The plant material Scutellariae baicalensis radix, which is rich in flavones (baicalin), possesses antibacterial, antifungal, antioxidant, and anti-inflammatory properties. This work aimed to develop a 3D-printed chitosan-based hydrogel rich in Scutellariae baicalensis extract as an innovative approach for the personalized treatment of periodontal diseases. Chitosan-based hydrogels were prepared, and the printability of the prepared hydrogels was determined. The hydrogel with 2.5% w/v of high molecular-weight chitosan (CS), 2% w/v gelatin (Gel), and 10% w/w of extract (Ex) presented the best printability, producing smooth and uniform scaffolds. It was proved that the CS/Gel/Ex hydrogel was stabilized by hydrogen bonds and remained in amorphous dispersion in the 3D-printed structures (confirmed by ATR-FTIR and XRPD). Due to the amorphization of the active substance, a significant increase in the release of baicalin in vitro was observed. It was demonstrated that there was an initial burst release and a continuous release profile (n = 3). Higuchi kinetic was the most likely baicalin release kinetic. The second fit, the Korsmeyer-Peppas kinetics model, showed coupled diffusion of the active ingredient in the hydrated matrix and polymer relaxation regulated release, with n values ranging from 0.45 to 0.89. The anti-inflammatory properties of 3D-printed scaffolds were assessed as the ability to inhibit the activity of the hyaluronidase enzyme. Activity was assessed as IC50 = 63.57 ± 4.98 mg hydrogel/mL (n = 6). Cytotoxicity tests demonstrated the biocompatibility of the material. After 24 h of exposure to the 2.5CS/2Gel/10Ex scaffold, fibroblasts migrated toward the scratch, closed the "wound" by 97.1%, and significantly accelerated the wound healing process. The results render the 3D-printed CS/Gel/extract scaffolds as potential candidates for treating periodontal diseases.
Silica-based ceramics doped with calcium and magnesium have been proposed as suitable materials for scaffold fabrication. Akermanite (Ca2MgSi2O7) has attracted interest for bone regeneration due to its controllable biodegradation rate, improved mechanical properties, and high apatite-forming ability. Despite the profound advantages, ceramic scaffolds provide weak fracture resistance. The use of synthetic biopolymers such as poly(lactic-co-glycolic acid) (PLGA) as coating materials improves the mechanical performance of ceramic scaffolds and tailors their degradation rate. Moxifloxacin (MOX) is an antibiotic with antimicrobial activity against numerous aerobic and anaerobic bacteria. In this study, silica-based nanoparticles (NPs) enriched with calcium and magnesium, as well as copper and strontium ions that induce angiogenesis and osteogenesis, respectively, were incorporated into the PLGA coating. The aim was to produce composite akermanite/PLGA/NPs/MOX-loaded scaffolds through the foam replica technique combined with the sol-gel method to improve the overall effectiveness towards bone regeneration. The structural and physicochemical characterizations were evaluated. Their mechanical properties, apatite forming ability, degradation, pharmacokinetics, and hemocompatibility were also investigated. The addition of NPs improved the compressive strength, hemocompatibility, and in vitro degradation of the composite scaffolds, resulting in them keeping a 3D porous structure and a more prolonged release profile of MOX that makes them promising for bone regeneration applications.
BACKGROUND: Aliphatic polyesters are widely used for biomedical, pharmaceutical and environmental applications due to their high biodegradability and cost-effective production. Recently, star and hyperbranched polyesters based on glycerol and ω-carboxy fatty diacids have gained considerable interest. Succinic acid and bio-based diacids similar to glycerol are regarded as safe materials according to the US Food and Drug Administration (FDA). Bioactive glass scaffolds utilized in bone tissue engineering are relatively brittle materials. However, their mechanical properties can be improved by using polymer coatings that can further control their degradation rate, tailor their biocompatibility and enhance their performance. The purpose of this study is to explore a new biopolyester poly(glycerol succinate) (PGSuc) reinforced with mesoporous bioactive nanoparticles (MSNs) as a novel coating material to produce hybrid scaffolds for bone tissue engineering. METHODS: Bioactive glass scaffolds were coated with neat PGSuc, PGSuc loaded with dexamethasone sodium phosphate (DexSP) and PGSuc loaded with DexSP-laden MSNs. The physicochemical, mechanical and biological properties of the scaffolds were also evaluated. RESULTS: Preliminary data are provided showing that polymer coatings with and without MSNs improved the physicochemical properties of the 1393 bioactive glass scaffolds and increased the ALP activity and alizarin red staining, suggesting osteogenic differentiation potential when cultured with adipose-derived mesenchymal stem cells. CONCLUSIONS: PGSuc with incorporated MSNs coated onto 1393 bioactive glass scaffolds could be promising candidates in bone tissue engineering applications.
Bioprinting is an emerging technology with various applications in developing functional tissue constructs for the replacement of harmed or damaged tissues and simultaneously controlled drug delivery systems (DDSs) for the administration of several active substances, such as growth factors, proteins, and drug molecules. It is a novel approach that provides high reproducibility and precise control over the fabricated constructs in an automated way. An ideal bioink should possess proper mechanical, rheological, and biological properties essential to ensure proper function. Chitosan is a promising natural-derived polysaccharide to be used as ink because of its attractive properties, such as biodegradability, biocompatibility, low cost, and non-immunogenicity. This review focuses on 3D bioprinting technology for the preparation of chitosan-based hydrogel scaffolds for the regeneration of tissues delivering either cells or active substances to promote restoration.
Humans have severely altered freshwater ecosystems globally, causing a loss of biodiversity. Regulatory frameworks, like the Water Framework Directive, have been developed to support actions that halt and reverse this loss. These frameworks use typology systems that summarize freshwater ecosystems into environmentally delineated types. Within types, ecosystems that are minimally impacted by human activities, i.e., in reference conditions, are expected to be similar concerning physical, chemical, and biological characteristics. This assumption is critical when water quality assessments rely on comparisons to type-specific reference conditions. Lyche Solheim et al. (2019) developed a pan-European river typology system, the Broad River Types, that unifies the national Water Framework Directive typology systems and is gaining traction within the research community. However, it is unknown how similar biological communities are within these individual Broad River Types. We used analysis of similarities and classification strength analysis to examine if the Broad River Types delineate distinct macroinvertebrate communities across Europe and whether they outperform two ecoregional approaches: the European Biogeographical Regions and Illies' Freshwater Ecoregions. We determined indicator and typical taxa for the types of all three typology systems and evaluated their distinctiveness. All three typology systems captured more variation in macroinvertebrate communities than random combinations of sites. The results were similar among typology systems, but the Broad River Types always performed worse than either the Biogeographic Regions or Illies' Freshwater Ecoregions. Despite reaching statistical significance, the statistics of analysis of similarity and classification strength were low in all tests indicating substantial overlap among the macroinvertebrate communities of different types. We conclude that the Broad River Types do not represent an improvement upon existing freshwater typologies when used to delineate macroinvertebrate communities and we propose future avenues for advancement: regionally constrained types, better recognition of intermittent rivers, and consideration of biotic communities.
Ecosystem , Rivers , Animals , Biodiversity , Environmental Monitoring/methods , Humans , Invertebrates
Engineered electrospun membranes have emerged as promising materials in guided tissue regeneration, as they provide an appropriate framework for the formation of new functional periodontal tissues. The development of multifunctional local drug delivery systems with sustained release of drugs for prolonged infection control can be used in periodontal surgical interventions to simultaneously prohibit epithelium downgrowth and ensure proper healing and regeneration of damaged periodontal tissues. The aim of the present study was the fabrication of novel composite membranes from PLGA/moxifloxacin-loaded mesoporous nanocarriers through electrospinning and the evaluation of their drug release profiles. The addition of moxifloxacin-loaded mesoporous nanocarriers in PLGA yielded a sustained and prolonged drug release, while maintaining satisfactory mechanical strength. The freshly fabricated membranes were found to be biocompatible at masses less than 1 mg after exposure to healthy erythrocytes. Increase in the amount of polymer led to more uniform fibers with large diameters and pores. The study of the parameters of the electrospinning process indicated that increase in the applied voltage value and rotation speed of the collector led to more uniform fibers with higher diameter and larger pores, suitable for tissue regeneration applications, such as periodontal tissue regeneration.
A new multimetric index (HeLLBI) based on littoral benthic macroinvertebrates is presented in this paper for classification of Greek natural lakes, in compliance with the requirements of Water Framework Directive (WFD). The method was developed based on the collection of littoral benthic invertebrate fauna and environmental data from 109 sampling sites in 21 natural lakes of the Greek National Water Monitoring Network. We focused the analysis on the effects of shore morphological alterations and eutrophication to the littoral invertebrate fauna, identified to family level, except oligochaetes, which were identified as a class, and more particularly to taxonomic composition and abundance, to taxa sensitivity, and to richness/diversity. Three metrics were included in the multimeric index: the relative abundance of Odonata classes, the Average Score per Taxon, and the Simpson's diversity index. The metrics were converted to ecological quality ratios and ecological class boundaries were defined. The final multimetric index HeLLBI is expressed as an arithmetic average of normalized ecological quality ratios of the above metrics and a final score was assigned to each lake. Pressure-response relationships of HeLLBI scores were statistically tested for morphological alterations, expressed as percentage of artificial shoreline, and eutrophication, expressed as total phosphorus. The HeLLBI scores correspond to ecological classes, according to WFD, and sampling sites with different ecological status contained distinct biological communities; those at high status where more diverse and with sensitive taxa and as the water quality deteriorated, macroinvertebrate assemblages consisted of fewer and more tolerant to degradation taxa. The HeLLBI method gave a reliable assessment of littoral benthic invertebrate fauna of Greek natural lakes and could be a useful tool for the classification of ecological status of other Mediterranean lakes.
Lakes , Water , Animals , Ecosystem , Environmental Monitoring , Invertebrates
The compound of chitin is the second most important and abundant natural biopolymer in the world. The main extraction and exploitation sources of this natural polysaccharide polymer are mainly crustaceans species, such as shrimps and crabs. Chitosan (CS) (poly-ß-(1 â 4)-2-amino-2-deoxy-d-glucose) can be derived from chitin and can be mentioned as a compound that has high value-added applications due to its wide variety of uses, including pharmaceutical, biomedical, and cosmetics applications, food etc. Furthermore, chitosan is a biopolymer that can be used for adsorption applications because it contains amino and hydroxyl groups in its chemical structure (molecules), resulting in possible interactions of adsorption between chitosan and pollutants (uranium, mercury, rare earth elements (REEs), phenols, etc.). However, adsorption is a very effective, fast, simple, and low-cost process. This review article places emphasis on recent demonstrated research papers (2014-2020) where the chemical modifications of CS are explained briefly (grafting, cross-linking etc.) for the uptake of uranium, mercury, and REEs in synthesized aqueous solutions. Finally, figures and tables from selected synthetic routes of CS are presented and the effects of pH and the best mathematical fitting of isotherm and kinetic equations are discussed. In addition, the adsorption mechanisms are discussed.
BACKGROUND: The human leukocyte antigen (HLA) class II molecules are constitutively expressed in some melanoma, but the underlying molecular mechanisms have not yet been characterized. METHODS: The expression of HLA class II antigen processing machinery (APM) components was determined in melanoma samples by qPCR, Western blot, flow cytometry and immunohistochemistry. Immunohistochemical and TCGA datasets were used for correlation of HLA class II expression to tumor grading, T-cell infiltration and patients' survival. RESULTS: The heterogeneous HLA class II expression in melanoma samples allowed us to characterize four distinct phenotypes. Phenotype I totally lacks constitutive HLA class II surface expression, which is inducible by interferon-gamma (IFN-γ); phenotype II expresses low basal surface HLA class II that is further upregulated by IFN-γ; phenotype III lacks constitutive and IFN-γ controlled HLA class II expression, but could be induced by epigenetic drugs; and in phenotype IV, lack of HLA class II expression is not recovered by any drug tested. High levels of HLA class II APM component expression were associated with an increased intra-tumoral CD4+ T-cell density and increased patients' survival. CONCLUSIONS: The heterogeneous basal expression of HLA class II antigens and/or APM components in melanoma cells is caused by distinct molecular mechanisms and has clinical relevance.
Novel chitosan copolymers (CS-g-SBMA) grafted with [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (SBMA) in various molar ratio 1.5:1, 5:1, 11.5:1 and 20:1, were synthesized in the present study. SBMA was selected as zwitterion molecule showing promising antibacterial properties. Grafted chitosan derivatives were fully characterized for their successful synthesis by NMR and FT-IR, for their crystallinity by XRD showing reduced crystallinity compared to CS alone. Furthermore, swelling studies were conducted with the grafted derivatives showing extensive swelling capacity (maximum degree of swelling up to 1800%) and water absorption was studied with differential scanning calorimetry and equilibrium water adsorption/desorption isotherms were analyzed. Caspofungin, a novel antifungal drug, was used to prepare a double-acting system, with both antibacterial and antifungal properties, proper for topical use. Drug loaded hydrogels were prepared with 10, 20 and 30 wt% drug content and the loaded hydrogels were fully characterized while antimicrobial studies showed enhanced properties. Caspofungin in vitro release showed an initial burst effect followed by a diffusion process while data analysis verified the initial burst release followed by a quasi Fickian diffusion-driven sustained release. Enhance antimicrobial properties was also observed in caspofungin-loaded hydrogels showing the successful fulfill of our scope; an amphiphilic system having great potential for the development of patches with inherent antimicrobial properties and prolonged antifungal properties.
Chitosan , Antifungal Agents , Caspofungin , Drug Carriers , Drug Liberation , Hydrogels , Hydrogen-Ion Concentration , Spectroscopy, Fourier Transform Infrared
Biglycan/genetics , Breast Neoplasms/genetics , Breast Neoplasms/immunology , MicroRNAs/genetics , Transforming Growth Factor beta/genetics , Animals , Biglycan/immunology , Biglycan/metabolism , Breast Neoplasms/metabolism , Disease Models, Animal , Female , Gene Expression Regulation, Neoplastic/genetics , Genes, MHC Class I/genetics , Genes, MHC Class I/immunology , Humans , Mice , MicroRNAs/immunology , MicroRNAs/metabolism , Signal Transduction/genetics , Signal Transduction/immunology , Transforming Growth Factor beta/immunology , Transforming Growth Factor beta/metabolism
Mesoporous silica-based nanoparticles (MSNs) are considered promising drug carriers because of their ordered pore structure, which permits high drug loading and release capacity. The dissolution of Si and Ca from MSNs can trigger osteogenic differentiation of stem cells towards extracellular matrix calcification, while Mg and Sr constitute key elements of bone biology and metabolism. The aim of this study was the synthesis and characterization of sol-gel-derived MSNs co-doped with Ca, Mg and Sr. Their physico-chemical properties were investigated by X-ray diffraction (XRD), scanning electron microscopy with energy dispersive X-ray analysis (SEM/EDX), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), X-ray fluorescence spectroscopy (XRF), Brunauer Emmett Teller and Brunauer Joyner Halenda (BET/BJH), dynamic light scattering (DLS) and ζ-potential measurements. Moxifloxacin loading and release profiles were assessed with high performance liquid chromatography (HPLC) cell viability on human periodontal ligament fibroblasts and their hemolytic activity in contact with human red blood cells (RBCs) at various concentrations were also investigated. Doped MSNs generally retained their textural characteristics, while different compositions affected particle size, hemolytic activity and moxifloxacin loading/release profiles. All co-doped MSNs revealed the formation of hydroxycarbonate apatite on their surface after immersion in simulated body fluid (SBF) and promoted mitochondrial activity and cell proliferation.
Drug Delivery Systems , Moxifloxacin/pharmacology , Nanoparticles/chemistry , Tissue Engineering , Cell Proliferation/drug effects , Dynamic Light Scattering , Humans , Magnesium/chemistry , Microscopy, Electron, Scanning , Moxifloxacin/chemistry , Osteogenesis/drug effects , Porosity , Silicon Dioxide/chemistry , X-Ray Diffraction
Tumor escape is often associated with abnormalities in the surface expression of the human leukocyte antigen class I (HLA-I) antigens thereby limiting CD8+ cytotoxic T cell responses. This impaired HLA-I surface expression can be mediated by deficient expression of components of the antigen processing and presentation machinery (APM) due to epigenetic, transcriptional and/or post-transcriptional processes. Since a discordant mRNA and protein expression pattern of APM components including the peptide transporter associated with antigen processing 1 (TAP1) has been frequently described in tumors of distinct origin, a post-transcriptional control of APM components caused by microRNAs (miR) was suggested. Using an in silico approach, miR-200a-5p has been identified as a candidate miR binding to the 3' untranslated region (UTR) of TAP1. Luciferase reporter assays demonstrated a specific binding of miR-200a-5p to the TAP1 3'-UTR. Furthermore, the miR-200a-5p expression is inversely correlated with the TAP1 protein expression in HEK293T cells and in a panel of melanoma cell lines as well as in primary melanoma lesions. High levels of miR-200a-5p expression were associated with a shorter overall survival of melanoma patients. Overexpression of miR-200a-5p reduced TAP1 levels, which was accompanied by a decreased HLA-I surface expression and an enhanced NK cell sensitivity of melanoma cells. These data show for the first time a miR-mediated control of the peptide transporter subunit TAP1 in melanoma thereby leading to a reduced HLA-I surface expression accompanied by an altered immune recognition and reduced patients' survival. Abbreviations: Ab: antibody; ACTB: ß-actin; APM: antigen processing and presentation machinery; ATCC: American tissue culture collection; ß2-m: ß2-microglobulin; BSA: bovine serum albumin; CTL: cytotoxic T lymphocyte; FCS: fetal calf serum; FFL: firefly luciferase; FFPE: formalin-fixed paraffin-embedded; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HC: heavy chain; HLA: human leukocyte antigen; HLA-I: HLA class I; HRP: horseradish peroxidase; IFN: interferon; im-miR: immune modulatory miRNA; LMP: low molecular weight protein; luc: luciferase; MFI: mean fluorescence intensity; MHC: major histocompatibility complex; miR: microRNA; NC: negative control; NK: natural killer; NSCLC: non-small cell lung carcinoma; OS: overall survival; PBMC: peripheral blood mononuclear cells; RBP: RNA-binding proteins; RL: Renilla; RLU: relative light units; TAP: transporter associated with antigen processing; tpn: tapasin; UTR: untranslated region.
Melanoma , MicroRNAs , ATP Binding Cassette Transporter, Subfamily B, Member 2/genetics , Antigen Presentation , HEK293 Cells , Humans , Leukocytes, Mononuclear , Melanoma/genetics , MicroRNAs/genetics , Peptide Transporter 1
The underlying molecular mechanisms of the aberrant expression of components of the HLA class I antigen processing and presentation machinery (APM) in tumors leading to evasion from T cell-mediated immune surveillance could be due to posttranscriptional regulation mediated by microRNAs (miRs). So far, some miRs controlling the expression of different APM components have been identified. Using in silico analysis and an miR enrichment protocol in combination with small RNA sequencing, miR-26b-5p and miR-21-3p were postulated to target the 3' untranslated region (UTR) of the peptide transporter TAP1, which was confirmed by high free binding energy and dual luciferase reporter assays. Overexpression of miR-26b-5p and miR-21-3p in melanoma cells downregulated the TAP1 protein and reduced expression of HLA class I cell surface antigens, which could be reverted by miR inhibitors. Moreover, miR-26b-5p overexpression induced a decreased T cell recognition. Furthermore, an inverse expression of miR-26b-5p and miR-21-3p with TAP1 was found in primary melanoma lesions, which was linked with the frequency of CD8+ T cell infiltration. Thus, miR-26-5p and miR-21-3p are involved in the HLA class I-mediated immune escape and might be used as biomarkers or therapeutic targets for HLA class Ilow melanoma cells.
Chitosan (CS) is a hemi-synthetic cationic linear polysaccharide produced by the deacetylation of chitin. CS is non-toxic, highly biocompatible, and biodegradable, and it has a low immunogenicity. Additionally, CS has inherent antibacterial properties and a mucoadhesive character and can disrupt epithelial tight junctions, thus acting as a permeability enhancer. As such, CS and its derivatives are well-suited for the challenging field of ocular drug delivery. In the present review article, we will discuss the properties of CS that contribute to its successful application in ocular delivery before reviewing the latest advances in the use of CS for the development of novel ophthalmic delivery systems. Colloidal nanocarriers (nanoparticles, micelles, liposomes) will be presented, followed by CS gels and lenses and ocular inserts. Finally, instances of CS coatings, aiming at conferring mucoadhesiveness to other matrixes, will be presented.
The aim of this work was to evaluate the effectiveness of neat chitosan (CS) and its derivatives with 2-acrylamido-2-methyl-1-propanesulfonic acid (AAMPS) and [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (MEDSP) as appropriate nanocarriers for the simultaneous ocular administration of dexamethasone sodium phosphate (DxP) and chloramphenicol (CHL). The derivatives CS-AAMPS and CS-MEDSP have been synthesized by free-radical polymerization and their structure has been proved by Fourier-Transformed Infrared Spectroscopy (FT-IR) spectroscopy. Both derivatives exhibited low cytotoxicity, enhanced mucoadhesive properties and antimicrobial activity against Staphylococcus aureus (S.aureus) and Escherichia coli (E. coli). Encapsulation was performed via ionic crosslinking gelation using sodium tripolyphosphate (TPP) as the crosslinking agent. Dynamic light scattering measurements (DLS) showed that the prepared nanoparticles had bimodal distribution and sizes ranging from 50-200 nm and 300-800 nm. Drugs were encapsulated in their crystalline (CHL) or amorphous (DexSP) form inside nanoparticles and their release rate was dependent on the used polymer. The CHL dissolution rate was substantially enhanced compared to the neat drug and the release time was extended up to 7 days. The release rate of DexSP was much faster than that of CHL and was prolonged up to 3 days. Drug release modeling unveiled that diffusion is the main release mechanism for both drugs. Both prepared derivatives and their drug-loaded nanoparticles could be used for extended and simultaneous ocular release formulations of DexSP and CHL drugs.
In the present study, a chitosan (CS) derivative with the 2-(Methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (SDAEM) zwitterionic monomer was prepared through chemical modification. The successful synthesis of CS-SDAEM was confirmed by Fourier-transform Infrared (FTIR) and Nuclear Magnetic Resonance (1H-NMR) spectroscopies. Its crystallinity was studied by X-ray Diffraction (XRD), while in vitro cytotoxicity and cell viability assays established its biocompatibility. Filtered fresh pomegranate juice (PJ) was loaded in nanoparticles of neat CS and its derivative via ionic gelation method. Dynamic Light Scattering (DLS) revealed nanoparticles sizes varying between 426 nm and 4.5 µm, indicating a size-dependence on the polymer concentration used during encapsulation. High-performance liquid chromatography coupled with photodiode array and electrospray ionization mass spectrometry detection (LC-PDA-ESI/MS) revealed that PJ active compounds were successfully and in sufficient amounts encapsulated in the nanoparticles interior, whereas XRD indicated a crystalline structure alteration after nanoencapsulation. The resulted PJ-loaded nanoparticles were further utilized for the preparation of innovative O/W cosmetic emulsions. All produced emulsions exhibited good pH and viscosity stability for up to 90 days, while the sun protection factor (SPF) was enhanced due to the presence of the PJ. Enhanced antioxidant and antimicrobial properties due to the phenolic compounds of PJ were also observed.
The major mechanisms of posttranscriptional gene regulation involve microRNAs (miRs) and RNA-binding proteins (RBPs). Recent studies not only identified functionally and characterized such factors, but rather investigated their use as biomarkers and suitability as biopharmaceuticals. Indeed, some miR-based drugs are currently tested in clinical studies as potential anti-viral and as anti-cancer agents. For the chemical application, a profound knowledge of the binding affinities of miRs and RBPs to their target RNA is essential. The authors recently identified several miRs regulating the non-classical human leukocyte antigen (HLA)-G, and characterized their binding affinity to the 3' untranslated region (UTR) of HLA-G. These miRs identified by miTRAP were classified into high affinity and low affinity miRs, which were either key regulators or fine tuners of HLA-G. While the miTRAP method has been described in detail, a novel modified miTRAP technique has been established, which completely consists of commercially available components and uses a simplified cloning strategy. This technique allows the identification and characterization of miRs and RBPs for any RNA sequence of interest.
MicroRNAs , 3' Untranslated Regions , Base Sequence , Chromatography, Affinity , Humans , MicroRNAs/genetics
By binding RNA in a sequence- and/or structure-dependent manner, RNA-binding proteins (RBPs) and their target RNA form a ribonucleoprotein complex involved in the RNA's fate. In this context, RBPs were shown to act as key players for post-transcriptional gene regulation by controlling RNA editing, splicing, polyadenylation, translocation, and stability. So far, over 1900 RBPs were identified and their deregulation has been associated with the development and progression of various disorders including cancer. Although a number of sophisticated approaches are available, our knowledge about direct RNA-RBP interactions is, however, quite limited. Here we present a protocol with restricted requirements for equipment and devices to identify RBPs. This approach is based on (i) the purification of biotinylated RNA, (ii) chromatographic separation of co-purified proteins, and (iii) their identification by mass spectrometry.
Neoplasms , RNA-Binding Proteins , Gene Expression Regulation , Humans , Neoplasms/genetics , RNA/genetics , RNA/metabolism , RNA Splicing , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism
The objective of this study was to develop chitosan (CS) nanoparticles (NPs) loaded with deferoxamine mesylate (DFO) for slow release of this iron-chelating drug. Drug nanoencapsulation was performed via ionic gelation of chitosan using sodium tripolyphosphate (TPP) as cross-linker. Nanoparticles with a size ranging between 150 and 400 nm were prepared for neat CS/TPP with a 2/1 molar ratio while their yield was directly dependent on the applied stirring rate during the preparation process. DFO at different content (20, 45 and 75 wt %) was encapsulated into these nanoparticles. We found that drug loading correlates with increasing DFO content while the entrapment efficiency has an opposite behavior due to the high solubility of DFO. Hydrogen-bonding between amino and hydroxyl groups of DFO with reactive groups of CS were detected using FT-IR spectroscopy while X-ray diffraction revealed that DFO was entrapped in amorphous form in the CS nanoparticles. DFO release is directly dependent on the content of loaded drug, while model analysis revealed that the release mechanism of DFO for the CS/TPP nanoparticles is by diffusion. Treatment of murine RAW 264.7 macrophages with nanoencapsulated DFO promoted an increased expression of transferrin receptor 1 (TfR1) mRNA, a typical homeostatic response to iron deficiency. These data provide preliminary evidence for release of pharmacologically active DFO from the chitosan nanoparticles.
