Sustained Release of Dexamethasone from 3D-Printed Scaffolds Modulates Macrophage Activation and Enhances Osteogenic Differentiation.

Enhancing osteogenesis via modulating immune cells is emerging as a new approach to address the current challenges in repairing bone defects and fractures. However, much remains unknown about the crosstalk between immune cells and osteolineage cells during bone formation. Moreover, biomaterial scaffold-based approaches to effectively modulate this crosstalk to favor bone healing are also lacking. This study is the first to investigate the interactions between macrophages and mesenchymal stem cells (MSCs) in co-cultures with the sustained release of an anti-inflammatory and pro-osteogenesis drug (dexamethasone) from three-dimensional (3D)-printed scaffolds. We successfully achieved the sustained release of dexamethasone from polycaprolactone (PCL) by adding the excipient-sucrose acetate isobutyrate (SAIB). Dexamethasone was released over 35 days in the 17-163 nM range. The osteogenic differentiation of MSCs was enhanced by M1 macrophages at early time points. The late-stage mineralization was dominated by dexamethasone, with little contribution from the macrophages. Besides confirming BMP-2 whose secretion was promoted by both dexamethasone and M1 macrophages as a soluble mediator for enhanced osteogenesis, IL-6 was found to be a possible new soluble factor that mediated osteogenesis in macrophage-MSC co-cultures. The phenotype switching from M1 to M2 was drastically enhanced by the scaffold-released dexamethasone but only marginally by the co-cultured MSCs. Our results offer new insight into macrophage-MSC crosstalk and demonstrate the potential of using drug-release scaffolds to both modulate inflammation and enhance bone regeneration.

Polymeric Reactor for the Synthesis of Superparamagnetic-Thermal Treatment of Breast Cancer.

Engineered superparamagnetic iron oxide nanoparticles (SPIONs) have been studied extensively for their localized homogeneous heat generation in breast cancer therapy. However, challenges such as aggregation and inability to produce sub-10 nm SPIONs limit their potential in magnetothermal ablation. We report a facile, efficient, and robust in situ method for the synthesis of SPIONs within a poly(ethylene glycol) (PEG) reactor adsorbed onto reduced graphene oxide nanosheets (rGO) via the microwave hydrothermal route. This promising modality yields crystalline, stable, biocompatible, and superparamagnetic PEGylated SPION-rGO nanocomposites (NCs) with uniform dispersibility. Our findings show that rGO acts as a breeding ground for the spatially distributed nanosites around which the ferrihydrite seeds accumulate to ultimately transform into immobilized SPIONs. PEG, in parallel, acts as a critical confining agent physically trapping the accumulated seeds to prevent their aggregation and create multiple domains on rGO for the synthesis of quantum-sized SPIONs (9 ± 1 nm in diameter). This dual functionality (rGO and PEG) exhibits a pronounced effect on reducing both the aggregation and the sizes of fabricated SPIONs as confirmed by the scanning transmission electron microscopy images, dynamic light scattering analyses, and the specific absorption rates (SARs). Reduced aggregation lowered the toxicity of NCs, where PEGylated SPION-rGO NCs are more biocompatible than PEGylated SPIONs, showing no significant induction of cell apoptosis, mitochondrial membrane injury, or oxidative stress. Significantly less lactate dehydrogenase release and hence less necrosis are observed after 48 h exposure to high doses of PEGylated SPION-rGO NCs compared with PEGylated SPIONs. NCs induce local heat generation with a SAR value of 1760 ± 97 W/g, reaching up to 43 ± 0.3 °C and causing significant MCF-7 breast tumor cell ablation of about 78 ± 10% upon applying an external magnetic field. Collectively, rGO and PEG functionalities have a synergistic effect on improving the synthesis, stability, biocompatibility, and magnetothermal properties of SPIONs.

Cellular responses of hyaluronic acid-coated chitosan nanoparticles.

In recent years, nanotechnology has been proven to offer promising biomedical applications for in vivo diagnostics and drug delivery, stressing the importance of thoroughly investigating the biocompatibility of potentially translatable nanoparticles (NPs). Herein, we report the cellular responses of uncoated chitosan NPs (CS NPs) and hyaluronic acid-coated chitosan NPs (HA-CS NPs) when introduced into Chinese hamster ovary cells (CHO-K1) in a dose-dependent manner (2.5, 0.25, 0.025, 0.0025, and 0.00025 mg mL-1) at two time points (24 and 48 h). MTS assay, cell proliferation, showed a decrease in the viability of cells when treated with 0.25 and 2.5 mg mL-1 CS NPs. When exposed to high doses of CS NPs, the lactate dehydrogenase (LDH) enzyme started to leak out of the cells and the cellular levels of mitochondrial potentials were significantly reduced accompanied by a high production of intracellular reactive oxygen species (ROS). Our study provides molecular evidence of the biocompatibility offered by HA-CS NPs, through ROS scavenging capabilities rescuing cells from the oxidative stress, showing no observed cellular stress and thereby revealing the promising effect of anionic hyaluronic acid to significantly reduce the cytotoxicity of CS NPs. Our findings are important to accelerate the translation and utilization of HA-CS NPs in drug delivery, demonstrating the pronounced effect of surface modifications on modulating the biological responses.

Efficacy of neutral and negatively charged liposome-loaded gentamicin on planktonic bacteria and biofilm communities.

We investigated the efficacy of liposomal gentamicin formulations of different surface charges against Pseudomonas aeruginosa and Klebsiella oxytoca. The liposomal gentamicin formulations were prepared by the dehydration-rehydration method, and their sizes and zeta potential were measured. Gentamicin encapsulation efficiency inside the liposomal formulations was determined by microbiologic assay, and stability of the formulations in biologic fluid was evaluated for a period of 48 h. The minimum inhibitory concentration and the minimum bactericidal concentration were determined, and the in vitro time kill studies of the free form of gentamicin and liposomal gentamicin formulations were performed. The activities of liposomal gentamicin in preventing and reducing biofilm-forming P. aeruginosa and K. oxytoca were compared to those of free antibiotic. The sizes of the liposomal formulations ranged from 625 to 806.6 nm in diameter, with the zeta potential ranging from -0.22 to -31.7 mV. Gentamicin encapsulation efficiency inside the liposomal formulation ranged from 1.8% to 43.6%. The liposomes retained >60% of their gentamicin content during the 48 h time period. The minimum inhibitory concentration of neutral formulation was lower than that of free gentamicin (0.25 versus 1 mg/L for P. aeruginosa and 0.5 versus 1 mg/L for K. oxytoca). The negatively charged formulation exhibited the same bacteriostatic concentration as that of free gentamicin. The minimum bactericidal concentration of neutral liposomes on planktonic bacterial culture was twofold lower than that of free gentamicin, whereas the negatively charged formulations were comparable to free gentamicin. The killing time curve values for the neutral negatively charged formulation against planktonic P. aeruginosa and K. oxytoca were better than those of free gentamicin. Furthermore, liposomal formulations prevent the biofilm-formation ability of these strains better than free gentamicin. In summary, liposomal formulations could be an effective lipid nanoparticle to combat acute infections where planktonic bacteria are predominant.

Hyaluronic Acid Coated Chitosan Nanoparticles Reduced the Immunogenicity of the Formed Protein Corona.

Studying the interactions of nanoparticles (NPs) with serum proteins is necessary for the rational development of nanocarriers. Optimum surface chemistry is a key consideration to modulate the formation of the serum protein corona (PC) and the resultant immune response. We investigated the constituent of the PC formed by hyaluronic acid-coated chitosan NPs (HA-CS NPs). Non-decorated chitosan NPs (CS NPs) and alginate-coated chitosan NPs (Alg-CS NPs) were utilized as controls. Results show that HA surface modifications significantly reduced protein adsorption relative to controls. Gene Ontology analysis demonstrates that HA-CS NPs were the least immunogenic nanocarriers. Indeed, less inflammatory proteins were adsorbed onto HA-CS NPs as opposed to CS and Alg-CS NPs. Interestingly, HA-CS NPs differentially adsorbed two unique anti-inflammatory proteins (ITIH4 and AGP), which were absent from the PC of both controls. On the other hand, CS and Alg-CS NPs selectively adsorbed a proinflammatory protein (Clusterin) that was not found on the surfaces of HA-CS NPs. While further studies are needed to investigate abilities of the PCs of only ITIH4 and AGP to modulate the interaction of NPs with the host immune system, our results suggest that this proof-of-concept could potentially be utilized to reduce the immunogenicity of a wide range of nanomaterials.

The occurrence of ESBL-producing Escherichia coli carrying aminoglycoside resistance genes in urinary tract infections in Saudi Arabia.

BACKGROUND: The infection and prevalence of extended-spectrum ß-lactamases (ESBLs) is a worldwide problem, and the presence of ESBLs varies between countries. In this study, we investigated the occurrence of plasmid-mediated ESBL/AmpC/carbapenemase/aminoglycoside resistance gene expression in Escherichia coli using phenotypic and genotypic techniques. METHODS: A total of 58 E. coli isolates were collected from hospitals in the city of Makkah and screened for the production of ESBL/AmpC/carbapenemase/aminoglycoside resistance genes. All samples were subjected to phenotypic and genotypic analyses. The antibiotic susceptibility of the E. coli isolates was determined using the Vitek-2 system and the minimum inhibitory concentration (MIC) assay. Antimicrobial agents tested using the Vitek 2 system and MIC assay included the expanded-spectrum (or third-generation) cephalosporins (e.g., cefoxitin, cefepime, aztreonam, cefotaxime, ceftriaxone, and ceftazidime) and carbapenems (meropenem and imipenem). Reported positive isolates were investigated using genotyping technology (oligonucleotide microarray-based assay and PCR). The genotyping investigation was focused on ESBL variants and the AmpC, carbapenemase and aminoglycoside resistance genes. E. coli was phylogenetically grouped, and the clonality of the isolates was studied using multilocus sequence typing (MLST). RESULTS: Our E. coli isolates exhibited different levels of resistance to ESBL drugs, including ampicillin (96.61%), cefoxitin (15.25%), ciprofloxacin (79.66%), cefepime (75.58%), aztreonam (89.83%), cefotaxime (76.27%), ceftazidime (81.36%), meropenem (0%) and imipenem (0%). Furthermore, the distribution of ESBL-producing E. coli was consistent with the data obtained using an oligonucleotide microarray-based assay and PCR genotyping against genes associated with ß-lactam resistance. ST131 was the dominant sequence type lineage of the isolates and was the most uropathogenic E. coli lineage. The E. coli isolates also carried aminoglycoside resistance genes. CONCLUSIONS: The evolution and prevalence of ESBL-producing E. coli may be rapidly accelerating in Saudi Arabia due to the high visitation seasons (especially to the city of Makkah). The health authority in Saudi Arabia should monitor the level of drug resistance in all general hospitals to reduce the increasing trend of microbial drug resistance and the impact on patient therapy.

Biodegradable Magnetic Silica@Iron Oxide Nanovectors with Ultra-Large Mesopores for High Protein Loading, Magnetothermal Release, and Delivery.

The delivery of large cargos of diameter above 15nm for biomedical applications has proved challenging since it requires biocompatible, stably-loaded, and biodegradable nanomaterials. In this study, we describe the design of biodegradable silica-iron oxide hybrid nanovectors with large mesopores for large protein delivery in cancer cells. The mesopores of the nanomaterials spanned from 20 to 60nm in diameter and post-functionalization allowed the electrostatic immobilization of large proteins (e.g. mTFP-Ferritin, ~534kDa). Half of the content of the nanovectors was based with iron oxide nanophases which allowed the rapid biodegradation of the carrier in fetal bovine serum and a magnetic responsiveness. The nanovectors released large protein cargos in aqueous solution under acidic pH or magnetic stimuli. The delivery of large proteins was then autonomously achieved in cancer cells via the silica-iron oxide nanovectors, which is thus a promising for biomedical applications.

Genome sequence of the date palm Phoenix dactylifera L.

Date palm (Phoenix dactylifera L.) is a cultivated woody plant species with agricultural and economic importance. Here we report a genome assembly for an elite variety (Khalas), which is 605.4 Mb in size and covers >90% of the genome (~671 Mb) and >96% of its genes (~41,660 genes). Genomic sequence analysis demonstrates that P. dactylifera experienced a clear genome-wide duplication after either ancient whole genome duplications or massive segmental duplications. Genetic diversity analysis indicates that its stress resistance and sugar metabolism-related genes tend to be enriched in the chromosomal regions where the density of single-nucleotide polymorphisms is relatively low. Using transcriptomic data, we also illustrate the date palm's unique sugar metabolism that underlies fruit development and ripening. Our large-scale genomic and transcriptomic data pave the way for further genomic studies not only on P. dactylifera but also other Arecaceae plants.