Biopolymer Lipid Hybrid Microcarrier for Transmembrane Inner Ear Delivery of Dexamethasone.

Dexamethasone is one of the most often used corticosteroid drugs for sensorineural hearing loss treatment, and is used either by intratympanic injection or through systemic delivery. In this study, a biopolymer lipid hybrid microcarrier was investigated for enhanced local drug delivery and sustained release at the round window membrane level of the middle ear for the treatment of sensorineural hearing loss (SNHL). Dexamethasone-loaded and dexamethasone-free microparticles were prepared using biopolymers (polysaccharide and protein, pectin and bovine serum albumin, respectively) combined with lipid components (phosphatidylcholine and Dimethyldioctadecylammonium bromide) in order to obtain a biopolymer-liposome hybrid system, with a complex structure combining to enhance performance in terms of physical and chemical stability. The structure of the microparticles was evaluated by FTIR, XRD, thermal analysis, optical microscopy, and scanning electron microscopy (SEM). The encapsulation efficiency determination and the in vitro Dexamethasone release study were performed using UV-Vis spectroscopy. The high value of encapsulation efficiency and the results of the release study indicated six days of sustained release, encouraging us to evaluate the in vitro cytotoxicity of Dexamethasone-loaded microparticles and their influence on the cytotoxicity induced by Cisplatin on auditory HEI-OC1 cells. The results show that the new particles are able to protect the inner ear sensory cells.

Current Concepts and Future Trends in Increasing the Benefits of Cochlear Implantation: A Narrative Review.

Hearing loss is the most common neurosensory disorder, and with the constant increase in etiological factors, combined with early detection protocols, numbers will continue to rise. Cochlear implantation has become the gold standard for patients with severe hearing loss, and interest has shifted from implantation principles to the preservation of residual hearing following the procedure itself. As the audiological criteria for cochlear implant eligibility have expanded to include patients with good residual hearing, more attention is focused on complementary development of otoprotective agents, electrode design, and surgical approaches. The focus of this review is current aspects of preserving residual hearing through a summary of recent trends regarding surgical and pharmacological fundamentals. Subsequently, the assessment of new pharmacological options, novel bioactive molecules (neurotrophins, growth factors, etc.), nanoparticles, stem cells, and gene therapy are discussed.

A comparative study of genotoxicity endpoints for women exposed to different levels of indoor radon concentrations.

BACKGROUND AND PURPOSE: Radon and its radioactive progenies are the most important source of ionizing radiation of natural origin, being classified as a Group 1 carcinogen. The aim of this study is to investigate the genotoxic effects of a wide range of indoor radon concentrations, as well as the kinetics of the process of repairing DNA-induced lesions by a challenging dose of gamma irradiation. MATERIAL AND METHODS: Female subjects residing in the Baita-Stei radon priority area were selected as the exposed group. The reference group was comprised of women from the same county (Bihor), but located in an area with an average indoor radon concentration typical of the county from which they were taken. Radon concentration values of 300 Bq/m3 and 148 Bq/m3, respectively, were chosen as a threshold in order to capture the impact of radon exposure between the groups. The alkaline comet assay was used in order to measure the DNA damage, as well as the repair kinetics at 2 and 24 h after 2 Gy challenging doses of gamma irradiation using peripheral blood lymphocytes. From the serum of the subjects, the oxidative damage by 8-hydroxydeoxyguanosine as well as the PARP induction was evaluated. The chromosomal aberrations were evaluated using the Cytokinesis Block MicroNucleus Assay. RESULTS: A statistically significant increase was observed in terms of DNA-induced lesions assessed by comet assay for the exposed group compared to the reference group. A positive correlation was obtained between DNA damage and the annual effective dose, respectively with the radon progenies concentrations. A statistically significant difference was also observed for the frequency of the micronuclei between the exposed - reference groups. Significantly faster repair kinetics of DNA-induced lesions was recorded for the first 2 h after gamma irradiation in the reference group compared to the exposed group. Using the threshold of 300 Bq/m3 for radon concentration, faster kinetics of DNA damage repair for people exposed to low radon concentrations, compared to those exposed to higher concentrations for the second phase of DNA repair kinetics was observed. CONCLUSION: An increased radiosensitivity of lymphocytes, as well as slower repair kinetics, may be associated with exposure to higher indoor radon concentrations.

Antibody-functionalized theranostic protein nanoparticles for the synergistic deep red fluorescence imaging and multimodal therapy of ovarian cancer.

Among women, ovarian cancer is the fifth most frequent type of cancer, and despite benefiting from current standard treatment plans, 90% of patients relapse in the subsequent 18 months and, eventually, perish. As a result, via embracing nanotechnological advancements in the field of medical science, researchers working in the areas of cancer therapy and imaging are looking for the next breakthrough treatment strategy to ensure lower cancer recurrence rates and improved outcomes for patients. Herein, we design a novel phototheranostic agent with optical features in the biological window of the electromagnetic spectrum via encapsulating a newly synthesized phthalocyanine dye within biocompatible protein nanoparticles, allowing the targeted fluorescence imaging and synergistic dual therapy of ovarian cancer. The nanosized agent displays great biocompatibility and enhanced aqueous biostability and photothermal activity, as well as high reactive-oxygen-species generation efficiency. To achieve the active targeting of the desired malignant tissue and suppress the rapid clearance of the photosensitive agent from the peritoneal cavity, the nanoparticles are biofunctionalized with an anti-folate receptor antibody. A2780 ovarian cancer cells are employed to confirm the improved targeting capabilities and the in vitro cytotoxic efficiency of the theranostic nanoparticles after exposure to a 660 nm LED lamp; upon measurement via MTT and flow cytometry assays, a significant 95% decrease in the total number of viable cells is seen. Additionally, the therapeutic performance of our newly designed nanoparticles was evaluated in vivo, via real-time thermal monitoring and histopathological assays, upon the irradiation of tumour-bearing mice with a 660 nm LED lamp (0.05 W cm-2). Foremost, separately from steady-state fluorescence imaging, we found that, via utilizing FLIM investigations, the differences in fluorescence lifetimes of antibody biofunctionalized and non-functionalized nanoparticles can be correlated to different intracellular localization and internalization pathways of the fluorescent agent, which is relevant for the development of a cutting-edge method for the detection of cancer cells that overexpress folate receptors at their surfaces.

Fluorescent Phthalocyanine-Encapsulated Bovine Serum Albumin Nanoparticles: Their Deployment as Therapeutic Agents in the NIR Region.

In recent times, researchers have aimed for new strategies to combat cancer by the implementation of nanotechnologies in biomedical applications. This work focuses on developing protein-based nanoparticles loaded with a newly synthesized NIR emitting and absorbing phthalocyanine dye, with photodynamic and photothermal properties. More precisely, we synthesized highly reproducible bovine serum albumin-based nanoparticles (75% particle yield) through a two-step protocol and successfully encapsulated the NIR active photosensitizer agent, achieving a good loading efficiency of 91%. Making use of molecular docking simulations, we confirm that the NIR photosensitizer is well protected within the nanoparticles, docked in site I of the albumin molecule. Encouraging results were obtained for our nanoparticles towards biomedical use, thanks to their negatively charged surface (-13.6 ± 0.5 mV) and hydrodynamic diameter (25.06 ± 0.62 nm), favorable for benefitting from the enhanced permeability and retention effect; moreover, the MTT viability assay upholds the good biocompatibility of our NIR active nanoparticles. Finally, upon irradiation with an NIR 785 nm laser, the dual phototherapeutic effect of our NIR fluorescent nanoparticles was highlighted by their excellent light-to-heat conversion performance (photothermal conversion efficiency 20%) and good photothermal and size stability, supporting their further implementation as fluorescent therapeutic agents in biomedical applications.

Zeaxanthin-Rich Extract from Superfood Lycium barbarum Selectively Modulates the Cellular Adhesion and MAPK Signaling in Melanoma versus Normal Skin Cells In Vitro.

The concern for implementing bioactive nutraceuticals in antioxidant-related therapies is of great importance for skin homeostasis in benign or malignant diseases. In order to elucidate some novel insights of Lycium barbarum (Goji berry) activity on skin cells, the present study focused on its active compound zeaxanthin. By targeting the stemness markers CD44 and CD105, with deep implications in skin oxidative stress mechanisms, we revealed, for the first time, selectivity in zeaxanthin activity. When applied in vitro on BJ human fibroblast cell line versus the A375 malignant melanoma cells, despite the moderate cytotoxicity, the zeaxanthin-rich extracts 1 and 2 were able to downregulate significantly the CD44 and CD105 membrane expression and extracellular secretion in A375, and to upregulate them in BJ cells. At mechanistic level, the present study is the first to demonstrate that the zeaxanthin-rich Goji extracts are able to influence selectively the mitogen-activated protein kinases (MAPK): ERK, JNK and p38 in normal BJ versus tumor-derived A375 skin cells. These results point out towards the applications of zeaxanthin from L. barbarum as a cytoprotective agent in normal skin and raises questions about its use as an antitumor prodrug alone or in combination with standard therapy.

Novel Phenothiazine-Bridged Porphyrin-(Hetero)aryl dyads: Synthesis, Optical Properties, In Vitro Cytotoxicity and Staining of Human Ovarian Tumor Cell Lines.

We report here the synthetic procedure applied for the preparation of new AB3-type and trans-A2B2 type meso-halogenophenothiazinyl-phenyl-porphyrin derivatives, their metal core complexation and their peripheral modification using Suzuki-Miyaura cross coupling reactions with various (hetero)aryl (phenothiazinyl, 7-formyl-phenothiazinyl, (9-carbazolyl)-phenyl and 4-formyl-phenyl, phenyl) boronic acid derivatives. The meso-phenothiazinyl-phenyl-porphyrin (MPP) dyes family was thus extended by a series of novel phenothiazine-bridged porphyrin-(hetero)aryl dyads characterized by UV-Vis absorption/emission properties typical to the porphyrin chromophore, slightly modulated by increasing the size of peripheral substituents. Three phenothiazine-bridged porphyrin-heteroaryl dyads with fluorescence emission above 655 nm were selected as fluorophores in red spectral region for applications in cellular staining of human ovarian tumors. In vitro experiments of cell metabolic activity displayed a moderate toxicity on human ovarian tumor cell lines (OVCAR-3, cisplatin-sensitive A2780 and cisplatin-resistant A2780cis respectively). Visualization of the stained living cells was performed both by fluorescence microscopy imaging and by fluorescence lifetime imaging under two photon excitation (TPE-FLIM), confirming their cellular uptake and the capability of staining the cell nucleus.

The expression of copper transporters associated with the ototoxicity induced by platinum-based chemotherapeutic agents.

BACKGROUND: Antitumor agents based on platinum have gained a well-established place in the treatment of several forms of cancer. Their efficiency is hampered by serious toxic effects against healthy tissues as well. Ototoxicity is a serious side effect leading to hearing impairment and represents an important issue affecting the patients' quality of life. The currently used platinum chemotherapeutics exert different toxicity towards cochlear cells. The aim of our study was to answer some questions regarding the differential uptake and cellular pharmacodynamics of Cisplatin (CDDP), Carboplatin (CBDCA) and Oxaliplatin (L-OHP) in the HEI-OC1 cochlear cell line. METHODS: We studied the expression of copper transporters CTR1, ATP7A and ATP7B which are presumably involved in the uptake, cellular transport and efflux of platinum compounds by immunofluorescence microscopy and flow-cytometry. The cellular uptake of the compounds was evaluated through the determination of intracellular platinum concentration by atomic absorption spectroscopy. The effects of the treatment of HEI-OC1 cells with platinum compounds were also evaluated: cytotoxicity with the Cell Titer Blue viability test, formation of reactive oxygen species with 2',7' -dichlorofluorescein diacetate, genotoxicity with the comet assay and apoptosis with the cleaved PARP ELISA test. RESULTS: CTR1, ATP7A and ATP7B were all expressed by HEI-OC1 cells. The treatment with the platinum compounds led to a modulation of their expression, manifested in a differential platinum uptake. Treatment with Cisplatin led to the highest intracellular concentration of platinum compared to Oxaliplatin and Carboplatin at the same dose. Treatment with CuSO4 reduced platinum uptake of all the compounds, significantly in the case of Cisplatin and Carboplatin. CDDP was the most cytotoxic against HEI-OC1 cells, with an IC50 = 65.79  µM, compared to 611.7 µM for L-OHP and 882.9 µM for CBDCA, at the same molar concentration. The production of ROS was the most intense after CDDP, followed by L-OHP and CBDCA. In the comet assay, at the 100 µM concentration, L-OHP and CBDCA induced DNA adducts while CDDP induced adducts as well as DNA strand breaks. CBDCA and L-OHP lead to a significant increase of cleaved PARP at 24h (p < 0.001), suggesting an important apoptotic process induced by these compounds at the used concentrations. CONCLUSIONS: The results obtained in the current study suggest that the modulation of copper transporters locally may represent a new strategy against platinum drugs ototoxicity.

Latest advances in chronic rhinosinusitis with nasal polyps endotyping and biomarkers, and their significance for daily practice.

The term chronic rhinosinusitis (CRS) comprises of an assortment of diseases that share a common feature: inflammation of the sinonasal mucosa. The phenotype classification of CRS, based on the presence of polyps, has failed to offer a curative treatment for the disease, particularly in refractory cases. Chronic rhinosinusitis with nasal polyps (CRSwNP) remains a challenging entity. Researchers have made efforts trying to characterize subtypes of the disease according to the endotypes, which are delineated by different immunological pathways, using biomarkers. Even if the inflammatory processes controlling CRSwNP are not fully understood, data suggested that the disease associated with a type 2 inflammatory mechanisms can be also linked to the type 1 or type 3 pathomechanism, being highly heterogeneous. Biomarkers for CRSwNP are proposed, such as: eosinophil count, cytokines, metalloproteinases, bitter and sweet taste receptors, and the nasal microbiome. For endotyping to be clinically applicable and simply determined, biomarkers referring to the intrinsic biomolecular mechanism still need to be found. Precision medicine is becoming the new standard of care, but innovative therapies such as biologics may be rather challenging for the clinicians in their daily practice. This new approach to CRSwNP implies patient selection and a simple algorithm for deciding the right treatment, easy to implement and adjust. Our review points out the ongoing new research on the pathophysiology of CRSwNP, biomarkers and treatment opportunities. It allows clinicians to keep abreast of current evidence-based knowledge and to individualize the management of CRSwNP, especially in refractory cases.

Low-dose radiations derived from cone-beam CT induce transient DNA damage and persistent inflammatory reactions in stem cells from deciduous teeth.

OBJECTIVES: Cone-beam CT (CBCT), a radiographic tool for diagnosis, treatment, and follow-up in dental practice, was introduced also in pediatric radiology, especially orthodontics. Such patients subjected to repetitive X-rays examinations may receive substantial levels of radiation doses. Ionizing radiation (IR), a recognized carcinogenic factor causing DNA double-strand breaks (DSBs) could be harmful to undifferentiated cells such as dental pulp stem cells (DPSCs) since inaccurately repaired or unrepaired DSBs may lead to malignant transformation. The H2AX and MRE11 proteins generated following DSBs formation and pro-inflammatory cytokines (CKs) secreted after irradiation are relevant candidates to monitor the cellular responses induced by CBCT. METHODS: DPSCs were extracted from human exfoliated deciduous teeth and their phenotype was assessed by immunocytochemistry and flow-cytometry. Cells were exposed to IR doses: 5.4-107.7 mGy, corresponding to 0.5-8 consecutive skull exposures, respectively. H2AX and MRE11 were detected in whole cells, while IL-1α, IL-6, IL-8, TNFα in supernatants, using enzyme-linked immunosorbent assay (ELISA) at different time points after exposure. RESULTS: The phosphorylation level of H2AX in DPSCs increased considerably at 0.5 h after exposure (p < 0.001 for 3, 5, 8 skull exposures and p < 0.05 for 1 skull exposure, respectively). MRE11 response could only be detected for the highest IR dose (p < 0.001) in the same interval. CKs secretion increased upon CBCT exposure according to doses and time. CONCLUSIONS: The DPSCs exposure to CBCT induces transient DNA damage and persistent inflammatory reaction in DPSCs drawing the attention on the potential risks of IR exposures and on the importance of dose monitoring in pediatric population.

Targeting MAPK (p38, ERK, JNK) and inflammatory CK (GDF-15, GM-CSF) in UVB-Activated Human Skin Cells with Vitis vinifera Seed Extract.

Ultraviolet B radiation (UVB) activates mitogen-activated protein kinases (MAPK): p38, extracellular signal regulated (ERK), and c-Jun N-terminal (JNK) kinases in human skin cells. Human keratinocytes (KC) exposed to UVB secrete several cytokines (CK), among which the growth differentiation factor-15 (GDF-15) is augmented in inflammatory and aging processes and the granulocyte macrophage-colony stimulating factor (GM-CSF) is involved in cell proliferation, differentiation, and survival, and both CK have implications in skin carcinogenesis. We assessed p38, ERK, JNK, GDF-15, and GM-CSF in UVB-exposed skin cells and a red grape (Vitis vinifera) seed extract's (GSE) capacities to regulate these pathways in UVB-exposed KC. Two concentrations of the GSE extract were selected: GSE1 (37.5 µgEqGA/mL) and GSE2 (75 µgEqGA/mL) and a UVB dose (100 mJ/cm2) within the physiological range. Molecules were assessed with ELISA, semiquantitative results being confirmed by Western blot. UVB triggered the signaling molecules' phosphorylation and the concentrations of CK. All molecules but GM-CSF increased early, at 2 h, from UVB exposure while GM-CSF increased later (at 8 h). MAPK and GDF-15 were regulated by GSE1; GM-CSF, by the higher concentration, GSE2. The amplitude and kinetics of the responses were diverse according to time point, molecules, and the extract's concentration. GSE exerted beneficial effects on MAPK and CK triggered by UVB in human skin cells: reduction of phosphorylation of the assessed signaling molecules and anti-inflammatory effects. Targeting MAPK and specific inflammatory mediators such as GDF-15 and GM-CSF with GSE in UVB-induced skin cells represents a novel and a promising starting point for future photoprotection strategies.

Biosynthesis of Silver Nanoparticles Using Ligustrum Ovalifolium Fruits and Their Cytotoxic Effects.

The present study reports for the first time the efficacy of bioactive compounds from Ligustrum ovalifolium L. fruit extract as reducing and capping agents of silver nanoparticles (AgNPs), developing a green, zero energetic, cost effective and simple synthesis method of AgNPs. The obtained nanoparticles were characterized by UV-Vis spectroscopy, transmission electron microscopy (TEM), X-ray diffraction (XRD) and Fourier Transform Infrared spectroscopy (FTIR), confirming that nanoparticles were crystalline in nature, spherical in shape, with an average size of 7 nm. The FTIR spectroscopy analysis demonstrated that the AgNPs were capped and stabilized by bioactive molecules from the fruit extract. The cytotoxicity of the biosynthesized AgNPs was in vitro evaluated against ovarian carcinoma cells and there were found to be effective at low concentration levels.

In vitro and in vivo anti-inflammatory properties of green synthesized silver nanoparticles using Viburnum opulus L. fruits extract.

A green, rapid and cost effective method for the bio-synthesis of silver nanoparticles (AgNPs), using polyphenols present in European cranberry bush fruit extracts was developed. The obtained AgNPs were characterized by ultra-violet visible spectroscopy (UV-VIS), Fourier transform - infrared spectroscopy (FT-IR), transmission electron microscopy (TEM) and X-ray diffraction patterns (XRD). The average size of the spherical AgNPs was found to be 25nm. The anti-inflammatory effect of the biomaterials was investigated, both in vitro (on HaCaT cell line, exposed to UVB radiation) and in vivo (on acute inflammation model in Wistar rats). Our results support the conclusion that the photosynthesized silver nanoparticles present a potent anti-inflammatory activity and could be successfully used as therapeutic tools for treatment of inflammation.

Novel Palladium(II) Complexes that Influence Prominin-1/CD133 Expression and Stem Cell Factor Release in Tumor Cells.

New Pd(II) complexes of 1,7-bis(2-methoxyphenyl)hepta-1,6-diene-3,5-dione were synthesized and structurally characterized. The complexes were tested in vitro on human colon and hepatic carcinoma cell lines, normal hepatic cells and hematopoietic progenitor cells. Biological tests proved that Pd(II) complexes 1 and 2 (containing a curcumin derivative) exhibit a strong in vitro antitumor effect against the cells derived from human colorectal carcinoma and the hepatic metastasis of a colorectal carcinoma. Complex 1 has an outstanding inhibitory effect against BRAF-mutant colon carcinoma and hepatocarcinoma cell growth; 1 and 2 are both more active than the free ligand and have the capacity to trigger early apoptotic processes. By flow cytometric measurements, an important decrease of prominin-1 (CD133) molecule expression on tumor cells membrane was identified in cell populations subjected to 1 and 2. Quantitative immune enzymatic assay proved restrictions in stem cell factor (SCF) release by treated tumor cells. Although less cytotoxic, the free ligand inhibits the surface marker CD133 expression in hepatocarcinoma cells, and in HT-29 colon carcinoma. The new synthesized Pd(II) complexes 1 and 2 exhibit an important potential through their selective cytotoxic activity and by targeting the stem-like tumor cell populations, which leads to the tumor growth arrest and prevention of metastasis.

Vitis vinifera seeds extract for the modulation of cytosolic factors BAX-α and NF-kB involved in UVB-induced oxidative stress and apoptosis of human skin cells.

BACKGROUND AND AIMS: The depletion of the ozone layer allows overexposure of the skin to UV radiation, which is prolonged due to the increasing life expectancy, together with inappropriate life habits contribute to the increasing incidence of cutaneous malignancies. Plant extracts with antioxidant capacities are frequently employed as a means to protect skin against ultraviolet (UV) radiations, thus preventing skin cancers. In the present study we assessed a red grape seed extract (GSE) potential capacities to reduce ultraviolet B (UVB) radiation-induced reactive oxygen species (ROS) and subsequent apoptosis in a human keratinocytes cell line (HaCaT). We identified molecules and pathways modulated by the GSE through which this may exert its photoprotective effect. METHODS: The GSE was standardized according to its polyphenolic content and the most important biologically active compounds, such as epigallocatechin and epicatechin, catechin hydrate, procyanidin B and gallic acid were evidenced by high-performance liquid chromatography. According to the plant extract cytotoxicity on the HaCaT cell line, two concentrations were selected for testing from the non-toxic range: GSE1 (37.5 µgEqGA/ml) and GSE2 (75 µgEqGA/ml). The level of ROS was evaluated with CM-H2DCFDA assay, while apoptosis, Bax-α and NF-kß p65 proteins with ELISA and confirmed by western-blot. RESULTS: Both concentrations of the extract decreased the level of ROS in UVB-irradiated keratinocytes (p<0.001), whereas apoptosis and Bax-α pro-apoptotic protein were only reduced by the higher concentration (GSE2). The NF-kB p65 protein level registered increasing values in time after UVB exposure of the cells, while the tested plant extract re-established its level when its smaller concentration was used (GSE1). CONCLUSION: These results encourage further studies on this extract in order to identify other molecules and pathways through which this extract might exert its beneficial effects and also recommend its use as a potential photoprotective agent.

Green synthesis, characterization and anti-inflammatory activity of silver nanoparticles using European black elderberry fruits extract.

This research aimed at reporting the synthesis, characterization and evaluation of the anti-inflammatory effects of some new biomaterials based on silver nanoparticles and polyphenols rich natural extracts. A fast and eco-friendly extracellular biosynthesis of silver nanoparticles (AgNPs), using European black elderberry (Sambucus nigra - SN, Adoxaceae family) fruit extracts was developed. The phytosynthesized nanoparticles exhibited an absorbance peak at 426nm, characteristic for AgNPs and their sizes were ranged from 20 to 80nm. The anti-inflammatory properties of AgNPs were assessed in vitro on HaCaT cells exposed to UVB radiation, in vivo on acute inflammation model and in humans on psoriasis lesions. In vitro, our results demonstrated the anti-inflammatory effects of functionalized AgNPs by the decrease of cytokines production induced by UVB irradiation. In vivo, the pre-administration of AgNPs reduced the edema and cytokines levels in the paw tissues, early after the induction of inflammation. The present study also demonstrated the possible use of synthesized AgNPs for the treatment of psoriasis lesions.

L-Leucine for gold nanoparticles synthesis and their cytotoxic effects evaluation.

This work reports the preparation of water-soluble leucine capped gold nanoparticles by two single-step synthesis methods. The first procedure involves a citrate reduction approach where the citrate is used as reducing agent and leucine as capping/stabilizing agent. Different sizes of gold nanoparticles, citrate reduced and stabilized by leucine, Leu-AuNPs-C, with the mean diameters in the range of 21-56 nm, were obtained by varying the macroscopic parameters such as: concentration of the gold precursor solution, Au (III):citrate molar ratio and leucine pH. In the second procedure, leucine acts both as reducing and stabilizing agent, allowing us to obtain spherical gold nanoparticles, Leu-AuNPs, with a majority of 80 % (with the mean diameter of 63 nm). This proves that leucine is an appropriate reductant for the formation of water-soluble and stable gold nanoparticles colloids. The characterization of the leucine coated gold nanoparticles was carried out by TEM, UV-Vis and FT-IR analysis. The cytotoxic effect of Leu-AuNPs-C and Leu-AuNPs was also evaluated.

Comparative in vitro study regarding the biocompatibility of titanium-base composites infiltrated with hydroxyapatite or silicatitanate.

BACKGROUND: The development of novel biomaterials able to control cell activities and direct their fate is warranted for engineering functional bone tissues. Adding bioactive materials can improve new bone formation and better osseointegration. Three types of titanium (Ti) implants were tested for in vitro biocompatibility in this comparative study: Ti6Al7Nb implants with 25% total porosity used as controls, implants infiltrated using a sol-gel method with hydroxyapatite (Ti HA) and silicatitanate (Ti SiO2). The behavior of human osteoblasts was observed in terms of adhesion, cell growth and differentiation. RESULTS: The two coating methods have provided different morphological and chemical properties (SEM and EDX analysis). Cell attachment in the first hour was slower on the Ti HA scaffolds when compared to Ti SiO2 and porous uncoated Ti implants. The Alamar blue test and the assessment of total protein content uncovered a peak of metabolic activity at day 8-9 with an advantage for Ti SiO2 implants. Osteoblast differentiation and de novo mineralization, evaluated by osteopontin (OP) expression (ELISA and immnocytochemistry), alkaline phosphatase (ALP) activity, calcium deposition (alizarin red), collagen synthesis (SIRCOL test and immnocytochemical staining) and osteocalcin (OC) expression, highlighted the higher osteoconductive ability of Ti HA implants. Higher soluble collagen levels were found for cells cultured in simple osteogenic differentiation medium on control Ti and Ti SiO2 implants. Osteocalcin (OC), a marker of terminal osteoblastic differentiation, was most strongly expressed in osteoblasts cultivated on Ti SiO2 implants. CONCLUSIONS: The behavior of osteoblasts depends on the type of implant and culture conditions. Ti SiO2 scaffolds sustain osteoblast adhesion and promote differentiation with increased collagen and non-collagenic proteins (OP and OC) production. Ti HA implants have a lower ability to induce cell adhesion and proliferation but an increased capacity to induce early mineralization. Addition of growth factors BMP-2 and TGFß1 in differentiation medium did not improve the mineralization process. Both types of infiltrates have their advantages and limitations, which can be exploited depending on local conditions of bone lesions that have to be repaired. These limitations can also be offset through methods of functionalization with biomolecules involved in osteogenesis.

Molecular biology of cholesteatoma.

Cholesteatoma is a non-neoplastic, keratinizing lesion, characterized by the proliferation of epithelium with aberrant micro-architecture into the middle ear and mastoid cavity. The exact pathogenic molecular mechanisms behind the formation and propagation of cholesteatoma remain unclear. Immunohistochemical examinations of the matrix and perimatrix have considerably improved the knowledge of cholesteatoma pathogenesis. In this review, the current concepts of cholesteatoma pathogenesis are discussed. Currently, the most widely acknowledged pathogenesis of acquired cholesteatoma is the theory that negative pressure, dysfunction of the Eustachian tube, causes a deepening retraction pocket that, when obstructed, desquamated keratin cannot be cleared from the recess, and a cholesteatoma results. Local infection leads to a disturbance of self-cleaning mechanisms, with cell debris and keratinocytes accumulate inside the retraction pocket, and this is followed by an immigration of immune cells, i.e., Langerhans' cells, T-cells, macrophages. There is an imbalance and a vicious circle of epithelial proliferation, keratinocyte differentiation and maturation, prolonged apoptosis, and disturbance of self-cleaning mechanisms. The inflammatory stimulus will induce an epithelial proliferation along with expression of lytic enzymes and cytokines. Bacteria inside the retraction pocket produce some antigens, which will activate different cytokines and lytic enzymes. These cytokines lead to activation and maturing of osteoclasts with the consequence of degradation of extracellular bone matrix and hyperproliferation, bone erosion and finally progression of the disease. Further research is necessary for a better understanding of the pathogenetic mechanisms and to expand the spectrum of therapeutic options.

Oxaliplatin induces different cellular and molecular chemoresistance patterns in colorectal cancer cell lines of identical origins.

BACKGROUND: Cancer cells frequently adopt cellular and molecular alterations and acquire resistance to cytostatic drugs. Chemotherapy with oxaliplatin is among the leading treatments for colorectal cancer with a response rate of 50%, inducing intrastrand cross-links on the DNA. Despite of this drug's efficiency, resistance develops in nearly all metastatic patients. Chemoresistance being of crucial importance for the drug's clinical efficiency this study aimed to contribute to the identification and description of some cellular and molecular alterations induced by prolonged oxaliplatin therapy. Resistance to oxaliplatin was induced in Colo320 (Colo320R) and HT-29 (HT-29R) colorectal adenocarcinoma cell lines by exposing the cells to increasing concentrations of the drug. Alterations in morphology, cytotoxicity, DNA cross-links formation and gene expression profiles were assessed in the parental and resistant variants with microscopy, MTT, alkaline comet and pangenomic microarray assays, respectively. RESULTS: Morphology analysis revealed epithelial-to-mesenchymal transition in the resistant vs parental cells suggesting alterations of the cells' adhesion complexes, through which they acquire increased invasiveness and adherence. Cytotoxicity measurements demonstrated resistance to oxaliplatin in both cell lines; Colo320 being more sensitive than HT-29 to this drug (P < 0.001). The treatment with oxaliplatin caused major DNA cross-links in both parental cell lines; in Colo320R small amounts of DNA cross-links were still detectable, while in HT-29R not. We identified 441 differentially expressed genes in Colo320R and 613 in HT-29R as compared to their parental counterparts (at least 1.5 -fold up- or down- regulation, p < 0.05). More disrupted functions and pathways were detected in HT-29R cell line than in Colo320R, involving genes responsible for apoptosis inhibition, cellular proliferation and epithelial-to-mesenchymal transition. Several upstream regulators were detected as activated in HT-29R cell line, but not in Colo320R. CONCLUSIONS: Our findings revealed a more resistant phenotype in HT-29R as compared to Colo320R and different cellular and molecular chemoresistance patterns induced by prolonged treatment with oxaliplatin in cell lines with identical origins (colorectal adenocarcinomas).