Complete substitution with modified nucleotides in self-amplifying RNA suppresses the interferon response and increases potency.

The use of modified nucleotides to suppress the interferon response and maintain translation of self-amplifying RNA (saRNA), which has been achieved for mRNA, has not yet succeeded. We identify modified nucleotides that, when substituted at 100% in saRNA, confer innate immune evasion and robust long-term protein expression, and when formulated as a vaccine, protect against lethal SARS-CoV-2 challenge in mice. This discovery advances saRNA therapeutics by enabling prolonged protein expression at low doses.

Drug Delivery Opportunities in Esophageal Cancer: Current Treatments and Future Prospects.

With one of the highest mortality rates of all malignancies, the 5-year survival rate for esophageal cancer is under 20%. Depending on the stage and extent of the disease, the current standard of care treatment paradigm includes chemotherapy or chemoradiotherapy followed by surgical esophagogastrectomy, with consideration for adjuvant immunotherapy for residual disease. This regimen has high morbidity, due to anatomic changes inherent in surgery, the acuity of surgical complications, and off-target effects of systemic chemotherapy and immunotherapy. We begin with a review of current treatments, then discuss new and emerging targets for therapies and advanced drug delivery systems. Recent and ongoing preclinical and early clinical studies are evaluating traditional tumor targets (e.g., human epidermal growth factor receptor 2), as well as promising new targets such as Yes-associated protein 1 or mammalian target of rapamycin to develop new treatments for this disease. Due the function and location of the esophagus, opportunities also exist to pair these treatments with a drug delivery strategy to increase tumor targeting, bioavailability, and intratumor concentrations, with the two most common delivery platforms being stents and nanoparticles. Finally, early results with antibody drug conjugates and chimeric antigenic receptor T cells show promise as upcoming therapies. This review discusses these innovations in therapeutics and drug delivery in the context of their successes and failures, with the goal of identifying those solutions that demonstrate the most promise to shift the paradigm in treating this deadly disease.

Tryptophan Metabolism in Alzheimer's Disease with the Involvement of Microglia and Astrocyte Crosstalk and Gut-Brain Axis.

Alzheimer's disease (AD) is an age-dependent neurodegenerative disease characterized by extracellular Amyloid Aß peptide (Aß) deposition and intracellular Tau protein aggregation. Glia, especially microglia and astrocytes are core participants during the progression of AD and these cells are the mediators of Aß clearance and degradation. The microbiota-gut-brain axis (MGBA) is a complex interactive network between the gut and brain involved in neurodegeneration. MGBA affects the function of glia in the central nervous system (CNS), and microbial metabolites regulate the communication between astrocytes and microglia; however, whether such communication is part of AD pathophysiology remains unknown. One of the potential links in bilateral gut-brain communication is tryptophan (Trp) metabolism. The microbiota-originated Trp and its metabolites enter the CNS to control microglial activation, and the activated microglia subsequently affect astrocyte functions. The present review highlights the role of MGBA in AD pathology, especially the roles of Trp per se and its metabolism as a part of the gut microbiota and brain communications. We (i) discuss the roles of Trp derivatives in microglia-astrocyte crosstalk from a bioinformatics perspective, (ii) describe the role of glia polarization in the microglia-astrocyte crosstalk and AD pathology, and (iii) summarize the potential of Trp metabolism as a therapeutic target. Finally, we review the role of Trp in AD from the perspective of the gut-brain axis and microglia, as well as astrocyte crosstalk, to inspire the discovery of novel AD therapeutics.

Advances in viscosupplementation and tribosupplementation for early-stage osteoarthritis therapy.

Joint kinematic instability, arising from congenital or acquired musculoskeletal pathoanatomy or from imbalances in anabolism and catabolism induced by pathophysiological factors, leads to deterioration of the composition, structure and function of cartilage and, ultimately, progression to osteoarthritis (OA). Alongside articular cartilage degeneration, synovial fluid lubricity decreases in OA owing to a reduction in the concentration and molecular weight of hyaluronic acid and surface-active mucinous glycoproteins that form a lubricating film over the articulating joint surfaces. Minimizing friction between articulating joint surfaces by lubrication is fundamental for decreasing hyaline cartilage wear and for maintaining the function of synovial joints. Augmentation with highly viscous supplements (that is, viscosupplementation) offers one approach to re-establishing the rheological and tribological properties of synovial fluid in OA. However, this approach has varied clinical outcomes owing to limited intra-articular residence time and ineffective mechanisms of chondroprotection. This Review discusses normal hyaline cartilage function and lubrication and examines the advantages and disadvantages of various strategies for restoring normal joint lubrication. These strategies include contemporary viscosupplements that contain antioxidants, anti-inflammatory drugs or platelet-rich plasma and new synthetic synovial fluid additives and cartilage matrix enhancers. Advanced biomimetic tribosupplements offer promise for mitigating cartilage wear, restoring joint function and, ultimately, improving patient care.

Acidic Nanoparticles Restore Lysosomal Acidification and Rescue Metabolic Dysfunction in Pancreatic ß-Cells under Lipotoxic Conditions.

Type 2 diabetes (T2D), a prevalent metabolic disorder lacking effective treatments, is associated with lysosomal acidification dysfunction, as well as autophagic and mitochondrial impairments. Here, we report a series of biodegradable poly(butylene tetrafluorosuccinate-co-succinate) polyesters, comprising a 1,4-butanediol linker and varying ratios of tetrafluorosuccinic acid (TFSA) and succinic acid as components, to engineer lysosome-acidifying nanoparticles (NPs). The synthesized NPs are spherical with diameters of ≈100 nm and have low polydispersity and good stability. Notably, TFSA NPs, which are composed entirely of TFSA, exhibit the strongest degradation capability and superior acidifying properties. We further reveal significant downregulation of lysosomal vacuolar (H+)-ATPase subunits, which are responsible for maintaining lysosomal acidification, in human T2D pancreatic islets, INS-1 ß-cells under chronic lipotoxic conditions, and pancreatic tissues of high-fat-diet (HFD) mice. Treatment with TFSA NPs restores lysosomal acidification, autophagic function, and mitochondrial activity, thereby improving the pancreatic function in INS-1 cells and HFD mice with lipid overload. Importantly, the administration of TFSA NPs to HFD mice reduces insulin resistance and improves glucose clearance. These findings highlight the therapeutic potential of lysosome-acidifying TFSA NPs for T2D.

Longitudinal in vivo cationic contrast-enhanced computed tomography classifies equine articular cartilage injury and repair.

Cationic contrast-enhanced computed tomography (CECT) capitalizes on increased contrast agent affinity to the charged proteoglycans in articular cartilage matrix to provide quantitative assessment of proteoglycan content with enhanced images. While high resolution microCT has demonstrated success, we investigate cationic CECT use in longitudinal in vivo imaging at clinical resolution. We hypothesize that repeated administration of CA4+ will have no adverse side effects or complications, and that sequential in vivo imaging assessments will distinguish articular cartilage repair tissue from early degenerative and healthy cartilage in critically sized chondral defects. In an established equine translational preclinical model, lameness and synovial effusion scores are similar to controls after repeated injections of CA4+ (eight injections over 16 weeks) compared to controls. Synovial fluid total protein, leukocyte concentration, and sGAG and PGE2 concentrations and articular cartilage and synovial membrane scores are also equivalent to controls. Longitudinal in vivo cationic CECT attenuation in repair tissue is significantly lower than peripheral to (adjacent) and distantly from defects (remote sites) by 4 weeks (p < 0.001), and this difference persists until 16 weeks. At the 6- and 8-week time points, the adjacent locations exhibit significantly lower cationic CECT attenuation compared with the remote sites, reflecting peri-defect degeneration (p < 0.01). Cationic CECT attenuation at clinical resolution significantly correlates with cationic CECT (microCT) (r = 0.69, p < 0.0001), sGAG (r = 0.48, p < 0.0001), and ICRS II histology score (r = 0.63, p < 0.0001). In vivo cationic CECT imaging at clinical resolution distinguishes fibrous repair tissue from degenerative and healthy hyaline cartilage and correlates with molecular tissue properties of articular cartilage.

Accessing Cyclobutane Polymers: Overcoming Synthetic Challenges via Efficient Continuous Flow [2 + 2] Photopolymerization.

We report an improved and efficient method to prepare well-defined, structurally complex truxinate cyclobutane polymers via a thioxanthone sensitized solution state [2 + 2] photopolymerization. Monomers with varying electron density and structure polymerize in good to excellent yields to afford a library of 42 polyesters. Monomers with internal olefin separation distances of greater than 5 Å undergo polymerization via intermolecular [2 + 2] photocycloaddition readily, as opposed to the intramolecular [2 + 2] photocycloaddition observed in monomers with olefins in closer proximity. Use of a continuous flow reactor decreases reaction time, increases polymer molecular weight, and decreases dispersity compared to batch reactions. Furthermore, under continuous flow, polymerization is readily scalable beyond what is possible with batch reactions. This advancement ushers truxinate cyclobutane-based polyesters, which have been historically limited to a few examples and only research scale quantities, to the forefront of development as new materials for potential use across industry sectors.

Immunotherapies for locally aggressive cancers.

Improving surgical resection outcomes for locally aggressive tumors is key to inducing durable locoregional disease control and preventing progression to metastatic disease. Macroscopically complete resection of the tumor is the standard of care for many cancers, including breast, ovarian, lung, sarcoma, and mesothelioma. Advancements in cancer diagnostics are increasing the number of surgically eligible cases through early detection. Thus, a unique opportunity arises to improve patient outcomes with decreased recurrence rates via intraoperative delivery treatments using local drug delivery strategies after the tumor has been resected. Of the current systemic treatments (e.g., chemotherapy, targeted therapies, and immunotherapies), immunotherapies are the latest approach to offer significant benefits. Intraoperative strategies benefit from direct access to the tumor microenvironment which improves drug uptake to the tumor and simultaneously minimizes the risk of drug entering healthy tissues thereby resulting in fewer or less toxic adverse events. We review the current state of immunotherapy development and discuss the opportunities that intraoperative treatment provides. We conclude by summarizing progress in current research, identifying areas for exploration, and discussing future prospects in sustained remission.

Relaxin as a treatment for musculoskeletal fibrosis: What we know and future directions.

Fibrotic changes in musculoskeletal diseases arise from the abnormal buildup of fibrotic tissue around the joints, leading to limited mobility, compromised joint function, and diminished quality of life. Relaxin (RLX) attenuates fibrosis by accelerating collagen degradation and inhibiting excessive extracellular matrix (ECM) production. Further, RLX disrupts myofibroblast activation by modulating the TGF-ß/Smads signaling pathways, which reduces connective tissue fibrosis. However, the mechanisms and effects of RLX in musculoskeletal pathologies are emerging as increasing research focuses on relaxin's impact on skin, ligaments, tendons, cartilage, joint capsules, connective tissues, and muscles. This review delineates the actions of relaxin within the musculoskeletal system and the challenges to its clinical application. Relaxin shows significant potential in both in vivo and in vitro studies for broadly managing musculoskeletal fibrosis; however, challenges such as short biological half-life and sex-specific responses may pose hurdles for clinical use.

Strategy, Design, and Fabrication of Electrochemical Biosensors: A Tutorial.

Advanced healthcare requires novel technologies capable of real-time sensing to monitor acute and long-term health. The challenge relies on converting a real-time quantitative biological and chemical signal into a desired measurable output. Given the success in detecting glucose and the commercialization of glucometers, electrochemical biosensors continue to be a mainstay of academic and industrial research activities. Despite the wealth of literature on electrochemical biosensors, reports are often specific to a particular application (e.g., pathogens, cancer markers, glucose, etc.), and most fail to convey the underlying strategy and design, and if it is transferable to detection of a different analyte. Here we present a tutorial review for those entering this research area that summarizes the basic electrochemical techniques utilized as well as discusses the designs and optimization strategies employed to improve sensitivity and maximize signal output.

Decreased Lung Metastasis in Triple Negative Breast Cancer Following Locally Delivered Supratherapeutic Paclitaxel-Loaded Polyglycerol Carbonate Nanoparticle Therapy.

Breast cancer is among the most prevalent malignancies, accounting for 685,000 deaths worldwide in 2020, largely due to its high metastatic potential. Depending on the stage and tumor characteristics, treatment involves surgery, chemotherapy, targeted biologics, and/or radiation therapy. However, current treatments are insufficient for treating or preventing metastatic disease. Herein, we describe supratherapeutic paclitaxel-loaded nanoparticles (81 wt % paclitaxel) to treat the primary tumor and reduce the risk of subsequent metastatic lesions in the lungs. Primary tumor volume and lung metastasis are reduced by day 30, compared to the paclitaxel clinical standard treatment. The ultrahigh levels of paclitaxel afford an immunotherapeutic effect, increasing natural killer cell activation and decreasing NETosis in the lung, which limits the formation of metastatic lesions.

A missed opportunity: A scoping review of the effect of sex and age on osteoarthritis using large animal models.

OBJECTIVE: The objective was to critically analyze the published literature accounting for sex differences and skeletal age (open vs. closed physis) in preclinical animal models of OA, including the disaggregation of data by sex and skeletal maturity when data is generated from combined sex and/or multi-aged cohorts without proper confounding. METHOD: A scoping literature review of PubMed, Web of Science, EMBASE, and SCOPUS was performed for studies evaluating the effect of sex and age in experimental studies and clinical trials utilizing preclinical large animal models of OA. RESULTS: A total of 9727 papers were identified in large animal (dog, pig, sheep, goat, horse) models for preclinical OA research, of which 238 ex vivo and/or in vivo studies disclosed model type, animal species, sex, and skeletal age sufficient to analyze their effect on outcomes. Dogs, followed by pigs, sheep, and horses, were the most commonly used models. A paucity of preclinical studies evaluated the effect of sex and age in large animal models of naturally occurring or experimentally induced OA: 26 total studies reported some kind of analysis of the effects of sex or age, with 4 studies discussing the effects of sex only, 11 studies discussing the effects of age only, and 11 studies analyzing both the effects of age and sex. CONCLUSION: Fundamental to translational research, OARSI is uniquely positioned to develop recommendations for conducting preclinical studies using large animal models of OA that consider biological mechanisms linked to sex chromosomes, skeletal age, castration, and gonadal hormones affecting OA pathophysiology and treatment response.

Triple contrast computed tomography reveals site-specific biomechanical differences in the human knee joint-A proof of concept study.

Cartilage and synovial fluid are challenging to observe separately in native computed tomography (CT). We report the use of triple contrast agent (bismuth nanoparticles [BiNPs], CA4+, and gadoteridol) to image and segment cartilage in cadaveric knee joints with a clinical CT scanner. We hypothesize that BiNPs will remain in synovial fluid while the CA4+ and gadoteridol will diffuse into cartilage, allowing (1) segmentation of cartilage, and (2) evaluation of cartilage biomechanical properties based on contrast agent concentrations. To investigate these hypotheses, triple contrast agent was injected into both knee joints of a cadaver (N = 1), imaged with a clinical CT at multiple timepoints during the contrast agent diffusion. Knee joints were extracted, imaged with micro-CT (µCT), and biomechanical properties of the cartilage surface were determined by stress-relaxation mapping. Cartilage was segmented and contrast agent concentrations (CA4+ and gadoteridol) were compared with the biomechanical properties at multiple locations (n = 185). Spearman's correlation between cartilage thickness from clinical CT and reference µCT images verifies successful and reliable segmentation. CA4+ concentration is significantly higher in femoral than in tibial cartilage at 60 min and further timepoints, which corresponds to the higher Young's modulus observed in femoral cartilage. In this pilot study, we show that (1) large BiNPs do not diffuse into cartilage, facilitating straightforward segmentation of human knee joint cartilage in a clinical setting, and (2) CA4+ concentration in cartilage reflects the biomechanical differences between femoral and tibial cartilage. Thus, the triple contrast agent CT shows potential in cartilage morphology and condition estimation in clinical CT.

Cationic tantalum oxide nanoparticle contrast agent for micro computed tomography reveals articular cartilage proteoglycan distribution and collagen architecture alterations.

OBJECTIVE: Cationic tantalum oxide nanoparticles (Ta2O5-cNPs), as a newly introduced contrast agent for computed tomography of cartilage, offer quantitative evaluation of proteoglycan (PG) content and biomechanical properties. However, knowledge on the depth-wise impact of cartilage constituents on nanoparticle diffusion, particularly the influence of the collagen network, is lacking. In this study, we aim to establish the depth-dependent relationship between Ta2O5-cNP diffusion and cartilage constituents (PG content, collagen content and network architecture). METHODS: Osteochondral samples (n = 30) were harvested from healthy equine stifle joints (N = 15) and the diffusion of 2.55 nm diameter cationic Ta2O5-cNPs into the cartilage was followed with micro computed tomography (µCT) imaging for up to 96 hours. The diffusion-related parameters, Ta2O5-cNP maximum partition (Pmax) and diffusion time constant, were compared against biomechanical and depth-wise structural properties. Biomechanics were assessed using stress-relaxation and sinusoidal loading protocols, whereas PG content, collagen content and collagen network architecture were determined using digital densitometry, Fourier-transform infrared spectroscopy and polarized light microscopy, respectively. RESULTS: The Pmax correlates with the depth-wise distribution of PGs (bulk Spearman's ρ = 0.87, p < 0.001). More open collagen network architecture at the superficial zone enhances intake of Ta2O5-cNPs, but collagen content overall decreases the intake. The Pmax values correlate with the equilibrium modulus (ρ = 0.80, p < 0.001) of articular cartilage. CONCLUSION: This study establishes the feasibility of Ta2O5-cNPs for the precise and comprehensive identification of biomechanical and structural changes in articular cartilage via contrast-enhanced µCT.

Transcription-Factor-Induced Aggregation of Biomimetic Oligonucleotide-b-Protein Micelles.

Polymeric micelles and especially those based on natural diblocks are of particular interest due to their advantageous properties in terms of molecular recognition, biocompatibility, and biodegradability. We herein report a facile and straightforward synthesis of thermoresponsive elastin-like polypeptide (ELP) and oligonucleotide (ON) diblock bioconjugates, ON-b-ELP, through copper-catalyzed azide-alkyne cycloaddition. The resulting thermosensitive diblock copolymer self-assembles above its critical micelle temperature (CMT ∼30 °C) to form colloidally stable micelles of ∼50 nm diameter. The ON-b-ELP micelles hybridize with an ON complementary strand and maintain their size and stability. Next, we describe the capacity of these micelles to bind proteins, creating more complex structures using the classic biotin-streptavidin pairing and the specific recognition between a transcription factor protein and the ON strand. In both instances, the micelles are intact, form larger structures, and retain their sensitivity to temperature.

A polymer network architecture provides superior cushioning and lubrication of soft tissue compared to a linear architecture.

We report the relationships between linear vs. network polymer architecture and biomechanical outcomes including lubrication and cushioning when the polymers are applied to the surface of articulating knee cartilage. Aqueous formulations of the bioinspired polymer poly(2-methacryloyloxylethyl phosphorylcholine) (pMPC) exhibit tuneable rheological properties, with network pMPC exhibiting increased elasticity and viscosity compared to linear pMPC. Application of a polymer network, compared to a linear one, to articulating tissue surfaces reduces friction, lessens tissue strain, minimizes wear, and protects tissue - thereby improving overall tissue performance. Administration of the network pMPC to the middle carpal joint of skeletally mature horses elicits a safe response similar to saline as monitored over a 70 day period.

High dose, dual-release polymeric films for extended surgical bed paclitaxel delivery.

While surgery represents a major therapy for most solid organ cancers, local recurrence is clinically problematic for cancers such as sarcoma for which adjuvant radiotherapy and systemic chemotherapy provide minimal local control or survival benefit and are dose-limited due to off-target side effects. We describe an implantable, biodegradable poly(1,2-glycerol carbonate) and poly(caprolactone) film with entrapped and covalently-bound paclitaxel enabling safe, controlled, and extended local delivery of paclitaxel achieving concentrations 10,000× tissue levels compared to systemic administration. Films containing entrapped and covalently-bound paclitaxel implanted in the tumor bed, immediately after resection of human cell line-derived chondrosarcoma and patient-derived xenograft liposarcoma and leiomyosarcoma in mice, improve median 90- or 200-day recurrence-free and overall survival compared to control mice. Furthermore, mice in the experimental film arm show no film-related morbidity. Continuous, extended, high-dose paclitaxel delivery via this unique polymer platform safely improves outcomes in three different sarcoma models and provides a rationale for future incorporation into human trials.

Complete substitution with modified nucleotides suppresses the early interferon response and increases the potency of self-amplifying RNA.

Self-amplifying RNA (saRNA) will revolutionize vaccines and in situ therapeutics by enabling protein expression for longer duration at lower doses. However, a major barrier to saRNA efficacy is the potent early interferon response triggered upon cellular entry, resulting in saRNA degradation and translational inhibition. Substitution of mRNA with modified nucleotides (modNTPs), such as N1-methylpseudouridine (N1mΨ), reduce the interferon response and enhance expression levels. Multiple attempts to use modNTPs in saRNA have been unsuccessful, leading to the conclusion that modNTPs are incompatible with saRNA, thus hindering further development. Here, contrary to the common dogma in the field, we identify multiple modNTPs that when incorporated into saRNA at 100% substitution confer immune evasion and enhance expression potency. Transfection efficiency enhances by roughly an order of magnitude in difficult to transfect cell types compared to unmodified saRNA, and interferon production reduces by >8 fold compared to unmodified saRNA in human peripheral blood mononuclear cells (PBMCs). Furthermore, we demonstrate expression of viral antigens in vitro and observe significant protection against lethal challenge with a mouse-adapted SARS-CoV-2 strain in vivo . A modified saRNA vaccine, at 100-fold lower dose than a modified mRNA vaccine, results in a statistically improved performance to unmodified saRNA and statistically equivalent performance to modified mRNA. This discovery considerably broadens the potential scope of self-amplifying RNA, enabling entry into previously impossible cell types, as well as the potential to apply saRNA technology to non-vaccine modalities such as cell therapy and protein replacement.

Intravital measurements of solid stresses in tumours reveal length-scale and microenvironmentally dependent force transmission.

In cancer, solid stresses impede the delivery of therapeutics to tumours and the trafficking and tumour infiltration of immune cells. Understanding such consequences and the origin of solid stresses requires their probing in vivo at the cellular scale. Here we report a method for performing volumetric and longitudinal measurements of solid stresses in vivo, and findings from its applicability to tumours. We used multimodal intravital microscopy of fluorescently labelled polyacrylamide beads injected in breast tumours in mice as well as mathematical modelling to compare solid stresses at the single-cell and tissue scales, in primary and metastatic tumours, in vitro and in mice, and in live mice and post-mortem tissue. We found that solid-stress transmission is scale dependent, with tumour cells experiencing lower stresses than their embedding tissue, and that tumour cells in lung metastases experience substantially higher solid stresses than those in the primary tumours. The dependence of solid stresses on length scale and the microenvironment may inform the development of therapeutics that sensitize cancer cells to such mechanical forces.

Synthesis of Polyethylene Glycol-Poly(glycerol carbonate) Block Copolymeric Micelles as Surfactant-Free Drug Delivery Systems.

We report the synthesis of block copolymers of monomethoxylated polyethylene glycol and poly(glycerol carbonate) (mPEG-b-PGC) via the ring-opening polymerization of benzyl glycidyl ether, monomethoxylated polyethylene glycol, and carbon dioxide using a cobalt salen catalyst. The resulting block copolymers display high polymer/cyclic carbonate selectivity (>99%) and, if two oxirane monomers are used, random incorporation into the polymer feed. The resulting diblock mPEG-b-PGC polymer shows promise as a nanocarrier for surfactant-free, sustained chemotherapeutic delivery. mPEG-b-PGC, with paclitaxel conjugated to the pendant primary alcohol of the glycerol polymer backbone, readily forms 175 nm diameter particles in solution and contains 4.6 wt % paclitaxel (PTX), which is released over 42 days. The mPEG-b-PGC polymer itself is noncytotoxic, whereas the PTX-loaded nanoparticles are cytotoxic to lung, breast, and ovarian cancer cell lines.