Perfluorooctanesulfonic Acid Alters Pro-Cancer Phenotypes and Metabolic and Transcriptional Signatures in Testicular Germ Cell Tumors.

The potential effects of poly- and perfluoroalkyl substances (PFAS) are a recently emergent human and environmental health concern. There is a consistent link between PFAS exposure and cancer, but the mechanisms are poorly understood. Although epidemiological evidence supporting PFAS exposure and cancer in general is conflicting, there is relatively strong evidence linking PFAS and testicular germ cell tumors (TGCTs). However, no mechanistic studies have been performed to date concerning PFAS and TGCTs. In this report, the effects of the legacy PFAS perfluorooctanesulfonic acid (PFOS) and the newer "clean energy" PFAS lithium bis(trifluoromethylsulfonyl)imide (LiTFSi, called HQ-115), on the tumorigenicity of TGCTs in mice, TGCT cell survival, and metabolite production, as well as gene regulation were investigated. In vitro, the proliferation and survival of both chemo-sensitive and -resistant TGCT cells were minimally affected by a wide range of PFOS and HQ-115 concentrations. However, both chemicals promoted the growth of TGCT cells in mouse xenografts at doses consistent with human exposure but induced minimal acute toxicity, as assessed by total body, kidney, and testis weight. PFOS, but not HQ-115, increased liver weight. Transcriptomic alterations of PFOS-exposed normal mouse testes were dominated by cancer-related pathways and gene expression alterations associated with the H3K27me3 polycomb pathway and DNA methylation, epigenetic pathways that were previously showed to be critical for the survival of TGCT cells after cisplatin-based chemotherapy. Similar patterns of PFOS-mediated gene expression occurred in PFOS-exposed cells in vitro. Metabolomic studies revealed that PFOS also altered metabolites associated with steroid biosynthesis and fatty acid metabolism in TGCT cells, consistent with the proposed ability of PFAS to mimic fatty acid-based ligands controlling lipid metabolism and the proposed role of PFAS as endocrine disrupters. Our data, is the first cell and animal based study on PFAS in TGCTs, support a pro-tumorigenic effect of PFAS on TGCT biology and suggests epigenetic, metabolic, and endocrine disruption as potential mechanisms of action that are consistent with the non-mutagenic nature of the PFAS class.

PFAS assessment in fish - Samples from Illinois waters.

Per- and Polyfluoroalkyl substances (PFAS) have been widely used in various industries, including pesticide production, electroplating, packaging, paper making, and the manufacturing of water-resistant clothes. This study investigates the levels of PFAS in fish tissues collected from four target waterways (15 sampling points) in the northwestern part of Illinois during 2021-2022. To assess accumulation, concentrations of 17 PFAS compounds were evaluated in nine fish species to potentially inform on exposure risks to local sport fishing population via fish consumption. At least four PFAS (PFHxA, PFHxS, PFOS, and PFBS) were detected at each sampling site. The highest concentrations of PFAS were consistently found in samples from the Rock River, particularly in areas near urban and industrial activities. PFHxA emerged as the most accumulated PFAS in the year 2022, while PFBS and PFOS dominated in 2021. Channel Catfish exhibited the highest PFAS content across different fish species, indicating its bioaccumulation potential across the food chain. Elevated levels of PFOS were observed in nearly all fish, indicating the need for careful consideration of fish consumption. Additional bioaccumulation data in the future years is needed to shed light on the sources and PFAS accumulation potential in aquatic wildlife in relation to exposures for potential health risk assessment.

Exosome-coated oxygen nanobubble-laden hydrogel augments intracellular delivery of exosomes for enhanced wound healing.

Wound healing is an obvious clinical concern that can be hindered by inadequate angiogenesis, inflammation, and chronic hypoxia. While exosomes derived from adipose tissue-derived stem cells have shown promise in accelerating healing by carrying therapeutic growth factors and microRNAs, intracellular cargo delivery is compromised in hypoxic tissues due to activated hypoxia-induced endocytic recycling. To address this challenge, we have developed a strategy to coat oxygen nanobubbles with exosomes and incorporate them into a polyvinyl alcohol/gelatin hybrid hydrogel. This approach not only alleviates wound hypoxia but also offers an efficient means of delivering exosome-coated nanoparticles in hypoxic conditions. The self-healing properties of the hydrogel, along with its component, gelatin, aids in hemostasis, while its crosslinking bonds facilitate hydrogen peroxide decomposition, to ameliorate wound inflammation. Here, we show the potential of this multifunctional hydrogel for enhanced healing, promoting angiogenesis, facilitating exosome delivery, mitigating hypoxia, and inhibiting inflammation in a male rat full-thickness wound model.

In vitro toxicity of Lithium bis(trifluoromethanesulfonyl)imide (LiTFSI) on Human Renal and Hepatoma Cells.

We evaluate the cytotoxicity, intracellular redox conditions, apoptosis, and methylation of DNMTs/TETs upon exposure to LiTFSI, a novel Per and Polyfluoroalkyl Substances (PFAS) commonly found in lithium-ion batteries, on human renal carcinoma cells (A498) and hepatoma cells (HepG2). The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay showed both Perfluorooctane sulfonate (PFOS) and Lithium bis(trifluoromethanesulfonyl)imide (LiTFSI) had a dose-dependent effect on A498 and HepG2, with LiTFSI being less toxic. Intracellular redox conditions were assessed with a microplate reader and confocal, which showed a significant decrease in Reactive Oxygen Species (ROS) levels and an increase in Superoxide dismutase (SOD) content in both cells. Exposure to LiTFSI enhanced cell apoptosis, with HepG2 being more susceptible than A498. Quantitative analysis of mRNA expression levels of 19 genes associated with kidney injury, methylation, lipid metabolism and transportation was performed. LiTFSI exposure impacted kidney function by downregulating smooth muscle alpha-actin (Acta2) and upregulating transforming growth factor beta 1 (Tgfb1), B-cell lymphoma 2-like 1) Bcl2l1, hepatitis A virus cellular receptor 1 (Harvcr1), nuclear factor erythroid 2-like 2 (Nfe2l2), and hairy and enhancer of split 1 (Hes1) expression. LiTFSI exposure also affected the abundance of transcripts associated with DNA methylation by the expression of ten-eleven translocation (TET) and DNA methyltransferase (DNMT) genes. Furthermore, LiTFSI exposure induced an increase in lipid anabolism and alterations in lipid catabolism in HepG2. Our results provide new insight on the potential role of a new contaminant, LiTFSI in the regulation of oxidative stress, apoptosis and methylation in human renal carcinoma and hepatoma cells.

Comparative hepatotoxicity of novel lithium bis(trifluoromethanesulfonyl)imide (LiTFSI, ie. HQ-115) and legacy Perfluorooctanoic acid (PFOA) in male mice: Insights into epigenetic mechanisms and pathway-specific responses.

Lithium Bis(trifluoromethanesulfonyl)imide (LiTFSI ie. HQ-115), a polymer electrolyte used in energy applications, has been detected in the environment, yet its health risks and environmental epigenetic effects remain unknown. This study aims to unravel the potential health risks associated with LiTFSI, investigate the role of DNA methylation-induced toxic mechanisms in its effects, and compare its hepatotoxic impact with the well-studied Perfluorooctanoic Acid (PFOA). Using a murine model, six-week-old male CD1 mice were exposed to 10 and 20 mg/kg/day of each chemical for 14 days as 14-day exposure and 1 and 5 mg/kg/day for 30 days as 30-day exposure. Results indicate that PFOA exposure induced significant hepatotoxicity, characterized by liver enlargement, and elevated serum biomarkers. In contrast, LiTFSI exposure showed lower hepatotoxicity, accompanied by mild liver injuries. Despite higher bioaccumulation of PFOA in serum, LiTFSI exhibited a similar range of liver concentrations compared to PFOA. Reduced Representative Bisulfite Sequencing (RRBS) analysis revealed distinct DNA methylation patterns between 14-day and 30-day exposure for the two compounds. Both LiTFSI and PFOA implicated liver inflammatory pathways and lipid metabolism. Transcriptional results showed that differentially methylated regions in both exposures are enriched with cancer/disease-related motifs. Furthermore, Peroxisome proliferator-activated receptor alpha (PPARα), a regulator of lipid metabolism, was upregulated in both exposures, with downstream genes indicating potential oxidative damages. Overall, LiTFSI exhibits distinct hepatotoxicity profiles, emphasizing the need for comprehensive assessment of emerging PFAS compounds.

Nano oxygen chamber by cascade reaction for hypoxia mitigation and reactive oxygen species scavenging in wound healing.

Hypoxia, excessive reactive oxygen species (ROS), and impaired angiogenesis are prominent obstacles to wound healing following trauma and surgical procedures, often leading to the development of keloids and hypertrophic scars. To address these challenges, a novel approach has been proposed, involving the development of a cascade enzymatic reaction-based nanocarriers-laden wound dressing. This advanced technology incorporates superoxide dismutase modified oxygen nanobubbles and catalase modified oxygen nanobubbles within an alginate hydrogel matrix. The oxygen nano chamber functions through a cascade reaction between superoxide dismutase and catalase, wherein excessive superoxide in the wound environment is enzymatically decomposed into hydrogen peroxide, and this hydrogen peroxide is subsequently converted into oxygen by catalase. This enzymatic cascade effectively controls wound inflammation and hypoxia, mitigating the risk of keloid formation. Concurrently, the oxygen nanobubbles release oxygen continuously, thus providing a sustained supply of oxygen to the wound site. The oxygen release from this dynamic system stimulates fibroblast proliferation, fosters the formation of new blood vessels, and contributes to the overall wound healing process. In the rat full-thickness wound model, the cascade reaction-based nano oxygen chamber displayed a notable capacity to expedite wound healing without scarring. Furthermore, in the pilot study of porcine full-thickness wound healing, a notable acceleration of tissue repair was observed in the conceived cascade reaction-based gel treated group within the 3 days post-surgery, which represents the proliferation stage of healing process. These achievements hold significant importance in ensuring the complete functional recovery of tissues, thereby highlighting its potential as a promising approach for enhancing wound healing outcomes.

Dynamic Effect of ß-Lactam Antibiotic Inactivation Due to the Inter- and Intraspecies Interaction of Drug-Resistant Microbes.

The presence of ß-lactamase positive microorganisms imparts a pharmacological effect on a variety of organisms that can impact drug efficacy by influencing the function or composition of bacteria. Although studies to assess dynamic intra- and interspecies communication with bacterial communities exist, the efficacy of drug treatment and quantitative assessment of multiorganism response is not well understood due to the lack of technological advances that can be used to study coculture interactions in a dynamic format. In this study, we investigate how ß-lactamase positive microorganisms can neutralize the effect of ß-lactam antibiotics in a dynamic format at the inter- and intraspecies level using microbial bead technology. Three interactive models for the biological compartmentalization of organisms were demonstrated to evaluate the effect of ß-lactam antibiotics on coculture systems. Our model at the intraspecies level attempts to mimic the biofilm matrix more closely as a community-level feature of microorganisms, which acknowledges the impact of nondrug-resistant species in shaping the dynamic response. In particular, the results of intraspecies studies are highly supportive of the biofilm mode of bacterial growth, which can provide structural support and protect the bacteria from an assault on host or environmental factors. Our findings also indicate that ß-lactamase positive bacteria can neutralize the cytotoxic effect of ß-lactam antibiotics at the interspecies level when cocultured with cancer cells. Results were validated using ß-lactamase positive bacteria isolated from environmental niches, which can trigger phenotypical alteration of ß-lactams when cocultured with other organisms. Our compartmentalization strategy acts as an independent ecosystem and provides a new avenue for multiscale studies to assess intra- and interspecies interactions.

Mucosal DNA methylome alteration in Crohn's disease: surgical and non-surgical groups.

Crohn's disease (CD) is characterized as a chronic, relapsing, and progressive disorder with a complex etiology involving interactions between host, microbiome, and the external environment. Genome wide association studies (GWAS) suggest several genetic variations in the diseased individuals but that explains only a small proportion of susceptibility to disease conditions. This indicates the possible role of epigenome which links environmental factors to the genetic variation in the disease etiology. The current study is focused on the DNA methylome evolution with disease progression. We performed Reduced Representation Bisulfite Sequencing (RRBS) to analyze differential DNA methylation in the diseased and healthy mucosal tissues of 2 different groups of CD patients: non-surgical and surgical, categorized based on the severity of disease and standard of care needed. Patients in both groups have unique DNA methylation signature compared to the healthy tissue. After removing single nucleotide polymorphisms (SNPs), 1,671 differentially methylated loci were found in the non-surgical and 3,334 in the surgical group of which only 206 were found overlapping in both groups. Furthermore, differential DNA methylation was noted in some of the GWAS associated genes implicated in CD. Also, functional enrichment analysis showed high representation of several key pathways where differential methylations were observed, and these can be implicated in CD pathogenesis. We identified specific DNA methylation patterns in the mucosal DNA of surgical and non-surgical CD patients which indicates evolution of the methylome as the disease progresses from initial to the advance stage. These unique patterns can be used as DNA methylation signatures to identify different stages of the disease.

Virus on surfaces: Chemical mechanism, influence factors, disinfection strategies, and implications for virus repelling surface design.

While SARS-CoV-2 is generally under control, the question of variants and infections still persists. Fundamental information on how the virus interacts with inanimate surfaces commonly found in our daily life and when in contact with the skin will be helpful in developing strategies to inhibit the spread of the virus. Here in, a critically important review of current understanding of the interaction between virus and surface is summarized from chemistry point-of-view. The Derjaguin-Landau-Verwey-Overbeek and extended Derjaguin-Landau-Verwey-Overbeek theories to model virus attachments on surfaces are introduced, along with the interaction type and strength, and quantification of each component. The virus survival and transfer are affected by a combination of biological, physical, and chemical parameters, as well as environmental parameters. The surface properties for virus and virus survival on typical surfaces such as metals, plastics, and glass are summarized. Attention is also paid to the transfer of virus to/from surfaces and skin. Typical virus disinfection strategies utilizing heat, light, chemicals, and ozone are discussed together with their disinfection mechanism. In the last section, design principles for virus repelling surface chemistry such as surperhydrophobic or surperhydrophilic surfaces are also introduced, to demonstrate how the integration of surface property control and advanced material fabrication can lead to the development of functional surfaces for mitigating the effect of viral infection upon contact.

Oxygen Nanobubbles-Embedded Hydrogel as Wound Dressing to Accelerate Healing.

Herein, we propose an oxygen nanobubbles-embedded hydrogel (ONB-G) with carbopol for oxygenation of wounds to accelerate the wound healing process. We integrate carbopol, hydrogel, and dextran-based oxygen nanobubbles (ONBs) to prepare ONB-G where ONBs can hold and release oxygen to accelerate wound healing. Oxygen release tests showed that the proposed ONB-G could encapsulate oxygen in the hydrogels for up to 34 days; meanwhile, fluorescence studies indicated that the ONB-G could maintain high oxygen levels for up to 4 weeks. The effect of carbopol concentration on the oxygen release capacity and rheological features of the ONB-G were also investigated along with the sterility of ONB-G. HDFa cell-based studies were first conducted to evaluate the viability, proliferation, and revival of cells in hypoxia. Scratch assay and mRNA expression studies indicated the potential benefit for wound closure. Histological evaluation of tissues with a pig model with incision and punch wounds showed that treatment with ONB-G exhibited improved healing compared with hydrogel without ONBs or treated without the gel. Our studies show that dextran-shell ONBs embedded in a gel (ONB-G) have the potential to accelerate wound healing, given its oxygen-holding capacity and release properties.

Oxygenated Wound Dressings for Hypoxia Mitigation and Enhanced Wound Healing.

Oxygen is a critical factor that can regulate the wound healing processes such as skin cell proliferation, granulation, re-epithelialization, angiogenesis, and tissue regeneration. However, hypoxia, a common occurrence in the wound bed, can impede normal healing processes. To enhance wound healing, oxygenation strategies that could effectively increase wound oxygen levels are effective. The present review summarizes wound healing stages and the role of hypoxia in wound healing and overviews current strategies to incorporate various oxygen delivery or generating materials for wound dressing, including catalase, nanoenzyme, hemoglobin, calcium peroxide, or perfluorocarbon-based materials, in addition to photosynthetic bacteria and hyperbaric oxygen therapy. Mechanism of action, oxygenation efficacy, and potential benefits and drawbacks of these dressings are also discussed. We conclude by highlighting the importance of design optimization in wound dressings to address the clinical needs to improve clinical outcomes.

Toxicity of per- and polyfluoroalkyl substances to microorganisms in confined hydrogel structures.

Per- and polyfluoroalkyl substances (PFAS) as a group of environmentally persistent synthetic chemicals has been widely used in industrial and consumer products. Bioaccumulation studies have documented the adverse effects of PFAS in various living organisms. Despite the large number of studies, experimental approaches to evaluate the toxicity of PFAS on bacteria in a biofilm-like niche as structured microbial communities are sparse. This study suggests a facile approach to query the toxicity of PFOS and PFOA on bacteria (Escherichia coli K12 MG1655 strain) in a biofilm-like niche provided by hydrogel-based core-shell beads. Our study shows that E. coli MG1655 upon complete confinement in hydrogel beads exhibit altered physiological characteristics of viability, biomass, and protein expression, compared to their susceptible counterpart cultivated under planktonic conditions. We find that soft-hydrogel engineering platforms may provide a protective role for microorganisms from environmental contaminants, depending on the size or thickness of the protective/barrier layer. We expect our study to provide insights on the toxicity of environmental contaminants on organisms under encapsulated conditions that could potentially be useful for toxicity screening and in evaluating ecological risk of soil, plant, and mammalian microbiome.

Sensor technologies for the detection and monitoring of endocrine-disrupting chemicals.

Endocrine-disrupting chemicals (EDCs) are a class of man-made substances with potential to disrupt the standard function of the endocrine system. These EDCs include phthalates, perchlorates, phenols, some heavy metals, furans, dimethoate, aromatic hydrocarbons, some pesticides, and per- and polyfluoroalkyl substances (PFAS). EDCs are widespread in the environment given their frequent use in daily life. Their production, usage, and consumption have increased many-fold in recent years. Their ability to interact and mimic normal endocrine functions makes them a potential threat to human health, aquatics, and wild life. Detection of these toxins has predominantly been done by mass spectroscopy and/or chromatography-based methods and to a lesser extent by advanced sensing approaches such as electrochemical and/or colorimetric methods. Instrument-based analytical techniques are often not amenable for onsite detection due to the lab-based nature of these detecting systems. Alternatively, analytical approaches based on sensor/biosensor techniques are more attractive because they are rapid, portable, equally sensitive, and eco-friendly. Advanced sensing systems have been adopted to detect a range of EDCs in the environment and food production systems. This review will focus on advances and developments in portable sensing techniques for EDCs, encompassing electrochemical, colorimetric, optical, aptamer-based, and microbial sensing approaches. We have also delineated the advantages and limitations of some of these sensing techniques and discussed future developments in sensor technology for the environmental sensing of EDCs.

Mechanomemory in protein diffusivity of chromatin and nucleoplasm after force cessation.

It is known that external mechanical forces can regulate structures and functions of living cells and tissues in physiology and diseases. However, after cessation of the force, how structures are altered in response to the dynamics of the chromatin and molecules in the nucleoplasm remains elusive. Here, using single-molecule imaging approaches, we show that exogenous local forces via integrins applied for 2 to 10 min decondensed the chromatin and increased chromatin and nucleoplasm protein mobility inside the nucleus, leading to elevated diffusivity of single protein molecules in the nucleoplasm, tens of minutes after the cessation of force. Diffusion experiments with fluorescence correlation spectroscopy in live single cells show that the mechanomemory in chromatin and nucleoplasm protein diffusivity was regulated by nuclear pore complexes. Protein molecular dynamics simulation recapitulated the experimental findings in live cells and showed that nucleoplasm protein diffusivity was regulated by the number of nuclear pore complexes. The mechanomemory in elevated protein diffusivity of the nucleoplasm after force cessation represents a physical process that reverses protein-protein condensation in phase separation via unjamming of the chromatin. Our findings of mechanomemory in chromatin and nucleoplasm protein diffusivity suggest that the effect of force on the nucleus remains tens of minutes after force cessation and thus is more far-reaching than previously anticipated.

Gut Microbiome-Host Metabolome Homeostasis upon Exposure to PFOS and GenX in Male Mice.

Alterations of the normal gut microbiota can cause various human health concerns. Environmental chemicals are one of the drivers of such disturbances. The aim of our study was to examine the effects of exposure to perfluoroalkyl and polyfluoroalkyl substances (PFAS)-specifically, perfluorooctane sulfonate (PFOS) and 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy) propanoic acid (GenX)-on the microbiome of the small intestine and colon, as well as on liver metabolism. Male CD-1 mice were exposed to PFOS and GenX in different concentrations and compared to controls. GenX and PFOS were found to have different effects on the bacterial community in both the small intestine and colon based on 16S rRNA profiles. High GenX doses predominantly led to increases in the abundance of Clostridium sensu stricto, Alistipes, and Ruminococcus, while PFOS generally altered Lactobacillus, Limosilactobacillus, Parabacteroides, Staphylococcus, and Ligilactobacillus. These treatments were associated with alterations in several important microbial metabolic pathways in both the small intestine and colon. Untargeted LC-MS/MS metabolomic analysis of the liver, small intestine, and colon yielded a set of compounds significantly altered by PFOS and GenX. In the liver, these metabolites were associated with the important host metabolic pathways implicated in the synthesis of lipids, steroidogenesis, and in the metabolism of amino acids, nitrogen, and bile acids. Collectively, our results suggest that PFOS and GenX exposure can cause major perturbations in the gastrointestinal tract, aggravating microbiome toxicity, hepatotoxicity, and metabolic disorders.

Characterization of Oxygen Nanobubbles and In Vitro Evaluation of Retinal Cells in Hypoxia.

Purpose: Vein or artery occlusion causes a hypoxic environment by preventing oxygen delivery and diffusion to tissues. Diseases such as retinal vein occlusion, central retinal artery occlusion, or diabetic retinopathy create a stroke-type condition that leads to functional blindness in the effected eye. We aim to develop an oxygen delivery system consisting of oxygen nanobubbles (ONBs) that can mitigate retinal ischemia during a severe hypoxic event such as central retinal artery occlusion. Methods: ONBs were synthesized to encapsulate oxygen saturated molecular medical grade water. Stability, oxygen release, biocompatibility, reactive oxygen species, superoxide, MTT, and terminal uridine nick-end labeling assays were performed. Cell viability was evaluated, and safety experiments were conducted in rabbits. Results: The ONBs were approximately 220 nm in diameter, with a zeta potential of -58.8 mV. Oxygen release studies indicated that 74.06 µg of O2 is released from the ONBs after 12 hours at 37°C. Cell studies indicated that ONBs are safe and cells are viable. There was no significant increase in reactive oxygen species, superoxide, or double-stranded DNA damage after ONB treatment. ONBs preserve mitochondrial function and viability. Histological sections from rabbit eyes indicated that ONBs were not toxic. Conclusions: The ONBs proposed have excellent oxygen holding and release properties to mitigate ischemic conditions in the retina. They are sterile, stable, and nontoxic. Translation Relevance: ONB technology was evaluated for its physical properties, oxygen release, sterility, stability, and safety. Our results indicate that ONBs could be a viable treatment approach to mitigate hypoxia during ischemic conditions in the eye upon timely administration.

Characterizing Spray-Dried Powders through NIR Spectroscopy: Effect of Two Preparation Strategies for Calibration Samples and Comparison of Two Types of NIR Spectrometers.

Emerging portable near infrared (NIR) spectroscopic approaches coupled with data analysis and chemometric techniques provide opportunities for the rapid characterization of spray-dried products and process optimization. This study aimed to enhance the understanding of applying NIR spectroscopy in spray-dried samples by comparing two sample preparation strategies and two spectrometers. Two sets of whey protein-maltodextrin matrixes, one with a protein content gradient and one with a consistent protein content, were spray-dried, and the effect of the two preparation strategies on NIR calibration model development was studied. Secondly, a portable NIR spectrometer (PEAK) was compared with a benchtop NIR spectrometer (CARY) for the moisture analysis of prepared samples. When validating models with the samples with focused protein contents, the best PLS protein models established from the two sample sets had similar performances. When comparing two spectrometers, although CARY outperformed PEAK, PEAK still demonstrated reliable performance for moisture analysis, indicating that it is capable as an inline sensor.

Hierarchical encapsulation of bacteria in functional hydrogel beads for inter- and intra- species communication.

To sequester prokaryotic cells in a biofilm-like niche, the creation of a pertinent and reliable microenvironment that reflects the heterogeneous nature of biological systems is vital for sustenance. Design of a microenvironment that is conducive for growth and survival of organisms, should account for factors such as mass transport, porosity, stability, elasticity, size, functionality, and biochemical characteristics of the organisms in the confined architecture. In this work we present an artificial long-term confinement model fabricated by natural alginate hydrogels that are structurally stable and can host organisms for over 10 days in physiologically relevant conditions. A unique feature of the confinement platform is the development of stratified habitats wherein bacterial cells can be entrapped in the core as well as in the shell layers, wherein the thickness and the number of shell layers are tunable at fabrication. We show that the hydrogel microenvironment in the beads can host complex subpopulations of organisms similar to that in a biofilm. Dynamic interaction of bacterial colonies encapsulated in different beads or within the core and stratified layers of single beads was demonstrated to show intra- species communication. Inter- species communication between probiotic bacteria and human colorectal carcinoma cells was also demonstrated to highlight a possible bidirectional communication between the organisms in the beads and the environment. STATEMENT OF SIGNIFICANCE: Bacteria confinement in a natural soft hydrogel structure has always been a challenge due to the collapse of hydrogel architectures. Alternative methods have been attempted to encapsulate microorganisms by employing various processes to avoid/minimize rupturing of hydrogel structures. However, most of the past approaches have been unfavorable in balancing cell proliferation and functionality upon confinement. Our study addresses the fundamental gap in knowledge necessary to create favorable and complex 3D biofilm mimics utilizing natural hydrogel for microbial colonization for long-term studies. Our approach represents a cornerstone in the development of 3D functional architectures not only to advance studies in microbial communication, host-microbe interaction but also to address basic and fundamental questions in biology.

Soft hydrogel-shell confinement systems as bacteria-based bioactuators and biosensors.

Genetically engineered (GE) bacteria were utilized for developing functional systems upon confinement within a restricted space. Use of natural soft hydrogel such as alginate, gelatin, and agarose, have been investigated as promising approaches to design functional architectures. Nevertheless, a challenge is to develop functional microenvironments that support biofilm-like confinement in a relevant three-dimensional (3D) format for long-term studies. We demonstrate a natural soft hydrogel bioactuator based on alginate core-shell structures (0.25-2 mm core and 50-300 µm shell thickness) that enables 3D microbial colonization upon confinement with high cell density. Specially, our study evaluates the efficiency of bacteria-functional system by recapitulating various GE bacteria which can produce common reporter proteins, to demonstrate their actuator functions as well as dynamic pair-wise interactions. The structural design of the hydrogel can endure continued growth of various bacteria colonies within the confined space for over 10 days. The total amount of cellular biomass upon hydrogel-shell confinement was increased 5-fold compared to conventional techniques without hydrogel-shell. Furthermore, the enzymatic activity increased 3.8-fold and bioluminescence signal by 8-fold compared to the responses from conventional hydrogel systems. The conceptualized platform and our workflow represent a reliable strategy with core-shell structures to develop artificial hydrogel habitats as bacteria-based functional systems for bioactuation.

Multifunctional Oxygenated Particles for Targeted Cancer Drug Delivery and Evaluation with Darkfield Hyperspectral Imaging.

We propose a novel multifunctional nanocarrier system for targeted drug delivery for lung cancer theranostics. Oxygenated particles (OPs) synthesized with an oxygen-encapsulating carboxymethyl cellulose shell were used as a platform to deliver oxygen to the hypoxic tumor microenvironment. The OPs synthesized could also be conjugated with ligands (e.g., antibodies) to target cancer cells expressing the corresponding antigens to deliver a drug, doxorubicin. In vitro testing of functionalized OPs showed increased efficacy of doxorubicin against the proliferation of lung cancer cells. Both confocal fluorescence imaging and darkfield microscopy hyperspectral imaging validated the OP complex and its efficient targeting of specific cells to deliver the therapeutic. The nanocarrier platform developed can also serve as a diagnostic imaging reagent as demonstrated by darkfield microscopy. Results show that the theranostic OPs developed with multifunctional modalities enabled targeted drug delivery with improved efficacy and tracking of drug delivery vehicles by imaging.