approach to bioconjugates could improve the delivery and acceptability of the interventions.

Getting to the Core of It All: Nanocapsules to Mitigate Infusion Reactions Can Promote Hemostasis and Be a Platform for Intravenous Therapies.

One of the significant challenges to translation of intravenously administered nanomaterials has been complement-mediated infusion reactions which can be lethal. Slow infusions can reduce infusion reactions, but slow infusions are not always possible in applications like controlling bleeding following trauma. Thus, avoiding complement activation and infusion responses is essential to manage bleeding. We identified nanocapsules based on polyurethane as candidates that did not activate C5a and explored their PEGylation and functionalization with the GRGDS peptide to create a new class of hemostatic nanomaterials. Using the clinically relevant rotational thromboelastography (ROTEM), we determined that nanocapsules promote faster clotting than controls and maintain the maximum clot firmness, which is critical for reducing bleeding. Excitingly, these polyurethane-based nanocapsules did not activate complement or the major pro-inflammatory cytokines. This work provides critical evidence for the role of modulating the core material in developing safer nanomedicines for intravenous applications.

Engineering PEGylated Polyester Nanoparticles to Reduce Complement-Mediated Infusion Reaction.

Translation of intravenously administered nanomaterials to the clinic is limited due to adverse infusion reactions. While these reactions are infrequent, with up to 10% prone to experiencing infusion reactions, the reactions can be severe and life-threatening. One of the innate immune pathways, the complement activation pathway, plays a significant role in mediating this response. Nanoparticle surface properties are a relevant design feature, as they control the blood proteins the nanoparticles interact with and allow the nanoparticles to evade the immune reaction. PEGylation of nanosurfaces is critical in improving the blood circulation of nanoparticles and reducing opsonization. Our goal was to understand whether modifying the surface architecture by varying the PEG density and architecture can impact the complement response in vitro. We utilized block copolymers of poly(lactic acid)-b-poly(ethylene glycol) prepared with poly(ethylene glycol) macroinitiators of molecular weights 3400 and 5000 Da. Tracking the complement biomarker C5a, we monitored the impact of changing PEGylation of the nanoparticles. We also investigated how the changing PEG length on the nanoparticle surface impacts further strengthening the stealth properties. Lastly, we determined which cytokines change upon blood incubation with nanoparticles in vitro to understand the extent to which inflammation may occur and the crosstalk between the complement and immune responses. Increasing PEGylation reduced the generation of complement-mediated anaphylatoxin C5a in vitro, with 5000 Da PEG more effectively reducing levels of C5a generated compared to 3400 Da PEG. The insights gathered regarding the impact of PEG density and PEG chain length would be critical in developing stealth nanoparticles that do not lead to infusion reactions upon intravenous administration.

Biomimetic Strategies To Treat Traumatic Brain Injury by Leveraging Fibrinogen.

There were over 27 million new cases of traumatic brain injuries (TBIs) in 2016 across the globe. TBIs are often part of complicated trauma scenarios and may not be diagnosed initially because the primary clinical focus is on stabilizing the patient. Interventions used to stabilize trauma patients may inadvertently impact the outcomes of TBIs. Recently, there has been a strong interest in the trauma community toward administrating fibrinogen-containing solutions intravenously to help stabilize trauma patients. While this interventional shift may benefit general trauma scenarios, fibrinogen is associated with potentially deleterious effects for TBIs. Here, we deconstruct what components of fibrinogen may be beneficial as well as potentially harmful following TBI and extrapolate this to biomimetic approaches to treat bleeding and trauma that may also lead to better outcomes following TBI.

The Chemistry of Lyophilized Blood Products.

With the development of new biologics and bioconjugates, storage and preservation have become more critical than ever before. Lyophilization is a method of cell and protein preservation by removing a solvent such as water from a substance followed by freezing. This technique has been used in the past and still holds promise for overcoming logistic challenges in safety net hospitals with limited blood banking resources, austere environments such as combat, and mass casualty situations where existing resources may be outstripped. This method allows for long-term storage and transport but requires the bioconjugation of preservatives to prevent cell destabilization. Trehalose is utilized as a bioconjugate in platelet and red blood cell preservation to maintain protein thermodynamics and stabilizing protein formulations in liquid and freeze-dried states. Biomimetic approaches have been explored as alternatives to cryo- and lyopreservation of blood components. Intravascular hemostats such as PLGA nanoparticles functionalized with PEG motifs, topical hemostats utilizing fibrinogen or chitosan, and liposomal encapsulated hemoglobin with surface modifications are effectively stored long-term through bioconjugation. In thinking about the best methods for storage and transport, we are focusing this topical review on blood products that have the longest track record of preservation and looking at how these methods can be applied to synthetic systems.

Engineering Intravenously Administered Nanoparticles to Reduce Infusion Reaction and Stop Bleeding in a Large Animal Model of Trauma.

Bleeding from traumatic injury is the leading cause of death for young people across the world, but interventions are lacking. While many agents have shown promise in small animal models, translating the work to large animal models has been exceptionally difficult in great part because of infusion-associated complement activation to nanomaterials that leads to cardiopulmonary complications. Unfortunately, this reaction is seen in at least 10% of the population. We developed intravenously infusible hemostatic nanoparticles that were effective in stopping bleeding and improving survival in rodent models of trauma. To translate this work, we developed a porcine liver injury model. Infusion of the first generation of hemostatic nanoparticles and controls 5 min after injury led to massive vasodilation and exsanguination even at extremely low doses. In naïve animals, the physiological changes were consistent with a complement-associated infusion reaction. By tailoring the zeta potential, we were able to engineer a second generation of hemostatic nanoparticles and controls that did not exhibit the complement response at low and moderate doses but did at the highest doses. These second-generation nanoparticles led to cessation of bleeding within 10 min of administration even though some signs of vasodilation were still seen. While the complement response is still a challenge, this work is extremely encouraging in that it demonstrates that when the infusion-associated complement response is managed, hemostatic nanoparticles are capable of rapidly stopping bleeding in a large animal model of trauma.

Intravenously administered nanoparticles increase survival following blast trauma.

Explosions account for 79% of combat-related injuries, leading to multiorgan hemorrhage and uncontrolled bleeding. Uncontrolled bleeding is the leading cause of death in battlefield traumas as well as in civilian life. We need to stop the bleeding quickly to save lives, but, shockingly, there are no treatments to stop internal bleeding. A therapy that halts bleeding in a site-specific manner and is safe, stable at room temperature, and easily administered is critical for the advancement of trauma care. To address this need, we have developed hemostatic nanoparticles that are administered intravenously. When tested in a model of blast trauma with multiorgan hemorrhaging, i.v. administration of the hemostatic nanoparticles led to a significant improvement in survival over the short term (1 h postblast). No complications from this treatment were apparent out to 3 wk. This work demonstrates that these particles have the potential to save lives and fundamentally change trauma care.

A hydrogel-endothelial cell implant mimics infantile hemangioma: modulation by survivin and the Hippo pathway.

Microvascular endothelial cells cultured in three-dimensional hydrogel scaffolds form a network of microvessel structures when implanted subcutaneously in mice, inosculate with host vessels, and over time remodel into large ectatic vascular structures resembling hemangiomas. When compared with infantile hemangiomas, similarities were noted, including a temporal progression from a morphological appearance of a proliferative phase to the appearance of an involuted phase, mimicking the proliferative and involutional phases of infantile hemangioma. Consistent with the progression of a proliferative phase to an involuted phase, both the murine implants and human biopsy tissue exhibit reduced expression of Ajuba, YAP, and Survivin labeling as they progressed over time. Significant numbers of CD45+, CD11b+, Mac3+ mononuclear cells were found at the 2-week time point in our implant model that correlated with the presence of CD45+, CD68+ mononuclear cells observed in biopsies of human proliferative-phase hemangiomas. At the 4-week time point in our implant model, only small numbers of CD45+ cells were detected, which again correlated with our findings of significantly diminished CD45+, CD68+ mononuclear cells in human involutional-phase hemangiomas. The demonstration of mononuclear cell infiltration transiently in the proliferative phase of these lesions suggests that the vascular proliferation and/or regression may be driven in part by an immune response. Gross and microscopic morphological appearances of human proliferative and involutional hemangiomas and our implant model correlate well with each other as do the expression levels of Hippo pathway components (Ajuba and YAP) and Survivin and correlate with proliferation in these entities. Inhibitors of Survivin and Ajuba (which we have demonstrated to inhibit proliferation and increase apoptosis in murine hemangioendothelioma cell tissue culture) may have potential as other beneficial treatments for proliferating infantile hemangiomas. This implant model may have potential as a modest through-put screen for testing and development of therapeutics targeted at the proliferative phase of infantile hemangiomas, reducing the subsequent postinvolutional scarring or deformities sometimes associated with these lesions.

Tuning ligand density on intravenous hemostatic nanoparticles dramatically increases survival following blunt trauma.

Targeted nanoparticles are being pursued for a range of medical applications. Here we utilized targeted nanoparticles (synthetic platelets) to halt bleeding in acute trauma. One of the major questions that arises in the field is the role of surface ligand density in targeted nanoparticles' performance. We developed intravenous hemostatic nanoparticles (GRGDS-NP1) and previously demonstrated their ability to reduce bleeding following femoral artery injury and increase survival after lethal liver trauma in the rat. These nanoparticles are made from block copolymers, poly(lactic-co-glycolic acid)-b-poly L-lysine-b-poly(ethylene glycol). Surface-conjugated targeting ligand density can be tightly controlled with this system, and here we investigated the effect of varying density on hemostasis and biodistribution. We increased the targeting peptide (GRGDS) concentration 100-fold (GRGDS-NP100) and undertook an in vitro dose-response study using rotational thromboelastometry, finding that GRGDS-NP100 hemostatic nanoparticles were efficacious at doses at least 10 times lower than the GRGDS-NP1. These results were recapitulated in vivo, demonstrating efficacy at eight-fold lower concentration after lethal liver trauma. 1 h survival increased to 92% compared with a scrambled peptide control, 45% (OR = 14.4, 95% CI = [1.36, 143]), a saline control, 47% (OR = 13.5, 95% CI = [1.42, 125]), and GRGDS-NP1, 80% (OR = 1.30, n.s.). This work demonstrates the impact of changing synthetic platelet ligand density on hemostasis and lays the foundation for methods to determine optimal ligand concentration parameters.

Nature vs. Manmade: Comparing Exosomes and Liposomes for Traumatic Brain Injury.

Traumatic brain injury (TBI) of all severities is a significant public health burden, causing a range of effects that can lead to death or a diminished quality of life. Liposomes and mesenchymal stem cell-derived exosomes are two drug delivery agents with potential to be leveraged in the treatment of TBI by increasing the efficacy of drug therapies as well as having additional therapeutic effects. They exhibit several physical similarities, but key differences affect their performances as nanocarriers. Liposomes can be produced commercially at scale, and liposomes achieve higher encapsulation efficiency. Meanwhile, the intrinsic cargo and targeting moieties of exosomes, which liposomes lack, give exosomes a greater ability to facilitate neural regeneration, and exosomes do not trigger the infusion reactions that liposomes can. However, there are concerns about both exosomes and liposomes regarding interactions with tumors. The same routes of administration can be used for both exosomes and liposomes, resulting in somewhat different distribution throughout the body. While the effect of the nanocarrier type on accumulation in the brain is not concrete, targeting leads to increased accumulation of both exosomes and liposomes in the brain, upon which on-demand release can be used for both drug deliverers. Although neither have been applied to TBI in humans, preclinical trials have shown their immense potential, as have clinical trials pertaining to other brain injuries and conditions. While questions remain, research thus far shows that the various differences make exosomes a better choice of nanocarrier for TBI.

Polyester Nanoparticles and Polyurethane Nanocapsules Deliver Pirfenidone To Reduce Fibrosis and Scarring.

Pirfenidone has been shown to reduce fibrosis and modulate inflammation associated with conditions from pulmonary fibrosis to rheumatoid arthritis. It may also be useful for ocular diseases as well. However, for pirfenidone to be effective, it needs to be delivered to the tissue of interest, which, in the case of the eye, in particular, motivates the need for a system that permits local, long-term delivery to address the continuing pathology of the, condition. We investigated a set of delivery systems to determine the impact of encapsulation materials on the loading and delivery of pirfenidone. While the polyester system based on poly(lactic-co-glycolic acid) (PLGA) nanoparticles exhibited higher loading than a polyurethane-based nanocapsule system, the delivery was short, with 85% of the drug being released in 24 h and no measurable drug after 7 days. Addition of different poloxamers impacted the loading but not the release of the drug. In contrast, the polyurethane nanocapsule system delivered 60% of the drug over the first 24 h and the remainder over the next 50 days. Furthermore, the polyurethane system permitted on-demand delivery via ultrasound. Being able to tune the amount of drug delivered via ultrasound has the potential to tailor the delivery of pirfenidone to modulate inflammation and fibrosis. We used a fibroblast scratch assay to confirm the bioactivity of the released drug. This work provides multiple platforms for the delivery of pirfenidone locally and over time in both passive and on-demand formulations with the potential to address a range of inflammatory and fibrotic conditions.

Intravenous hemostatic nanoparticles increase survival following blunt trauma injury.

Trauma is the leading cause of death for people ages 1-44, with blood loss comprising 60-70% of mortality in the absence of lethal CNS or cardiac injury. Immediate intervention is critical to improving chances of survival. While there are several products to control bleeding for external and compressible wounds, including pressure dressings, tourniquets, or topical materials (e.g., QuikClot, HemCon), there are no products that can be administered in the field for internal bleeding. There is a tremendous unmet need for a hemostatic agent to address internal bleeding in the field. We have developed hemostatic nanoparticles (GRGDS-NPs) that reduce bleeding times by ~50% in a rat femoral artery injury model. Here, we investigated their impact on survival following administration in a lethal liver resection injury in rats. Administration of these hemostatic nanoparticles reduced blood loss following the liver injury and dramatically and significantly increased 1 h survival from 40 and 47% in controls (inactive nanoparticles and saline, respectively) to 80%. Furthermore, we saw no complications following administration of these nanoparticles. We further characterized the nanoparticles' effect on clotting time (CT) and maximum clot firmness (MCF) using rotational thromboelastometry (ROTEM), a clinical measurement of whole-blood coagulation. Clotting time is significantly reduced, with no change in MCF. Administration of these hemostatic nanoparticles after massive trauma may help staunch bleeding and improve survival in the critical window following injury, and this could fundamentally change trauma care.

Azithromycin Protects Retinal Glia Against Oxidative Stress-Induced Morphological Changes, Inflammation, and Cell Death.

The reactivity of retinal glia in response to oxidative stress has a significant effect on retinal pathobiology. The reactive glia change their morphology and secret cytokines and neurotoxic factors in response to oxidative stress associated with retinal neurovascular degeneration. Therefore, pharmacological intervention to protect glial health against oxidative stress is crucial for maintaining homeostasis and the normal function of the retina. In this study, we explored the effect of azithromycin, a macrolide antibiotic with antioxidant, immunomodulatory, anti-inflammatory, and neuroprotective properties against oxidative stress-induced morphological changes, inflammation, and cell death in retinal microglia and Müller glia. Oxidative stress was induced by H2O2, and the intracellular oxidative stress was measured by DCFDA and DHE staining. The change in morphological characteristics such as the surface area, perimeter, and circularity was calculated using ImageJ software. Inflammation was measured by enzyme-linked immunosorbent assays for TNF-α, IL-1ß, and IL-6. Reactive gliosis was characterized by anti-GFAP immunostaining. Cell death was measured by MTT assay, acridine orange/propidium iodide, and trypan blue staining. Pretreatment of azithromycin inhibits H2O2-induced oxidative stress in microglial (BV-2) and Müller glial (MIO-M1) cells. We observed that azithromycin inhibits oxidative stress-induced morphological changes, including the cell surface area, circularity, and perimeter in BV-2 and MIO-M1 cells. It also inhibits inflammation and cell death in both the glial cells. Azithromycin could be used as a pharmacological intervention on maintaining retinal glial health during oxidative stress.

Biocompatible Nanocapsules for Self-Healing Dental Resins and Bone Cements.

Bone cements and dental resins are methacrylate-based materials that have been in use for many years, but their failure rates are quite high with essentially all dental resins failing within 10 years and 25% of all prosthetic implants will undergo aseptic loosening. There are significant healthcare costs and impacts on quality of life of patients. Self-healing bone cements and resins could improve the lifespan of these systems, reduce costs, and improve patient outcomes, but they have been limited by efficacy and toxicity of the components. To address these issues, we developed a self-healing system based on a dual nanocapsule system. Two nanocapsules were synthesized, one containing an initiator and one encapsulating a monomer, both in polyurethane shells. The monomer used was triethylene glycol dimethacrylate. The initiator capsules synthesized contained benzoyl peroxide and butylated hydroxytoluene. Resins containing the nanocapsules were tested in tension until failure, and the fractured surfaces were placed together. 33% of the samples showed self-healing behaviors to the point where they could be reloaded and tested in tension. Furthermore, the capsules and their components showed good biocompatibility with Caco-2 cells, a human epithelial cell line suggesting that they would be well tolerated in vivo.