Leaky gut model of the human intestinal mucosa for testing siRNA-based nanomedicine targeting JAK1.

Complex in vitro models of human immune cells and intestinal mucosa may have a translation-assisting role in the assessment of anti-inflammatory compounds. Chronic inflammation of the gastrointestinal tract is a hallmark of inflammatory bowel diseases (IBD). In both IBD entities, Crohn's disease and ulcerative colitis, impaired immune cell activation and dysfunctional epithelial barrier are the common pathophysiology. Current therapeutic approaches are targeting single immune modulator molecules to stop disease progression and reduce adverse effects. Such molecular targets can be difficult to assess in experimental animal models of colitis, due to the disease complexity and species differences. Previously, a co-culture model based on human epithelial cells and monocytes arranged in a physiological microenvironment was used to mimic inflamed mucosa for toxicological and permeability studies. The leaky gut model described here, a co-culture of Caco-2, THP-1 and MUTZ-3 cells, was used to mimic IBD-related pathophysiology and for combined investigations of permeability and target engagement of two Janus kinase (JAK) inhibitors, tofacitinib (TOFA) and a JAK1-targeting siRNA nanomedicine. The co-culture just before reaching confluency of the epithelium was used to mimic the compromised intestinal barrier. Delivery efficacy and target engagement against JAK1 was quantified via downstream analysis of STAT1 protein phosphorylation after IFN-γ stimulation. Compared to a tight barrier, the leaky gut model showed 92 ± 5% confluence, a barrier function below 200 Ω*cm2, and enhanced immune response to bacteria-derived lipopolysaccharides. By confocal microscopy we observed an increased accumulation of siJAK1-nanoparticles within the sub-confluent regions leading to uptake into immune cells near the epithelium. A concentration-dependent downregulation of JAK/STAT pathway was observed for siJAK1-nanoparticles (10 ± 12% to 16 ± 12%), whereas TOFA inhibition was 86 ± 2%, compared to untreated cells. By mimicking the status of severely damaged epithelium, like in IBD, the leaky gut model holds promise as a human in vitro system to evaluate the efficacy of anti-inflammatory drugs and nanomedicines.

Innovative transdermal delivery of insulin using gelatin methacrylate-based microneedle patches in mice and mini-pigs.

Painless and controlled on-demand drug delivery is the ultimate goal for the management of various chronic diseases, including diabetes. To achieve this purpose, microneedle patches are gaining increased attention. While degradable microneedle (MN) arrays are widely employed, the use of non-dissolving MN patches remains a challenge to overcome. In this study, we demonstrate that crosslinking gelatin methacrylate with polyethylene glycol diacrylate (PEGDA) is potent for engineering non-dissolving MN arrays. Incorporation of MoS2 nanosheets as a photothermal component into MN hydrogels results in MNs featuring on-demand release properties. An optimized MoS2-MN array patch formed using a hydrogel solution containing 500 µg mL-1 of MoS2 and photochemically crosslinked for 5 min shows required mechanical behavior under a normal compressive load to penetrate the stratum corneum of mice or pig skin and allows the delivery of macromolecular therapeutics such as insulin upon swelling. Using ex vivo and in vivo models, we show that the MoS2-MN patches can be used for loading and releasing insulin for therapeutic purposes. Indeed, transdermal administration of insulin loaded into MoS2-MN patches reduces blood glucose levels in C57BL/6 mice and mini-pigs comparably to subcutaneously injected insulin. We believe that this on-demand delivery system might alter the current insulin therapies and might be a potential approach for delivery of other proteins.

Tuning the Immunostimulation Properties of Cationic Lipid Nanocarriers for Nucleic Acid Delivery.

Nonviral systems, such as lipid nanoparticles, have emerged as reliable methods to enable nucleic acid intracellular delivery. The use of cationic lipids in various formulations of lipid nanoparticles enables the formation of complexes with nucleic acid cargo and facilitates their uptake by target cells. However, due to their small size and highly charged nature, these nanocarrier systems can interact in vivo with antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages. As this might prove to be a safety concern for developing therapies based on lipid nanocarriers, we sought to understand how they could affect the physiology of APCs. In the present study, we investigate the cellular and metabolic response of primary macrophages or DCs exposed to the neutral or cationic variant of the same lipid nanoparticle formulation. We demonstrate that macrophages are the cells affected most significantly and that the cationic nanocarrier has a substantial impact on their physiology, depending on the positive surface charge. Our study provides a first model explaining the impact of charged lipid materials on immune cells and demonstrates that the primary adverse effects observed can be prevented by fine-tuning the load of nucleic acid cargo. Finally, we bring rationale to calibrate the nucleic acid load of cationic lipid nanocarriers depending on whether immunostimulation is desirable with the intended therapeutic application, for instance, gene delivery or messenger RNA vaccines.

Introduction of a model of skin lesions on rats and testing of dissolving microneedles containing 5-aminolevulinic acid.

Topical photodynamic therapy (PDT) is widely used to treat non melanoma skin cancers. It consists of topically applying on the skin lesions a cream containing a prodrug (5-aminolevulinic acid (5-ALA) or methyl aminolevulinate (MAL)) that is then metabolized to the photosensitizer protoporphyrin IX (PpIX). Light irradiation at PpIX excitation wavelength combined with oxygen then lead to a photochemical reaction inducing cell death. Nevertheless, this conventional PDT treatment is currently restricted to superficial skin lesions since the penetration depth of the prodrug is limited and hampers the production of PpIX in deep seated lesions. To overcome this problem, dissolving microneedles (MNs) included in a square flexible patch were developed. This easy-to-handle MN-patch is composed of 5-ALA mixed with hyaluronic acid (HA) and has the ability to dissolve after skin application. To evaluate the efficiency of this MN-patch in vivo, a skin lesion model has been developed on rats by applying UV-B illuminations. After 40 UV-B illuminations, histological and pharmacokinetic controls confirmed that the rats presented skin lesions. Once the rat skin lesion model has been validated, it was demonstrated that the MNs penetrated into the skin and fully dissolved in one hour on most of the rats. After one hour, the fluorescence images showed that the MN-patch produced a consequent and homogeneous level of PpIX. Overall, the dissolving MN-patch is a recent technology that has interesting features and several preclinical investigations should be led to compare its efficiency to that of the conventional treatment for PDT of non melanoma skin cancers.

A facile fabrication of dissolving microneedles containing 5-aminolevulinic acid.

Photodynamic therapy induced by protoporphyrin IX (PpIX) is widely used to treat precancerous skin lesions. The penetration depth of the prodrug 5-aminolevulinic acid (5-ALA) using topical application is currently limited, which hampers the production of PpIX in deep seated lesions. To enhance 5-ALA delivery in deep skin layers, a soluble microneedles patch (MN-patch) containing 5-ALA has been successfully developed by using a fast solvent casting molding method which could be easily up-scaled. The shape, number and height of the needles have been designed according to the medical application and the mechanical strain necessary for skin insertion. Hyaluronic acid (HA) has been chosen as the needle materials due to its biocompatibility, fast solubility and biodegradation and was mixed with 5-ALA prior to casting. HA-based MN-patch containing 5-ALA have exhibited mechanical properties enabling a good insertion into the skin without significant damages to MN. Interactions between HA and 5-ALA were evaluated by Fourier transform infrared spectroscopy (FTIR) and carbon nuclear magnetic resonance (13C NMR), stability of 5-ALA in the MN-patch was monitored by proton nuclear magnetic resonance (1H NMR) and exhibited a good stability over 5 months after manufacturing. Dissolution rate of the whole patch was completed in 1 hour in ex vivo rat skin without cytotoxicity. Overall, the MN-patch can be a promising technique to enhance 5-ALA penetration and produce PpIX in deeper skin lesions.

ß-CD-Functionalized Microdevice for Rapid Capture and Release of Bacteria.

Most procedures for detecting pathogens in liquid media require an initial concentration step. In this regard, carbohydrates have proven to be attractive affinity ligands for the solid-phase capture of bacteria that use lectins for adhesion to host cell membranes. However, the use of cyclodextrin-immobilized substrates to selectively trap bacteria has not been explored before. Here, using quartz-crystal microbalance with dissipation monitoring experiments, we demonstrate that functionalization of surfaces by ß-cyclodextrin (ß-CD) can not only allow for rapid and efficient capture of bacterial cells in liquid but also their facile elution with an aqueous solution of a selectively methylated ß-CD derivative as a competitive molecule. This capture/elution strategy, which is based on host-guest interactions between membrane components of the bacterial cell and the CD cavities, is performed in physiological conditions and can be integrated in a microchip. Indeed, proof-of-concept studies showed the potential of ß-CD-modified micropillar-integrated microfluidic devices for concentration of bacteria. The results obtained with Escherichia coli suggest that this approach could be broadly applicable among Gram-negative bacteria, which share common cell membrane structures.

Point-of-care diagnostics for ricin exposure.

A long-sought milestone in the defense against bioterrorism is the development of rapid, simple, and near-patient assays for diagnostic and theranostic purposes. Here, we present a powerful test based on a host response to a biological weapon agent, namely the ricin toxin. A signature for exposure to ricin was extracted and characterized in mice and then integrated into a plastic microfluidic cartridge. This enabled early diagnosis of exposure to ricin in mice using a drop of whole blood in less than 1 h and 30 min. The cartridge stores the reagents and implements all of the steps of the analysis, including mRNA extraction from a drop of blood, followed by tens of parallel RT-qPCR reactions. The simple and low-cost microfluidic cartridge developed here may find other applications in point-of-care diagnostics.

Single bacteria identification by Raman spectroscopy.

We report on rapid identification of single bacteria using a low-cost, compact, Raman spectroscope. We demonstrate that a 60-s procedure is sufficient to acquire a comprehensive Raman spectrum in the range of 600 to 3300 cm⁻¹. This time includes localization of small bacteria aggregates, alignment on a single individual, and spontaneous Raman scattering signal collection. Fast localization of small bacteria aggregates, typically composed of less than a dozen individuals, is achieved by lensfree imaging over a large field of view of 24 mm². The lensfree image also allows precise alignment of a single bacteria with the probing beam without the need for a standard microscope. Raman scattered light from a 34-mW continuous laser at 532 nm was fed to a customized spectrometer (prototype Tornado Spectral Systems). Owing to the high light throughput of this spectrometer, integration times as low as 10 s were found acceptable. We have recorded a total of 1200 spectra over seven bacterial species. Using this database and an optimized preprocessing, classification rates of ~90% were obtained. The speed and sensitivity of our Raman spectrometer pave the way for high-throughput and nondestructive real-time bacteria identification assays. This compact and low-cost technology can benefit biomedical, clinical diagnostic, and environmental applications.

Macro to microfluidics system for biological environmental monitoring.

Biological environmental monitoring (BEM) is a growing field of research which challenges both microfluidics and system automation. The aim is to develop a transportable system with analysis throughput which satisfies the requirements: (i) fully autonomous, (ii) complete protocol integration from sample collection to final analysis, (iii) detection of diluted molecules or biological species in a large real life environmental sample volume, (iv) robustness and (v) flexibility and versatility. This paper discusses all these specifications in order to define an original fluidic architecture based on three connected modules, a sampling module, a sample preparation module and a detection module. The sample preparation module highly concentrates on the pathogens present in a few mL samples of complex and unknown solutions and purifies the pathogens' nucleic acids into a few µL of a controlled buffer. To do so, a two-step concentration protocol based on magnetic beads is automated in a reusable macro-to-micro fluidic system. The detection module is a PCR based miniaturized platform using digital microfluidics, where reactions are performed in 64 nL droplets handled by electrowetting on dielectric (EWOD) actuation. The design and manufacture of the two modules are reported as well as their respective performances. To demonstrate the integration of the complete protocol in the same system, first results of pathogen detection are shown.