Novel air-liquid interface culture model to investigate stiffness-dependent behaviors of alveolar epithelial cells.

Pulmonary alveoli are functional units in gas exchange in the lung, and their dysfunctions in lung diseases such as interstitial pneumonia are accompanied by fibrotic changes in structure, elevating the stiffness of extracellular matrix components. The present study aimed to test the hypothesis that such changes in alveoli stiffness induce functional alteration of epithelial cell functions, exacerbating lung diseases. For this, we have developed a novel method of culturing alveolar epithelial cells on polyacrylamide gel with different elastic modulus at an air-liquid interface. It was demonstrated that A549 cells on soft gels, mimicking the modulus of a healthy lung, upregulated mRNA expression and protein synthesis of surfactant protein C (SFTPC). By contrast, the cells on stiff gels, mimicking the modulus of the fibrotic lung, exhibited upregulation of SFTPC gene expression but not at the protein level. Cell morphology, as well as cell nucleus volume, were also different between the two types of gels.

Labile assembly of a tardigrade protein induces biostasis.

Tardigrades are microscopic animals that survive desiccation by inducing biostasis. To survive drying tardigrades rely on intrinsically disordered CAHS proteins, which also function to prevent perturbations induced by drying in vitro and in heterologous systems. CAHS proteins have been shown to form gels both in vitro and in vivo, which has been speculated to be linked to their protective capacity. However, the sequence features and mechanisms underlying gel formation and the necessity of gelation for protection have not been demonstrated. Here we report a mechanism of fibrillization and gelation for CAHS D similar to that of intermediate filament assembly. We show that in vitro, gelation restricts molecular motion, immobilizing and protecting labile material from the harmful effects of drying. In vivo, we observe that CAHS D forms fibrillar networks during osmotic stress. Fibrillar networking of CAHS D improves survival of osmotically shocked cells. We observe two emergent properties associated with fibrillization; (i) prevention of cell volume change and (ii) reduction of metabolic activity during osmotic shock. We find that there is no significant correlation between maintenance of cell volume and survival, while there is a significant correlation between reduced metabolism and survival. Importantly, CAHS D's fibrillar network formation is reversible and metabolic rates return to control levels after CAHS fibers are resolved. This work provides insights into how tardigrades induce reversible biostasis through the self-assembly of labile CAHS gels.

In-situ gel: A smart carrier for drug delivery.

In-situ gel technology is a promising drug delivery strategy that undergoes a 'sol to gel' transition upon administration, providing controlled and prolonged drug release. These gels are composed of cross-linked 3D networks of polymers, with hydrogels being a specific type of absorbing water while retaining their shape. Gelation can be triggered by various stimuli, such as temperature, pH, ions, and light. They offer several advantages like improved patient compliance, extended drug residence time, localized drug delivery, etc, but also have some disadvantages like drug degradation and limited mechanical strength. In-situ gel falls into three categories: temperature-sensitive, ion-sensitive, and pH-sensitive, but multi-responsive gels that respond to multiple stimuli have better drug release characteristics. The mechanism of in-situ gel formation involves physical and chemical mechanisms. There are various applications of in-situ gel, like ocular drug delivery, nose-to-brain delivery, etc. In this review, we have discussed the types, and mechanisms of in-situ gel & use of in-situ gel in the treatment of different diseases through various routes like buccal, vaginal, ocular, nasal, etc., along with its use in targeted drug delivery.

Investigating the Role of Extracellular Matrix Stiffness in Modulating the Ferroptosis Process in Hepatocellular Carcinoma Cells via Scanning Electrochemical Microscopy.

Extracellular matrix (ECM) stiffness modulates a variety of cellular processes, including ferroptosis, a process with significant potential implications for hepatocellular carcinoma (HCC) fibrosis and cirrhosis. However, the exact relationship between ECM stiffness and HCC ferroptosis is yet unclarified, partially due to the lack of in situ information on key parameters of the ferroptosis process of living HCC cells. This study pioneers the use of in vitro mechanical microenvironment models of HCC and the scanning electrochemical microscopy (SECM) technique for understanding this interplay. We first cultured HuH7 cells on 4.0, 18.0, and 44.0 kPa polyacrylamide (PA) gels to simulate early, intermediate, and advanced HCC ECM stiffness, respectively. Then, we used SECM to in situ monitor changes in cell membrane permeability, respiratory activity, and reactive oxygen species (ROS) levels of erastin-induced HuH7 cells on PA gels, finding that increasing ECM stiffness potentiates ferroptosis, including increased membrane permeabilization and H2O2 release as well as reduced respiratory activity. Through further transcriptome sequencing and molecular biology measurements, we identified a critical role for focal adhesion kinase (FAK)-mediated yes-associated protein (YAP) in regulating the ferroptosis process dependent on ECM stiffness, which provides novel insights into the mechanical regulation of ferroptosis in HCC cells and may pave the way for innovative therapeutic strategies.

Tocopherol succinate-loaded ethosomal gel synthesized by cold method technique: Deeper biophysical characterizations for translational application on human skin.

BACKGROUND: Tocopherols are well-known antioxidant and moisturizing agent. Tocopherol succinate (TS) are widely used in many skin products especially used in anti-aging and skin whitening product formulation. AIM: We previously reported the successful synthesis and preliminary characterizations of stable TS ethosomal gels (TSEG) (DOI: 10.1111/jocd.14907). Herein, we develop and further characterize TSEG to enhance the stability of the developed formulation with increased permeation through skin. METHODS: Cold method technique was used to prepare TS ethosomes. The developed ethosomal vesicle size was 250 nm, which allowed TS to penetrate through the stratum corneum layer and act on melanocytes. For stability study was assessed by thermogravimetric analysis (TGA) by placing TSEG and unloaded/control ethosomal gel (CEG) at various temperature conditions, that is, 8°C, 25°C, 40°C, and 40°C ± 75% RH for 3 months. Organoleptic evaluation was done in terms of color, odor, and phase separation. Transmission electron microscopy (TEM), Fourier Transform infrared spectroscopy (FTIR), x-ray diffraction spectroscopy (XRD), zeta potential (ZP) and particle size (PS) was used for TSEG physical characterizations. In vitro dissolution and ex-vivo permeation studies (using Franz diffusion cell) were performed for both TSEG and CEG formulations. Human women (N = 34) were used to evaluate in vivo biophysical parameters including erythema, melanin, moisture content, sebum level, and skin elasticity. RESULTS: Developed formulation was highly thermostable during the 3 months. Erythema, melanin, and sebum level decreased while marked improvement (p < 0.05) in moisture content and elasticity have been observed for the developed TSEG. CONCLUSION: The developed TSEG formulation was found to be efficient, safe (no adverse effects observed), stable (at least for 3 months), and easy to use for topical application with improved skin complexation and skin integrity.

Ultra-Rapid and Specific Gelation of Collagen Molecules for Transparent and Tough Gels by Transition Metal Complexation.

Collagen is the most abundant protein in the human body and one of the main components of stromal tissues in tumors which have a high elastic modulus of over 50 kPa. Although collagen has been widely used as a cell culture scaffold for cancer cells, there have been limitations when attempting to fabricate a tough collagen gel with cells like a cancer stroma. Here, rapid gelation of a collagen solution within a few minutes by transition metal complexation is demonstrated. Type I collagen solution at neutral pH shows rapid gelation with a transparency of 81% and a high modulus of 1,781 kPa by mixing with K2 PtCl4 solution within 3 min. Other transition metal ions also show the same rapid gelation, but not basic metal ions. Interestingly, although type I to IV collagen molecules show rapid gelation, other extracellular matrices  do not exhibit this phenomenon. Live imaging of colon cancer organoids in 3D culture indicates a collective migration property with modulating high elastic modulus, suggesting activation for metastasis progress. This technology will be useful as a new class of 3D culture for cells and organoids due to its facility for deep-live observation and mechanical stiffness adjustment.

Topical treatment of tea saponin stabilized silybin nanocrystal gel reduced oxidative stress in UV-induced skin damage.

UV-induced peroxidation is a significant factor in skin damage. Some natural products have been utilized to protect the skin. However, most of them suffer from issues such as poor bioavailability. A promising strategy is to prepare them as safe and convenient gels. In this study, we constructed Silybin Nanocrystal Gel (SIL-NG). Tea saponin, a spatial stabilizer that we have previously reported, was used to prepare SIL-NS and subsequently combined with xanthan gum to prepare SIL-NG with an excellent safety profile. This nanogel with a natural stabilizer has a suitable ductility and shows a good safety profile in vitro and in vivo. In L929 cells, SIL-NG was able to reduce H2O2-induced ROS levels. In addition, SIL-NG exhibited better antioxidant activity compared to SIL-NS. SIL-NG was able to reduce UVB irradiation-induced oxidative damage in mice, significantly increase SOD activity, and reduce MDA levels. In conclusion, our work gives a new perspective on the treatment of UV skin damage using natural ingredients.

Sinapic acid-loaded gel accelerates diabetic wound healing process by promoting re-epithelialization and attenuating oxidative stress in rats.

Impaired wound healing is a critical health concern for individuals with diabetes. Sinapic acid, a phyto-compound, has wound-healing potential owing to its various bioactivities. In this study, we explored the wound-healing ability of sinapic acid in diabetes. Full-thickness excisional wounds were created in streptozotocin-induced diabetic rats. Sinapic acid-loaded gels (1%, 2%, and 3%) were prepared and applied topically to diabetic skin wounds. On day 7 post-wounding, rats were sacrificed, and macroscopic, histopathological, and oxidative markers of wound healing activity were evaluated in the collected wound tissues. Sinapic acid-loaded gels showed better recovery in re-epithelialization (p < 0.05) and angiogenesis (p < 0.05) compared to the negative control group. Sinapic acid-loaded gels (1%, 2%, and 3%) showed 87.46%, 79.53%, and 68.78% wound contraction, respectively. They increased collagen content (28.05 ± 1.66, 17.30 ± 2.19, and 11.64 ± 1.25, respectively) and decreased malondialdehyde (MDA) levels (17.49 ± 1.61, 18.44 ± 1.24, and 19.16 ± 1.77, respectively) compared to the negative control group (6.76 ± 0.89, and 43.58 ± 3.70, respectively) (p < 0.05). Moreover, sinapic acid-loaded gel groups demonstrated enhanced antioxidant capacity (approximately 2-2.5-fold) compared to the negative control group (p < 0.05). Sinapic acid 1% loaded gel showed the best effect on the diabetic healing process, whereas sinapic acid 2% loaded gel and reference drug showed similar effects. The results of this study, for the first time, suggest that the topical application of sinapic acid can promote diabetic wound healing, especially at low doses.

Adipose-Derived Stem Cells in Reinforced Collagen Gel: A Comparison between Two Approaches to Differentiation towards Smooth Muscle Cells.

Scaffolds made of degradable polymers, such as collagen, polyesters or polysaccharides, are promising matrices for fabrication of bioartificial vascular grafts or patches. In this study, collagen isolated from porcine skin was processed into a gel, reinforced with collagen particles and with incorporated adipose tissue-derived stem cells (ASCs). The cell-material constructs were then incubated in a DMEM medium with 2% of FS (DMEM_part), with added polyvinylalcohol nanofibers (PVA_part sample), and for ASCs differentiation towards smooth muscle cells (SMCs), the medium was supplemented either with human platelet lysate released from PVA nanofibers (PVA_PL_part) or with TGF-ß1 + BMP-4 (TGF + BMP_part). The constructs were further endothelialised with human umbilical vein endothelial cells (ECs). The immunofluorescence staining of alpha-actin and calponin, and von Willebrand factor, was performed. The proteins involved in cell differentiation, the extracellular matrix (ECM) proteins, and ECM remodelling proteins were evaluated by mass spectrometry on day 12 of culture. Mechanical properties of the gels with ASCs were measured via an unconfined compression test on day 5. Gels evinced limited planar shrinkage, but it was higher in endothelialised TGF + BMP_part gel. Both PVA_PL_part samples and TGF + BMP_part samples supported ASC growth and differentiation towards SMCs, but only PVA_PL_part supported homogeneous endothelialisation. Young modulus of elasticity increased in all samples compared to day 0, and PVA_PL_part gel evinced a slightly higher ratio of elastic energy. The results suggest that PVA_PL_part collagen construct has the highest potential to remodel into a functional vascular wall.

Intranasally administered thermosensitive gel for brain-targeted delivery of rhynchophylline to treat Parkinson's disease.

The aim of this study is to overcome the obstacle of the blood-brain barrier (BBB) in therapeutic drugs of Parkinson's disease (PD), like rhynchophylline (RIN) entry by intranasal administration and to solve the problem of short residence time of drugs in the nasal cavity by the dosage form design of thermosensitive gel. We first conducted a study of the screening of absorption enhancers and 3% hydroxypropyl-ß-cyclodextrin (HP-ß-CD) was effective to improve the nasal mucosal permeability of RIN. By adjusting the ratio of different components in order to make the gel with adhesion and rapid gelation which were determined to be Poloxamer 407 (P407) 20%, Poloxamer 188 (P188) 1%, polyethylene glycol 6000 (PEG-6000) 1% and HP-ß-CD 3%. In addition, the characterization showed that the thermosensitive gel was network cross-linked, rapidly gelation upon entry into the nasal cavity and was stable as semi-solid state with adhesion as well as sustained release properties. Moreover, pharmacokinetic study was performed to evaluate the bioavailability and brain targeting of RIN thermosensitive gel and which were 1.6 times and 2.1 times higher than those of oral administration. We also evaluated the anti-PD effects of RIN thermosensitive gel in-vitro as well as in-vivo. The results showed that RIN thermosensitive gel was effective in repairing the motor function impairment, dysregulated expression levels of oxidative stress factors, and positive neuronal damage within the substantia nigra and dopamine caused by PD. The constructed intranasal drug administration strategy through thermosensitive gel provided a new choice for targeted treatment of PD together with other central nervous system diseases.

Improvement of the cell viability of hepatocytes cultured in three-dimensional collagen gels using pump-free perfusion driven by water level difference.

Cell-containing collagen gels are one of the materials employed in tissue engineering and drug testing. A collagen gel is a useful three-dimensional (3D) scaffold that improves various cell functions compared to traditional two-dimensional plastic substrates. However, owing to poor nutrient availability, cells are not viable in thick collagen gels. Perfusion is an effective method for supplying nutrients to the gel. In this study, we maintained hepatocytes embedded in a 3D collagen gel using a simple pump-free perfusion cell culture system with ordinary cell culture products. Flow was generated by the difference in water level in the culture medium. Hepatocytes were found to be viable in a collagen gel of thickness 3.26 (± 0.16 S.E.)-mm for 3 days. In addition, hepatocytes had improved proliferation and gene expression related to liver function in a 3D collagen gel compared to a 2D culture dish. These findings indicate that our perfusion method is useful for investigating the cellular functions of 3D hydrogels.

Exploring the difference in the mechanics of vascular smooth muscle cells from wild-type and apolipoprotein-E knockout mice.

Atherosclerosis-related cardiovascular diseases are a leading cause of mortality worldwide. Vascular smooth muscle cells (VSMCs) comprise the medial layer of the arterial wall and undergo phenotypic switching during atherosclerosis to a synthetic phenotype capable of proliferation and migration. The surrounding environment undergoes alterations in extracellular matrix (ECM) stiffness and composition and an increase in cholesterol content. Using an atherosclerotic murine model, we analyzed how the mechanics of VSMCs isolated from Western diet-fed apolipoprotein-E knockout (ApoE-/-) and wild-type (WT) mice were altered during atherosclerosis. Increased stiffness of ApoE-/- VSMCs correlated with a greater degree of stress fiber alignment, as evidenced by atomic force microscopy (AFM)-generated force maps and stress fiber topography images. On type-1 collagen (COL1)-coated polyacrylamide (PA) gels (referred to as substrate) of varying stiffness, ApoE-/- VSMCs had lower adhesion forces to COL1 and N-cadherin (N-Cad) compared with WT cells. ApoE-/- VSMC stiffness was significantly greater than that of WT cells. Cell stiffness increased with increasing substrate stiffness for both ApoE-/- and WT VSMCs. In addition, ApoE-/- VSMCs showed an enhanced migration capability on COL1-coated substrates and a general decreasing trend in migration capacity with increasing substrate stiffness, correlating with lowered adhesion forces as compared with WT VSMCs. Altogether, these results demonstrate the potential contribution of the alteration in VSMC mechanics in the development of atherosclerosis.

Osteoclast-mediated acidic hydrolysis of thermally gelled curdlan component of the bone scaffolds: Is it possible?

Many biomaterials for bone regeneration have recently been produced using thermally gelled curdlan (1,3-ß-d-glucan) as a binder for bioceramics. As the human organism does not produce enzymes having the ability to degrade curdlan, it is not clear what is the fate of curdlan gel after its implantation in the bone. To clarify this point, in this research osteoclasts were cultured on the curdlan gel to show its degradation by acidic hydrolysis. The studies clearly demonstrated microstructural (AFM and SEM imaging) and chemical changes (Raman spectroscopy) on the curdlan surface caused by osteoclast culture. Moreover, degradation test in a cell-free system using HCl solution (pH = 4.5), mimicking environment in the resorption lacuna, showed great weight loss of the sample, release of glucose, and chemical changes typical of curdlan degradation. Thus, the presented research for the first time provides a strong evidence of osteoclast-mediated acidic hydrolysis of thermally obtained curdlan gel.

12-O-Tetradecanoylphorbol 13-acetate promotes proliferation and epithelial-mesenchymal transition in HHUA cells cultured on collagen type I gel: A feasible model to find new therapies for endometrial diseases.

HHUA endometrial adenocarcinoma cells aggregated into spheroids when cultured on collagen type I gels. 12-O-Tetradecanoylphorbol 13-acetate, a PKC activator, disassembled the spheroids through epithelial-mesenchymal transition and increased their proliferation rate, while inducing cell death under monolayer culture conditions. These unusual behaviors of endometrial epithelial cells with collagen fibers could be a target for the treatment of some endometrial diseases.

Penetration Enhancement Strategies for Intradermal Delivery of Cromolyn Sodium.

This study aimed to explore the use of chemical and physical enhancement strategies for the intradermal delivery of cromolyn sodium (CS) for treatment of atopic dermatitis. CS gels were formulated to individually contain 2.5 and 9% salcaprozate sodium (SNAC) as a potential chemical enhancer. The effect of microneedles, alone and in combination with SNAC, was investigated via in vitro permeation studies. Skin impedance and FTIR evaluation of SNAC-treated stratum corneum (SC) was done and compared to the control. The amount of drug delivered in the dermis after 24 h by the 2.5% and 9% SNAC gels was 23.29 ± 1.89 µg/cm2 and 35.87 ± 2.23 µg/cm2, respectively, which were significantly higher than the control (p < 0.05) but were not remarkably different from each other (p > 0.05). Microneedles enhanced permeation in both the control and 2.5% SNAC groups (p < 0.05); however, no synergistic enhancement was observed when microneedle and SNAC treatments were combined (p > 0.05). Over 24 h of treating the SC with 2.5% SNAC, FTIR evaluation showed stretches on the CH2 asymmetric and symmetric stretching vibrations observed at 2920.23 cm-1 and 2850.79 cm-1 respectively in untreated SC, which shifted to higher wavenumbers and indicated some lipid fluidizing effect. However, no significant drop in skin impedance was seen with SNAC as compared to the control (p > 0.05). SNAC was concluded to have skin permeation enhancement effect on CS, while microneedles effectively enhanced CS permeation even in the absence of SNAC.

A spectrofluorometric analysis to evaluate transcutaneous biodistribution of fluorescent nanoparticulate gel formulations.

The investigation of the absorption of drug delivery systems, designed for the transport of therapeutic molecules inside the body, could be relatively simplified by the fluorophore association and tracking by means of bio-imaging techniques (i.e., optical in vivo imaging or confocal and multiphoton microscopy). However, when a fluorescence signal comes out from the skin, its specific detection can be problematic. Skin high autofluorescence can hinder the observation of administered exogenous fluorophores conjugated to drug delivery systems, making it more challenging to detect their biodistribution. In the present study, we have developed a method based on the spectrofluorometric analysis of skin samples to discriminate the fluorescent signal coming from administered fluorescent molecules from the background. Moreover, we gave a semi-quantitative evaluation of the signal intensity. Thus, we distinguished two gel formulations loading the fluorophore rhodamine B (called GEL RHO and GEL SLN-RHO). The two formulations of gels, one of which containing solid lipid nanoparticles (GEL RHO-SLN), were administered on skin explants incubated in a bioreactor, and the penetration was evaluated at different time points (2 and 6 hours). Cryostatic sections of skin samples were observed with confocal laser scanning microscopy, and a spectrofluorometric analysis was performed. Significantly higher signal intensity in the samples administered with SLN-RHO GEL, with a preferential accumulation in the hair bulbs, was found. Reaching also the deeper layers of the hair shaft after 6 hours, the solid lipid nanoparticles thickened with polymer represent a suitable drug delivery system for transcutaneous administration.

Electromechanical response performance of a reinforced biomass gel artificial muscle based on natural polysaccharide of sodium alginate doped with an ionic liquid for micro-nano regulation.

In this paper, a reinforced Biomass Gel Artificial Muscle (BGAM) was fabricated by natural polysaccharide of Sodium Alginate (SA) doped with an Ionic Liquid (IL) of 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIm][BF4]). Micro-nano regulation effect and reinforcement mechanism of IL doping content on electromechanical response performance of BGAM were researched during a single cycle and repeated cycles. Then, a green fabrication process and a set of valid test methods for BGAM were proposed in detail. The experimental results showed that when IL doping content was 4 mL, the BGAM achieved optimal modification, with a porosity of 70.47%, where it internally adopted the porous polymer structure of ion channels. Additionally, specific capacitance of BGAM attained a maximum value of 126.98 mF/g, and the inner resistance and elastic modulus reached minimum values of 2.018 Ω and 1.871 MPa, separately. Thus, the optimal working life and output-force density values, namely, 1720 s and 13.072 mN/g, respectively, were also determined for the BGAM.

DNA-Controlled Spatiotemporal Patterning of a Cytoskeletal Active Gel.

Living cells move and change their shape because signaling chemical reactions modify the state of their cytoskeleton, an active gel that converts chemical energy into mechanical forces. To create life-like materials, it is thus key to engineer chemical pathways that drive active gels. Here we describe the preparation of DNA-responsive surfaces that control the activity of a cytoskeletal active gel composed of microtubules: A DNA signal triggers the release of molecular motors from the surface into the gel bulk, generating forces that structure the gel. Depending on the DNA sequence and concentration, the gel forms a periodic band pattern or contracts globally. Finally, we show that the structuration of the active gel can be spatially controlled in the presence of a gradient of DNA concentration. We anticipate that such DNA-controlled active matter will contribute to the development of life-like materials with self-shaping properties.

Self-immobilization of coacervate droplets by enzyme-mediated hydrogelation.

An artificial protocell model mimicking stimuli-triggered extracellular matrix formation is demonstrated based on the self-immobilization of coacervate microdroplets. Endogenous enzyme activity within the microdroplets results in the release of Ca2+ ions that trigger hydrogelation throughout the external environment, which in turn mechanically supports and chemically stabilizes the protocells.

Effect of high-intensity ultrasound soymilk pretreatment on the physicochemical properties of microbial transglutaminase-catalyzed tofu gel.

Tofu prepared by conventional methods often has a bitter taste and poor water-holding capacity (WHC). To improve the quality of the product, alternative processes must be developed. Herein, the effect of ultrasound pretreatment on the properties of soymilk and tofu gel derived thereof were investigated. Treatment of soymilk with ultrasound gave rise to a reduction in the particle size and an enhancement in the surface hydrophobicity, whereby optimum values were obtained after 15 min treatment. Subsequently, microbial transglutaminase (MTG) was added to ultrasound-treated soymilk to promote the soy protein crosslinking. The gel strength, WHC, and nonfreezable water content of MTG-catalyzed tofu gel obtained from treated soymilk increased with the extension of the ultrasound pretreatment time, whereas the free sulfhydryl content decreased because of the formation of disulfide bonds. Fourier transform infrared spectroscopy demonstrated variations in the secondary structure of MTG-catalyzed tofu gel. Furthermore, soymilk's exposure to high-intensity ultrasound pretreatment led to a tofu gel with a dense, homogenous, and stable network structure, as evidenced by scanning electron microscopy. Therefore, this study answers for the theoretical support of the industrial production of MTG-catalyzed tofu gel from ultrasound-treated soymilk. PRACTICAL APPLICATION: High-intensity ultrasound pretreatment improved the texture properties of MTG-catalyzed tofu gel. The resulting MTG-catalyzed tofu gel has potential application in industrial production.