Mitochondria dysregulation contributes to secondary neurodegeneration progression post-contusion injury in human 3D in vitro triculture brain tissue model.

Traumatic Brain injury-induced disturbances in mitochondrial fission-and-fusion dynamics have been linked to the onset and propagation of neuroinflammation and neurodegeneration. However, cell-type-specific contributions and crosstalk between neurons, microglia, and astrocytes in mitochondria-driven neurodegeneration after brain injury remain undefined. We developed a human three-dimensional in vitro triculture tissue model of a contusion injury composed of neurons, microglia, and astrocytes and examined the contributions of mitochondrial dysregulation to neuroinflammation and progression of injury-induced neurodegeneration. Pharmacological studies presented here suggest that fragmented mitochondria released by microglia are a key contributor to secondary neuronal damage progression after contusion injury, a pathway that requires astrocyte-microglia crosstalk. Controlling mitochondrial dysfunction thus offers an exciting option for developing therapies for TBI patients.

Functional bioengineered tissue models of neurodegenerative diseases.

Aging-associated neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases remain poorly understood and no disease-modifying treatments exist despite decades of investigation. Predominant in vitro (e.g., 2D cell culture, organoids) and in vivo (e.g., mouse) models of these diseases are insufficient mimics of human brain tissue structure and function and of human neurodegenerative pathobiology, and have thus contributed to this collective translational failure. This has been a longstanding challenge in the field, and new strategies are required to address both fundamental and translational needs. Bioengineered tissue culture models constitute a class of promising alternatives, as they can overcome the low cell density, poor nutrient exchange, and long term culturability limitations of existing in vitro models. Further, they can reconstruct the structural, mechanical, and biochemical cues of native brain tissue, providing a better mimic of human brain tissues for in vitro pathobiological investigation and drug development. We discuss bioengineering techniques for the generation of these neurodegenerative tissue models, including biomaterials-, organoid-, and microfluidics-based approaches, and design considerations for their construction. To aid the development of the next generation of functional neurodegenerative disease models, we discuss approaches to incorporate greater cellular diversity and simulate aging processes within bioengineered brain tissues.

Data analytics-guided rational design of antimicrobial nanomedicines against opportunistic, resistant pathogens.

Nanoparticle carriers can improve antibiotic efficacy by altering drug biodistribution. However, traditional screening is impracticable due to a massive dataspace. A hybrid informatics approach was developed to identify polymer, antibiotic, and particle determinants of antimicrobial nanomedicine activity against Burkholderia cepacia, and to model nanomedicine performance. Polymer glass transition temperature, drug octanol-water partition coefficient, strongest acid dissociation constant, physiological charge, particle diameter, count and mass mean polydispersity index, zeta potential, fraction drug released at 2 h, and fraction release slope at 2 h were highly correlated with antimicrobial performance. Graph analysis provided dimensionality reduction while preserving nonlinear descriptor-property relationships, enabling accurate modeling of nanomedicine performance. The model successfully predicted particle performance in holdout validation, with moderate accuracy at rank-ordering. This data analytics-guided approach provides an important step toward the development of a rational design framework for antimicrobial nanomedicines against resistant infections by selecting appropriate carriers and payloads for improved potency.

Screening neuroprotective compounds in herpes-induced Alzheimer's disease cell and 3D tissue models.

Alzheimer's Disease (AD) is a neurodegenerative disorder that can cause life-altering and debilitating cognitive decline. AD's etiology is poorly understood, and no disease-modifying therapeutics exist. Here, we describe the use of 2D and 3D tissue culture models of herpesvirus-induced AD, which recapitulate hallmark disease features of plaque formation, gliosis, neuroinflammation, and impaired neuronal signaling, to screen a panel of 21 medications, supplements, and nutraceuticals with purported neuroprotective benefits. This screen identified green tea catechins and resveratrol as having strong anti-plaque properties, functional neuroprotective benefits, and minimal neurotoxicity, providing support for their further investigation as AD preventives and therapies. Two other candidates, citicoline and metformin, reduced plaque formation and were minimally toxic, but did not protect against virus-induced impairments in neuronal signaling. This study establishes a simple platform for rapidly screening and characterizing AD compounds of interest in 2D and 3D human cortical tissue models representing physiologically relevant disease features.

Bovine NK-lysin peptides exert potent antimicrobial activity against multidrug-resistant Salmonella outbreak isolates.

Multidrug-resistant (MDR) Salmonella is a threat to public health. Non-antibiotic therapies could serve as important countermeasures to control MDR Salmonella outbreaks. In this study, antimicrobial activity of cationic α-helical bovine NK-lysin-derived antimicrobial peptides was evaluated against MDR Salmonella outbreak isolates. NK2A and NK2B strongly inhibited MDR Salmonella growth while NK1 and NK2C showed minimum-to-no growth inhibition. Scrambled-NK2A, which is devoid of α-helicity but has the same net positive charge as NK2A, also failed to inhibit bacterial growth. Incubation of negatively charged MDR Salmonella with NK2A showed increased Zeta potential, indicating bacterial-peptide electrostatic attraction. Confocal and transmission electron microscopy studies revealed NK2A-mediated damage to MDR Salmonella membranes. LPS inhibited NK2A-mediated growth suppression in a dose-dependent response, suggesting irreversible NK2A-LPS binding. LPS-NK2A binding and bacterial membrane disruption was also confirmed via electron microscopy using gold nanoparticle-NK2A conjugates. Finally, NK2A-loaded polyanhydride nanoparticles showed sustained peptide delivery and anti-bacterial activity. Together, these findings indicate that NK2A α-helicity and positive charge are prerequisites for antimicrobial activity and that MDR Salmonella killing is mediated by direct interaction of NK2A with LPS and the inner membrane, leading to bacterial membrane permeabilization. With further optimization using nano-carriers, NK2A has the potential to become a potent anti-MDR Salmonella agent.

Biodistribution of degradable polyanhydride particles in Aedes aegypti tissues.

Insecticide resistance poses a significant threat to the control of arthropods that transmit disease agents. Nanoparticle carriers offer exciting opportunities to expand the armamentarium of insecticides available for public health and other pests. Most chemical insecticides are delivered by contact or feeding, and from there must penetrate various biological membranes to reach target organs and kill the pest organism. Nanoparticles have been shown to improve bioactive compound navigation of such barriers in vertebrates, but have not been well-explored in arthropods. In this study, we explored the potential of polyanhydride micro- and nanoparticles (250 nm- 3 µm), labeled with rhodamine B to associate with and/or transit across insect biological barriers, including the cuticle, epithelium, midgut and ovaries, in female Ae. aeygpti mosquitoes. Mosquitoes were exposed using conditions to mimic surface contact with a residual spray or paint, topical exposure to mimic contact with aerosolized insecticide, or per os in a sugar meal. In surface contact experiments, microparticles were sometimes observed in association with the exterior of the insect cuticle. Nanoparticles were more uniformly distributed across exterior tissues and present at higher concentrations. Furthermore, by surface contact, topical exposure, or per os, particles were detected in internal organs. In every experiment, amphiphilic polyanhydride nanoparticles associated with internal tissues to a higher degree than hydrophobic nanoparticles. In vitro, nanoparticles associated with Aedes aegypti Aag2 cells within two hours of exposure, and particles were evident in the cytoplasm. Further studies demonstrated that particle uptake is dependent on caveolae-mediated endocytosis. The propensity of these nanoparticles to cross biological barriers including the cuticle, to localize in target tissue sites of interest, and to reach the cytoplasm of cells, provides great promise for targeted delivery of insecticidal candidates that cannot otherwise reach these cellular and subcellular locations.

High-Throughput Synthesis and Screening of Rapidly Degrading Polyanhydride Nanoparticles.

Combinatorial techniques can accelerate the discovery and development of polymeric nanodelivery devices by pairing high-throughput synthesis with rapid materials characterization. Biodegradable polyanhydrides demonstrate tunable release, high cellular internalization, and dose sparing properties when used as nanodelivery devices. This nanoparticle platform shows promising potential for small molecule drug delivery, but the pace of understanding and rational design of these nanomedicines is limited by the low throughput of conventional characterization. This study reports the use of a high-throughput method to synthesize libraries of a newly synthesized, rapidly eroding polyanhydride copolymer based on 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and sebacic acid (SA) monomers. The high-throughput method enabled efficient screening of copolymer microstructure, revealing weak block-type and alternating architectures. The high-throughput method was adapted to synthesize nanoparticle libraries encapsulating hydrophobic model drugs. Drug release from these nanoparticles was rapid, with a majority of the payload released within 3 days. Drug release was dramatically slowed at acidic pH, which could be useful for oral drug delivery. Rhodamine B (RhoB) release kinetics generally followed patterns of polymer erosion kinetics, while Coomassie brilliant blue (CBB) released the fastest from the slowest degrading polymer chemistry and vice versa. These differences in trends between copolymer chemistry and release kinetics were hypothesized to arise from differences in mixing thermodynamics. A high-throughput method was developed to synthesize polymer-drug film libraries and characterize mixing thermodynamics by melting point depression. Rhodamine B had a negative χ for all copolymers with <30 mol % CPTEG tested, indicating a tendency toward miscibility. By contrast, CBB χ increased, eventually becoming positive near 15:85 CPTEG:SA, with increasing CPTEG content. This indicates an increasing tendency toward phase separation in CPTEG-rich copolymers. These in vitro results screening polymer-drug interactions showed good agreement with in silico predictions from Hansen solubility parameter estimation and were able to explain the observed differences in model drug release trends.

Data Analytics Approach for Rational Design of Nanomedicines with Programmable Drug Release.

Drug delivery vehicles can improve the functional efficacy of existing antimicrobial therapies by improving biodistribution and targeting. A critical property of such nanomedicine formulations is their ability to control the release kinetics of their payloads. The combination of (and interactions among) polymer, drug, and nanoparticle properties gives rise to nonlinear behavioral relationships and large data space. These factors complicate both first-principles modeling and screening of nanomedicine formulations. Predictive analytics may offer a more efficient approach toward the rational design of nanomedicines by identifying key descriptors and correlating them to nanoparticle release behavior. In this work, antibiotic release kinetics data were generated from polyanhydride nanoparticle formulations with varying copolymer compositions, encapsulated drug type, and drug loading. Four antibiotics, doxycycline, rifampicin, chloramphenicol, and pyrazinamide, were used. Linear manifold learning methods were used to relate drug release properties with polymer, drug, and nanoparticle properties, and key descriptors were identified that are highly correlated with release properties. However, these linear methods could not predict release behavior. Nonlinear multivariate modeling based on graph theory was then used to deconvolute the governing relationships between these properties, and predictive models were generated to rapidly screen lead nanomedicine formulations with desirable release properties with minimal nanoparticle characterization. Release kinetics predictions of two drugs containing atoms not included in the model showed good agreement with experimental results, validating the model and indicating its potential to virtually explore new polymer and drug pairs not included in the training data set. The models were shown to be robust after the inclusion of these new formulations, in that the new inclusions did not significantly change model regression. This approach provides the first step toward the development of a framework that can be used to rationally design nanomedicine formulations by selecting the appropriate carrier for a drug payload to program desirable release kinetics.

Nanotherapeutic provides dose sparing and improved antimicrobial activity against Brucella melitensis infections.

New therapies are needed to treat chronic bacterial diseases and intracellular pathogens, in particular, are very difficult to manage. The use of nanotherapeutics represents an approach to exploit size and charge of biological membranes to overcome barriers for treatment of intracellular pathogens including Brucella melitensis. In this work, polyanhydride nanoparticles comprised of copolymers of sebacic acid, 1,6-bis(p-carboxyphenoxy)hexane, and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane were synthesized to encapsulate antimicrobial compounds doxycycline and rifampicin. The nanoparticles demonstrated sustained release of rifampicin for a week with the antimicrobial activity peaking at 72 h and lasting up to a week. Treatment of B. melitensis infected macrophages with rifampicin-containing nanoparticles rapidly eliminated viable intracellular bacteria following 48 h of treatment and pretreatment with the nano-formulations prevented intracellular infection in contrast to soluble drug controls. Treatment of infected BALB/c mice with a nanoparticle cocktail containing doxycycline and rifampicin for five days decreased bacterial burden by three log10 in the liver. Extended release of antibiotics was demonstrated in vivo by treating B. melitensis infected mice with the standard therapy of daily 0.5 mg doxycycline dose or single 0.5 mg doxycycline-encapsulated nanoparticles delivered once a week. After 3 weeks, bacterial counts in spleen and liver were statistically equal between animals treated with the weekly nano-formulation and daily soluble drug, representing a seven-fold dose sparing. Altogether, these results demonstrated that the use of nanotherapeutics was successful at increasing antimicrobial efficacy and improving in vivo activity through a combination of intracellular delivery, dose sparing, and extended release in treating chronic bacterial infections. This platform technology can also provide benefits for drug delivery against other chronic intracellular bacterial pathogens, including Mycobacterium and Burkholderia species, including treatments against antibiotic-resistant infections.

Automated High-Throughput Synthesis of Protein-Loaded Polyanhydride Nanoparticle Libraries.

The development of high-throughput techniques and combinatorial libraries can facilitate rapid synthesis and screening of biomaterial-based nanocarriers for drug and vaccine delivery. This study describes a high-throughput method using an automated robot for synthesizing polyanhydride nanoparticles encapsulating proteins. Polyanhydrides are a class of safe and biodegradable polymers that have been widely used as drug and vaccine delivery vehicles. The robot contains a multiplexed homogenizer and has the capacity to handle parallel streams of monomer or polymer solutions to synthesize polymers and/or nanoparticles. Copolymer libraries were synthesized using the monomers sebacic acid, 1,6-bis( p-carboxyphenoxy)hexane, and 1,8-bis( p-carboxyphenoxy)-3,6-dioxactane and compared to conventionally synthesized copolymers. Nanoparticle libraries of varying copolymer compositions encapsulating the model antigen ovalbumin were synthesized using flash nanoprecipitation. The amount of the surfactant Span 80 was varied to test its effect on protein encapsulation efficiency as well as antigen release kinetics. It was observed that, although the amount of surfactant did not significantly affect protein release rate, its presence enhanced protein encapsulation efficiency. Protein burst and release kinetics from conventionally and combinatorially synthesized nanoparticles were similar even though particles synthesized using the high-throughput technique were smaller. Finally, it was demonstrated that the high-throughput method could be adapted to functionalize the surface of particle libraries to aid in the design and screening of targeted drug and vaccine delivery systems. These results suggest that the new high-throughput method is a viable alternative to conventional methods for synthesizing and screening protein and vaccine delivery vehicles.

Ligand-cascading nano-delivery devices to enable multiscale targeting of anti-neurodegenerative therapeutics.

Neurodegenerative diseases are a debilitating set of conditions that affect a significant fraction of the world's population, and this fraction is expected to increase as the population ages. Many therapeutic strategies have been explored to treat the pathological mechanisms of neurodegenerative diseases, but multiple sequential hurdles to central nervous system (CNS) delivery, including the blood-brain barrier (BBB), diseased neuronal membranes, and the organelle barrier, make drug delivery challenging and necessitate the use of innovative strategies to target and cross each barrier. Advances in drug delivery technology have the potential to improve the standard of treatment for neurodegenerative diseases by enhancing local drug concentration at the pathologically relevant cells and organelles. Furthermore, ligand-cascading nano-delivery devices could address these issues by sequentially presenting targeting ligands for crossing each of the aforementioned hurdles. In this review, we provide an overview of ligand technologies that enable BBB transcytosis, localization to or internalization in diseased neuronal cells, and localization at the organelle of interest. We summarize recent strategies for sequentially presenting pertinent ligands at each hurdle to CNS delivery. These ligand-cascade strategies will enable rational design of nano-delivery devices for multiscale targeting of anti-neurodegenerative therapeutics.

Repurposing endogenous type I CRISPR-Cas systems for programmable gene repression.

CRISPR-Cas systems have shown tremendous promise as heterologous tools for genome editing and transcriptional regulation. Because these RNA-directed immune systems are found in most prokaryotes, an opportunity exists to harness the endogenous systems as convenient tools in these organisms. Here, we report that the Type I-E CRISPR-Cas system in Escherichia coli can be co-opted for programmable transcriptional repression. We found that deletion of the signature cas3 gene converted this immune system into a programmable gene regulator capable of reversible gene silencing of heterologous and endogenous genes. Targeting promoter regions yielded the strongest repression, whereas targeting coding regions showed consistent strand bias. Furthermore, multi-targeting CRISPR arrays could generate complex phenotypes. This strategy offers a simple approach to convert many endogenous Type I systems into transcriptional regulators, thereby expanding the available toolkit for CRISPR-mediated genetic control while creating new opportunities for genome-wide screens and pathway engineering.