Targeted Biodegradable Near-Infrared Fluorescent Nanoparticles for Colorectal Cancer Imaging.

Colorectal cancer (CRC) is the third leading cause of cancer death in the U.S., and early detection and diagnosis are essential for effective treatment. Current methods are inadequate for rapid detection of early disease, revealing flat lesions, and delineating tumor margins with accuracy and molecular specificity. Fluorescence endoscopy can generate wide field-of-view images enabling detection of CRC lesions and margins; increased signal intensity and improved signal-to-noise ratios can increase both speed and sensitivity of cancer detection. For this purpose, we developed targeted near-infrared (NIR) fluorescent silica nanoparticles (FSNs). We tuned their size to 50-200 nm and conjugated their surface with an antibody to carcinoembryonic antigen (CEA) to prepare CEA-FSNs. The physicochemical properties and biodegradable profiles of CEA-FSN were characterized, and molecular targeting was verified in culture using HT29 (CEA positive) and HCT116 (CEA negative) cells. CEA-FSNs bound to the HT29 cells to a greater extent than to the HCT116 cells, and smaller CEA-FSNs were internalized into HT29 cells more efficiently than larger CEA-FSNs. After intravenous administration of CEA-FSNs, a significantly greater signal was observed from the CEA-positive HT29 than the CEA-negative HCT116 tumors in xenografted mice. In F344-PIRC rats, polyps in the intestine were detected by white-light endoscopy, and NIR fluorescent signals were found in the excised intestinal tissue after topical application of CEA-FSNs. Immunofluorescence imaging of excised tissue sections demonstrated that the particle signals coregistered with signals for both CRC and CEA. These results indicate that CEA-FSNs have potential as a molecular imaging marker for early diagnosis of CRC.

Shape Anisotropy-Governed High-Performance Nanomagnetosol for In Vivo Magnetic Particle Imaging of Lungs.

Caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), coronavirus disease 2019 (COVID-19) has shown extensive lung manifestations in vulnerable individuals, putting lung imaging and monitoring at the forefront of early detection and treatment. Magnetic particle imaging (MPI) is an imaging modality, which can bring excellent contrast, sensitivity, and signal-to-noise ratios to lung imaging for the development of new theranostic approaches for respiratory diseases. Advances in MPI tracers would offer additional improvements and increase the potential for clinical translation of MPI. Here, a high-performance nanotracer based on shape anisotropy of magnetic nanoparticles is developed and its use in MPI imaging of the lung is demonstrated. Shape anisotropy proves to be a critical parameter for increasing signal intensity and resolution and exceeding those properties of conventional spherical nanoparticles. The 0D nanoparticles exhibit a 2-fold increase, while the 1D nanorods have a > 5-fold increase in signal intensity when compared to VivoTrax. Newly designed 1D nanorods displayed high signal intensities and excellent resolution in lung images. A spatiotemporal lung imaging study in mice revealed that this tracer offers new opportunities for monitoring disease and guiding intervention.

Flavinated SDHA underlies the change in intrinsic optical properties of oral cancers.

The molecular basis of reduced autofluorescence in oral squamous cell carcinoma (OSCC) cells relative to normal cells has been speculated to be due to lower levels of free flavin adenine dinucleotide (FAD). This speculation, along with differences in the intrinsic optical properties of extracellular collagen, lies at the foundation of the design of currently-used clinical optical detection devices. Here, we report that free FAD levels may not account for differences in autofluorescence of OSCC cells, but that the differences relate to FAD as a co-factor for flavination. Autofluorescence from a 70 kDa flavoprotein, succinate dehydrogenase A (SDHA), was found to be responsible for changes in optical properties within the FAD spectral region, with lower levels of flavinated SDHA in OSCC cells. Since flavinated SDHA is required for functional complexation with succinate dehydrogenase B (SDHB), decreased SDHB levels were observed in human OSCC tissue relative to normal tissues. Accordingly, the metabolism of OSCC cells was found to be significantly altered relative to normal cells, revealing vulnerabilities for both diagnosis and targeted therapy. Optimizing non-invasive tools based on optical and metabolic signatures of cancers will enable more precise and early diagnosis leading to improved outcomes in patients.

Polylactide Degradation Activates Immune Cells by Metabolic Reprogramming.

Polylactide (PLA) is the most widely utilized biopolymer in medicine. However, chronic inflammation and excessive fibrosis resulting from its degradation remain significant obstacles to extended clinical use. Immune cell activation has been correlated to the acidity of breakdown products, yet methods to neutralize the pH have not significantly reduced adverse responses. Using a bioenergetic model, delayed cellular changes were observed that are not apparent in the short-term. Amorphous and semi-crystalline PLA degradation products, including monomeric l-lactic acid, mechanistically remodel metabolism in cells leading to a reactive immune microenvironment characterized by elevated proinflammatory cytokines. Selective inhibition of metabolic reprogramming and altered bioenergetics both reduce these undesirable high cytokine levels and stimulate anti-inflammatory signals. The results present a new biocompatibility paradigm by identifying metabolism as a target for immunomodulation to increase tolerance to biomaterials, ensuring safe clinical application of PLA-based implants for soft- and hard-tissue regeneration, and advancing nanomedicine and drug delivery.

Glycolytic reprogramming in macrophages and MSCs during inflammation.

Background: Dysregulated inflammation is associated with many skeletal diseases and disorders, such as osteolysis, non-union of fractures, osteonecrosis, osteoarthritis and orthopaedic infections. We previously showed that continuous infusion of lipopolysaccharide (LPS) contaminated polyethylene particles (cPE) caused prolonged inflammation and impaired bone formation. However, the metabolic and bioenergetic processes associated with inflammation of bone are unknown. Mitochondria are highly dynamic organelles that modulate cell metabolism and orchestrate the inflammatory responses that involve both resident and recruited cells. Glycolytic reprogramming, the shift from oxidative phosphorylation (OXPHOS) to glycolysis causes inappropriate cell activation and function, resulting in dysfunctional cellular metabolism. We hypothesized that impaired immunoregulation and bone regeneration from inflammatory states are associated with glycolytic reprogramming and mitochondrial dysfunction in macrophages (Mφ) and mesenchymal stromal cells (MSCs). Methods: We used the Seahorse XF96 analyzer and real-time qPCR to study the bioenergetics of Mφ and MSCs exposed to cPE. To understand the oxygen consumption rate (OCR), we used Seahorse XF Cell Mito Stress Test Kit with Seahorse XF96 analyzer. Similarly, Seahorse XF Glycolytic Rate Assay Kit was used to detect the extracellular acidification rate (ECAR) and Seahorse XF Real-Time ATP Rate Assay kit was used to detect the real-time ATP production rates from OXPHOS and glycolysis. Real-time qPCR was performed to analyze the gene expression of key enzymes in glycolysis and mitochondrial biogenesis. We further detected the gene expression of proinflammatory cytokines in Mφ and genes related to cell differentiation in MSC during the challenge of cPE. Results: Our results demonstrated that the oxidative phosphorylation of Mφ exposed to cPE was significantly decreased when compared with the control group. We found reduced basal, maximal and ATP-production coupled respiration rates, and decreased proton leak in Mφ during challenge with cPE. Meanwhile, Mφ showed increased basal glycolysis and proton efflux rates (PER) when exposed to cPE. The percentage (%) of PER from glycolysis was higher in Mφ exposed to cPE, indicating that the contribution of the glycolytic pathway to total extracellular acidification was elevated during the challenge of cPE. In line with the results of OCR and ECAR, we found Mφ during cPE challenge showed higher glycolytic ATP (glycoATP) production rates and lower mitochondrial ATP (mitoATP) production rates which is mainly from OXPHOS. Interestingly, MSCs showed enhanced glycolysis during challenge with cPE, but no significant changes in oxygen consumption rates (OCR). In accordance, seahorse assay of real-time ATP revealed glycoATP rates were elevated while mitoATP rates showed no significant differences in MSC during challenge with cPE. Furthermore, Mφ and MSCs exposed to cPE showed upregulated gene expression levels of glycolytic regulators and Mφ exposed to cPE expressed higher levels of pro-inflammatory cytokines. Conclusion: This study demonstrated the dysfunctional bioenergetic activity of bone marrow-derived Mφ and MSCs exposed to cPE, which could impair the immunoregulatory properties of cells in the bone niche. The underlying molecular defect related to disordered mitochondrial function could represent a potential therapeutic target during the resolution of inflammation.

Flavinated SDHA Underlies the Change in Intrinsic Optical Properties of Oral Cancers.

The molecular basis of reduced autofluorescence in oral squamous cell carcinoma (OSCC) cells relative to normal cells has been speculated to be due to lower levels of free flavin adenine dinucleotide (FAD). This speculation, along with differences in the intrinsic optical properties of extracellular collagen, lie at the foundation of the design of currently-used clinical optical detection devices. Here, we report that free FAD levels may not account for differences in autofluorescence of OSCC cells, but that the differences relate to FAD as a co-factor for flavination. Autofluorescence from a 70 kDa flavoprotein, succinate dehydrogenase A (SDHA), was found to be responsible for changes in optical properties within the FAD spectral region with lower levels of flavinated SDHA in OSCC cells. Since flavinated SDHA is required for functional complexation with succinate dehydrogenase B (SDHB), decreased SDHB levels were observed in human OSCC tissue relative to normal tissues. Accordingly, the metabolism of OSCC cells was found to be significantly altered relative to normal cells, revealing vulnerabilities for both diagnosis and targeted therapy. Optimizing non-invasive tools based on optical and metabolic signatures of cancers will enable more precise and early diagnosis leading to improved outcomes in patients.

Gla-domain mediated targeting of externalized phosphatidylserine for intracellular delivery.

Phosphatidylserine (PS) is a negatively charged phospholipid normally localized to the inner leaflet of the plasma membrane of cells but is externalized onto the cell surface during apoptosis as well as in malignant and infected cells. Consequently, PS may comprise an important molecular target in diagnostics, imaging, and targeted delivery of therapeutic agents. While an array of PS-binding molecules exist, their utility has been limited by their inability to internalize diagnostic or therapeutic payloads. We describe the generation, isolation, characterization, and utility of a PS-binding motif comprised of a carboxylated glutamic acid (GLA) residue domain that both recognizes and binds cell surface-exposed PS, and then unlike other PS-binding molecules is internalized into these cells. Internalization is independent of the traditional endosomal-lysosomal pathway, directly entering the cytosol of the target cell rapidly. We demonstrate that this PS recognition extends to stem cells and that GLA-domain-conjugated probes can be detected upon intravenous administration in animal models of infectious disease and cancer. GLA domain binding and internalization offer new opportunities for specifically targeting cells with surface-exposed PS for imaging and delivery of therapeutics.

Glycolytic reprogramming underlies immune cell activation by polyethylene wear particles.

Primary total joint arthroplasties (TJAs) are widely and successfully applied reconstructive procedures to treat end-stage arthritis. Nearly 50 % of TJAs are now performed in young patients, posing a new challenge: performing TJAs which last a lifetime. The urgency is justified because subsequent TJAs are costlier and fraught with higher complication rates, not to mention the toll taken on patients and their families. Polyethylene particles, generated by wear at joint articulations, drive aseptic loosening by inciting insidious inflammation associated with surrounding bone loss. Down modulating polyethylene particle-induced inflammation enhances integration of implants to bone (osseointegration), preventing loosening. A promising immunomodulation strategy could leverage immune cell metabolism, however, the role of immunometabolism in polyethylene particle-induced inflammation is unknown. Our findings reveal that immune cells exposed to sterile or contaminated polyethylene particles show fundamentally altered metabolism, resulting in glycolytic reprogramming. Inhibiting glycolysis controlled inflammation, inducing a pro-regenerative phenotype that could enhance osseointegration.

Bioadhesives with Antimicrobial Properties.

Bioadhesives with antimicrobial properties enable easier and safer treatment of wounds as compared to the traditional methods such as suturing and stapling. Composed of natural or synthetic polymers, these bioadhesives seal wounds and facilitate healing while preventing infections through the activity of locally released antimicrobial drugs, nanocomponents, or inherently antimicrobial polers. Although many different materials and strategies are employed to develop antimicrobial bioadhesives, the design of these biomaterials necessitates a prudent approach as achieving all the required properties including optimal adhesive and cohesive properties, biocompatibility, and antimicrobial activity can be challenging. Designing antimicrobial bioadhesives with tunable physical, chemical, and biological properties will shed light on the path for future advancement of bioadhesives with antimicrobial properties. In this review, the requirements and commonly used strategies for developing bioadhesives with antimicrobial properties are discussed. In particular, different methods for their synthesis and their experimental and clinical applications on a variety of organs are reviewed. Advances in the design of bioadhesives with antimicrobial properties will pave the way for a better management of wounds to increase positive clinical outcomes.

Metabolic profile of mesenchymal stromal cells and macrophages in the presence of polyethylene particles in a 3D model.

BACKGROUND: Continuous cross talk between MSCs and macrophages is integral to acute and chronic inflammation resulting from contaminated polyethylene particles (cPE); however, the effect of this inflammatory microenvironment on mitochondrial metabolism has not been fully elucidated. We hypothesized that (a) exposure to cPE leads to impaired mitochondrial metabolism and glycolytic reprogramming and (b) macrophages play a key role in this pathway. METHODS: We cultured MSCs with/without uncommitted M0 macrophages, with/without cPE in 3-dimensional gelatin methacrylate (3D GelMA) constructs/scaffolds. We evaluated mitochondrial function (membrane potential and reactive oxygen species-ROS production), metabolic pathways for adenosine triphosphate (ATP) production (glycolysis or oxidative phosphorylation) and response to stress mechanisms. We also studied macrophage polarization toward the pro-inflammatory M1 or the anti-inflammatory M2 phenotype and the osteogenic differentiation of MSCs. RESULTS: Exposure to cPE impaired mitochondrial metabolism of MSCs; addition of M0 macrophages restored healthy mitochondrial function. Macrophages exposed to cPE-induced glycolytic reprogramming, but also initiated a response to this stress to restore mitochondrial biogenesis and homeostatic oxidative phosphorylation. Uncommitted M0 macrophages in coculture with MSC polarized to both M1 and M2 phenotypes. Osteogenesis was comparable among groups after 21 days. CONCLUSION: This work confirmed that cPE exposure triggers impaired mitochondrial metabolism and glycolytic reprogramming in a 3D coculture model of MSCs and macrophages and demonstrated that macrophages cocultured with MSCs undergo metabolic changes to maintain energy production and restore homeostatic metabolism.

Biomimetic cell membrane-coated poly(lactic-co-glycolic acid) nanoparticles for biomedical applications.

Poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) are commonly used for drug delivery because of their favored biocompatibility and suitability for sustained and controlled drug release. To prolong NP circulation time, enable target-specific drug delivery and overcome physiological barriers, NPs camouflaged in cell membranes have been developed and evaluated to improve drug delivery. Here, we discuss recent advances in cell membrane-coated PLGA NPs, their preparation methods, and their application to cancer therapy, management of inflammation, treatment of cardiovascular disease and control of infection. We address the current challenges and highlight future research directions needed for effective use of cell membrane-camouflaged NPs.

Stereochemistry Determines Immune Cellular Responses to Polylactide Implants.

Repeating l- and d-chiral configurations determine polylactide (PLA) stereochemistry, which affects its thermal and physicochemical properties, including degradation profiles. Clinically, degradation of implanted PLA biomaterials promotes prolonged inflammation and excessive fibrosis, but the role of PLA stereochemistry is unclear. Additionally, although PLA of varied stereochemistries causes differential immune responses in vivo, this observation has yet to be effectively modeled in vitro. A bioenergetic model was applied to study immune cellular responses to PLA containing >99% l-lactide (PLLA), >99% d-lactide (PDLA), and a 50/50 melt-blend of PLLA and PDLA (stereocomplex PLA). Stereocomplex PLA breakdown products increased IL-1ß, TNF-α, and IL-6 protein levels but not MCP-1. Expression of these proinflammatory cytokines is mechanistically driven by increases in glycolysis in primary macrophages. In contrast, PLLA and PDLA degradation products selectively increase MCP-1 protein expression. Although both oxidative phosphorylation and glycolysis are increased with PDLA, only oxidative phosphorylation is increased with PLLA. For each biomaterial, glycolytic inhibition reduces proinflammatory cytokines and markedly increases anti-inflammatory (IL-10) protein levels; differential metabolic changes in fibroblasts were observed. These findings provide mechanistic explanations for the diverse immune responses to PLA of different stereochemistries and underscore the pivotal role of immunometabolism in the biocompatibility of biomaterials applied in medicine.

Erratum: Engineering Extracellular Vesicles to Target Pancreatic Tissue In Vivo: Erratum.

[This corrects the article DOI: 10.7150/ntno.54879.].

A Biodegradable Bioactive Glass-Based Hydration Sensor for Biomedical Applications.

Monitoring changes in edema-associated intracranial pressure that complicates trauma or surgery would lead to improved outcomes. Implantable pressure sensors have been explored, but these sensors require post-surgical removal, leading to the risk of injury to brain tissue. The use of biodegradable implantable sensors would help to eliminate this risk. Here, we demonstrate a bioactive glass (BaG)-based hydration sensor. Fluorine (CaF2) containing BaG (BaG-F) was produced by adding 5, 10 or 20 wt.% of CaF2 to a BaG matrix using a melting manufacturing technique. The structure, morphology and electrical properties of the resulting constructs were evaluated to understand the physical and electrical behaviors of this BaG-based sensor. Synthesis process for the production of the BaG-F-based sensor was validated by assessing the structural and electrical properties. The structure was observed to be amorphous and dense, the porosity decreased and grain size increased with increasing CaF2 content in the BaG matrix. We demonstrated that this BaG-F chemical composition is highly sensitive to hydration, and that the electrical sensitivity (resistive-capacitive) is induced by hydration and reversed by dehydration. These properties make BaG-F suitable for use as a humidity sensor to monitor brain edema and, consequently, provide an alert for increased intracranial pressure.

Harnessing insect olfactory neural circuits for detecting and discriminating human cancers.

There is overwhelming evidence that presence of cancer alters cellular metabolic processes, and these changes are manifested in emitted volatile organic compound (VOC) compositions of cancer cells. Here, we take a novel forward engineering approach by developing an insect olfactory neural circuit-based VOC sensor for cancer detection. We obtained oral cancer cell culture VOC-evoked extracellular neural responses from in vivo insect (locust) antennal lobe neurons. We employed biological neural computations of the antennal lobe circuitry for generating spatiotemporal neuronal response templates corresponding to each cell culture VOC mixture, and employed these neuronal templates to distinguish oral cancer cell lines (SAS, Ca9-22, and HSC-3) vs. a non-cancer cell line (HaCaT). Our results demonstrate that three different human oral cancers can be robustly distinguished from each other and from a non-cancer oral cell line. By using high-dimensional population neuronal response analysis and leave-one-trial-out methodology, our approach yielded high classification success for each cell line tested. Our analyses achieved 76-100% success in identifying cell lines by using the population neural response (n = 194) collected for the entire duration of the cell culture study. We also demonstrate this cancer detection technique can distinguish between different types of oral cancers and non-cancer at different time-matched points of growth. This brain-based cancer detection approach is fast as it can differentiate between VOC mixtures within 250 ms of stimulus onset. Our brain-based cancer detection system comprises a novel VOC sensing methodology that incorporates entire biological chemosensory arrays, biological signal transduction, and neuronal computations in a form of a forward-engineered technology for cancer VOC detection.

Magnetothermal Control of Temperature-Sensitive Repressors in Superparamagnetic Iron Nanoparticle-Coated Bacillus subtilis.

Superparamagnetic iron oxide nanoparticles (SPIONs) are used as contrast agents in magnetic resonance imaging (MRI) and magnetic particle imaging (MPI), and resulting images can be used to guide magnetothermal heating. Alternating magnetic fields (AMF) cause local temperature increases in regions with SPIONs, and we investigated the ability of magnetic hyperthermia to regulate temperature-sensitive repressors (TSRs) of bacterial transcription. The TSR, TlpA39, was derived from a Gram-negative bacterium and used here for thermal control of reporter gene expression in Gram-positive, Bacillus subtilis. In vitro heating of B. subtilis with TlpA39 controlling bacterial luciferase expression resulted in a 14.6-fold (12 hours; h) and 1.8-fold (1 h) increase in reporter transcripts with a 10.0-fold (12 h) and 12.1-fold (1 h) increase in bioluminescence. To develop magnetothermal control, B. subtilis cells were coated with three SPION variations. Electron microscopy coupled with energy dispersive X-ray spectroscopy revealed an external association with, and retention of, SPIONs on B. subtilis. Furthermore, using long duration AMF we demonstrated magnetothermal induction of the TSRs in SPION-coated B. subtilis with a maximum of 5.6-fold increases in bioluminescence. After intramuscular injections of SPION-coated B. subtilis, histology revealed that SPIONs remained in the same locations as the bacteria. For in vivo studies, 1 h of AMF is the maximum exposure due to anesthesia constraints. Both in vitro and in vivo, there was no change in bioluminescence after 1 h of AMF treatment. Pairing TSRs with magnetothermal energy using SPIONs for localized heating with AMF can lead to transcriptional control that expands options for targeted bacteriotherapies.

Tracking the fates of iron-labeled tumor cells in vivo using magnetic particle imaging.

The use of imaging to detect and monitor the movement and accumulation of cells in living subjects can provide significant insights that can improve our understanding of metastasis and guide therapeutic development. For cell tracking using Magnetic Resonance Imaging (MRI), cells are labeled with iron oxides and the effects of the iron on water provides contrast. However, due to low specificity and difficulties in quantification with MRI, other modalities and approaches need to be developed. Magnetic Particle Imaging (MPI) is an emerging imaging technique which directly detects iron, allowing for a specific, quantitative and sensitive readout. Here, we use MPI to image iron-labeled tumor cells longitudinally, from implantation and growth at a primary site to movement to distant anatomic sites. In vivo bioluminescent imaging (BLI) was used to localize tumor metastases and computed tomography (CT) allowed for correlation of these signals to anatomic locations. These three imaging modalities provide information on immune escape and metastasis of iron-labeled, and unlabeled, tumor cells, and the accumulation of cell-free iron contrast over time. We localized iron signals by MPI and tumor cells via BLI, and correlated these positive contrast images with CT scans to reveal the anatomic sites with cancer cells; histologic analysis confirmed the presence of iron-labeled tumor cells in the tissues, suggesting that the metastatic cells retained enough iron for MPI detection. The use of multi-modality cell tracking reveals the movement, accumulation and fates of labeled cells that will be helpful understanding cancer progression and guiding the development of targeted therapies.

Engineered endosymbionts that alter mammalian cell surface marker, cytokine and chemokine expression.

Developing modular tools that direct mammalian cell function and activity through controlled delivery of essential regulators would improve methods of guiding tissue regeneration, enhancing cellular-based therapeutics and modulating immune responses. To address this challenge, Bacillus subtilis was developed as a chassis organism for engineered endosymbionts (EES) that escape phagosome destruction, reside in the cytoplasm of mammalian cells, and secrete proteins that are transported to the nucleus to impact host cell response and function. Two synthetic operons encoding either the mammalian transcription factors Stat-1 and Klf6 or Klf4 and Gata-3 were recombined into the genome of B. subtilis expressing listeriolysin O (LLO) from Listeria monocytogenes and expressed from regulated promoters. Controlled expression of the mammalian proteins from B. subtilis LLO in the cytoplasm of J774A.1 macrophage/monocyte cells altered surface marker, cytokine and chemokine expression. Modulation of host cell fates displayed some expected patterns towards anti- or pro-inflammatory phenotypes by each of the distinct transcription factor pairs with further demonstration of complex regulation caused by a combination of the EES interaction and transcription factors. Expressing mammalian transcription factors from engineered intracellular B. subtilis as engineered endosymbionts comprises a new tool for directing host cell gene expression for therapeutic and research purposes.