Transport of CLCA2 to the nucleus by extracellular vesicles controls keratinocyte survival and migration.

Chloride channel accessory 2 (CLCA2) is a transmembrane protein, which promotes adhesion of keratinocytes and their survival in response to hyperosmotic stress. Here we show that CLCA2 is transported to the nucleus of keratinocytes via extracellular vesicles. The nuclear localization is functionally relevant, since wild-type CLCA2, but not a mutant lacking the nuclear localization signal, suppressed migration of keratinocytes and protected them from hyperosmotic stress-induced cell death. In the nucleus, CLCA2 bound to and activated ß-catenin, resulting in enhanced expression of Wnt target genes. Mass-spectrometry-based interaction screening and functional rescue studies identified RNA binding protein 3 as a key effector of nuclear CLCA2. This is of likely relevance in vivo because both proteins co-localize in the human epidermis. Together, these results identify an unexpected nuclear function of CLCA2 in keratinocytes under homeostatic and stress conditions and suggest a role of extracellular vesicles and their nuclear transport in the control of key cellular activities.

A Bioinspired and Cost-Effective Device for Minimally Invasive Blood Sampling.

Conventional venipuncture is invasive and challenging in low and middle-income countries. Conversely, point-of-care devices paired with fingersticks, although less invasive, suffer from high variability and low blood volume collection. Recently approved microsampling devices address some of these issues but remain cost-prohibitive for resource-limited settings. In this work, a cost-effective microsampling device is described for the collection of liquid blood with minimal invasiveness and sufficient volume retrieval for laboratory analyses or immediate point-of-care testing. Inspired by the anatomy of sanguivorous leeches, the single-use device features a storage compartment for blood collection and a microneedle patch hidden within a suction cup. Finite Element Method simulations, corroborated by mechanical analyses, guide the material selection for device fabrication and design optimization. In piglets, the device successfully collects ≈195 µL of blood with minimal invasiveness. Additionally, a tailor-made lid and adapter enable safe fluid transportation and integration with commercially available point-of-care systems for on-site analyses, respectively. Taken together, the proposed platform holds significant promise for enhancing healthcare in the pediatric population by improving patient compliance and reducing the risk of needlestick injuries through concealed microneedles. Most importantly, given its cost-effective fabrication, the open-source microsampling device may have a meaningful impact in resource-limited healthcare settings.

The intracellular visualization of exogenous DNA in fluorescence microscopy.

In the development of non-viral gene delivery vectors, it is essential to reliably localize and quantify transfected DNA inside the cell. To track DNA, fluorescence microscopy methods are commonly applied. These mostly rely on fluorescently labeled DNA, DNA binding proteins fused to a fluorescent protein, or fluorescence in situ hybridization (FISH). In addition, co-stainings are often used to determine the colocalization of the DNA in specific cellular compartments, such as the endolysosomes or the nucleus. We provide an overview of these DNA tracking methods, advice on how they should be combined, and indicate which co-stainings or additional methods are required to draw precise conclusions from a DNA tracking experiment. Some emphasis is given to the localization of exogenous DNA inside the nucleus, which is the last step of DNA delivery. We argue that suitable tools which allow for the nuclear detection of faint signals are still missing, hampering the rational development of more efficient non-viral transfection systems.

Loss of Dnmt3a increased self-renewal and resistance to pegIFNα in JAK2-V617F-positive myeloproliferative neoplasms.

Pegylated interferon alpha (pegIFNα) can induce molecular remissions in JAK2-V617F-positive myeloproliferative neoplasms (MPN) patients by targeting long-term hematopoietic stem cells (LT-HSCs). Additional somatic mutations in genes regulating LT-HSC self-renewal, such as DNMT3A, have been reported to have poorer responses to pegIFNα. We investigated if DNMT3A loss leads to alterations in JAK2-V617F LT-HSCs functions conferring resistance to pegIFNα treatment in a mouse model of MPN and in hematopoietic progenitors from MPN patients. Long-term treatment with pegIFNα normalized blood parameters, reduced splenomegaly and JAK2-V617F-chimerism in single-mutant JAK2-V617F (VF) mice. However, pegIFNα in VF;Dnmt3aΔ/Δ (VF;DmΔ/Δ) mice worsened splenomegaly and failed to reduce JAK2-V617F-chimerism. Furthermore, LT-HSCs from VF;DmΔ/Δ mice compared to VF were less prone to accumulate DNA damage and exit dormancy upon pegIFNα treatment. RNA-sequencing showed that IFNα induced stronger upregulation of inflammatory pathways in LT-HSCs from VF;DmΔ/Δ compared to VF mice, indicating that the resistance of VF;DmΔ/Δ LT-HSC was not due to failure in IFNα signaling. Transplantations of bone marrow from pegIFNα treated VF;DmΔ/Δ mice gave rise to more aggressive disease in secondary and tertiary recipients. Liquid cultures of hematopoietic progenitors from MPN patients with JAK2-V617F and DNMT3A mutation showed increased percentages of JAK2-V617F-positive colonies upon IFNα exposure, whereas in patients with JAK2-V617F alone the percentages of JAK2-V617F-positive colonies decreased or remained unchanged. PegIFNα combined with 5-azacytidine only partially overcame resistance in VF;DmΔ/Δ mice. However, this combination strongly decreased the JAK2-mutant allele burden in mice carrying VF mutation only, showing potential to inflict substantial damage preferentially to the JAK2-mutant clone.

Cyclodextrin microneedles for the delivery of a nanoparticle-based peptide antigen vaccine.

In recent years, microneedles (MNs) have gained considerable interest in drug formulation due to their non-invasive and patient-friendly nature. Dissolving MNs have emerged as a promising approach to enhance drug delivery across the skin in a painless manner without generating sharp waste and providing the possibility for self-administration. Cyclodextrins, a group of cyclic oligosaccharides, are well-established in pharmaceutical products due to their safety and unique ability to form inclusion complexes with various drug molecules. In this manuscript, we report the development and characterization of dissolving MNs composed of cyclodextrins for intradermal delivery of a cyclodextrin-based nanoparticulate vaccine. Different cyclodextrins were tested and the most promising candidates were fabricated into MNs by micromolding. The MNs' piercing effectiveness and drug permeation across the skin were tested ex vivo. Furthermore, in vivo studies were carried out to assess the skin's tolerance to cyclodextrin-based MNs, and to evaluate the immune response using a model peptide antigen in a mouse model. The data revealed that the MNs were well-tolerated and effective, even leading to dose-sparing effects. This study highlights the potential of cyclodextrin-based dissolving MNs as a versatile platform for intradermal vaccine delivery, providing a compatible matrix for nanoparticulate formulations to enhance immune responses.

Inhibition of urease-mediated ammonia production by 2-octynohydroxamic acid in hepatic encephalopathy.

Hepatic encephalopathy is a neuropsychiatric complication of liver disease which is partly associated with elevated ammonemia. Urea hydrolysis by urease-producing bacteria in the colon is often mentioned as one of the main routes of ammonia production in the body, yet research on treatments targeting bacterial ureases in hepatic encephalopathy is limited. Herein we report a hydroxamate-based urease inhibitor, 2-octynohydroxamic acid, exhibiting improved in vitro potency compared to hydroxamic acids that were previously investigated for hepatic encephalopathy. 2-octynohydroxamic acid shows low cytotoxic and mutagenic potential within a micromolar concentration range as well as reduces ammonemia in rodent models of liver disease. Furthermore, 2-octynohydroxamic acid treatment decreases cerebellar glutamine, a product of ammonia metabolism, in male bile duct ligated rats. A prototype colonic formulation enables reduced systemic exposure to 2-octynohydroxamic acid in male dogs. Overall, this work suggests that urease inhibitors delivered to the colon by means of colonic formulations represent a prospective approach for the treatment of hepatic encephalopathy.

pH-dependent pressure-sensitive colonic capsules for the delivery of aqueous bacterial suspensions.

Microbiome-based therapies hold great promise for treating various diseases, but the efficient delivery of live bacteria to the colon remains a challenge. Furthermore, current oral formulations, such as lyophilized bacterial capsules or tablets, are produced using processes that can decrease bacterial viability. Consequently, high dosages are required to achieve efficacy. Herein, we report the design of pressure-sensitive colonic capsules for the encapsulation and delivery of aqueous suspensions of live bacteria. The capsules consisted of 2 functional thin-films (hydrophobic and enteric) of ethyl cellulose and Eudragit S100 dip-coated onto hydroxypropyl methylcellulose molds. The capsules could be loaded with aqueous media and provide protection against acidic fluids and, to some extent, oxygen diffusion, suggesting their potential suitability for delivering anaerobic bacterial strains. Disintegration and mechanical studies indicated that the capsules could withstand transit through the stomach and upper/proximal small intestinal segments and rupture in the ileum/colon. In vitro studies showed that bacterial cells (anaerobic and aerobic commensals) remained highly viable (74-98%) after encapsulation and exposure to the simulated GI tract conditions. In vivo studies with a beagle dog model revealed that 67% of the capsules opened after 3.5 h, indicating content release in the distal gastrointestinal tract. These data demonstrate that live aqueous bacterial suspensions comprised of both aerobic and anaerobic commensals can be encapsulated and in the future might be efficiently delivered to the distal gastrointestinal tract, suggesting the practical applications of these capsules in microbiome-based therapies.

Boosting systemic absorption of peptides with a bioinspired buccal-stretching patch.

Biopharmaceuticals, including proteins and peptides, have revolutionized the treatment of a wide range of diseases, from diabetes and cardiovascular disorders to virus infections and cancer. Despite their efficacy, most of these macromolecular drugs require parenteral administration because of their high molecular weight and relative instability. Over the past 40 years, only a few oral peptide drugs have entered clinical trials, even when formulated with substantial amounts of permeation enhancers. To overcome the epithelial barrier, devices that inject drugs directly into the gastrointestinal mucosa have been proposed recently. However, the robustness and safety of those complex systems are yet to be assessed. In this study, we introduced an innovative technology to boost drug absorption by synergistically combining noninvasive stretching of the buccal mucosa with permeation enhancers. Inspired by the unique structural features of octopus suckers, a self-applicable suction patch was engineered, enabling strong adhesion to and effective mechanical deformation of the mucosal tissue. In dogs, this suction patch achieved bioavailability up to two orders of magnitude higher than those of the commercial tablet formulation of desmopressin, a peptide drug known for its poor oral absorption. Moreover, systemic exposure comparable to that of the approved oral semaglutide tablet was achieved without further optimization. Last, a first-in-human study involving 40 healthy participants confirmed the dosage form's acceptability, thereby supporting the clinical translatability of this simple yet effective platform technology.

Get out or die trying: Peptide- and protein-based endosomal escape of RNA therapeutics.

RNA therapeutics offer great potential to transform the biomedical landscape, encompassing the treatment of hereditary conditions and the development of better vaccines. However, the delivery of RNAs into the cell is hampered, among others, by poor endosomal escape. This major hurdle is often tackled using special lipids, polymers, or protein-based delivery vectors. In this review, we will focus on the most prominent peptide- and protein-based endosomal escape strategies with focus on RNA drugs. We discuss cell penetrating peptides, which are still incorporated into novel transfection systems today to promote endosomal escape. However, direct evidence for enhanced endosomal escape by the action of such peptides is missing and their transfection efficiency, even in permissive cell culture conditions, is rather low. Endosomal escape by the help of pore forming proteins or phospholipases, on the other hand, allowed to generate more efficient transfection systems. These are, however, often hampered by considerable toxicity and immunogenicity. We conclude that the perfect enhancer of endosomal escape has yet to be devised. To increase the chances of success, any new transfection system should be tested under relevant conditions and guided by assays that allow direct quantification of endosomal escape.

Ultrafast and Controlled Capturing, Loading, and Release of Extracellular Vesicles by a Portable Microstructured Electrochemical Fluidic Device.

Extracellular vesicles (EVs) are secreted by all living cells and are found in body fluids. They exert numerous physiological and pathological functions and serve as cargo shuttles. Due to their safety and inherent bioactivity, they have emerged as versatile therapeutic agents, biomarkers, and potential drug carriers. Despite the growing interest in EVs, current progress in this field is, in part, limited by relatively inefficient isolation techniques. Conventional methods are indeed slow, laborious, require specialized laboratory equipment, and may result in low yield and purity. This work describes an electrochemically controlled "all-in-one" device enabling capturing, loading, and releasing of EVs. The device is composed of a fluidic channel confined within antibody-coated microstructured electrodes. It rapidly isolates EVs with a high level of purity from various biofluids. As a proof of principle, the device is applied to isolate EVs from skin wounds of healthy and diabetic mice. Strikingly, it is found that EVs from healing wounds of diabetic mice are enriched in mitochondrial proteins compared to those of healthy mice. Additionally, the device improves the loading protocol of EVs with polyplexes, and may therefore find applications in nucleic acid delivery. Overall, the electrochemical device can greatly facilitate the development of EVs-based technologies.

Improving extracellular vesicles production through a Bayesian optimization-based experimental design.

With the growing demand and diversity of biological drugs, developing optimal processes for their accelerated production with minimal resource utilization is a pressing challenge. Typically, such optimization involves multiple target properties, such as production yield, biological activity, and product purity. Therefore, strategic experimental design techniques that can characterize the parameter space while simultaneously arriving at the optimal process satisfying multiple target properties are required. To achieve this, we propose the use of a multi-objective batch Bayesian optimization (MOBBO) algorithm and illustrate its successful application for the production of extracellular vesicles (EVs) from a 3D culture of mesenchymal stem cells (MSCs) considering three objectives, namely to maximize the vesicle-to-protein ratio, maximize the enzymatic activity of the MSC-EV protein CD73, and minimize the amount of calregulin impurities. We show that the optimal combination of the process parameters to address the intended objectives could be achieved with only 32 experiments. For the four parameters considered (i.e., microcarrier concentration, seeding density, centrifugation time, and impeller speed), this number of experiments is comparable to or lower than the classical design of experiments (DoE) and the traditional one-factor-at-a-time (OFAT) approach. We illustrate how the algorithm adaptively samples in the process parameter space, selectively excluding unfavorable regions, thus minimizing the number of experiments required to reach optimal conditions. Finally, we compare the obtained solutions to the literature data and present possible applications of the collected data for other modeling activities such as Quality by Design, process monitoring, control, and scale-up.

Engineering Lipid Spherulites for the Sustained Release of Highly Dosed Small Hydrophilic Compounds.

Currently, there is a lack of parenteral sustained release formulations for the delivery of highly dosed small hydrophilic drugs. Therefore, parenteral lipid spherulites are engineered capable of entrapping large amounts of such compounds and spontaneously releasing them in a sustained fashion. A library of spherulites is prepared with a simple green process, using phosphatidylcholine (PC) and/or phosphatidylethanolamine (PE), nonionic surfactants and water. The vesicle formulations exhibiting appropriate size distribution and morphology are selected and loaded with 4,6-di-O-(methoxy-diethyleneglycol)-myo-inositol-1,2,3,5-tetrakis(phosphate), ((OEG2 )2 -IP4), an inositol phosphate derivative currently under clinical evaluation for the treatment of aortic valve stenosis. The loading efficiency of spherulites is up to 12.5-fold higher than that of liposomes produced with the same materials. While the PC-containing vesicles showed high stability, the PE spherulites gradually lost their multilayer organization upon dilution, triggering the active pharmaceutical ingredient (API) release over time. In vitro experiments and pharmacokinetic studies in rats demonstrated the ability of PE spherulites to increase the systemic exposure of (OEG2 )2 -IP4 up to 3.1-fold after subcutaneous injection, and to completely release their payload within 3-4 d. In conclusion, PE spherulites represent a promising lipid platform for the extravascular parenteral administration of highly dosed small hydrophilic drugs.

3D printing of a controlled urea delivery device for the prevention of tooth decay.

Dental caries is one of the most widespread chronic infectious diseases in the world. It is mainly caused by the production of acid in the biofilm from the bacterial metabolism of carbohydrates. Nowadays, the prevention of caries is mainly based on the use of topical formulations containing fluoride. However, effective fluoride supplementation may not be sufficient in high-risk individuals, leading to the exploration of alternative strategies such as the neutralization of acid in the oral cavity. Urea is hydrolyzed into ammonia by oral bacteria, leading to a local alkalization that may counteract tooth decay. Herein, we report the fabrication of 3D printed personalized dental trays with a local and prolonged release of urea. Composite filaments with tunable urea release kinetics were produced by hot melt extrusion of poly(ε-caprolactone) and poly(vinyl alcohol) or poly(ethylene glycol) blends mixed with urea. The filaments were further used to 3D print by fused deposition modeling objects capable of releasing urea in a sustained and spatially controlled manner. In vitro studies performed in the presence of Streptococcus salivarius demonstrated the ability of urea released from a 3D printed model toothguards to reduce the pH drop induced by carbohydrates. This study showed the potential of urea-loaded devices to reduce cariogenic acidification of the environment surrounding the enamel by delivering urea directly to the tooth surface.

Digital Light 3D Printed Bioresorbable and NIR-Responsive Devices with Photothermal and Shape-Memory Functions.

Digital light processing (DLP) 3D printing is a promising technique for the rapid manufacturing of customized medical devices with high precision. To be successfully translated to a clinical setting, challenges in the development of suitable photopolymerizable materials have yet to be overcome. Besides biocompatibility, it is often desirable for the printed devices to be biodegradable, elastic, and with a therapeutic function. Here, a multifunctional DLP printed material system based on the composite of gold nanorods and polyester copolymer is reported. The material demonstrates robust near-infrared (NIR) responsiveness, allowing rapid and stable photothermal effect leading to the time-dependent cell death. NIR light-triggerable shape transformation is demonstrated, resulting in a facilitated insertion and expansion of DLP printed stent ex vivo. The proposed strategy opens a promising avenue for the design of multifunctional therapeutic devices based on nanoparticle-polymer composites.

3D printed elastomers with Sylgard-184-like mechanical properties and tuneable degradability.

The 3D printing of biodegradable elastomers with high mechanical strength is of great interest for personalized medicine, but rather challenging. In this study, we propose a dual-polymer resin formulation for digital light processing of biodegradable elastomers with tailorable mechanical properties comparable to those of Sylgard-184.

3D printing of a controlled fluoride delivery device for the prevention and treatment of tooth decay.

Dental decay is a highly prevalent chronic disease affecting people from all ages. Clinically, fluoride supplementation is the primary strategy in the prevention of dental decay. However, the current existing self-application formulations such as gels or mouthwashes are rapidly cleared after administration, resulting in modest efficacy even after repeated applications. Therefore, a user-friendly formulation that can provide sustained fluoride release in the oral cavity is of great interest for dental decay prevention. Herein, we report the utilization of fused deposition modelling to fabricate personalised mouthguards, which allow local and prolonged fluoride elution. Composite filaments comprising sodium fluoride and polymers with tuneable hydrophobicity were produced using blends of poly(ε-caprolactone) (PCL) and poly(vinyl alcohol) or poly(ethylene glycol) (PEG). The materials exhibited suitable mechanical properties for dental devices as well as different release kinetics depending on their composition. Ex vivo studies were performed on decayed human teeth using the 3D printed tooth caps that precisely fit the complex geometries of each specimen. A significant elevation of fluoride content in the lesion mineral in contact with the PCL/PEG tooth caps was achieved compared to the ones in contact with solutions mimicking dental care products. In conclusion, this study suggested that a sustained localized drug release of fluoride from personalised 3D printed mouthguards at the device-enamel interface can improve the incorporation of fluoride in the tooth matrix and prevent lesion progression.

Light-Based Printing of Leachable Salt Molds for Facile Shaping of Complex Structures.

3D printing is a powerful manufacturing technology for shaping materials into complex structures. While the palette of printable materials continues to expand, the rheological and chemical requisites for printing are not always easy to fulfill. Here, a universal manufacturing platform is reported for shaping materials into intricate geometries without the need for their printability, but instead using light-based printed salt structures as leachable molds. The salt structures are printed using photocurable resins loaded with NaCl particles. The printing, debinding, and sintering steps involved in the process are systematically investigated to identify ink formulations enabling the preparation of crack-free salt templates. The experiments reveal that the formation of a load-bearing network of salt particles is essential to prevent cracking of the mold during the process. By infiltrating the sintered salt molds and leaching the template in water, complex-shaped architectures are created from diverse compositions such as biomedical silicone, chocolate, light metals, degradable elastomers, and fiber composites, thus demonstrating the universal, cost-effective, and sustainable nature of this new manufacturing platform.

Activity-Based Approach for Selective Molecular CO2 Sensing.

Carbon dioxide (CO2) impacts every aspect of life, and numerous sensing technologies have been established to detect and monitor this ubiquitous molecule. However, its selective sensing at the molecular level remains an unmet challenge, despite the tremendous potential of such an approach for understanding this molecule's role in complex environments. In this work, we introduce a unique class of selective fluorescent carbon dioxide molecular sensors (CarboSen) that addresses these existing challenges through an activity-based approach. Besides the design, synthesis, and evaluation of these small molecules as CO2 sensors, we demonstrate their utility by tailoring their reactivity and optical properties, allowing their use in a broad spectrum of multidisciplinary applications, including atmospheric sensing, chemical reaction monitoring, enzymology, and live-cell imaging. Collectively, these results showcase the potential of CarboSen sensors as broadly applicable tools to monitor and visualize carbon dioxide across multiple disciplines.

The TFAMoplex-Conversion of the Mitochondrial Transcription Factor A into a DNA Transfection Agent.

Non-viral gene delivery agents, such as cationic lipids, polymers, and peptides, mainly rely on charge-based and hydrophobic interactions for the condensation of DNA molecules into nanoparticles. The human protein mitochondrial transcription factor A (TFAM), on the other hand, has evolved to form nanoparticles with DNA through highly specific protein-protein and protein-DNA interactions. Here, the properties of TFAM are repurposed to create a DNA transfection agent by means of protein engineering. TFAM is covalently fused to Listeria monocytogenes phospholipase C (PLC), an enzyme that lyses lipid membranes under acidic conditions, to enable endosomal escape and human vaccinia-related kinase 1 (VRK1), which is intended to protect the DNA from cytoplasmic defense mechanisms. The TFAM/DNA complexes (TFAMoplexes) are stabilized by cysteine point mutations introduced rationally in the TFAM homodimerization site, resulting in particles, which show maximal activity when formed in 80% serum and transfect HeLa cells in vitro after 30 min of incubation under challenging cell culture conditions. The herein developed TFAM-based DNA scaffolds combine interesting characteristics in an easy-to-use system and can be readily expanded with further protein factors. This makes the TFAMoplex a promising tool in protein-based gene delivery.