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.

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.

PEGylated Polyester Nanoparticles Trigger Adverse Events in a Large Animal Model of Trauma and in Naïve Animals: Understanding Cytokine and Cellular Correlations with These Events.

Intravenously infusible nanoparticles to control bleeding have shown promise in rodents, but translation into preclinical models has been challenging as many of these nanoparticle approaches have resulted in infusion responses and adverse outcomes in large animal trauma models. We developed a hemostatic nanoparticle technology that was screened to avoid one component of the infusion response: complement activation. We administered these hemostatic nanoparticles, control nanoparticles, or saline volume controls in a porcine polytrauma model. While the hemostatic nanoparticles promoted clotting as marked by a decrease in prothrombin time and both the hemostatic nanoparticles and controls did not active complement, in a subset of the animals, hard thrombi were found in uninjured tissues in both the hemostatic and control nanoparticle groups. Using data science methods that allow one to work across heterogeneous data sets, we found that the presence of these thrombi correlated with changes in IL-6, INF-alpha, lymphocytes, and neutrophils. While these findings might suggest that this formulation would not be a safe one for translation for trauma, they provide guidance for developing screening tools to make nanoparticle formulations in the complex milieux of trauma as well as for therapeutic interventions more broadly. This is important as we look to translate intravenously administered nanoparticle formulations for therapies, particularly considering the vascular changes seen in a subset of patients following COVID-19. We need to understand adverse events like thrombi more completely and screen for these events early to make nanomaterials as safe and effective as possible.

Hydroporphyrin-Doped Near-Infrared-Emitting Polymer Dots for Cellular Fluorescence Imaging.

Near-infrared (NIR) fluorescent semiconductor polymer dots (Pdots) have shown great potential for fluorescence imaging due to their exceptional chemical and photophysical properties. This paper describes the synthesis of NIR-emitting Pdots with great control and tunability of emission peak wavelength. The Pdots were prepared by doping poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-co-(1,4-benzo-(2,1',3)-thiadiazole)] (PFBT), a semiconducting polymer commonly used as a host polymer in luminescent Pdots, with a series of chlorins and bacteriochlorins with varying functional groups. Chlorins and bacteriochlorins are ideal dopants due to their high hydrophobicity, which precludes their use as molecular probes in aqueous biological media but on the other hand prevents their leakage when doped into Pdots. Additionally, chlorins and bacteriochlorins have narrow deep red to NIR-emission bands and the wide array of synthetic modifications available for modifying their molecular structure enables tuning their emission predictably and systematically. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) measurements show the chlorin- and bacteriochlorin-doped Pdots to be nearly spherical with an average diameter of 46 ± 12 nm. Efficient energy transfer between PFBT and the doped chlorins or bacteriochlorins decreases the PFBT donor emission to near baseline level and increases the emission of the doped dyes that serve as acceptors. The chlorin- and bacteriochlorin-doped Pdots show narrow emission bands ranging from 640 to 820 nm depending on the doped dye. The paper demonstrates the utility of the systematic chlorin and bacteriochlorin synthesis approach by preparing Pdots of varying emission peak wavelength, utilizing them to visualize multiple targets using wide-field fluorescence microscopy, binding them to secondary antibodies, and determining the binding of secondary antibody-conjugated Pdots to primary antibody-labeled receptors in plant cells. Additionally, the chlorin- and bacteriochlorin-doped Pdots show a blinking behavior that could enable their use in super-resolution imaging methods like STORM.

Finding the sweet spot: a library of hydrogels with tunable degradation for tissue model development.

In vitro models are valuable tools for applications including understanding cellular mechanisms and drug screening. Hydrogel biomaterials facilitate in vitro models by mimicking the extracellular matrix and in vivo microenvironment. However, it can be challenging for cells to form tissues in hydrogels that do not degrade. In contrast, if hydrogels degrade too much or too quickly, tissue models may be difficult to assess in a high throughput manner. In this paper, we present a poly(allylamine) (PAA) based synthetic hydrogel system which can be tuned to control the mechanical and chemical cues provided by the hydrogel scaffold. PAA is a polycation with several biomedical applications, including the delivery of small molecules, nucleic acids, and proteins. Based on PAA and poly(ethylene glycol) (PEG), we developed a synthetic non-degradable system with potential applications for long-term cultures. We then created a second set of gels that combined PAA with poly-L-lysine (PLL) to generate a library of semi-degradable gels with unique degradation kinetics. In this work, we present the hydrogel systems' synthesis, characterization, and degradation profiles along with cellular data demonstrating that a subset of gels supports the formation of endothelial cell cord-like structures.

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.

Unaffected ex vivo clotting cascade by experimental hemostatic nanoparticles when introduced in the presence of recombinant tissue plasminogen activator.

CONTEXT: Hemostatic nanoparticles (hNPs) have shown efficacy in decreasing intracerebral hemorrhage (ICH) in animal models and are suggested to be of use to counter tissue plasminogen activator (tPA)-induced acute ICH. AIMS: The objective of this study was to test the ability of an hNP preparation to alter the clotting properties of blood exposed to tPA ex vivo. MATERIALS AND METHODS: Fresh blood samples were obtained from normal male Sprague-Dawley rats (~300 g; n = 6) and prepared for coagulation assays by thromboelastography (TEG) methods. Samples were untreated, exposed to tPA, or exposed to tPA and then to hNP. TEG parameters included reaction time (R, time in minutes elapsed from test initiation to initial fibrin formation), coagulation time (K, time in minutes from R until initial clot formation), angle (α, a measure in degrees of the rate of clot formation), maximum amplitude (MA, the point when the clot reaches its MA in mm), lysis at 30 min after MA (LY30, %), and clot strength (G, dynes/cm2), an index of clot strength. STATISTICAL ANALYSIS USED: Kruskal-Wallis test was employed to compare TEG parameters measured for untreated control samples versus those exposed to tPA and to compare tPA-exposed samples to samples treated with tPA + hNPs. Significances were inferred at P ≤ 0.05. RESULTS: Compared to untreated samples, tPA-treated samples showed a trend toward decreased angle and G suggesting potentially clot formation rate and clot strength. The addition of hNP did not affect any of these or other measured indices. CONCLUSIONS: The data demonstrated no hemostatic effects when the hNP was used in the presence of tPA. The lack of change in any of the TEG parameters measured in the present study may indicate limitations of the hNPs to reverse the thrombolytic cascade initiated by tPA.

Screen Printing Tissue Models Using Chemically Cross-Linked Hydrogel Systems: A Simple Approach To Efficiently Make Highly Tunable Matrices.

In vitro models provide a good starting point for drug screening and understanding various cellular mechanisms corresponding to different conditions. 3D cultures have drawn significant interest to mimic the in vivo microenvironment better and overcome the limitations of the 2D monolayered cultures. We previously reported a technique based on the screen printing process to pattern live mammalian cells using gelatin as the bioink. Even though gelatin is an inexpensive scaffolding material with various tissue engineering applications, it might not be the ideal hydrogel material to provide various mechanical and chemical cues to the cells. In this paper, we discuss the synthesis and characterization of two synthetic chemically cross-linked hydrogel systems based on poly(ethylene glycol) (PEG) and poly-l-lysine (PLL) to be used as the bioink in the screen printing process. These hydrogels are suitable as the bioinks for the screen printing process and serve as the barebone materials that can be tuned mechanically and augmented chemically to create a suitable in vitro microenvironment for the cells. This paper presents the synthesis, mechanical testing, and characterization of the hydrogel systems and their applications in the screen printing process.

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.

A non-invasive nanoparticle-based sustained dual-drug delivery system as an eyedrop for endophthalmitis.

Endophthalmitis is an infectious disease that affects the entire eye spreading to the internal retinal layers and the vitreous and causes severe sight-threatening conditions. Current treatment strategies rely on intraocular injections of antibiotics that are invasive, may lead to procedural complications and, ultimately, blindness. In this study, we developed a non-invasive strategy as an eyedrop containing nanoparticle-based dual-drug delivery system in which the hydrophobic poly-L-lactide core was loaded with azithromycin or triamcinolone acetonide, and the hydrophilic shell was made of chitosan. The developed nanoparticles were ~200-250 nm in size, spherical in shape, moderately hydrophilic, lysozyme tolerant, cytocompatible, and hemocompatible. Application of these chitosan-coated nanoparticles as eye drops to C57BL/6 mice showed higher bioavailability in choroid and retina when compared to the uncoated nanoparticles. The delivery system showed sustained release of drug for 300 h and exhibited antimicrobial effects against Gram-positive and Gram-negative bacteria and anti-inflammatory effects on activated microglial cells. Interestingly, the combination of the nanoparticles loaded with azithromycin and the nanoparticles loaded with triamcinolone acetonide acted synergistically as compared to either of the nanoparticles/drugs alone. Overall, the developed dual-drug delivery system is non-invasive, has antimicrobial and anti-inflammatory effects, and shows potential as an eye drop formulation against endophthalmitis.

Highlighting the usage of polymeric nanoparticles for the treatment of traumatic brain injury: A review study.

There are very limited options for treating traumatic brain injury (TBI). Nanoparticles offer the potential of targeting specific cell types, and, potentially, crossing the BBB under the right conditions making them an area of active research for treating TBI. This review focuses on polymeric nanoparticles and the impact of their chemistry, size, and surface groups on their interactions with the vasculature and cells of the brain following injury. The vast majority of the work in the field focuses on acute injury, and when the work is looked at closely, it suggests that nanoparticles rely on interactions with vascular and immune cells to alter the environment of the brain. Nonetheless, there are promising results from a number of approaches that lead to behavioral improvements coupled with neuroprotection that offer promise for therapeutic outcomes. The majority of approaches have been tested immediately following injury. It is not entirely clear what impact these approaches will have in chronic TBI, but being able to modulate inflammation specifically may have a role both during and after the acute phase of injury.

Thermo in the Time of COVID-19: Using Improvisation to Foster Discussion and Translating the Experience to Online Learning.

This paper covers teaching a graduate thermodynamics class as a seminar and using improvisational activities to foster community and discussion. The paper includes the experience of piloting improvisational activities online to help foster community for an entirely virtual version of the thermodynamics seminar class. Improvisational activities were found to help foster discussion in a thermodynamics seminar class, and some of these improvisational activities can be translated online in ways that may help to foster connection and community across the curriculum including online.

Development of a Sensitive Assay to Screen Nanoparticles in vitro for Complement Activation.

Nanomedicines are often recognized by the innate immune system as a threat, leading to unwanted clearance due to complement activation. This adverse reaction not only alters the bioavailability of the therapeutic but can also cause cardiopulmonary complications and death in a portion of the population. There is a need for tools for assessing complement response in the early stage of development of nanomedicines. Currently, quantifying complement-mediated response in vitro is limited due to differences between in vitro and in vivo responses for the same precursors, differences in the complement systems in different species, and lack of highly sensitive tools for quantifying the changes. Hence, we have worked on developing complement assay conditions and sample preparation techniques that can be highly sensitive in assessing the complement-mediated response in vitro mimicking the in vivo activity. We are screening the impact of incubation time, nanoparticle dosage, anticoagulants, and species of the donor in both blood and blood components. We have validated the optimal assay conditions by replicating the impact of zeta potential seen in vivo on complement activation in vitro. As observed in our previous in vivo studies, where nanoparticles with neutral zeta-potential were able to suppress complement response, the change in the complement biomarker was least for the neutral nanoparticles as well through our developed guidelines. These assay conditions provide a vital tool for assessing the safety of intravenously administered nanomedicines.