Unveiling the role of hydroxyapatite and hydroxyapatite/silver composite in osteoblast-like cell mineralization: An exploration through their viscoelastic properties.

Mechanical properties are becoming fundamental for advancing the comprehension of cellular processes. This study addresses the relationship between viscoelastic properties and the cellular mineralization process. Osteoblast-like cells treated with an osteogenic medium were employed for this purpose. Additionally, the study explores the impact of hydroxyapatite (HA) and hydroxyapatite/silver (HA/Ag) composite on this process. AFM relaxation experiments were conducted to extract viscoelastic parameters using the Fractional Zener (FZ) and Fractional Kelvin (FK) models. Our findings revealed that the main phases of mineralization are associated with alterations in the viscoelastic properties of osteoblast-like cells. Furthermore, HA and HA/Ag treatments significantly influenced changes in the viscoelastic properties of these cells. In particular, the HA/Ag treatment demonstrated a marked enhancement in cell fluidity, suggesting a possible role of silver in accelerating the mineralization process. Moreover, the study underscores the independence observed between fluidity and stiffness, indicating that modifications in one parameter may not necessarily correspond to changes in the other. These findings shed light on the factors involved in the cellular mineralization process and emphasize the importance of using viscoelastic properties to discern the impact of treatments on cells.

Poly(vinyl alcohol)/Silk Fibroin/Ag-NPs Composite Nanofibers as a Substrate for MG-63 Cells' Growth.

Nanofiber scaffolds of polyvinyl alcohol, silk fibroin from Bombyx mori cocoons, and silver nanoparticles were developed as a substrate for MG-63 growth. The fiber morphology, mechanical properties, thermal degradation, chemical composition, and water contact angle were investigated. In vitro tests were performed by the cell viability MTS test of MG-63 cells on electrospun PVA scaffolds, mineralization was analyzed by alizarin red, and the alkaline phosphatase (ALP) assay was evaluated. At higher PVA concentrations, Young's modulus (E) increased. The addition of fibroin and silver nanoparticles improved the thermal stability of PVA scaffolds. FTIR spectra indicated characteristic absorption peaks related to the chemical structures of PVA, fibroin, and Ag-NPs, demonstrating good interactions between them. The contact angle of the PVA scaffolds decreased with the incorporation of fibroin and showed hydrophilic characteristics. In all concentrations, MG-63 cells on PVA/fibroin/Ag-NPs scaffolds had higher cell viability than PVA pristine. On day ten of culture, PVA18/SF/Ag-NPs showed the highest mineralization, observed by the alizarin red test. PVA10/SF/Ag-NPs presented the highest alkaline phosphatase activity after an incubation time of 37 h. The achievements indicate the potential of the nanofibers of PVA18/SF/Ag-NPs as a possible substitute for bone tissue engineering (BTE).

Development of a new generation of miniemulsion based on cottonseed oil with α-tocopherol and ZnO and evaluation of its adjuvant activity.

Background: Emulsions have been widely used as immunological adjuvants. But the use of materials derived from plants such as cottonseed oil, alpha-tocopherol, or minerals such as zinc, as well as their use at the nanometric scale has been little explored. In this study, we develop a new miniemulsion and evaluated its antioxidant and phagocytic capacity, as well as parameters related to immune response stimulation by cytokine expression and antibodies production in a mice model. Methods: Formulated CN (cottonseed oil miniemulsion) and CNZ (cottonseed oil miniemulsion whit zinc oxide nanoparticles) miniemulsions were characterized by scanning electronic microscopy SEM, DLS and FT-IR. In murine macrophages, splenocytes and thymocytes primary cultures safety and cytotoxicity were determined by MTT. In macrophages the antioxidant and phagocytic capacity was evaluated. In BALB/c mice, the stimulation of the immune system was determined by the expression of cytokines and the production of antibodies. Results: The CN and CNZ presented stability for 90 days. Immediately after preparation, the CN presented a higher particle size (543.1 nm) than CNZ (320 nm). FT-IR demonstrated the correct nanoparticle synthesis by the absence of sulfate groups. CN and CNZ (1.25 to 10 µL/mL) had no toxic effect on macrophages (p = 0.108), splenocytes (p = 0.413), and thymocytes (p = 0.923). All CN and CNZ doses tested induced nitric oxide and antioxidants production in dose dependent manner when compared with control. CN-ovalbumin and CNZ-ovalbumin treatments in femoral subcutaneous tissue area showed inflammation with higher leukocyte infiltration compared with FCA. The intraperitoneal administration with CN, CNZ, and FCA showed a higher total intraperitoneal cells recruitment (CD14+) after 24 h of inoculation than control (p = 0.0001). CN and CNZ increased the phagocyte capacity with respect to untreated macrophages in the Candida albicans-phagocytosis assay. The evaluation of residual CFU indicated that only CN significantly decreased (p = 0.004) this value at 3 h. By other side, only CN increased (p = 0.002) the nitric oxide production. CNZ stimulated a major INFγ secretion compared with FCA at day 7. A major IL-2 secretion was observed at days 7 and 14, stimulated with CN and CNZ. Both miniemulsions did not affect the antibody isotypes production (IgG1, IgG2a, IgG3, IgA and IgM) at days 7, 14, 28, and 42. CN induced a significant IgG production against OVA, but lesser than FCA. Conclusions: The two new miniemulsions with adjuvant and antioxidant capacity, were capable of generating leukocyte infiltration and increased cytokines and antibodies production.

Antitumor efficacy of silver nanoparticles reduced with ß-D-glucose as neoadjuvant therapy to prevent tumor relapse in a mouse model of breast cancer.

Introduction: Neoadjuvant therapy constitutes a valuable modality for diminishing tumor volume prior to surgical resection. Nonetheless, its application encounters limitations in the context of recurrent tumors, which manifest resistance to conventional treatments. Silver nanoparticles (AgNPs) have emerged as a promising alternative for cancer treatment owing to their cytotoxic effects. Methods: Cellular viability was assessed by Alamar blue assay in 4T1 breast cancer cell line. Silver biodistribution was detected by an inductively coupled plasma optical emission spectrometer in an in vivo mice model. For neoadjuvant evaluation, mice were randomized and treated intratumoral with AgNPs-G or intraperitoneally with doxorubicin (DOX) as a control. Recurrence was determined after 170 days by counting lung metastatic nodules (dyed with Bouin solution) with histological confirmation by H&E. Masson's stain, Ki67 immunohistochemistry, and a TUNEL assay were performed in lungs from treated mice. Results: AgNPs-G reduced 4T1 cell viability and in an ex vivo assay the AgNPs-G decreased the tumor cell viability. After intravenous administration of AgNPs-G were detected in different organs. After intratumor administration, AgNPs-G are retained. The AgNPs-G treatment significantly reduced tumor volume before its surgical resection. AgNPs-G reduced the development of lung metastatic nodules and the expression of Ki67. TUNEL assay indicated that AgNPs-G didn't induce apoptosis. Conclusions: We concluded that intratumor administration of AgNPs-G reduced tumor volume before surgical resection, alongside a reduction in lung metastatic nodules, and Ki67 expression. These findings provide valuable insights into the AgNPs-G potential for intratumor and neoadjuvant cancer therapies. However, further research is needed to explore their full potential and optimize their use in clinical settings.

Chitosan-G-Glycidyl Methacrylate/Au Nanocomposites Promote Accelerated Skin Wound Healing.

Herein, we report the synthesis of Au nanoparticles (AuNPs) in chitosan (CTS) solution by chemically reducing HAuCl4. CTS was further functionalized with glycidyl methacrylate (chitosan-g-glycidyl methacrylate/AuNP, CTS-g-GMA/AuNP) to improve the mechanical properties for cellular regeneration requirements of CTS-g-GMA/AuNP. Our nanocomposites promote excellent cellular viability and have a positive effect on cytokine regulation in the inflammatory and anti-inflammatory response of skin cells. After 40 days of nanocomposite exposure to a skin wound, we showed that our films have a greater skin wound healing capacity than a commercial film (TheraForm®), and the presence of the collagen allows better cosmetic ave aspects in skin regeneration in comparison with a nanocomposite with an absence of this protein. Electrical percolation phenomena in such nanocomposites were used as guiding tools for the best nanocomposite performance. Our results suggest that chitosan-based Au nanocomposites show great potential for skin wound repair.

Cytotoxicity and Genotoxicity of Azobenzene-Based Polymeric Nanocarriers for Phototriggered Drug Release and Biomedical Applications.

Drug nanoencapsulation increases the availability, pharmacokinetics, and concentration efficiency for therapeutic regimes. Azobenzene light-responsive molecules experience a hydrophobicity change from a polar to an apolar tendency by trans-cis photoisomerization upon UV irradiation. Polymeric photoresponse nanoparticles (PPNPs) based on azobenzene compounds and biopolymers such as chitosan derivatives show prospects of photodelivering drugs into cells with accelerated kinetics, enhancing their therapeutic effect. PPNP biocompatibility studies detect the safe concentrations for their administration and reduce the chance of side effects, improving the effectiveness of a potential treatment. Here, we report on a PPNP biocompatibility evaluation of viability and the first genotoxicity study of azobenzene-based PPNPs. Cell line models from human ventricular cardiomyocytes (RL14), as well as mouse fibroblasts (NIH3T3) as proof of concept, were exposed to different concentrations of azobenzene-based PPNPs and their precursors to evaluate the consequences on mitochondrial metabolism (MTT assay), the number of viable cells (trypan blue exclusion test), and deoxyribonucleic acid (DNA) damage (comet assay). Lethal concentrations of 50 (LC50) of the PPNPs and their precursors were higher than the required drug release and synthesis concentrations. The PPNPs affected the cell membrane at concentrations higher than 2 mg/mL, and lower concentrations exhibited lesser damage to cellular genetic material. An azobenzene derivative functionalized with a biopolymer to assemble PPNPs demonstrated biocompatibility with the evaluated cell lines. The PPNPs encapsulated Nile red and dofetilide separately as model and antiarrhythmic drugs, respectively, and delivered upon UV irradiation, proving the phototriggered drug release concept. Biocompatible PPNPs are a promising technology for fast drug release with high cell interaction opening new opportunities for azobenzene biomedical applications.

Tuning HAuCl4/Sodium Citrate Stoichiometry to Fabricate Chitosan-Au Nanocomposites.

Nanocomposite engineering of biosensors, biomaterials, and flexible electronics demand a highly tunable synthesis of precursor materials to achieve enhanced or desired properties. However, this process remains limited due to the need for proper synthesis-property strategies. Herein, we report on the ability to synthesize chitosan-gold nanocomposite thin films (CS/AuNP) with tunable properties by chemically reducing HAuCl4 in chitosan solutions and different HAuCl4/sodium citrate molar relationships. The structure, electrical, and relaxation properties of nanocomposites have been investigated as a function of HAuCl4/sodium citrate molar relation. It was shown that gold particle size, conductivity, Vogel temperature (glass transition), and water content strongly depend upon HAuCl4/sodium citrate relationships. Two relaxation processes have been observed in nanocomposites; the α-relaxation process, related to a glass transition in wet CS/AuNP films, and the σ-relaxation related to the local diffusion process of ions in a disordered system. The ability to fine-tune both α- and σ-relaxations may be exploited in the proper design of functional materials for biosensors, biomaterials, and flexible electronics applications.

Relaxation Phenomena in Chitosan-Au Nanoparticle Thin Films.

Chitosan-gold nanoparticle (CS/AuNP) thin films were synthesized through the chemical reduction of HAuCl4 in sodium citrate/chitosan solutions. The dielectric and dynamic mechanical behaviors of CS/AuNP films have been investigated as a function of moisture and HAuCl4 content. Two relaxation processes in the nanocomposites have been observed. The α-relaxation process is related to a glass transition in wet CS/AuNP films. However, in dry composites (with 0.2 wt% of moisture content), the glass transition vanished. A second relaxation process was observed from 70 °C to the onset of thermal degradation (160 °C) in wet films and from 33 °C to the onset of degradation in dry films. This relaxation is identified as the σ-relaxation and may be related to the local diffusion process of ions between high potential barriers in disordered systems. The α- and σ-relaxation processes are affected by the HAuCl4 content of the solutions from which films were obtained because of the interaction between CS, sodium succinate, and gold nanoparticles. With about 0.6 mM of HAuCl4, the conductivity of both wet and dry films sharply increased by six orders, corresponding to the percolation effect, which may be related to the appearance of a conductivity pathway between AuNPs, HAuCl4, and NaCl.

Chitosan-Coated Gold Nanoparticles Induce Low Cytotoxicity and Low ROS Production in Primary Leucocytes, Independent of Their Proliferative Status.

(1) Background: Chitosan-coated gold nanoparticles (CH-AuNPs) have important theranostic applications in biomedical sciences, including cancer research. However, although cell cytotoxicity has been studied in cancerous cells, little is known about their effect in proliferating primary leukocytes. Here, we assessed the effect of CH-AuNPs and the implication of ROS on non-cancerous endothelial and fibroblast cell lines and in proliferative lymphoid cells. (2) Methods: The Turkevich method was used to synthetize gold nanoparticles. We tested cell viability, cell death, ROS production, and cell cycle in primary lymphoid cells, compared with non-cancer and cancer cell lines. Concanavalin A (ConA) or lipopolysaccharide (LPS) were used to induce proliferation on lymphoid cells. (3) Results: CH-AuNPs presented high cytotoxicity and ROS production against cancer cells compared to non-cancer cells; they also induced a different pattern of ROS production in peripheral blood mononuclear cells (PBMCs). No significant cell-death difference was found in PBMCs, splenic mononuclear cells, and bone marrow cells (BMC) with or without a proliferative stimuli. (4) Conclusions: Taken together, our results highlight the selectivity of CH-AuNPs to cancer cells, discarding a consistent cytotoxicity upon proliferative cells including endothelial, fibroblast, and lymphoid cells, and suggest their application in cancer treatment without affecting immune cells.

Virucidal Activity of Gold Nanoparticles Synthesized by Green Chemistry Using Garlic Extract.

Measles virus (MeV) is a paramyxovirus that infects humans, principally children. Despite the existence of an effective and safe vaccine, the number of cases of measles has increased due to lack of vaccination coverage. The World Health Organization (WHO) reports that the number of cases worldwide multiplied fourfold between January and March 2019, to 112,000. Today, there is no treatment available for MeV. In recent years, it has been demonstrated that natural extracts (herbal or algal) with antiviral activity can also work as reducing agents that, in combination with nanotechnology, offer an innovative option to counteract viral infections. Here, we synthetized and evaluated the antiviral activity of gold nanoparticles using garlic extract (Allium sativa) as a reducing agent (AuNPs-As). These nanoparticles actively inhibited MeV replication in Vero cells at a 50% effective concentration (EC50) of 8.829 µg/mL, and the selectivity index (SI) obtained was 16.05. AuNPs-As likely inhibit viral infection by blocking viral particles directly, showing a potent virucidal effect. Gold nanoparticles may be useful as a promising strategy for treating and controlling the infection of MeV and other related enveloped viruses.

Chitosan gold nanoparticles induce different ROS-dependent cell death modalities in leukemic cells.

BACKGROUND: Nanotechnology proposes the use of gold nanoparticles (AuNPs) for drug delivery, diagnosis, and treatment of cancer. Leukemia is a type of hematopoietic cancer that results from the malignant transformation of white blood cells. Chitosan-coated AuNPs (CH-AuNPs) are cell death inductors in HeLa and MCF-7 cancer cells without affecting peripheral blood mononuclear cells (PBMC). Considering the selectivity and versatile cytotoxicity of CH-AuNPs, we evaluated whether their selectivity is due to the cell lineage or the characteristics of the cancer cells, by assessing its cytotoxicity in leukemic cells. Moreover, we further examined the cell death mechanism and assessed the implication of nuclear damage, autophagosome formation, and the cell death mechanism induced in leukemic cells. MATERIALS AND METHODS: We synthesized CH-AuNPs by chemical methods and analyzed their cell death capacity in a T-acute lymphocytic leukemia cell line (CEM), in a chronic myeloid leukemia cell line (K562), and in healthy cells from the same lineage (PBMC and bone marrow, BM, cells). Then, we assessed ROS generation and mitochondrial and nuclear damage. Finally, we evaluated whether cell death occurred by autophagy, apoptosis, or necroptosis, and the role of ROS in this mechanism. RESULTS: We found that CH-AuNPs did not affect PBMC and BM cells, whereas they are cytotoxic in a dose-dependent manner in leukemic cells. ROS production leads to mitochondrial and nuclear damage, and cell death. We found that CH-AuNPs induce apoptosis in CEM and necroptosis in K562, both undergoing autophagy as a pro-survival mechanism. CONCLUSION: CH-AuNPs are selective cell death inductors in hematologic cancer cells, without affecting their healthy counterparts. Cell death induced by CH-AuNPs is independent of the cancer cell type; however, its mechanism is different depending on the type of leukemic cells.

Chitosan gold nanoparticles induce cell death in HeLa and MCF-7 cells through reactive oxygen species production.

BACKGROUND: Nanotechnology has gained important interest, especially in the development of new therapies; the application of gold nanoparticles (AuNPs) in the treatment and detection of diseases is a growing trend in this field. As cancer represents a serious health problem around the world, AuNPs are studied as potential drugs or drug carriers for anticancer agents. Recent studies show that AuNPs stabilized with chitosan (CH) possess interesting biological activities, including potential antitumor effects that could be selective to cancer cells. MATERIALS AND METHODS: In this study, we synthesized sodium citrate-AuNPs and CH-capped AuNPs of 3-10 nm, and analyzed their cytotoxicity in cervical (HeLa) and breast (MCF-7) cancer cells, and in peripheral blood mononuclear cells (PBMCs). Then, we evaluated the clonogenic potential, cell cycle, nuclear alterations, caspase dependence, and reactive oxygen species (ROS) production in HeLa and MCF-7 cells after chitosan gold nanoparticles (CH-AuNPs) exposure. RESULTS: Our data showed that CH-AuNPs are cytotoxic in a dose-dependent manner in the cancer cell lines tested, while they induce low cytotoxicity in PBMCs. Sodium citrate gold nanoparticles did not show cytotoxic effects. In both HeLa and MCF-7 cell lines, CH-AuNPs inhibit clonogenic potential without inducing cell cycle arrest or nuclear alterations. The cell death mechanism is specific for the type of cancer cell line tested, as it depends on caspase activation in HeLa cells, whereas it is caspase independent in MCF-7 cells. In all cases, ROS production is mandatory for cell death induction by CH-AuNPs, as ROS inhibition with N-acetyl cysteine inhibits cell death. CONCLUSION: Our results show that CH-AuNPs are selective for HeLa and MCF-7 cancer cells, rather than normal PBMCs, and that ROS production seems to be a conserved feature of the cell death mechanism induced by CH-AuNPs. These results improve the knowledge of CH-AuNPs and open the way to the design of new pharmacological strategies using these agents against cancer.

Novel gigahertz frequency dielectric relaxations in chitosan films.

Molecular relaxations of chitosan films have been investigated in the wide frequency range of 0.1 to 3 × 10(9) Hz from -10 °C to 110 °C using dielectric spectroscopy. For the first time, two high-frequency relaxation processes (in the range 10(8) to 3 × 10(9) Hz) are reported in addition to the low frequency relaxations α and ß. These two relaxation processes are related to the vibrations of OH and NH2/NH3(+), respectively. The high-frequency relaxations exhibit Arrhenius-type dependencies in the temperature range 10 °C to 54 °C with negative activation energy; this observation is traceable to hydrogen bonding reorientation. At temperatures above the glass transition temperature (54 °C), the activation energy changes from negative to positive values due to breaking of hydrogen bonding and water loss. Upon cooling in a sealed environment, the activation energies of two relaxation processes are nearly zero. FTIR and XRD analyses reveal associated structural changes upon heating and cooling. These two new high-frequency relaxation processes can be attributed to the interaction of bound water with OH and NH2/NH3(+), respectively. A plausible scenario for these high-frequency relaxations is discussed in light of impedance spectroscopy, TGA, FTIR and XRD measurements.