Synthetic extremophiles via species-specific formulations improve microbial therapeutics.

Microorganisms typically used to produce food and pharmaceuticals are now being explored as medicines and agricultural supplements. However, maintaining high viability from manufacturing until use remains an important challenge, requiring sophisticated cold chains and packaging. Here we report synthetic extremophiles of industrially relevant gram-negative bacteria (Escherichia coli Nissle 1917, Ensifer meliloti), gram-positive bacteria (Lactobacillus plantarum) and yeast (Saccharomyces boulardii). We develop a high-throughput pipeline to define species-specific materials that enable survival through drying, elevated temperatures, organic solvents and ionizing radiation. Using this pipeline, we enhance the stability of E. coli Nissle 1917 by more than four orders of magnitude over commercial formulations and demonstrate its capacity to remain viable while undergoing tableting and pharmaceutical processing. We further show, in live animals and plants, that synthetic extremophiles remain functional against enteric pathogens and as nitrogen-fixing plant supplements even after exposure to elevated temperatures. This synthetic, material-based stabilization enhances our capacity to apply microorganisms in extreme environments on Earth and potentially during exploratory space travel.

Drinkable in situ-forming tough hydrogels for gastrointestinal therapeutics.

Pills are a cornerstone of medicine but can be challenging to swallow. While liquid formulations are easier to ingest, they lack the capacity to localize therapeutics with excipients nor act as controlled release devices. Here we describe drug formulations based on liquid in situ-forming tough (LIFT) hydrogels that bridge the advantages of solid and liquid dosage forms. LIFT hydrogels form directly in the stomach through sequential ingestion of a crosslinker solution of calcium and dithiol crosslinkers, followed by a drug-containing polymer solution of alginate and four-arm poly(ethylene glycol)-maleimide. We show that LIFT hydrogels robustly form in the stomachs of live rats and pigs, and are mechanically tough, biocompatible and safely cleared after 24 h. LIFT hydrogels deliver a total drug dose comparable to unencapsulated drug in a controlled manner, and protect encapsulated therapeutic enzymes and bacteria from gastric acid-mediated deactivation. Overall, LIFT hydrogels may expand access to advanced therapeutics for patients with difficulty swallowing.

Oral Biologic Delivery: Advances Toward Oral Subunit, DNA, and mRNA Vaccines and the Potential for Mass Vaccination During Pandemics.

Oral vaccination enables pain-free and self-administrable vaccine delivery for rapid mass vaccination during pandemic outbreaks. Furthermore, it elicits systemic and mucosal immune responses. This protects against infection at mucosal surfaces, which may further enhance protection and minimize the spread of disease. The gastrointestinal (GI) tract presents a number of prospective mucosal inductive sites for vaccine targeting, including the oral cavity, stomach, and small intestine. However, currently available oral vaccines are effectively limited to live-attenuated and inactivated vaccines against enteric diseases. The GI tract poses a number of challenges,including degradative processes that digest biologics and mucosal barriers that limit their absorption. This review summarizes the approaches currently under development and future opportunities for oral vaccine delivery to established (intestinal) and relatively new (oral cavity, stomach) mucosal targets. Special consideration is given to recent advances in oral biologic delivery that offer promise as future platforms for the administration of oral vaccines.

Development of a long-acting direct-acting antiviral system for hepatitis C virus treatment in swine.

Chronic hepatitis C virus (HCV) infection is a leading cause of cirrhosis worldwide and kills more Americans than 59 other infections, including HIV and tuberculosis, combined. While direct-acting antiviral (DAA) treatments are effective, limited uptake of therapy, particularly in high-risk groups, remains a substantial barrier to eliminating HCV. We developed a long-acting DAA system (LA-DAAS) capable of prolonged dosing and explored its cost-effectiveness. We designed a retrievable coil-shaped LA-DAAS compatible with nasogastric tube administration and the capacity to encapsulate and release gram levels of drugs while resident in the stomach. We formulated DAAs in drug-polymer pills and studied the release kinetics for 1 mo in vitro and in vivo in a swine model. The LA-DAAS was equipped with ethanol and temperature sensors linked via Bluetooth to a phone application to provide patient engagement. We then performed a cost-effectiveness analysis comparing LA-DAAS to DAA alone in various patient groups, including people who inject drugs. Tunable release kinetics of DAAs was enabled for 1 mo with drug-polymer pills in vitro, and the LA-DAAS safely and successfully provided at least month-long release of sofosbuvir in vivo. Temperature and alcohol sensors could interface with external sources for at least 1 mo. The LA-DAAS was cost-effective compared to DAA therapy alone in all groups considered (base case incremental cost-effectiveness ratio $39,800). We believe that the LA-DAA system can provide a cost-effective and patient-centric method for HCV treatment, including in high-risk populations who are currently undertreated.

Preferences of Persons With or at Risk for Hepatitis C for Long-Acting Treatments.

BACKGROUND: Whereas safe, curative treatments for hepatitis C virus (HCV) have been available since 2015, there are still 58 million infected persons worldwide, and global elimination may require new paradigms. We sought to understand the acceptability of approaches to long-acting HCV treatment. METHODS: A cross-sectional, 43-question survey was administered to 1457 individuals with or at risk of HCV at 28 sites in 9 countries to assess comparative interest in a variety of long-acting strategies in comparison with oral pills. RESULTS: Among HCV-positive participants, 37.7% most preferred an injection, 5.6% an implant, and 6% a gastric residence device, as compared with 50.8% who stated they would most prefer taking 1-3 pills per day. When compared directly to taking pills, differences were observed in the relative preference for an injection based on age (P<.001), location (P<.001), and prior receipt of HCV treatment (P=.005) but not sex. When an implant was compared with pills, greater preference was represented by women (P=.01) and adults of younger ages (P=.01 per 5 years). Among participants without HCV, 49.5% believed that injections are stronger than pills and 34.7% preferred taking injections to pills. Among those at-risk participants who had received injectable medications in the past, 123 of 137 (89.8%) expressed willingness to receive one in the future. CONCLUSIONS: These data point to high acceptability of long-acting treatments, which for a substantial minority might even be preferred to pills for the treatment of HCV infection. Long-acting treatments for HCV infection might contribute to global efforts to eliminate hepatitis C.

Kirigami-inspired stents for sustained local delivery of therapeutics.

Implantable drug depots have the capacity to locally meet therapeutic requirements by maximizing local drug efficacy and minimizing potential systemic side effects. Tubular organs including the gastrointestinal tract, respiratory tract and vasculature all manifest with endoluminal disease. The anatomic distribution of localized drug delivery for these organs using existing therapeutic modalities is limited. Application of local depots in a circumferential and extended longitudinal fashion could transform our capacity to offer effective treatment across a range of conditions. Here we report the development and application of a kirigami-based stent platform to achieve this. The stents comprise a stretchable snake-skin-inspired kirigami shell integrated with a fluidically driven linear soft actuator. They have the capacity to deposit drug depots circumferentially and longitudinally in the tubular mucosa of the gastrointestinal tract across millimetre to multi-centimetre length scales, as well as in the vasculature and large airways. We characterize the mechanics of kirigami stents for injection, and their capacity to engage tissue in a controlled manner and deposit degradable microparticles loaded with therapeutics by evaluating these systems ex vivo and in vivo in swine. We anticipate such systems could be applied for a range of endoluminal diseases by simplifying dosing regimens while maximizing drug on-target effects through the sustained release of therapeutics and minimizing systemic side effects.

Mucus interaction to improve gastrointestinal retention and pharmacokinetics of orally administered nano-drug delivery systems.

Oral delivery of therapeutics is the preferred route of administration due to ease of administration which is associated with greater patient medication adherence. One major barrier to oral delivery and intestinal absorption is rapid clearance of the drug and the drug delivery system from the gastrointestinal (GI) tract. To address this issue, researchers have investigated using GI mucus to help maximize the pharmacokinetics of the therapeutic; while mucus can act as a barrier to effective oral delivery, it can also be used as an anchoring mechanism to improve intestinal residence. Nano-drug delivery systems that use materials which can interact with the mucus layers in the GI tract can enable longer residence time, improving the efficacy of oral drug delivery. This review examines the properties and function of mucus in the GI tract, as well as diseases that alter mucus. Three broad classes of mucus-interacting systems are discussed: mucoadhesive, mucus-penetrating, and mucolytic drug delivery systems. For each class of system, the basis for mucus interaction is presented, and examples of materials that inform the development of these systems are discussed and reviewed. Finally, a list of FDA-approved mucoadhesive, mucus-penetrating, and mucolytic drug delivery systems is reviewed. In summary, this review highlights the progress made in developing mucus-interacting systems, both at a research-scale and commercial-scale level, and describes the theoretical basis for each type of system.

Powering Implantable and Ingestible Electronics.

Implantable and ingestible biomedical electronic devices can be useful tools for detecting physiological and pathophysiological signals, and providing treatments that cannot be done externally. However, one major challenge in the development of these devices is the limited lifetime of their power sources. The state-of-the-art of powering technologies for implantable and ingestible electronics is reviewed here. The structure and power requirements of implantable and ingestible biomedical electronics are described to guide the development of powering technologies. These powering technologies include novel batteries that can be used as both power sources and for energy storage, devices that can harvest energy from the human body, and devices that can receive and operate with energy transferred from exogenous sources. Furthermore, potential sources of mechanical, chemical, and electromagnetic energy present around common target locations of implantable and ingestible electronics are thoroughly analyzed; energy harvesting and transfer methods befitting each energy source are also discussed. Developing power sources that are safe, compact, and have high volumetric energy densities is essential for realizing long-term in-body biomedical electronics and for enabling a new era of personalized healthcare.

Identification of bile acid and fatty acid species as candidate rapidly bactericidal agents for topical treatment of gonorrhoea.

BACKGROUND: Novel therapeutic strategies are urgently needed for Neisseria gonorrhoeae, given its increasing antimicrobial resistance. Treatment of oropharyngeal N. gonorrhoeae infections has proven particularly challenging, with most reported treatment failures of the first-line drug ceftriaxone occurring at this site and lower cure rates in recent trials of new antibiotics reported for oropharyngeal infections compared with other sites of infection. However, the accessibility of the oropharynx to topical therapeutics provides an opportunity for intervention. Local delivery of a therapeutic at a high concentration would enable the use of non-traditional antimicrobial candidates, including biological molecules that exploit underlying chemical sensitivities of N. gonorrhoeae but lack the potency or pharmacokinetic profiles required for effective systemic administration. METHODS: Two classes of molecules that are thought to limit gonococcal viability in vivo, bile acids and short- and medium-chain fatty acids, were examined for rapid bactericidal activity. RESULTS: The bile acids deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA), but not other bile acid species, exerted extremely rapid bactericidal properties against N. gonorrhoeae, reducing viability more than 100 000-fold after 1 min. The short-chain fatty acids formic acid and hexanoic acid shared this rapid bactericidal activity. All four molecules are effective against a phylogenetically diverse panel of N. gonorrhoeae strains, including clinical isolates with upregulated efflux pumps and resistance alleles to the most widely used classes of existing antimicrobials. DCA and CDCA are both approved therapeutics for non-infectious indications and are well-tolerated by cultured epithelial cells. CONCLUSIONS: DCA and CDCA are attractive candidates for further development as anti-gonococcal agents.

Genotype-targeted local therapy of glioma.

Aggressive neurosurgical resection to achieve sustained local control is essential for prolonging survival in patients with lower-grade glioma. However, progression in many of these patients is characterized by local regrowth. Most lower-grade gliomas harbor isocitrate dehydrogenase 1 (IDH1) or IDH2 mutations, which sensitize to metabolism-altering agents. To improve local control of IDH mutant gliomas while avoiding systemic toxicity associated with metabolic therapies, we developed a precision intraoperative treatment that couples a rapid multiplexed genotyping tool with a sustained release microparticle (MP) drug delivery system containing an IDH-directed nicotinamide phosphoribosyltransferase (NAMPT) inhibitor (GMX-1778). We validated our genetic diagnostic tool on clinically annotated tumor specimens. GMX-1778 MPs showed mutant IDH genotype-specific toxicity in vitro and in vivo, inducing regression of orthotopic IDH mutant glioma murine models. Our strategy enables immediate intraoperative genotyping and local application of a genotype-specific treatment in surgical scenarios where local tumor control is paramount and systemic toxicity is therapeutically limiting.

Historical Evolution and Provider Awareness of Inactive Ingredients in Oral Medications.

PURPOSE: A multitude of different versions of the same medication with different inactive ingredients are currently available. It has not been quantified how this has evolved historically. Furthermore, it is unknown whether healthcare professionals consider the inactive ingredient portion when prescribing medications to patients. METHODS: We used data mining to track the number of available formulations for the same medication over time and correlate the number of available versions in 2019 to the number of manufacturers, the years since first approval, and the number of prescriptions. A focused survey among healthcare professionals was conducted to query their consideration of the inactive ingredient portion of a medication when writing prescriptions. RESULTS: The number of available versions of a single medication have dramatically increased in the last 40 years. The number of available, different versions of medications are largely determined by the number of manufacturers producing this medication. Healthcare providers commonly do not consider the inactive ingredient portion when prescribing a medication. CONCLUSIONS: A multitude of available versions of the same medications provides a potentially under-recognized opportunity to prescribe the most suitable formulation to a patient as a step towards personalized medicine and mitigate potential adverse events from inactive ingredients.

Ultrasound-Mediated Delivery of RNA to Colonic Mucosa of Live Mice.

BACKGROUND & AIMS: It is a challenge to deliver nucleic acids to gastrointestinal (GI) tissues due to their size and need for intracellular delivery. They are also extremely susceptible to degradation by nucleases, which are ubiquitous in the GI tract. We investigated whether ultrasound, which can permeabilize tissue through a phenomenon known as transient cavitation, can be used to deliver RNA to the colonic mucosa of living mice. METHODS: We investigated delivery of fluorescently labeled permeants to colon tissues of Yorkshire pigs ex vivo and mice in vivo. Colon tissues were collected and fluorescence was measured by confocal microscopy. We then evaluated whether ultrasound is effective in delivering small interfering (si)RNA to C57BL/6 mice with dextran sodium sulfate-induced colitis. Some mice were given siRNA against tumor necrosis factor (Tnf) mRNA for 6 days; colon tissues were collected and analyzed histologically and TNF protein levels measured by enzyme-linked immunosorbent assay. Feces were collected and assessed for consistency and occult bleeding. We delivered mRNA encoding firefly luciferase to colons of healthy C57BL/6 mice. RESULTS: Exposure of ex vivo pig colon tissues to 20 kHz ultrasound for 1 minute increased the level of delivery of 3 kDa dextran 7-fold compared with passive diffusion (P = .037); 40 kHz ultrasound application for 0.5 seconds increased the delivery 3.3-fold in living mice (P = .041). Confocal microscopy analyses of colon tissues from pigs revealed regions of punctuated fluorescent dextran signal, indicating intracellular delivery of macromolecules. In mice with colitis, ultrasound delivery of unencapsulated siRNA against Tnf mRNA reduced protein levels of TNF in colon tissues, compared with mice with colitis given siRNA against Tnf mRNA without ultrasound (P ≤ .014), and reduced features of inflammation (P ≤ 4.1 × 10-5). Separately, colons of mice administered an mRNA encoding firefly luciferase with ultrasound and the D-luciferin substrate had levels of bioluminescence 11-fold greater than colons of mice given the mRNA alone (P = .0025). Ultrasound exposures of 40 kHz ultrasound for 0.5 seconds were well tolerated, even in mice with acute colitis. CONCLUSIONS: Ultrasound can be used to deliver mRNAs and siRNAs to the colonic mucosa of mice and knock down expression of target mRNAs.

Convergence for Translation: Drug-Delivery Research in Multidisciplinary Teams.

The void between drug discovery and successful translation to humans is vast. In order to develop effective solutions, convergence of a multidisciplinary team with a range of expertise is required. In this Essay, examples of successful sustained delivery systems for antimalarials and antiretrovirals, as well as to the gastrointestinal tract are discussed.

Whole-Exome Sequencing Analyses of Inflammatory Bowel Disease-Associated Colorectal Cancers.

BACKGROUND & AIMS: A long duration of inflammatory bowel disease (IBD) increases the risk for colorectal cancer. Mutation analysis of limited numbers of genes has indicated that colorectal tumors that develop in patients with IBD differ from those of patients without IBD. We performed whole-exome sequencing analyses to characterize the genetic landscape of these tumors. METHODS: We collected colorectal tumor and non-neoplastic tissues from 31 patients with IBD and colorectal cancer (15 with ulcerative colitis, 14 with Crohn's disease, and 2 with indeterminate colitis) and performed whole-exome sequencing analyses of the microdissected tumor and matched nontumor tissues. We identified somatic alterations by comparing matched specimens. The prevalence of mutations in sporadic colorectal tumors was obtained from previously published exome-sequencing studies. RESULTS: Two specimens had somatic mutations in the DNA proofreading or mismatch repair genes POLE, MLH1, and MSH6 and the tumor cells had a hypermutable phenotype. The remaining tumors had, on average, 71 alterations per sample. TP53 was the most commonly mutated gene, with prevalence similar to that of sporadic colorectal tumors (63% of cases). However, tumors from the patients with IBD had a different mutation spectrum. APC and KRAS were mutated at significantly lower rates in tumors from patients with IBD than in sporadic colorectal tumors (13% and 20% of cases, respectively). Several genes were mutated more frequently or uniquely in tumors from patients with IBD, including SOX9 and EP300 (which encode proteins in the WNT pathway), NRG1 (which encodes an ERBB ligand), and IL16 (which encodes a cytokine). Our study also revealed recurrent mutations in components of the Rho and Rac GTPase network, indicating a role for noncanonical WNT signaling in development of colorectal tumors in patients with IBD. CONCLUSIONS: Colorectal tumors that develop in patients with IBD have distinct genetic features from sporadic colorectal tumors. These findings could be used to develop disease-specific markers for diagnosis and treatment of patients with IBD and colorectal cancer.

Simple battery armor to protect against gastrointestinal injury from accidental ingestion.

Inadvertent battery ingestion in children and the associated morbidity and mortality results in thousands of emergency room visits every year. Given the risk for serious electrochemical burns within hours of ingestion, the current standard of care for the treatment of batteries in the esophagus is emergent endoscopic removal. Safety standards now regulate locked battery compartments in toys, which have resulted in a modest reduction in inadvertent battery ingestion; specifically, 3,461 ingestions were reported in 2009, and 3,366 in 2013. Aside from legislation, minimal technological development has taken place at the level of the battery to limit injury. We have constructed a waterproof, pressure-sensitive coating, harnessing a commercially available quantum tunneling composite. Quantum tunneling composite coated (QTCC) batteries are nonconductive in the low-pressure gastrointestinal environment yet conduct within the higher pressure of standard battery housings. Importantly, this coating technology enables most battery-operated equipment to be powered without modification. If these new batteries are swallowed, they limit the external electrolytic currents responsible for tissue injury. We demonstrate in a large-animal model a significant decrease in tissue injury with QTCC batteries compared with uncoated control batteries. In summary, here we describe a facile approach to increasing the safety of batteries by minimizing the risk for electrochemical burn if the batteries are inadvertently ingested, without the need for modification of most battery-powered devices.

A pH-responsive supramolecular polymer gel as an enteric elastomer for use in gastric devices.

Devices resident in the stomach-used for a variety of clinical applications including nutritional modulation for bariatrics, ingestible electronics for diagnosis and monitoring, and gastric-retentive dosage forms for prolonged drug delivery-typically incorporate elastic polymers to compress the devices during delivery through the oesophagus and other narrow orifices in the digestive system. However, in the event of accidental device fracture or migration, the non-degradable nature of these materials risks intestinal obstruction. Here, we show that an elastic, pH-responsive supramolecular gel remains stable and elastic in the acidic environment of the stomach but can be dissolved in the neutral-pH environment of the small and large intestines. In a large animal model, prototype devices with these materials as the key component demonstrated prolonged gastric retention and safe passage. These enteric elastomers should increase the safety profile for a wide range of gastric-retentive devices.

Microneedle Sensors for Point-of-Care Diagnostics.

Point-of-care (POC) has the capacity to support low-cost, accurate and real-time actionable diagnostic data. Microneedle sensors have received considerable attention as an emerging technique to evolve blood-based diagnostics owing to their direct and painless access to a rich source of biomarkers from interstitial fluid. This review systematically summarizes the recent innovations in microneedle sensors with a particular focus on their utility in POC diagnostics and personalized medicine. The integration of various sensing techniques, mostly electrochemical and optical sensing, has been established in diverse architectures of "lab-on-a-microneedle" platforms. Microneedle sensors with tailored geometries, mechanical flexibility, and biocompatibility are constructed with a variety of materials and fabrication methods. Microneedles categorized into four types: metals, inorganics, polymers, and hydrogels, have been elaborated with state-of-the-art bioengineering strategies for minimally invasive, continuous, and multiplexed sensing. Microneedle sensors have been employed to detect a wide range of biomarkers from electrolytes, metabolites, polysaccharides, nucleic acids, proteins to drugs. Insightful perspectives are outlined from biofluid, microneedles, biosensors, POC devices, and theragnostic instruments, which depict a bright future of the upcoming personalized and intelligent health management.

Thiol Coordination Softens Liquid Metal Particles To Improve On-Demand Conductivity.

Achieving tunable rupturing of eutectic gallium indium (EGaIn) particles holds great significance in flexible electronic applications, particularly pressure sensors. We tune the mechanosensitivity of EGaIn particles by preparing them in toluene with thiol surfactants and demonstrate an improvement over typical preparations in ethanol. We observe, across multiple length scales, that thiol surfactants and the nonpolar solvent synergistically reduce the applied stress requirements for electromechanical actuation. At the nanoscale, dodecanethiol and propanethiol in toluene suppress gallium oxide growth, as characterized by transmission electron microscopy and X-ray photoelectron spectroscopy. Quantitative AFM imaging produces force-indentation curves and height images, while conductive AFM measures current while probing individual EGaIn particles. As the applied force increases, thiolated particles demonstrate intensified softening, rupturing, and stress-induced electrical activation at forces 40% lower than those for bare particles in ethanol. To confirm that thiolation facilitates rupturing at the macroscale, a laser is used to ablate samples of EGaIn particles. Scanning electron microscopy and resistance measurements across macroscopic samples confirm that thiolated EGaIn particles coalesce to exhibit electrical activation at 0.1 W. Particles prepared in ethanol, however, require 3 times higher laser power to demonstrate a similar behavior. This unique collection of advanced techniques demonstrates that our particle synthesis conditions can facilitate on-demand functionality to benefit electronic applications.

Percutaneous Intratumoral Immunoadjuvant Gel Increases the Abscopal Effect of Cryoablation for Checkpoint Inhibitor Resistant Cancer.

Percutaneous cryoablation is a common clinical therapy for metastatic and primary cancer. There are rare clinical reports of cryoablation inducing regression of distant metastases, known as the "abscopal" effect. Intratumoral immunoadjuvants may be able to augment the abscopal rate of cryoablation, but existing intratumoral therapies suffer from the need for frequent injections and inability to confirm target delivery, leading to poor clinical trial outcomes. To address these shortcomings, an injectable thermoresponsive gel-based controlled release formulation is developed for the FDA-approved Toll-like-receptor 7 (TLR7) agonist imiquimod ("Imigel") that forms a tumor-resident depot upon injection and contains a contrast agent for visualization under computed tomography (CT). The poly-lactic-co-glycolic acid-polyethylene glycol-poly-lactic-co-glycolic acid (PLGA-PEG-PLGA)-based amphiphilic copolymer gel's underlying micellar nature enables high drug concentration and a logarithmic release profile that is additive with the neo-antigen release from cryoablation, requiring only a single injection. Rheological testing demonstrated the thermoresponsive increase in viscosity at body temperature and radio-opacity via microCT. Its ability to significantly augment the abscopal rate of cryoablation is demonstrated in otherwise immunotherapy resistant metastatic tumors in two aggressive colorectal and breast cancer dual tumor models with an all or nothing response, responders generally demonstrating complete regression of bilateral tumors in 90-day survival studies.