Enhancing Osteoblastic Cell Cultures with Gelatin Methacryloyl, Bovine Lactoferrin, and Bioactive Mesoporous Glass Scaffolds Loaded with Distinct Parsley Extracts.

The increasing interest in innovative solutions for addressing bone defects has driven research into the use of Bioactive Mesoporous Glasses (MBGs). These materials, distinguished by their well-ordered mesoporous structure, possess the capability to accommodate plant extracts with well-established osteogenic properties, including bovine lactoferrin (bLF), as part of their 3D scaffold composition. This harmonizes seamlessly with the ongoing advancements in the field of biomedicine. In this study, we fabricated 3D scaffolds utilizing MBGs loaded with extracts from parsley leaves (PL) and embryogenic cultures (EC), rich in bioactive compounds such as apigenin and kaempferol, which hold potential benefits for bone metabolism. Gelatin Methacryloyl (GelMa) served as the polymer, and bLF was included in the formulation. Cytocompatibility, Runx2 gene expression, ALP enzyme activity, and biomineralization were assessed in preosteoblastic MC3T3-E1 cell cultures. MBGs effectively integrated PL and EC extracts with loadings between 22.6 ± 0.1 and 43.6 ± 0.3 µM for PL and 26.3 ± 0.3 and 46.8 ± 0.4 µM for EC, ensuring cell viability through a release percentage between 28.3% and 59.9%. The incorporation of bLF in the 3D scaffold formulation showed significant differences compared to the control in all assays, even at concentrations below 0.2 µM. Combinations, especially PL + bLF at 0.19 µM, demonstrated additive potential, with superior biomineralization compared to EC. In summary, this study highlights the effectiveness of MBGs in incorporating PL and EC extracts, along with bLF, into 3D scaffolds. The results underscore cytocompatibility, osteogenic activity, and biomineralization, offering exciting potential for future in vivo applications.

Nanodevices based on mesoporous glass nanoparticles enhanced with zinc and curcumin to fight infection and regenerate bone.

Nanotechnology-based approaches are emerging as promising strategies to treat different bone pathologies such as infection, osteoporosis or cancer. To this end, several types of nanoparticles are being investigated, including those based on mesoporous bioactive glasses (MGN) which exhibit exceptional structural and textural properties and whose biological behaviour can be improved by including therapeutic ions in their composition and loading them with biologically active substances. In this study, the bone regeneration capacity and antibacterial properties of MGNs in the SiO2-CaO-P2O5 system were evaluated before and after being supplemented with 2.5% or 4% ZnO and loaded with curcumin. in vitro studies with preosteoblastic cells and mesenchymal stem cells allowed determining the biocompatible MGNs concentrations range. Moreover, the bactericidal effect of MGNs with zinc and curcumin against S. aureus was demonstrated, as a significant reduction of bacterial growth was detected in both planktonic and sessile states and the degradation of a pre-formed bacterial biofilm in the presence of the nanoparticles also occurred. Finally, MC3T3-E1 preosteoblastic cells and S. aureus were co-cultured to investigate competitive colonisation between bacteria and cells in the presence of the MGNs. Preferential colonisation and survival of osteoblasts and effective inhibition of both bacterial adhesion and biofilm formation of S. aureus in the co-culture system were detected. Our study demonstrated the synergistic antibacterial effect of zinc ions combined with curcumin and the enhancement of the bone regeneration characteristics of MGNs containing zinc and curcumin to obtain systems capable of simultaneously promoting bone regeneration and controlling infection. STATEMENT OF SIGNIFICANCE: In search of a new approach to regenerate bone and fight infections, a nanodevice based on mesoporous SiO2-CaO-P2O5 glass nanoparticles enriched with Zn2+ ions and loaded with curcumin was designed. This study demonstrates the synergistic effect of the simultaneous presence of zinc ions and curcumin in the nanoparticles that significantly reduces the bacterial growth in planktonic state and is capable to degrade pre-formed S. aureus biofilms whereas the nanosystem exhibits a cytocompatible behaviour in the presence of preosteoblasts and mesenchymal stem cells. Based on these results, the designed nanocarrier represents a promising alternative for the treatment of acute and chronic infections in bone tissues, while avoiding the significant current problem of bacterial resistance to antibiotics.

A versatile multicomponent mesoporous silica nanosystem with dual antimicrobial and osteogenic effects.

In this manuscript, we propose a simple and versatile methodology to design nanosystems based on biocompatible and multicomponent mesoporous silica nanoparticles (MSNs) for infection management. This strategy relies on the combination of antibiotic molecules and antimicrobial metal ions into the same nanosystem, affording a significant improvement of the antibiofilm effect compared to that of nanosystems carrying only one of these agents. The multicomponent nanosystem is based on MSNs externally functionalized with a polyamine dendrimer (MSN-G3) that favors internalization inside the bacteria and allows the complexation of multiactive metal ions (MSN-G3-Mn+). Importantly, the selection of both the antibiotic and the cation may be done depending on clinical needs. Herein, levofloxacin and Zn2+ ion, chosen owing to both its antimicrobial and osteogenic capability, have been incorporated. This dual biological role of Zn2+ could have and adjuvant effect thought destroying the biofilm in combination with the antibiotic as well as aid to the repair and regeneration of lost bone tissue associated to osteolysis during infection process. The versatility of the nanosystem has been demonstrated incorporating Ag+ ions in a reference nanosystem. In vitro antimicrobial assays in planktonic and biofilm state show a high antimicrobial efficacy due to the combined action of levofloxacin and Zn2+, achieving an antimicrobial efficacy above 99% compared to the MSNs containing only one of the microbicide agents. In vitro cell cultures with MC3T3-E1 preosteoblasts reveal the osteogenic capability of the nanosystem, showing a positive effect on osteoblastic differentiation while preserving the cell viability. STATEMENT OF SIGNIFICANCE: A simple and versatile methodology to design biocompatible and multicomponent MSNs based nanosystems for infection management is proposed. These nanosystems, containing two antimicrobial agents, levofloxacin and Zn2+, have been synthetized by external functionalization of MSNs with a polycationic dendrimer (MSNs-G3), which favours its internalization inside the bacteria and lead the complexation with metal ions through the amines of the dendrimer. The nanosystems offer a notable improvement of the antibiofilm effect (above 99%) than both components separately as well as osteogenic capability with positive effect on the osteoblastic differentiation and preserved cell viability.

Metabolomic response of osteosarcoma cells to nanographene oxide-mediated hyperthermia.

Nanographene oxide (nGO)-mediated hyperthermia has been increasingly investigated as a localized, minimally invasive anticancer therapeutic approach. Near InfraRed (NIR) light irradiation for inducing hyperthermia is particularly attractive, because biological systems mostly lack chromophores that absorb in this spectral window, facilitating the selective heating and destruction of cells which have internalized the NIR absorbing-nanomaterials. However, little is known about biological effects accompanying nGO-mediated hyperthermia at cellular and molecular levels. In this work, well-characterized pegylated nGO sheets with a hydrodynamic size of 300 nm were incubated with human Saos-2 osteosarcoma cells for 24 h and their internalization verified by flow cytometry and confocal microscopy. No effect on cell viability was observed after nGO uptake by Saos-2 cells. However, a proliferation delay was observed due to the presence of nGO sheets in the cytoplasm. 1H NMR metabolomics was employed to screen for changes in the metabolic profile of cells, as this could help to improve understanding of cellular responses to nanomaterials and provide new endpoint markers of effect. Cells internalizing nGO sheets showed noticeable changes in several metabolites compared to control cells, including decreased levels of several amino acids, taurine and creatine and increased levels of phosphocholine and uridine/adenosine nucleotides. After NIR irradiation, cells showed decreases in glutamate and uridine nucleotides, together with increases in glycerophosphocholine and adenosine monophosphate. Overall, this study has shown that the cellular metabolome sensitively responded to nGO exposure and nGO-mediated hyperthermia and that NMR metabolomics is a powerful tool to investigate treatment responses.

Beyond Traditional Hyperthermia: In Vivo Cancer Treatment with Magnetic-Responsive Mesoporous Silica Nanocarriers.

In this study, we present an innovation in the tumor treatment in vivo mediated by magnetic mesoporous silica nanoparticles. This device was built with iron oxide magnetic nanoparticles embedded in a mesoporous silica matrix and coated with an engineered thermoresponsive polymer. The magnetic nanoparticles act as internal heating sources under an alternating magnetic field (AMF) that increase the temperature of the surroundings, provoking the polymer transition and consequently the release of a drug trapped inside the silica pores. By a synergic effect between the intracellular hyperthermia and chemotherapy triggered by AMF application, significant tumor growth inhibition was achieved in 48 h after treatment. Furthermore, the small magnetic loading used in the experiments indicates that the treatment is carried out without a global temperature rise of the tissue, which avoids the problem of the necessity to employ large amounts of magnetic cores, as is common in current magnetic hyperthermia.

Mesoporous silica nanoparticles decorated with polycationic dendrimers for infection treatment.

This work aims to provide an effective and novel solution for the treatment of infection by using nanovehicles loaded with antibiotics capable of penetrating the bacterial wall, thus increasing the antimicrobial effectiveness. These nanosystems, named "nanoantibiotics", are composed of mesoporous silica nanoparticles (MSNs), which act as nanocarriers of an antimicrobial agent (levofloxacin, LEVO) localized inside the mesopores. To provide the nanosystem of bacterial membrane interaction capability, a polycationic dendrimer, concretely the poly(propyleneimine) dendrimer of third generation (G3), was covalently grafted to the external surface of the LEVO-loaded MSNs. After physicochemical characterization of this nanoantibiotic, the release kinetics of LEVO and the antimicrobial efficacy of each released dosage were evaluated. Besides, internalization studies of the MSNs functionalized with the G3 dendrimer were carried out, showing a high penetrability throughout Gram-negative bacterial membranes. This work evidences that the synergistic combination of polycationic dendrimers as bacterial membrane permeabilization agents with LEVO-loaded MSNs triggers an efficient antimicrobial effect on Gram-negative bacterial biofilm. These positive results open up very promising expectations for their potential application in new infection therapies. STATEMENT OF SIGNIFICANCE: Seeking new alternatives to current available treatments of bacterial infections represents a great challenge in nanomedicine. This work reports the design and optimization of a new class of antimicrobial agent, named "nanoantibiotic", based on mesoporous silica nanoparticles (MSNs) decorated with polypropyleneimine dendrimers of third generation (G3) and loaded with levofloxacin (LEVO) antibiotic. The covalently grafting of these G3 dendrimers to MSNs allows an effective internalization in Gram-negative bacteria. Furthermore, the LEVO loaded into the mesoporous cavities is released in a sustained manner at effective antimicrobial dosages. The novelty and originality of this manuscript relies on proving that the synergistic combination of bacteria-targeting and antimicrobial agents into a unique nanosystem provokes a remarkable antimicrobial effect against bacterial biofilm.

Multifunctional pH sensitive 3D scaffolds for treatment and prevention of bone infection.

Multifunctional-therapeutic three-dimensional (3D) scaffolds have been prepared. These biomaterials are able to destroy the S. aureus bacterial biofilm and to allow bone regeneration at the same time. The present study is focused on the design of pH sensitive 3D hierarchical meso-macroporous 3D scaffolds based on MGHA nanocomposite formed by a mesostructured glassy network with embedded hydroxyapatite nanoparticles, whose mesopores have been loaded with levofloxacin (Levo) as antibacterial agent. These 3D platforms exhibit controlled and pH-dependent Levo release, sustained over time at physiological pH (7.4) and notably increased at infection pH (6.7 and 5.5), which is due to the different interaction rate between diverse Levo species and the silica matrix. These 3D systems are able to inhibit the S. aureus growth and to destroy the bacterial biofilm without cytotoxic effects on human osteoblasts and allowing an adequate colonization and differentiation of preosteoblastic cells on their surface. These findings suggest promising applications of these hierarchical MGHA nanocomposite 3D scaffolds for the treatment and prevention of bone infection. STATEMENT OF SIGNIFICANCE: Multifunctional 3D nanocomposite scaffolds with the ability for loading and sustained delivery of an antimicrobial agent, to eliminate and prevent bone infection and at the same time to contribute to bone regeneration process without cytotoxic effects on the surrounding tissue has been proposed. These 3D scaffolds exhibit a sustained levofloxacin delivery at physiological pH (pH 7.4), which increasing notably when pH decreases to characteristic values of bone infection process (pH 6.7 and pH 5.5). In vitro competitive assays between preosteoblastic and bacteria onto the 3D scaffold surface demonstrated an adequate osteoblast colonization in entire scaffold surface together with the ability to eliminate bacteria contamination.

Auranofin-loaded nanoparticles as a new therapeutic tool to fight streptococcal infections.

Drug-loaded nanoparticles (NPs) can improve infection treatment by ensuring drug concentration at the right place within the therapeutic window. Poly(lactic-co-glycolic acid) (PLGA) NPs are able to enhance drug localization in target site and to sustainably release the entrapped molecule, reducing the secondary effects caused by systemic antibiotic administration. We have loaded auranofin, a gold compound traditionally used for treatment of rheumatoid arthritis, into PLGA NPs and their efficiency as antibacterial agent against two Gram-positive pathogens, Streptococcus pneumoniae and Streptococcus pyogenes was evaluated. Auranofin-PLGA NPs showed a strong bactericidal effect as cultures of multiresistant pneumococcal strains were practically sterilized after 6 h of treatment with such auranofin-NPs at 0.25 µM. Moreover, this potent bactericidal effect was also observed in S. pneumoniae and S. pyogenes biofilms, where the same concentration of auranofin-NPs was capable of decreasing the bacterial population about 4 logs more than free auranofin. These results were validated using a zebrafish embryo model demonstrating that treatment with auranofin loaded into NPs achieved a noticeable survival against pneumococcal infections. All these approaches displayed a clear superiority of loaded auranofin PLGA nanocarriers compared to free administration of the drug, which supports their potential application for the treatment of streptococcal infections.

In vitro evaluation of glass-glass ceramic thermoseed-induced hyperthermia on human osteosarcoma cell line.

The use of biomaterials as implantable thermoseeds under the action of an external magnetic field is a very interesting methodology to focus the heat into the target tumors as osteosarcoma. In this study, biocompatible and bioactive G15GC85 thermoseeds, tailored through the combination of sol-gel glasses (G) with a magnetic glass ceramic (GC), were used to induce hyperthermia on cultured human osteosarcoma cells after exposition to alternating magnetic field (MF, 100 kHz/200 Oe). G15GC85 magnetic glass-glass ceramic thermoseeds induced in vitro effective hyperthermia with drastic reduction in proliferation of human osteosarcoma Saos-2 cells and high increase of apoptotic cells after two 40 min consecutive sessions of MF. Deep cell morphology alterations were observed after this hyperthermic treatment, and the proteomic analysis revealed modification of gamma actin molecular properties related to cytoskeleton alterations. These results indicate that G15GC85 thermoseeds allow to induce in vitro effective hyperthermia on human osteosarcoma cells.

Smart drug delivery through DNA/magnetic nanoparticle gates.

Mesoporous silica nanoparticles can be modified to perform on-demand stimuli-responsive dosing of therapeutic molecules. The silica network was loaded with iron oxide superparamagnetic nanocrystals, providing the potential to perform targeting and magnetic resonance imaging. Single-stranded DNA was immobilized onto the material surface. The complementary DNA sequence was then attached to magnetic nanoparticles. The present work demonstrates that DNA/magnetic nanoparticle conjugates are able to cap the pores of the magnetic silica particles upon hybridization of both DNA strands. Progressive double-stranded DNA melting as a result of temperature increase gave rise to uncapping and the subsequent release of a mesopore-filled model drug, fluorescein. The reversibility of DNA linkage results in an "on-off" release mechanism. Moreover, the magnetic component of the whole system allows reaching hyperthermic temperatures (42-47 °C) under an alternating magnetic field. This feature leaves open the possibility of a remotely triggered drug delivery. Furthermore, due to its capacity to increase the temperature of the surrounding media, this multifunctional device could play an important role in the development of advanced drug delivery systems for thermochemotherapy against cancer.

In vitro positive biocompatibility evaluation of glass-glass ceramic thermoseeds for hyperthermic treatment of bone tumors.

A new kind of magnetic thermoseed for bone tissue engineering has been synthesized. The materials used are specially designed to restore bone tissue after tumor extirpation, because they exhibit bioactive behavior and the ability to act as thermoseeds for cancer treatment using hyperthermia. The L929 cell line of mouse fibroblasts has been used in a wide biocompatibility study concerning cell proliferation and morphology studies, mitochondrial function determination, lactate dehydrogenase measurement, and flow cytometry studies, including cell cycle analysis, cell size and complexity, and intracellular reactive oxygen species content. The results presented in this work indicate that these bioactive magnetic materials are highly biocompatible and show greater cell response for thermoseeds with a higher magnetic phase content. There were no significant alterations detected in the cell cycle, and the interaction between fibroblasts and the different mixtures did not induce significant apoptosis.

Release evaluation of drugs from ordered three-dimensional silica structures.

Cubic mesoporous structures with Ia3d symmetry, such as MCM-48 and large pore Ia3d material (LP-Ia3d), which present different pore size (3.6 and 5.7nm, respectively), have been prepared, characterized and used as drug delivery systems. Ibuprofen and erythromycin have been chosen as drug models for delivery studies. The influence of the pore size at these structures has been studied and the results show that the delivery rate of drugs decreases with the pore size of the matrix. The influence of chemical nature of the pore surface on the delivery process has been also studied. In this case, the hydrophilic pore surface has been modified with hydrocarbon chains (C8 and C18 moieties) and the effect upon drug delivery of hydrophobic drugs like erythromycin has been studied. The results show a noticeable decrease of the delivery rate when the surface of the matrices is modified.