Injectable and self-healable nano-architectured hydrogel for NIR-light responsive chemo- and photothermal bacterial eradication.

Hydrogels with multifunctional properties activated at specific times have gained significant attention in the biomedical field. As bacterial infections can cause severe complications that negatively impact wound repair, herein, we present the development of a stimuli-responsive, injectable, and in situ-forming hydrogel with antibacterial, self-healing, and drug-delivery properties. In this study, we prepared a Pluronic F-127 (PF127) and sodium alginate (SA)-based hydrogel that can be targeted to a specific tissue via injection. The PF127/SA hydrogel was incorporated with polymeric short-filaments (SFs) containing an anti-inflammatory drug - ketoprofen, and stimuli-responsive polydopamine (PDA) particles. The hydrogel, after injection, could be in situ gelated at the body temperature, showing great in vitro stability and self-healing ability after 4 h of incubation. The SFs and PDA improved the hydrogel injectability and compressive strength. The introduction of PDA significantly accelerated the KET release under near-infrared light exposure and extended its release validity period. The excellent composites' photo-thermal performance led to antibacterial activity against representative Gram-positive and Gram-negative bacteria, resulting in 99.9% E. coli and S. aureus eradication after 10 min of NIR light irradiation. In vitro, fibroblast L929 cell studies confirmed the materials' biocompatibility and paved the way toward further in vivo and clinical application of the system for chronic wound treatments.

Influence of C60 Nanofilm on the Expression of Selected Markers of Mesenchymal-Epithelial Transition in Hepatocellular Carcinoma.

The epithelial-mesenchymal transition (EMT) is a process in which epithelial cells acquire the ability to actively migrate via a change to the mesenchymal phenotype. This mechanism occurs in an environment rich in cytokines and reactive oxygen species but poor in nutrients. The aim of this study was to demonstrate that the use of a fullerene C60 nanofilm can inhibit liver cancer cell invasion by restoring their non-aggressive, epithelial phenotype. We employed epithelial and mesenchymal HepG2 and SNU-449 liver cancer cells and non-cancerous mesenchymal HFF2 cells in this work. We used enzyme-linked immunosorbent assays (ELISAs) to determine the content of glutathione and transforming growth factor (TGF) in cells. We measured the total antioxidant capacity with a commercially available kit. We assessed cell invasion based on changes in morphology, the scratch test and the Boyden chamber invasion. In addition, we measured the effect of C60 nanofilm on restoring the epithelial phenotype at the protein level with protein membranes, Western blotting and mass spectrometry. C60 nanofilm downregulated TGF and increased glutathione expression in SNU-449 cells. When grown on C60 nanofilm, invasive cells showed enhanced intercellular connectivity; reduced three-dimensional invasion; and reduced the expression of key invasion markers, namely MMP-1, MMP-9, TIMP-1, TIMP-2 and TIMP-4. Mass spectrometry showed that among the 96 altered proteins in HepG2 cells grown on C60 nanofilm, 41 proteins are involved in EMT and EMT-modulating processes such as autophagy, inflammation and oxidative stress. The C60 nanofilm inhibited autophagy, showed antioxidant and anti-inflammatory properties, increased glucose transport and regulated the ß-catenin/keratin/Smad4/snail+slug and MMP signalling pathways. In conclusion, the C60 nanofilm induces a hybrid mesenchymal-epithelial phenotype and could be used in the prevention of postoperative recurrences.

Nanostructured graphene oxide enriched with metallic nanoparticles as a biointerface to enhance cell adhesion through mechanosensory modifications.

Nanostructuring is a process involving surface manipulation at the nanometric level, which improves the mechanical and biological properties of biomaterials. Specifically, it affects the mechanotransductive perception of the microenvironment of cells. Mechanical force conversion into an electrical or chemical signal contributes to the induction of a specific cellular response. The relationship between the cells and growth surface induces a biointerface-modifying cytophysiology and consequently a therapeutic effect. In this study, we present the fabrication of graphene oxide (GO)-based nanofilms decorated with metallic nanoparticles (NPs) as potential coatings for biomaterials. Our investigation showed the effect of decorating GO with metallic NPs for the modification of the physicochemical properties of nanostructures in the form of nanoflakes and nanofilms. A comprehensive biocompatibility screening panel revealed no disturbance in the metabolic activity of human fibroblasts (HFFF2) and bone marrow stroma cells (HS-5) cultivated on the GO nanofilms decorated with gold and copper NPs, whereas a significant cytotoxic effect of the GO nanocomplex decorated with silver NPs was demonstrated. The GO nanofilm decorated with gold NPs beneficially managed early cell adhesion as a result of the transient upregulation of α1ß5 integrin expression, acceleration of cellspreading, and formation of elongated filopodia. Additionally, the cells, sensing the substrate derived from the nanocomplex enriched with gold NPs, showed reduced elasticity and altered levels of vimentin expression. In the future, GO nanocomplexes decorated with gold NPs can be incorporated in the structure of architecturally designed biomimetic biomaterials as biocompatible nanostructuring agents with proadhesive properties.

The cytocompatibility of graphene oxide as a platform to enhance the effectiveness and safety of silver nanoparticles through in vitro studies.

The increasing emergence of antibiotic-resistant bacteria and the need to reduce the use of antibiotics call for the development of safe alternatives, such as silver nanoparticles. However, their potential cytotoxic effect needs to be addressed. Graphene oxide provides a large platform that can increase the effectiveness and safety of silver nanoparticles. Graphene oxide and silver nanoparticles complex applied as a part of an innovative material might have direct contact with human tissues, such as skin, or might be inhaled from aerosol or exfoliated pieces of the complex. Thereby, the safety of the prepared complex has to be evaluated carefully, employing a range of methods. We demonstrated the high cytocompatibility of graphene oxide and the graphene oxide-silver nanoparticles complex toward human cell lines, fetal foreskin fibroblasts (HFFF2), and lung epithelial cells (A549). The supporting platform of graphene oxide also neutralized the slight toxicity of bare silver nanoparticles. Finally, in studies on Staphylococcus aureus and Pseudomonas aeruginosa, the number of bacteria reduction was observed after incubation with silver nanoparticles and the graphene oxide-silver nanoparticles complex. Our findings confirm the possibility of employing a graphene oxide-silver nanoparticles complex as a safe agent with reduced silver nanoparticles' cytotoxicity and antibacterial properties.

Delivery of Melittin as a Lytic Agent via Graphene Nanoparticles as Carriers to Breast Cancer Cells.

Melittin, as an agent to lyse biological membranes, may be a promising therapeutic agent in the treatment of cancer. However, because of its nonspecific actions, there is a need to use a delivery method. The conducted research determined whether carbon nanoparticles, such as graphene and graphene oxide, could be carriers for melittin to breast cancer cells. The studies included the analysis of intracellular pH, the potential of cell membranes, the type of cellular transport, and the expression of receptor proteins. By measuring the particle size, zeta potential, and FT-IT analysis, we found that the investigated nanoparticles are connected by electrostatic interactions. The level of melittin encapsulation with graphene was 86%, while with graphene oxide it was 78%. A decrease in pHi was observed for all cell lines after administration of melittin and its complex with graphene. The decrease in membrane polarization was demonstrated for all lines treated with melittin and its complex with graphene and after exposure to the complex of melittin with graphene oxide for the MDA-MB-231 and HFFF2 lines. The results showed that the investigated melittin complexes and the melittin itself act differently on different cell lines (MDA-MB-231 and MCF-7). It has been shown that in MDA-MD-231 cells, melittin in a complex with graphene is transported to cells via caveolin-dependent endocytosis. On the other hand, the melittin-graphene oxide complex can reach breast cancer cells through various types of transport. Other differences in protein expression changes were also observed for tumor lines after exposure to melittin and complexes.

Laser-Assisted Fabrication of Injectable Nanofibrous Cell Carriers.

The use of injectable biomaterials for cell delivery is a rapidly expanding field which may revolutionize the medical treatments by making them less invasive. However, creating desirable cell carriers poses significant challenges to the clinical implementation of cell-based therapeutics. At the same time, no method has been developed to produce injectable microscaffolds (MSs) from electrospun materials. Here the fabrication of injectable electrospun nanofibers is reported on, which retain their fibrous structure to mimic the extracellular matrix. The laser-assisted micro-scaffold fabrication has produced tens of thousands of MSs in a short time. An efficient attachment of cells to the surface and their proliferation is observed, creating cell-populated MSs. The cytocompatibility assays proved their biocompatibility, safety, and potential as cell carriers. Ex vivo results with the use of bone and cartilage tissues proved that NaOH hydrolyzed and chitosan functionalized MSs are compatible with living tissues and readily populated with cells. Injectability studies of MSs showed a high injectability rate, while at the same time, the force needed to eject the load is no higher than 25 N. In the future, the produced MSs may be studied more in-depth as cell carriers in minimally invasive cell therapies and 3D bioprinting applications.

Comparison of the Toxicity of Pristine Graphene and Graphene Oxide, Using Four Biological Models.

There are numerous applications of graphene in biomedicine and they can be classified into several main areas: delivery systems, sensors, tissue engineering and biological agents. The growing biomedical field of applications of graphene and its derivates raises questions regarding their toxicity. We will demonstrate an analysis of the toxicity of two forms of graphene using four various biological models: zebrafish (Danio rerio) embryo, duckweed (Lemna minor), human HS-5 cells and bacteria (Staphylococcus aureus). The toxicity of pristine graphene (PG) and graphene oxide (GO) was tested at concentrations of 5, 10, 20, 50 and 100 µg/mL. Higher toxicity was noted after administration of high doses of PG and GO in all tested biological models. Hydrophilic GO shows greater toxicity to biological models living in the entire volume of the culture medium (zebrafish, duckweed, S. aureus). PG showed the highest toxicity to adherent cells growing on the bottom of the culture plates-human HS-5 cells. The differences in toxicity between the tested graphene materials result from their physicochemical properties and the model used. Dose-dependent toxicity has been demonstrated with both forms of graphene.

Multifunctional Platform Based on Electrospun Nanofibers and Plasmonic Hydrogel: A Smart Nanostructured Pillow for Near-Infrared Light-Driven Biomedical Applications.

Multifunctional nanomaterials with the ability to respond to near-infrared (NIR) light stimulation are vital for the development of highly efficient biomedical nanoplatforms with a polytherapeutic approach. Inspired by the mesoglea structure of jellyfish bells, a biomimetic multifunctional nanostructured pillow with fast photothermal responsiveness for NIR light-controlled on-demand drug delivery is developed. We fabricate a nanoplatform with several hierarchical levels designed to generate a series of controlled, rapid, and reversible cascade-like structural changes upon NIR light irradiation. The mechanical contraction of the nanostructured platform, resulting from the increase of temperature to 42 °C due to plasmonic hydrogel-light interaction, causes a rapid expulsion of water from the inner structure, passing through an electrospun membrane anchored onto the hydrogel core. The mutual effects of the rise in temperature and water flow stimulate the release of molecules from the nanofibers. To expand the potential applications of the biomimetic platform, the photothermal responsiveness to reach the typical temperature level for performing photothermal therapy (PTT) is designed. The on-demand drug model penetration into pig tissue demonstrates the efficiency of the nanostructured platform in the rapid and controlled release of molecules, while the high biocompatibility confirms the pillow potential for biomedical applications based on the NIR light-driven multitherapy strategy.