Mucosomes: Intrinsically Mucoadhesive Glycosylated Mucin Nanoparticles as Multi-Drug Delivery Platform

Mucus is a complex barrier for pharmacological treatments and overcoming it is one of the major challenges faced during transmucosal drug delivery. To tackle this issue, a novel class of glycosylated nanoparticles, named "mucosomes," which are based on the most important protein constituting mucus, the mucin, is introduced. Mucosomes are designed to improve drug absorption and residence time on the mucosal tissues. Mucosomes are produced (150-300 nm), functionalized with glycans, and loaded with the desired drug in a single one-pot synthetic process and, with this method, a wide range of small and macro molecules can be loaded with different physicochemical properties. Various in vitro models are used to test the mucoadhesive properties of mucosomes. The presence of functional glycans is indicated by the interaction with lectins. Mucosomes are proven to be storable at 4 degrees C after lyophilization, and administration through a nasal spray does not modify the morphology of the mucosomes. In vitro and in vivo tests indicate mucosomes do not induce adverse effects under the investigated conditions. This study proposes mucosomes as a ground-breaking nanosystem that can be applied in several pathological contexts, especially in mucus-related disorders

Hep3Gel: A Shape-Shifting Extracellular Matrix-Based, Three-Dimensional Liver Model Adaptable to Different Culture Systems

Drug-induced hepatotoxicity is a leading cause of clinical trial withdrawal. Therefore, in vitro modeling the hepatic behavior and functionalities is not only crucial to better understand physiological and pathological processes but also to support drug development with reliable high-throughput platforms. Different physiological and pathological models are currently under development and are commonly implemented both within platforms for standard 2D cultures and within tailor-made chambers. This paper introduces Hep3Gel: a hybrid alginate-extracellular matrix (ECM) hydrogel to produce 3D in vitro models of the liver, aiming to reproduce the hepatic chemomechanical niche, with the possibility of adapting its shape to different manufacturing techniques. The ECM, extracted and powdered from porcine livers by a specifically set-up procedure, preserved its crucial biological macromolecules and was embedded within alginate hydrogels prior to crosslinking. The viscoelastic behavior of Hep3Gel was tuned, reproducing the properties of a physiological organ, according to the available knowledge about hepatic biomechanics. By finely tuning the crosslinking kinetics of Hep3Gel, its dualistic nature can be exploited either by self-spreading or adapting its shape to different culture supports or retaining the imposed fiber shape during an extrusion-based 3D-bioprinting process, thus being a shape-shifter hydrogel. The self-spreading ability of Hep3Gel was characterized by combining empirical and numerical procedures, while its use as a bioink was experimentally characterized through rheological a priori printability evaluations and 3D printing tests. The effect of the addition of the ECM was evident after 4 days, doubling the survival rate of cells embedded within control hydrogels. This study represents a proof of concept of the applicability of Hep3Gel as a tool to develop 3D in vitro models of the liver

Toward 3D-Bioprinted Models of the Liver to Boost Drug Development

The main problems in drug development are connected to enormous costs related to the paltry success rate. The current situation empowered the development of high-throughput and reliable instruments, in addition to the current golden standards, able to predict the failures in the early preclinical phase. Being hepatotoxicity responsible for the failure of 30% of clinical trials, and the 21% of withdrawal of marketed drugs, the development of complex in vitro models (CIVMs) of liver is currently one of the hottest topics in the field. Among the different fabrication techniques, 3D-bioprinting is emerging as a powerful ally for their production, allowing the manufacture of three-dimensional constructs characterized by computer-controlled and customized geometry, and inter-batches reproducibility. Thanks to these, it is possible to rapidly produce tailored cell-laden constructs, to be cultured within static and dynamic systems, thus reaching a further degree of personalization when designing in vitro models. This review highlights and prioritizes the most recent advances related to the development of CIVMs of the hepatic environment to be specifically applied to pharmaceutical research, with a special focus on 3D-bioprinting, since the liver is primarily involved in the metabolism of drugs

Internally crosslinked alginate-based bioinks for the fabrication of in vitro hepatic tissue models

Bioprinting is a key technique to fabricate cell-laden volumetric constructs with controlled geometry. It can be used not only to replicate the architecture of a target organ but also to produce shapes that allow for the mimicry, in vitro, of specific desired features. Among the various materials suitable to be processed with this technique, sodium alginate is currently considered one of the most appealing because of its versatility. To date, the most widespread strategies to print alginate-based bioinks exploit external gelation as a primary process, by directly extruding the hydrogel-precursor solution into a crosslinking bath or within a sacrificial crosslinking hydrogel, where the gelation takes place. In this work, we describe the print optimization and the processing of Hep3Gel: an internally crosslinked alginate and ECM-based bioink for the production of volumetric hepatic tissue models. We adopted an unconventional strategy, by moving from the reproduction of the geometry and the architecture of liver tissue to the use of bioprinting to fabricate structures that can promote a high degree of oxygenation, as is the case with hepatic tissue. To this end, the design of structures was optimized by employing computational methods. The printability of the bioink was then studied and optimized through a combination of different a priori and a posteriori analyses. We produced 14-layered constructs, thus highlighting the possibility to exploit internal gelation alone to directly print self-standing structures with finely controlled viscoelastic properties. Constructs loaded with HepG2 cells were successfully printed and cultured in static conditions for up to 12 d, underlining the suitability of Hep3Gel to support mid/long-term cultures

Drug-induced hepatotoxicity studied in a 3D, in vitro model of the liver

Introduction In the context of drug development, liver plays a crucial role, and it is primarily involved in drug metabolism. However, the direct exposition to the active principles and their metabolites may damage hepatocytes, triggering a macrophages-mediated cascade, culminating with the differentiation of hepatic stellate cells in myofibroblast, and producing fibrous matrix. The threshold beyond which a certain molecule become hepatotoxic varies within individuals sub-populations, since it is strictly dependant by the pharmacokinetics [1], and therefore it is difficult to be uniquely predicted. This work will present the first developmental steps of a tailorable and standardizable in vitro 3D-model of the liver, to assess drugs hepatotoxicity. Materials and Methods ECM was obtained from decellularized porcine liver, by a combination of different methods [2]. The decellularization buffer was injected in multiple sites of 0.5 cm cubes of liver and then used to incubate the samples while under orbital stirring up to 7 days. Lyophilized cubes were grinded after freezing in liquid N2. ECM powder (1.4% w/v) was added in an alginate (ALG) solution (3.5% w/v) in complete medium. The hydrogel was characterized by rheological testing and the stability in medium was evaluated up to 14 days. For cell loaded hydrogels, HepG2 cells were suspended in the ALG-ECM suspension (2x106 cell/ml) prior crosslinking [3]. MTT test and confocal microscopy, with live/dead kit were employed to evaluate viability and spatial distribution. Results and Discussion The produced hydrogel shows rheological characteristics reproducing the ones of the liver tissue and is stable up to 12 days. Additionally, further rheological analyses demonstrated the suitability of the produced hydrogel to be processed via 3D bioprinting. Both qualitative (CLSM) and quantitative (MTT assay) viability analyses revealed that the number of cells within the hydrogel have increased through time. In particular, it has been demonstrated the feasibility of maintaining viable cells in culture up to 8 days. Additionally, CLSM analyses allowed to observe the formation of three-dimensional cellular clusters, similar to the ones in which hepatocytes are organized in vivo [4]. The model is now being employed for the study of hepatoxicity to the administration of various drugs. Acetaminophen, whose hepatotoxic effects are known and well described, has been exploited to benchmark the model, and to calibrate it for the study of other substances (i.e., midazolam, chlordiazepoxide). Conclusions This study demonstrated the possibility to realize a drug-induced hepatotoxicity model by synergically combining the chemical features of liver-derived ECM with the structural characteristic of an alginate hydrogel, mimicking the in vivo liver microenvironment. The presence of ECM positively impacted cell viability