The intra-articular administration of drugs has attracted great interest in recent decades for the treatment of osteoarthritis. The use of modified drugs has also attracted interest in recent years... Show moreThe intra-articular administration of drugs has attracted great interest in recent decades for the treatment of osteoarthritis. The use of modified drugs has also attracted interest in recent years because their intra-articular administration has demonstrated encouraging results. The objective of this work was to prepare injectable-thermosensitive hydrogels for the intra-articular administration of Etanercept (ETA), an inhibitor of tumor necrosis factor-alpha. Hydrogels were prepared from the physical mixture of chitosan and Pluronic F127 with beta-glycerolphosphate (BGP). Adding beta-glycerolphosphate to the system reduced the gelation time and also modified the morphology of the resulting material. In vitro studies were carried out to determine the cytocompatibility of the prepared hydrogels for the human chondrocyte line C28/I2. The in vitro release study showed that the incorporation of BGP into the system markedly modified the release of ETA. In the in vivo studies, it was verified that the hydrogels remained inside the implantation site in the joint until the end of the study. Furthermore, ETA was highly concentrated in the blood of the study mice 48 h after the loaded material was injected. Histological investigation of osteoarthritic knees showed that the material promotes cartilage recovery in osteoarthritic mice. The results demonstrate the potential of ETA-loaded injectable hydrogels for the localized treatment of joints. Show less
Jorquera-Cordero, C.; Lara, P.; Cruz, L.J.; Schomann, T.; Hofslot, A. van; Carvalho, T.G. de; ... ; Araujo, R.F. de 2022
One of the main reasons for cancer's low clinical response to chemotherapeutics is the highly immunosuppressive tumor microenvironment (TME). Tumor-ass ociated M2 macrophages (M2-TAMs) orchestrate... Show moreOne of the main reasons for cancer's low clinical response to chemotherapeutics is the highly immunosuppressive tumor microenvironment (TME). Tumor-ass ociated M2 macrophages (M2-TAMs) orchestrate the immunosuppression, which favors tumor progression. Extracellular vesicles (EVs) have shown great potential for targeted therapies as, depending on their biological origin, they can present different therapeutic properties, such as enhanced accumulation in the target tissue or modulation of the immune system. In the current study, EVs were isolated from M1-macrophages (M1-EVs) pre-treated with hyaluronic acid (HA) and the beta-blocker carvedilol (CV). The resulting modulated-M1 EVs (MM1-EVs) were further loaded with doxorubicin (MM1-DOX) to assess their effect in a mouse model of metastatic tumor growth. The cell death and cell migration profile were evaluated in vitro in 4T1 cells. The polarization of the RAW 264.7 murine macrophage cell line was also analyzed to evaluate the effects on the TME. Tumors were investigated by qRT-PCR and immunohistochemistry. MM1-DOX reduced the primary tumor size and metastases. NF-kappa B was the major gene downregulated by MM1-DOX. Furthermore, MM1-DOX reduced the expression of M2-TAM (CD-163) in tumors, which resulted in increased apoptosis (FADD) as well as decreased expression of MMP-2 and TGF-beta. These results suggest a direct effect in tumors and an upregulation in the TME immunomodulation, which corroborate with our in vitro data that showed increased apoptosis, modulation of macrophage polarization, and reduced cell migration after treatment with M1-EVs combined with HA and CV. Our results indicate that the M1-EVs enhanced the antitumor effects of DOX, especially if combined with HA and CV in an animal model of metastatic cancer. Show less
Cartilage diseases currently affect a high percentage of the world's population. Almost all of these diseases, such as osteoarthritis (OA), cause inflammation of this soft tissue. However, this... Show moreCartilage diseases currently affect a high percentage of the world's population. Almost all of these diseases, such as osteoarthritis (OA), cause inflammation of this soft tissue. However, this could be controlled with biomaterials that act as an anti-inflammatory delivery system, capable of dosing these drugs over time in a specific area. The objective of this study was to incorporate etanercept (ETA) into porous three-layer scaffolds to decrease the inflammatory process in this soft tissue. ETA is a blocker of pro-inflammatory cytokines, such as tumour necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6). For this reason, the scaffold was built based on natural polymers, including chitosan and type I collagen. The scaffold was grafted next to subchondral bone using hydroxyapatite as filler. One of the biomaterials obtained was also crosslinked to compare its mechanical properties with the non-treated one. Both samples' physicochemical properties were studied with SEM, micro-CT and photoacoustic imaging, and their rheological properties were also compared. The cell viability and proliferation of the human chondrocyte C28/I2 cell line were studied in vitro. An in vitro and in vivo controlled release study was evaluated in both specimens. The ETA anti-inflammatory effect was also studied by in vitro TNF-alpha and IL-6 production. The crosslinked and non-treated scaffolds had rheological properties suitable for this application. They were non-cytotoxic and favoured the in vitro growth of chondrocytes. The in vitro and in vivo ETA release showed desirable results for a drug delivery system. The TNF-alpha and IL-6 production assay showed that this drug was effective as an anti-inflammatory agent. In an in vivo OA mice model, safranin-O and fast green staining was carried out. The OA cartilage tissue improved when the scaffold with ETA was grafted in the damaged area. These results demonstrate that this type of biomaterial has high potential for clinical applications in tissue engineering and as a controlled drug delivery system in OA articular cartilage. Show less
Photodynamic therapy (PDT) is a promising and clinically approved method for the treatment of cancer. However, the efficacy of PDT is often limited by the poor selectivity and distribution of the... Show morePhotodynamic therapy (PDT) is a promising and clinically approved method for the treatment of cancer. However, the efficacy of PDT is often limited by the poor selectivity and distribution of the photosensitizers (PS) toward the malignant tumors, resulting in prolonged periods of skin photosensitivity. In this work, we present a simple and straightforward strategy to increase the tumor distribution, selectivity, and efficacy of lipophilic PS zinc phthalocyanine (ZnPc) in colon cancer by their stabilization in purified, naturally secreted extracellular vesicles (EVs). The PS ZnPc was incorporated in EVs (EV-ZnPc) by a direct incubation strategy that did not affect size distribution or surface charge. By using co-culture models simulating a tumor microenvironment, we determined the preferential uptake of EV-ZnPc toward colon cancer cells when compared with macrophages and dendritic cells. We observed that PDT promoted total tumor cell death in normal and immune cells, but showed selectivity against cancer cells in co-culture models. In vivo assays showed that after a single intravenous or intratumoral injection, EV-ZnPc were able to target the tumor cells and strongly reduce tumor growth over 15 days. These data expose opportunities to enhance the potential and efficacy of PDT using simple non-synthetic strategies that might facilitate translation into clinical practice. Show less
Background Extracellular vesicles (EVs) have shown great potential for targeted therapy, as they have a natural ability to pass through biological barriers and, depending on their origin, can... Show moreBackground Extracellular vesicles (EVs) have shown great potential for targeted therapy, as they have a natural ability to pass through biological barriers and, depending on their origin, can preferentially accumulate at defined sites, including tumors. Analyzing the potential of EVs to target specific cells remains challenging, considering the unspecific binding of lipophilic tracers to other proteins, the limitations of fluorescence for deep tissue imaging and the effect of external labeling strategies on their natural tropism. In this work, we determined the cell-type specific tropism of B16F10-EVs towards cancer cell and metastatic tumors by using fluorescence analysis and quantitative gold labeling measurements. Surface functionalization of plasmonic gold nanoparticles was used to promote indirect labeling of EVs without affecting size distribution, polydispersity, surface charge, protein markers, cell uptake or in vivo biodistribution. Double-labeled EVs with gold and fluorescent dyes were injected into animals developing metastatic lung nodules and analyzed by fluorescence/computer tomography imaging, quantitative neutron activation analysis and gold-enhanced optical microscopy. Results We determined that B16F10 cells preferentially take up their own EVs, when compared with colon adenocarcinoma, macrophage and kidney cell-derived EVs. In addition, we were able to detect the preferential accumulation of B16F10 EVs in small metastatic tumors located in lungs when compared with the rest of the organs, as well as their precise distribution between tumor vessels, alveolus and tumor nodules by histological analysis. Finally, we observed that tumor EVs can be used as effective vectors to increase gold nanoparticle delivery towards metastatic nodules. Conclusions Our findings provide a valuable tool to study the distribution and interaction of EVs in mice and a novel strategy to improve the targeting of gold nanoparticles to cancer cells and metastatic nodules by using the natural properties of malignant EVs. Show less
