Complex Evaluation of Nanocomposite-Based Hydroxyapatite for Biomedical Applications.

A magnesium-doped hydroxyapatite in chitosan matrix (MgHApC) sample was developed as a potential platform for numerous applications in the pharmaceutical, medical, and food industries. Magnesium-doped hydroxyapatite suspensions in the chitosan matrix were obtained by the coprecipitation technique. The surface shape and morphological features were determined by scanning electron microscopy (SEM). The hydrodynamic diameter of the suspended particles was determined by Dynamic light scattering (DLS) measurements. The stability of MgHApC suspensions was evaluated by ultrasonic measurements. The hydrodynamic diameter of the MgHApC particles in suspension was 29.5 nm. The diameter of MgHApC particles calculated from SEM was 12.5 ± 2 nm. Following the SEM observations, it was seen that the MgHApC particles have a spherical shape. The Fourier-transform infrared spectroscopy (FTIR) studies conducted on MgHApC proved the presence of chitosan and hydroxyapatite in the studied specimens. In vitro antimicrobial assays were performed on Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, and Candida albicans ATCC 10231 microbial strains. The antimicrobial experiments showed that MgHApC exhibited very good antimicrobial properties against all the tested microorganisms. More than that, the results of the in vitro studies revealed that the antimicrobial properties of the samples depend on the incubation time. The evaluation of the sample's cytotoxicity was performed using the human colon cancer (HCT-8) cell line. Our results suggested the great potential of MgHApC to be used in future applications in the field of biomedical applications (e.g., dentistry, orthopedics, etc.).

Predicting enzymatic function of protein sequences with attention.

MOTIVATION: There is a growing number of available protein sequences, but only a limited amount has been manually annotated. For example, only 0.25% of all entries of UniProtKB are reviewed by human annotators. Further developing automatic tools to infer protein function from sequence alone can alleviate part of this gap. In this article, we investigate the potential of Transformer deep neural networks on a specific case of functional sequence annotation: the prediction of enzymatic classes. RESULTS: We show that our EnzBert transformer models, trained to predict Enzyme Commission (EC) numbers by specialization of a protein language model, outperforms state-of-the-art tools for monofunctional enzyme class prediction based on sequences only. Accuracy is improved from 84% to 95% on the prediction of EC numbers at level two on the EC40 benchmark. To evaluate the prediction quality at level four, the most detailed level of EC numbers, we built two new time-based benchmarks for comparison with state-of-the-art methods ECPred and DeepEC: the macro-F1 score is respectively improved from 41% to 54% and from 20% to 26%. Finally, we also show that using a simple combination of attention maps is on par with, or better than, other classical interpretability methods on the EC prediction task. More specifically, important residues identified by attention maps tend to correspond to known catalytic sites. Quantitatively, we report a max F-Gain score of 96.05%, while classical interpretability methods reach 91.44% at best. AVAILABILITY AND IMPLEMENTATION: Source code and datasets are respectively available at https://gitlab.inria.fr/nbuton/tfpc and https://doi.org/10.5281/zenodo.7253910.

Dextran-Coated Iron Oxide Nanoparticles Loaded with 5-Fluorouracil for Drug-Delivery Applications.

This study aims to design and test different formulations composed of dextran-coated iron oxide nanoparticles (IONPs) loaded with 5-Fluorouracil (5-FU) with varying nanoparticle:drug ratios on colorectal cancer cells. The stable suspension of IONPs s was synthesized by the adapted co-precipitation method. The stable suspension of IONPs was mixed with a solution of dextran and 5-FU solubilized in a saline solution. The final suspensions with optimized ratios of IONP:5-FU in the final suspension were 0.5:1, 1:1, and 1.5:1. The information on the morphology and size distribution of the IONPs suspension and IONP loads with 5-FU was obtained using scanning electron microscopy (SEM). The presence of 5-FU and dextran on the surface of the IONPs was highlighted by energy-dispersive X-ray spectroscopy (EDS) studies. The determination of the surface charge of the nanoparticles in the final suspensions of IONP:5-FU was achieved by measuring the zeta potential (ζ). The hydrodynamic diameter of the resulting suspensions of IONP:5-FU was determined by dynamic light scattering (DLS). A cytocompatibility analysis was performed using Caco-2 (human epithelial colorectal adenocarcinoma) cells. In this research, our goal was to find a relationship between the formulation ratio of nanoparticles and drug, and the cellular response after exposure, as a strategy to increase the efficacy of this drug-delivery system. The nanoparticle uptake and antitumor activity, including modulation of oxidative stress, apoptosis, and proliferation biomarkers, were analyzed. The present study showed that the nanoformulation with the ratio IONP:5-FU 1.5:1 had the highest anti-tumor efficiency. Moreover, decreased MCM-2 expression in Caco-2 cells exposed to dextran-coated iron oxide nanoparticles loaded with 5-FU was demonstrated for the first time.

Response of the Endogenous Antioxidant Defense System Induced in RAW 264.7 Macrophages upon Exposure to Dextran-Coated Iron Oxide Nanoparticles.

Presently, iron oxide nanoparticles are the only ones approved for clinical use as contrast agents in magnetic resonance imaging (MRI). Even though there is a high demand for these types of nanoparticles both for clinical use as well as for research, there are difficulties in obtaining stable nanoparticles with reproducible properties. In this context, in this study, we report the obtaining by an adapted coprecipitation method of dextran-coated maghemite nanoparticles (ɤ-Fe2O3 NPs). The morphology and structure of the dextran-coated maghemite nanoparticles (ɤ-Fe2O3 NPs) were determined using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The TEM and SEM micrographs highlighted the obtaining of particles of nanometric size and spherical shape morphology. Furthermore, the high-resolution transmission electron microscopy (HRTEM), as well as selected area diffraction (SAED), revealed that the obtained samples presented the structure of cubic maghemite. In this study, we also explored the effects of the co-precipitation synthesized dextran-coated maghemite nanoparticles (ɤ-Fe2O3 NPs) on the redox status of macrophages. For cytotoxicity evaluation of these NPs, murine macrophages (RAW 264.7 cell line) were exposed to different concentrations of dextran-coated maghemite nanoparticles (ɤ-Fe2O3 NPs) corresponding to 0-500 µg Fe3+/mL and incubated for 24, 48, and 72 h. Intracellular iron uptake, changes in the oxidative stress parameters (reactive oxygen species production and malondialdehyde level), and the activity of antioxidant enzymes, as well as GSH concentration in cells, were evaluated after incubation with a lower (50 µg Fe3+/mL) and higher (500 µg Fe3+/mL) dose of NPs. The results indicated a significant decrease in RAW 264.7 cell viability after 72 h in the presence of NPs at concentrations above 25 µg Fe3+/mL. An important accumulation of NPs, dependent on dose and exposure time, was detected in macrophages, but it induced only a limited raise in the oxidative status. We showed here that the antioxidant capacity of RAW 264.7 macrophages was efficient in counteracting dextran-coated maghemite nanoparticles (ɤ-Fe2O3 NPs) toxicity even at higher doses.

Development of Novel Tetracycline and Ciprofloxacin Loaded Silver Doped Hydroxyapatite Suspensions for Biomedical Applications.

The objective of this study consisted of the development of new materials with antimicrobial properties at the nanometric scale that could lead to an increase in therapeutic efficacy and reduction of toxic side effects. This work focuses on obtaining and characterizing stable suspensions with narrow size distribution with antimicrobial properties. The stability of the suspensions obtained by an adapted co-precipitation method was evaluated by ultrasonic measurements. The size and size distribution of the particle populations were determined using scanning electron microscopy (SEM), and dynamic light scattering (DLS). Both methods of analysis showed a narrow distribution of particles. DLS gave a monomodal distribution with hydrodynamic diameters around 38 nm for ciprofloxacin embedded in silver doped hydroxyapatite (AgHA-C) and 45.7 nm for tetracycline embedded in silver doped hydroxyapatite (AgHA-T). The average diameters calculated from SEM were 17 nm for AgHA-C and 19 nm for AgHA-T. Both Ciprofloxacin and Tetracycline influenced the hydroxyapatite structure, which led to the appearance of new vibrational bands characteristic of the specific chemical composition in the FTIR spectrum. The antimicrobial properties of the AgHA-C and AgHA-T suspensions were assessed using the most common reference microbial strains Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Candida albicans ATCC 10231. The results of the in vitro antimicrobial assays determined that the AgHA-C and AgHA-T suspensions exhibited exceptional antimicrobial activity. Moreover, the data revealed that the antimicrobial activity increased with the increase of the incubation time.

Synthesis, Characterization, and Antimicrobial Activity of Magnesium-Doped Hydroxyapatite Suspensions.

Obtaining nanoscale materials has allowed for the miniaturization of components, which has led to the possibility of achieving more efficient devices with faster functions and much lower costs. While hydroxyapatite [HAp, Ca10(PO4)6(OH)2] is considered the most widely used material for medical applications in orthopedics, dentistry, and general surgery, the magnesium (Mg) is viewed as a promising biodegradable and biocompatible implant material. Furthermore, Mg is regarded as a strong candidate for developing medical implants due to its biocompatibility and antimicrobial properties against gram-positive and gram-negative bacteria. For this study, magnesium-doped hydroxyapatite (Ca10-xMgx (PO4)6 (OH)2, xMg = 0.1), 10MgHAp, suspensions were successfully obtained by an adapted and simple chemical co-precipitation method. The information regarding the stability of the nanosized 10MgHAp particles suspension obtained by ζ-potential analysis were confirmed for the first time by a non-destructive ultrasound-based technique. Structural and morphological studies of synthesized 10MgHAp were conducted by X-ray diffraction (XRD), Fourier-transform infrared (FTIR) spectroscopy in attenuated total reflectance (ATR) mode and scanning electron microscopy (SEM). The XRD analysis of the 10MgHAp samples confirmed that a single crystalline phase associated to HAp with an average grain size about 93.3 nm was obtained. The FTIR-ATR spectra revealed that the 10MgHAp sample presented broader IR bands with less visible peaks when compared to a well-crystallized pure HAp. The SEM results evidenced uniform MgHAp nanoparticles with spherical shape. The antimicrobial activity of the 10MgHAp suspension against gram-positive strains (Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212), gram-negative strains (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853), as well as a fungal strain (Candida albicans ATCC 90029) were evaluated.

Evaluation of Antibacterial Activity of Zinc-Doped Hydroxyapatite Colloids and Dispersion Stability Using Ultrasounds.

This study proves that the new developed zinc-doped hydroxyapatite (ZnHAp) colloids by an adapted sol-gel method can be widely used in the pharmaceutical, medical, and environmental industries. ZnHAp nanoparticles were stabilized in an aqueous solution, and their colloidal dispersions have been characterized by different techniques. Scanning Electron Microscopy (SEM) was used to get information on the morphology and composition of the investigated samples. Energy-dispersive X-ray spectroscopy (EDX) analysis confirmed the elemental compositions of ZnHAp colloidal dispersions. The homogeneous and uniform distribution of constituent elements (zinc, calcium, phosphorus, oxygen) was highlighted by the obtained elemental mapping results. The X-ray diffraction (XRD) results of the obtained samples showed a single phase corresponding to the hexagonal hydroxyapatite. The characteristic bands of the hydroxyapatite structure were also evidenced by Fourier-transform infrared spectroscopy (FTIR) analysis. For a stability assessment of the colloidal system, ζ-potential for the ZnHAp dispersions was estimated. Dynamic light scattering (DLS) was used to determine particles dispersion and hydrodynamic diameter (DHYD). The goal of this study was to provide for the first time information on the stability of ZnHAp particles in solutions evaluated by non-destructive ultrasound-based technique. In this work, the influence of the ZnHAp colloidal solutions stability on the development of bacteria, such as Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), was also established for the first time. The antimicrobial activity of ZnHAp solutions was strongly influenced by both the stability of the solutions and the amount of Zn.

Antimicrobial Activity of New Materials Based on Lavender and Basil Essential Oils and Hydroxyapatite.

This study presents, for the first-time, the results of a study on the hydrodynamic diameter of essential oils (EOs) of basil and lavender in water, and solutions of EOs of basil (B) and lavender (L) and hydroxyapatite (HAp). The possible influence of basil and lavender EOs on the size of hydroxyapatite nanoparticles was analyzed by Scanning Electron Microscopy (SEM). We also investigated the in vitro antimicrobial activity of plant EOs and plant EOs hydroxyapatite respectively, against Gram-positive bacteria (methicillin-resistant Staphylococcus aureus1144 (MRSA 1144) and S. aureus 1426) and Gram-negative bacteria (Escherichia coli ATCC 25922 and Escherichia coli ESBL 4493). From the autocorrelation function, obtained by Dynamic Light Scattering (DLS) measurements it was observed that basil yielded one peak at an average hydrodynamic diameter of 354.16 nm, while lavender yielded one peak at an average hydrodynamic diameter of 259.76 nm. In the case of HAp nanoparticles coated with basil (HApB) and lavender (HApL) essential oil, the aggregation was minimal. We found that the lavender EO exhibited a very good inhibitory growth activity (MIC values ranging from <0.1% for E. coli reference strain to 0.78% for S. aureus strains). The biological studies indicated that HapL material displayed an enhanced antimicrobial activity, indicating the potential use of HAp as vehicle for low concentrations of lavender EO with antibacterial properties. Flow cytometry analysis (FCM) allowed us to determine some of the potential mechanisms of the antimicrobial activities of EOs, suggesting that lavender EO was active against E. coli by interfering with membrane potential, the membrane depolarization effect being increased by incorporation of the EOs into the microporous structure of HAp. These findings could contribute to the development of new antimicrobial agents that are urgently needed for combating the antibiotic resistance phenomena.

Textural, Structural and Biological Evaluation of Hydroxyapatite Doped with Zinc at Low Concentrations.

The present work was focused on the synthesis and characterization of hydroxyapatite doped with low concentrations of zinc (Zn:HAp) (0.01 < xZn < 0.05). The incorporation of low concentrations of Zn2+ ions in the hydroxyapatite (HAp) structure was achieved by co-precipitation method. The physico-chemical properties of the samples were characterized by X-ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), Scanning Electron Microscopy (SEM), zeta-potential, and DLS and N2-BET measurements. The results obtained by XRD and FTIR studies demonstrated that doping hydroxyapatite with low concentrations of zinc leads to the formation of a hexagonal structure with lattice parameters characteristic to hydroxyapatite. The XRD studies have also shown that the crystallite size and lattice parameters of the unit cell depend on the substitutions of Ca2+ with Zn2+ in the apatitic structure. Moreover, the FTIR analysis revealed that the water content increases with the increase of zinc concentration. Furthermore, the Energy Dispersive X-ray Analysis (EDAX) and XPS analyses showed that the elements Ca, P, O, and Zn were found in all the Zn:HAp samples suggesting that the synthesized materials were zinc doped hydroxyapatite, Ca10-xZnx(PO4)6(OH), with 0.01 ≤ xZn ≤ 0.05. Antimicrobial assays on Staphylococcus aureus and Escherichia coli bacterial strains and HepG2 cell viability assay were carried out.

Structural Characterization and Antifungal Studies of Zinc-Doped Hydroxyapatite Coatings.

The present study is focused on the synthesis, characterization and antifungal evaluation of zinc-doped hydroxyapatite (Zn:HAp) coatings. The Zn:HAp coatings were deposited on a pure Si (Zn:HAp_Si) and Ti (Zn:HAp_Ti) substrate by a sol-gel dip coating method using a zinc-doped hydroxyapatite nanogel. The nature of the crystal phase was determined by X-ray diffraction (XRD). The crystalline phase of the prepared Zn:HAp composite was assigned to hexagonal hydroxyapatite in the P63/m space group. The colloidal properties of the resulting Zn:HAp (xZn = 0.1) nanogel were analyzed by Dynamic Light Scattering (DLS) and zeta potential. Scanning Electron Microscopy (SEM) was used to investigate the morphology of the zinc-doped hydroxyapatite (Zn:HAp) nanogel composite and Zn:HAp coatings. The elements Ca, P, O and Zn were found in the Zn:HAp composite. According to the EDX results, the degree of Zn substitution in the structure of Zn:HAp composite was 1.67 wt%. Moreover, the antifungal activity of Zn:HAp_Si and Zn:HAp_Ti against Candida albicans (C. albicans) was evaluated. A decrease in the number of surviving cells was not observed under dark conditions, whereas under daylight and UV light illumination a major decrease in the number of surviving cells was observed.