Apatite Nanoparticles Research Articles

Surface Modification of Ti-6Al-4V Alloy by Composite Coating of Polycaprolactone Nanofibers-Fluoroapatite Nanoparticles Doped with Silicon and Magnesium

The purpose of this research was to create polycaprolactone nanocomposite coating - fluor apatite nanoparticles doped with silicon and magnesium, as well as polycaprolactone coating on the alloy in order to improve and modify the biological properties of this alloy. For this purpose, nano composite coating and polycaprolactone coating were first created by immersion methode. Then the physical, corrosion and biological properties of the coating created by different methods were investigated. The results indicated the creation of a uniform nanocomposite coating with a thickness of about 6.26 micrometers, with appropriate structure and phases, and an increase in roughness by adding nanoparticles to the polycaprolactone coating. Electrochemical measurements Ti<sub>6</sub>Al<sub>4</sub>V showed that the sample coated with polycaprolactone with nanoparticles has polarization R<SUB>P</SUB>=5.349×10<sup>5</sup> Ωcm<sup>2</sup> resistance, which is higher than the sample coated with caprolactone with polarization resistance R<SUB>P</SUB>=1.191×10<sup>5</sup> Ωcm<sup>2 </sup>and the sample without coating with polarization resistance R<SUB>P</SUB>=5.2453×104 Ωcm<sup>2</sup>. Cytotoxicity test showed the non-cytotoxicity of the coatings. Also, the cell growth and proliferation of the sample with nano composite coating compared to the sample without coating has a statistically significant difference. Cell adhesion on the sample with nanocomposite coating was also much better than the sample without coating and the sample with polycaprolactone coating.

Snapshot of a Paleoarchean seafloor: Evidence from 3.43-3.35 Ga Strelley Pool chert-pebble conglomerate for deposition, silicification, and erosion of hydrothermal greenalite-apatite precipitates

Some of the oldest, well-preserved exhalative sedimentary rocks (ferruginous cherts and jaspilites) occur in volcanic-dominated Paleoarchean sequences in the northern Pilbara Craton, Australia. Jaspilites contain fine-grained hematite particles that have been interpreted to have formed following seawater oxidation of vent-derived Fe2+(aq) by photoautotrophs. However, recent studies suggest that most of the iron in the jaspilites was originally deposited as Fe(II)-rich silicates such as greenalite, sparking renewed debate about how iron was precipitated in the early oceans. Here we show that rounded clasts of dusty ferruginous chert in basal conglomerates of the 3.43–3.35 Ga Strelley Pool Formation, North Pole Dome, Pilbara Craton, Australia, contain abundant nanoparticles of greenalite and apatite that are texturally, mineralogically and chemically near-identical to occurrences in Neoarchean banded iron formations in the Hamersley Group, Australia. Hematite where present occurs in trace amounts and its distribution along the edges of clasts implies that at least some of the hematite formed during weathering. The greenalite-rich clasts display a pronounced positive shale-normalized Eu anomaly, small positive Y anomaly and lack a positive La anomaly, features typical of Paleoarchean exhalites. The co-occurrence of greenalite- and apatite-rich chert-pebbles with clasts of hydrothermal black chert and silicified felsic volcaniclastic sedimentary rocks points to a volcanically active provenance with vigorous hydrothermal activity, consistent with derivation from the underlying 3.43 Ga Panorama Formation. We argue that the greenalite and apatite nanoparticles precipitated during mixing between hydrothermal vent fluids and anoxic seawater and were silicified on the seafloor. The round to oval shape of the chert clasts indicates that the silica cement had recrystallized prior to erosion, consistent with the presence of polygonal shrinkage structures in the dusty chert clasts. Our findings imply that greenalite, contrary to recent suggestions, not only formed in Paleoarchean seawater, but was also stable during deposition, diagenesis, and resedimentation. The precipitation and non-dissolution of apatite nanoparticles in Paleoarchean seawater points to elevated phosphate concentrations, whereas the absence of primary hematite is consistent with models advocating a role for abiotic iron deposition during the Archean.

Tuning Mg Doping and Features of Bone-like Apatite Nanoparticles Obtained via Hydrothermal Synthesis.

Nanocrystalline apatites have been intensively studied for decades, not only for their well-known mimesis of bone apatite but also for applicative purposes, whether as biomaterials for skeletal repair or more recently for a variety of nanomedical applications enabled by their peculiar surface characteristics. Particularly, ion-doped apatites are of great interest because the incorporation of foreign ions in the composition of apatite (nano)crystals alters the bulk and surface properties, modifying their ability to interact with the external environment. This is clearly seen in the physiology of bone tissue, whose mineral phase, a low crystallinity apatitic phase, can dynamically exchange ions with cells, thus driving bone metabolism. Taking bone mineral as a model, the present work describes the development of Mg-doped hydroxyapatite nanoparticles, exploiting hydrothermal synthesis to achieve extents of Mg2+ doping hardly achieved before and using citrate to develop stable apatite colloidal dispersions. Morphological and physicochemical analyses, associated with in-depth investigation of ions populating the apatitic lattice and the nonapatitic surface layer, concurred to demonstrate the cooperative presence of Mg2+ and citrate ions, affecting the dynamic ion retention/release mechanisms. Achieving high Mg2+ doping rates and understanding how Mg doping translates into surface activation of apatite-based nanoparticles is expected to foster the design of novel smart and tunable devices, to adsorb and release ionic species and cargo molecules, with potential innovations in the biomedical field or even beyond, as in catalysis or for environmental remediation.

A nontoxic strontium nanoparticle that holds the potential to act upon osteocompetent cells: An in vitro and in vivo characterization.

Estrogen deficiency, long-term immobilization, and/or aging are commonly related to bone mass loss, thus increasing the risk of fractures. One option for bone replacement in injuries caused by either traumas or pathologies is the use of orthopedic cement based on polymethylmethacrylate (PMMA). Nevertheless, its reduced bioactivity may induce long-term detachment from the host tissue, resulting in the failure of the implant. In view of this problem, we developed an alternative PMMA-based porous cement (pPMMA) that favors cell invasion and improves osteointegration with better biocompatibility. The cement composition was changed by adding bioactive strontium-nanoparticles that mimic the structure of bone apatite. The nanoparticles were characterized regarding their physical-chemical properties, and their effects on osteoblasts and osteoclast cultures were assessed. Initial in vivo tests were also performed using 16 New Zealand rabbits as animal models, in which the pPMMA-cement containing the strontium nanoparticles were implanted. We showed that the apatite nanoparticles in which 90% of Ca2+ ions were substituted by Sr2+ (NanoSr 90%) upregulated TNAP activity and increased matrix mineralization. Moreover, at the molecular level, NanoSr 90% upregulated the mRNA expression levels of, Sp7, and OCN. Runx2 was increased at both mRNA and protein levels. In parallel, in vivo tests revealed that pPMMA-cement containing NanoSr 90%, upregulated two markers of bone maturation, OCN and BMP2, as well as the formation of apatite minerals after implantation in the femur of rabbits. The overall data support that strontium nanoparticles hold the potential to up-regulate mineralization in osteoblasts when associated with synthetic biomaterials.

Dentinal Tubule Occluding Efficacy Of Er: YAG Laser And Universal Adhesive Loaded With Nano-Carbonated Apatite.

ER:YAG laser and experimental resin-based dental adhesive loaded with functionalized carbonated apatite filler were used in this study to evaluate the dentin interaction in terms of penetration and occlusion of the dentinal tubules aiding in the control of dentin hypersensitivity (DH). Spheroidal Carbonated apatite nanoparticles (N-CAP), with an average size of 20±5 nm diameter, were synthesized, characterized, and incorporated in a universal adhesive "All Bond Universal, Bisco, USA", in (2% weight) concentration. Er:YAG laser "Lightwalker, FOTONA, EU" was adjusted to an energy output of 40mJ/ pulse and pulse repetition of 10 Hz for 10 seconds. Dentin specimens were prepared from the buccal surface of 75 extracted sound human molars. The specimens were randomly divided into five groups (n=15) according to the surface treatment: Group (L): Laser only; Group (LB): Laser in combination with adhesive; Group (LBN): Laser in combination with adhesive loaded with N-CAP; Group (B): adhesive only; and Group (BN): adhesive loaded with N-CAP. Depth of penetration and occlusion of the dentinal tubules were assessed using Environmental Scanning Electron Microscope Examination (ESEM). One-way ANOVA was used to compare groups, followed by a pairwise test for multiple comparisons (α=0.05). Groups (LB), and (LBN) showed the highest mean of dentinal tubules' penetration, with a non-significant difference between them. In contrast, the specimens treated with laser only (L) showed the most minor penetration. The employment of ER-YAG laser irradiation with the adhesive loaded with N-CAP was evaluated to be effective in penetrating and occluding the opened dentinal tubules.

Delivery of siRNAs Against Selective Ion Channels and Transporter Genes Using Hyaluronic Acid-coupled Carbonate Apatite Nanoparticles Synergistically Inhibits Growth and Survival of Breast Cancer Cells.

Dysregulated calcium homeostasis and consequentially aberrant Ca2+ signalling could enhance survival, proliferation and metastasis in various cancers. Despite rapid development in exploring the ion channel functions in relation to cancer, most of the mechanisms accounting for the impact of ion channel modulators have yet to be fully clarified. Although harnessing small interfering RNA (siRNA) to specifically silence gene expression has the potential to be a pivotal approach, its success in therapeutic intervention is dependent on an efficient delivery system. Nanoparticles have the capacity to strongly bind siRNAs. They remain in the circulation and eventually deliver the siRNA payload to the target organ. Afterward, they interact with the cell surface and enter the cell via endocytosis. Finally, they help escape the endo-lysosomal degradation system prior to unload the siRNAs into cytosol. Carbonate apatite (CA) nanocrystals primarily is composed of Ca2+, carbonate and phosphate. CA possesses both anion and cation binding domains to target negatively charged siRNA molecules. Hybrid CA was synthesized by complexing CA NPs with a hydrophilic polysaccharide - hyaluronic acid (HA). The average diameter of the composite particles was determined using Zetasizer and FE-SEM and their zeta potential values were also measured. The stronger binding affinity and cellular uptake of a fluorescent siRNA were observed for HA-CA NPs as compared to plain CA NPs. Hybrid CA was electrostatically bound individually and combined with three different siRNAs to silence expression of calcium ion channel and transporter genes, TRPC6, TRPM8 and SLC41A1 in a human breast cancer cell line (MCF-7) and evaluate their potential for treating breast cancer. Hybrid NPs carrying TRPC6, TRPM8 and SLC41A1 siRNAs could significantly enhance cytotoxicity both in vitro and in vivo. The resultant composite CA influenced biodistribution of the delivered siRNA, facilitating reduced off target distribution and enhanced breast tumor targetability.

One-Pot Synthesis of Bioinspired Peptide-Decorated Apatite Nanoparticles for Nanomedicine.

Hybrid organic-inorganic bio-inspired apatite nanoparticles (NPs) are attractive for biomedical applications and especially in nanomedicine. Unfortunately, their applications in nanomedicine are limited by their broad particle size distributions and uncontrolled drug loading due to their multistep synthesis process. Besides, very few attempts at exposing bioactive peptides on apatite NPs are made. In this work, an original one-pot synthesis of well-defined bioactive hybrid NPs composed of a mineral core of bioinspired apatite surrounded by an organic corona of bioactive peptides is reported. Dual stabilizing-bioactive agents, phosphonated polyethylene glycol-peptide conjugates, are prepared and directly used during apatite precipitation i) to form the organic corona during apatite precipitation, driving the size and shape of resulting hybrid NPs with colloidal stabilization and ii) to expose peptide moieties (RGD or YIGSR sequences) at the NPs periphery in view of conferring additional surface properties to enhance their interaction with cells. Here, the success of this approach is demonstrated, the functionalized NPs are fully characterized by Fourier-transform infrared, Raman, X-ray diffraction, solid and liquid state NMR, transmission electron microscopy, and dynamic light scattering, and their interaction with fibroblast cells is followed, unveiling a synergistic proliferative effect.

Super Carbonate Apatite-miR-497a-5p Complex Is a Promising Therapeutic Option against Inflammatory Bowel Disease.

The incidence of inflammatory bowel disease (IBD) is increasing worldwide. It is reported that TGF-β/Smad signal pathway is inactivated in patients with Crohn's disease by overexpression of Smad 7. With expectation of multiple molecular targeting by microRNAs (miRNAs), we currently attempted to identify certain miRNAs that activate TGF-β/Smad signal pathway and aimed to prove in vivo therapeutic efficacy in mouse model. Through Smad binding element (SBE) reporter assays, we focused on miR-497a-5p. This miRNA is common between mouse and human species and enhanced the activity of TGF-β/Smad signal pathway, decreased Smad 7 and/or increased phosphorylated Smad 3 expression in non-tumor cell line HEK293, colorectal cancer cell line HCT116 and mouse macrophage J774a.1 cells. MiR-497a-5p also suppressed the production of inflammatory cytokines TNF-α, IL-12p40, a subunit of IL-23, and IL-6 when J774a.1 cells were stimulated by lipopolysaccharides (LPS). In a long-term therapeutic model for mouse dextran sodium sulfate (DSS)-induced colitis, systemic delivery of miR-497a-5p load on super carbonate apatite (sCA) nanoparticle as a vehicle restored epithelial structure of the colonic mucosa and suppressed bowel inflammation compared with negative control miRNA treatment. Our data suggest that sCA-miR-497a-5p may potentially have a therapeutic ability against IBD although further investigation is essential.

Abstract 2698: Improved calcium phosphate delivery system cNaD to aim clinical application

Abstract Super carbonate apatite (sCA) nanoparticle is a superb calcium phosphate delivery system for miRNA and siRNA to tumors (PlosOne 2015). We reported that sCA delivered efficiently siRNA for survivin and PTBP as well as miR-340 (Mol Cancer Ther 2015, 2015) to colon tumors implanted into nude mice. MiR-4689 and miR-29b-1-5p suppressed KRAS mutated colon cancer cells (Mol Ther NA2015, 2018) and miR-302 and miR-369 realized in vivo cancer reprograming (PlosOne 2015). In every case the RNA function was fully realized. Moreover, effect of influenza vaccination was augmented by sCA plus CpG nucleic acid (NA) (Frontiers in Immunol 2018). sCA coupled with miR-29a or miR-29b prevented the DSS (dextran sodium sulfate)-induced inflammatory bowel disease (Mol Ther NA2018). We also applied the sCA-ICG complex to minimally invasive treatment of photodynamic therapy (Mol Cancer Ther 2018). Following these outstanding findings, we tried and succeeded to establish a clinically available novel calcium phosphate delivery system, named cNaD, which controls the nano-particle size and avoids accumulation in normal organs including liver and spleen. It is characterized by its ability to efficiently deliver siRNA and microRNA to cancers and provided prominent therapeutic effect in various mouse models. Safety test has been accomplished in rats and monkeys based on GLP standards. Also, the scale-up manufacturing system >1000 folds has been attained. Our cNaD system will accelerate the development of NA medicine in the world. Citation Format: Hirofumi Yamamoto. Improved calcium phosphate delivery system cNaD to aim clinical application [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2698.

Retarding breast tumor growth with nanoparticle-facilitated intravenous delivery of BRCA1 and BRCA2 tumor suppressor genes

Gene augmentation therapy entails replacement of the abnormal tumor suppressor genes in cancer cells. In this study, we performed gene augmentation for BRCA1/2 tumor suppressors in order to retard tumor development in breast cancer mouse model. We formulated inorganic carbonate apatite (CA) nanoparticles (NPs) to carry and deliver the purified BRCA1/2 gene- bearing plasmid DNA both in vitro and in vivo. The outcome of BRCA1/2 plasmid-loaded NPs delivery on cellular viability of three breast cancer cell lines such as MCF-7, MDA-MB-231 and 4T1 were evaluated by MTT assay. The result in MCF-7 cell line exhibited that transfection of BRCA 1/2 plasmids with CA NPs significantly reduced cancer cell growth in comparison to control group. Moreover, we noticed a likely pattern of cellular cytotoxicity in 4T1 murine cancer cell line. Following transfection with BRCA1 plasmid-loaded NPs, and Western blot analysis, a notable reduction in the phospho-MAPK protein of MAPK signaling pathway was detected, revealing reduced growth signal. Furthermore, in vivo study in 4T1 induced breast cancer mouse model showed that the tumor growth rate and final volume were decreased significantly in the mouse group treated intravenously with BRCA1 + NPs and BRCA2 + NPs formulations. Our results established that BRCA1/2 plasmids incorporated into CA NPs mitigated breast tumor growth, signifying their application in the therapy for breast cancer.

Thiourea-Capped Nanoapatites Amplify Osmotic Stress Tolerance in Zea mays L. by Conserving Photosynthetic Pigments, Osmolytes Biosynthesis and Antioxidant Biosystems.

Salinity is one of the most prevalent abiotic stresses which not only limits plant growth and yield, but also limits the quality of food products. This study was conducted on the surface functionalization of phosphorus-rich mineral apatite nanoparticles (ANPs), with thiourea as a source of nitrogen (TU–ANPs) and through a co-precipitation technique for inducing osmotic stress tolerance in Zea mays. The resulting thiourea-capped apatite nanostructure (TU–ANP) was characterized using complementary analytical techniques, such as EDX, SEM, XRD and IR spectroscopy. The pre-sowing of soaked seeds of Zea mays in 1.00 µg/mL, 5.00 µg/mL and 10 µg/mL of TU–ANPs yielded growth under 0 mM, 60 mM and 100 mM osmotic stress of NaCl. The results show that Ca and P salt acted as precursors for the synthesis of ANPs at an alkaline pH of 10–11. Thiourea as a source of nitrogen stabilized the ANPs’ suspension medium, leading to the synthesis of TU–ANPs. XRD diffraction analysis validated the crystalline nature of TU–ANPs with lattice dimensions of 29 nm, calculated from FWHM using the Sherrer equation. SEM revealed spherical morphology with polydispersion in size distribution. EDS confirmed the presence of Ca and P at a characteristic KeV, whereas IR spectroscopy showed certain stretches of binding functional groups associated with TU–ANPs. Seed priming with TU–ANPs standardized germination indices (T50, MGT, GI and GP) which were significantly declined by NaCl-based osmotic stress. Maximum values for biochemical parameters, such as sugar (39.8 mg/g at 10 µg/mL), protein (139.8 mg/g at 10 µg/mL) and proline (74.1 mg/g at 10 µg/mL) were recorded at different applied doses of TU–ANP. Antioxidant biosystems in the form of EC 1.11.1.6 catalase (11.34 IU/g FW at 10 µg/mL), EC 1.11.1.11 APX (0.95 IU/G FW at 10 µg/mL), EC 1.15.1.1 SOD (1.42 IU/g FW at 5 µg/mL), EC 1.11.1.7 POD (0.43 IU/g FW at 5 µg/mL) were significantly restored under osmotic stress. Moreover, photosynthetic pigments, such as chlorophyll A (2.33 mg/g at 5 µg/mL), chlorophyll B (1.99 mg/g at 5 µg/mL) and carotenoids (2.52 mg/g at 10 µg/mL), were significantly amplified under osmotic stress via the application of TU–ANPs. Hence, the application of TU–ANPs restores the growth performance of plants subjected to induced osmotic stress.

In Vivo Effects of Nanotechnologically Synthesized and Characterized Fluoridated Strontium Apatite Nanoparticles in the Surgical Treatment of Endodontic Bone Lesions

In this study, fluoridated strontium apatite (SAP) nanoparticles with different mole percentages (5%, 10%, 30%, and 50%) synthesized using a hydrothermal method were used as biomaterials. The in vivo biocompatibility of the synthesized nanoparticles was investigated by embedding them as biomaterials in bone defects created in rat tibiae. Through the hematoxylin-eosin staining method, a histopathological analysis was performed for new bone formation, osteoblast density, and fibrotic tissue formation. Fluorine (F) addition affected the structural and morphological properties of the nanoparticles. With the F doping, the shapes of the nanoparticles changed from nano-rods to almost spherical. The Sr/P ratios, with a stoichiometric value of 1.67, were 1.76, 1.53, 1.54, 1.68, and 1.79 in pure, 5%, 10%, 30%, and 50% F-doped nanoparticles, respectively. The F/Sr ratios of 5%, 10%, 30%, and 50% F-doped nanoparticles were 0.05, 0.13, 0.16, and 0.20, respectively. The highest values in terms of fibrotic tissue formation were obtained in the group containing pure SAP. The best results in terms of new bone formation and osteoblast density in bone defects were observed in the groups with higher F ratios (30% and 50% F-doped). Pure and F-doped strontium apatite nanoparticles showed good results for new bone formation and osteoblast levels compared to the control group. It was observed that an increase in the fluorine ratio resulted in better bone healing. The results showed that pure and F-doped SAP nanoparticles synthesized by a hydrothermal method can be used as biomaterials in orthopedics and dentistry, especially in the surgical treatment of endodontic lesions.

Improved systemic half-life of glucagon-like peptide-1-loaded carbonate apatite nanoparticles in rats.

BACKGROUNDGlucagon-like peptide-1 (GLP1) is an endogenous peptide that regulates blood glucose level. But its susceptibility to rapid metabolic degradation limits its therapeutic use. AIMTo prepare GLP1-encapsulated nanosize particle with controlled release property to improve the systemic half-life of GLP1. METHODSGLP1 nanoparticles were prepared by complexation of GLP1 with carbonate apatite nanoparticles (CA NPs). The physicochemical properties of the CA NPs, the effects of GLP1-loaded CA NPs on cell viability, and the systemic bioavailability of GLP1 after CA NPs administration were determined. RESULTSThe GLP1-loaded CA NPs was within 200 nm in size and stable in fetal bovine serum. The formulation did not affect the viability of human cell lines suggesting that the accumulation of CA NPs in target tissues is safe. In Sprague Dawley rats, the plasma GLP1 Levels as measured from the GLP1-loaded CA NPs-treated rats, were significantly higher than that of the control rats and free GLP1-treated rats at 1 h post-treatment (P < 0.05), and the level remained higher than the other two groups for at least 4 h. CONCLUSIONThe GLP1-loaded CA NPs improved the plasma half-life of GLP1. The systemic bioavailability of GLP1 is longer than other GLP1 nanoparticles reported to date.

In Vivo Evaluation of the Effects of B-Doped Strontium Apatite Nanoparticles Produced by Hydrothermal Method on Bone Repair.

In the present study, the structural, morphological, and in vivo biocompatibility of un-doped and boron (B)-doped strontium apatite (SrAp) nanoparticles were investigated. Biomaterials were fabricated using the hydrothermal process. The structural and morphological characterizations of the fabricated nanoparticles were performed by XRD, FT-IR, FE-SEM, and EDX. Their biocompatibility was investigated by placing them in defects in rat tibiae in vivo. The un-doped and B-doped SrAp nanoparticles were successfully fabricated. The produced nanoparticles were in the shape of nano-rods, and the dimensions of the nano-rods decreased as the B ratio increased. It was observed that the structural and morphological properties of strontium apatite nanoparticles were affected by the contribution of B. A stoichiometric Sr/P ratio of 1.67 was reached in the 5% B-doped sample (1.68). The average crystallite sizes were 34.94 nm, 39.70 nm, 44.93 nm, and 48.23 nm in un-doped, 1% B-doped, 5% B-doped, and 10% B-doped samples, respectively. The results of the in vivo experiment revealed that the new bone formation and osteoblast density were higher in the groups with SrAp nanoparticles doped with different concentrations of B than in the control group, in which the open defects were untreated. It was observed that this biocompatibility and the new bone formation were especially elevated in the B groups, which added high levels of strontium were added. The osteoblast density was higher in the group in which the strontium element was placed in the opened bone defect compared with the control group. However, although new bone formation was slightly higher in the strontium group than in the control group, the difference was not statistically significant. Furthermore, the strontium group had the highest amount of fibrotic tissue formation. The produced nanoparticles can be used in dental and orthopedic applications as biomaterials.

Biodegradable Carbonate Apatite Nanoparticle as a Delivery System to Promote Afatinib Delivery for Non-Small Cell Lung Cancer Treatment.

Nanomedicine-based drug-delivery systems have significant interest in cancer treatment, such as improving the stabilities and biocompatibilities, precise targeting, and reducing toxicities for non-cancerous cells. Herein, this study presents the synthesis and characterisation of carbonate apatite nanoparticles (nCA) and encapsulated afatinib (AFA) as promising drug delivery candidates for lung cancer treatment. nCA/AFA was synthesised and physicochemically characterised, then the encapsulation capacity, drug loading, and cumulative drug release profile were evaluated. Powder X-ray diffraction (PXRD) confirmed that the synthesised nCA is apatite. Fourier-transform infrared spectroscopy (FTIR) results confirmed the drug loading into the nanoparticles. High-resolution transmission electron microscopy (HR-TEM) determined the morphology of nCA and nCA/AFA and the diameters of 47.36 ± 3.16 and 42.97 ± 2.78 nm, respectively, without an unaltered nCA phase. Encapsulation efficiency (%) and drug loading (%) were 55.08% ± 1.68% and 8.19% ± 0.52%. Brunauer–Emmett–Teller (BET) and dynamic light-scattering (DLS) results revealed that the synthesised nCA is mesoporous, with a surface area of 55.53 m2/g, and is negatively charged. Atomic force microscopy (AFM) showed increasing roughness of nCA/AFA compared to nCA. The drug release from the nano-formulation nCA/AFA demonstrated slow and sustained release compared to the pure drug. Accordingly, nCA/AFA represents a promising drug delivery system for NSCLC treatment.

Fabrication of bioactive poly(ether ether ketone) by laser melt infiltration of poly(ether ether ketone) inside the strontium apatite coatings

A versatile method to immobilize apatite nanoparticles onto PEEK substrate by using CO2 laser was demonstrated. Various strontium apatites were synthesized and dispersed in ethanol at a low concentration of citric acid. This dispersion of strontium apatites was coated on the surface of the PEEK substrate. The apatite-coated PEEK substrate was subsequently irradiated by CO2 laser with repeated multiple scanning mode. The surface temperature was monitored in real time and controlled at approximately the melting point of PEEK. The thermally fixed apatite coatings were unified with the PEEK substrate and unable to separate off. Dissolution of apatite layer in dilute acid solution revealed highly rugged surface morphologies of the PEEK substrate hidden inside the apatite coatings. From these observations we concluded that the heat-fixed apatite layer consisted of nanocomposite of apatite and PEEK. We propose a possible mechanism for the laser bonding processes of this apatite coating, that is, when the coating surface is irradiated by a CO2 laser with rapid periodic scanning mode, this irradiation initiates periodic temperature change with thermal expansion of the air on top of the apatite layer and may induce a photoacoustic effect on them, which may generate pressure waves to hold down the apatite matrix to the PEEK substrate. The heat on the apatite matrix also initiates melting of the adjacent PEEK surface and the melted PEEK polymer intrudes into the voids of the apatite matrix by capillary forces to produce nanocomposite of them. This surface-selective formation of PEEK/apatite nanocomposites on the PEEK substrate may be seen as an alternative method to the conventional surface coatings or various PEEK composite materials.The bioactivity of the apatite-coated PEEK substrate was affirmed through biomimetic deposition of calcium phosphate from simulated body fluid and dissolution behaviors of the apatite coatings in water was also evaluated.

Luminescent Citrate-Functionalized Terbium-Substituted Carbonated Apatite Nanomaterials: Structural Aspects, Sensitized Luminescence, Cytocompatibility, and Cell Uptake Imaging.

This work explores the preparation of luminescent and biomimetic Tb3+-doped citrate-functionalized carbonated apatite nanoparticles. These nanoparticles were synthesized employing a citrate-based thermal decomplexing precipitation method, testing a nominal Tb3+ doping concentration between 0.001 M to 0.020 M, and a maturation time from 4 h to 7 days. This approach allowed to prepare apatite nanoparticles as a single hydroxyapatite phase when the used Tb3+ concentrations were (i) ≤ 0.005 M at all maturation times or (ii) = 0.010 M with 4 h of maturation. At higher Tb3+ concentrations, amorphous TbPO4·nH2O formed at short maturation times, while materials consisting of a mixture of carbonated apatite prisms, TbPO4·H2O (rhabdophane) nanocrystals, and an amorphous phase formed at longer times. The Tb3+ content of the samples reached a maximum of 21.71 wt%. The relative luminescence intensity revealed an almost linear dependence with Tb3+ up to a maximum of 850 units. Neither pH, nor ionic strength, nor temperature significantly affected the luminescence properties. All precipitates were cytocompatible against A375, MCF7, and HeLa carcinogenic cells, and also against healthy fibroblast cells. Moreover, the luminescence properties of these nanoparticles allowed to visualize their intracellular cytoplasmic uptake at 12 h of treatment through flow cytometry and fluorescence confocal microscopy (green fluorescence) when incubated with A375 cells. This demonstrates for the first time the potential of these materials as nanophosphors for living cell imaging compatible with flow cytometry and fluorescence confocal microscopy without the need to introduce an additional fluorescence dye. Overall, our results demonstrated that Tb3+-doped citrate-functionalized apatite nanoparticles are excellent candidates for bioimaging applications.

Biomimetic Citrate-Coated Luminescent Apatite Nanoplatforms for Diclofenac Delivery in Inflammatory Environments.

Luminescent nanoparticles are innovative tools for medicine, allowing the imaging of cells and tissues, and, at the same time, carrying and releasing different types of molecules. We explored and compared the loading/release ability of diclofenac (COX-2 antagonist), in both undoped- and luminescent Terbium3+ (Tb3+)-doped citrate-coated carbonated apatite nanoparticles at different temperatures (25, 37, 40 °C) and pHs (7.4, 5.2). The cytocompatibility was evaluated on two osteosarcoma cell lines and primary human osteoblasts. Biological effects of diclofenac-loaded-nanoparticles were monitored in an in vitro osteoblast’s cytokine–induced inflammation model by evaluating COX-2 mRNA expression and production of PGE2. Adsorption isotherms fitted the multilayer Langmuir-Freundlich model. The maximum adsorbed amounts at 37 °C were higher than at 25 °C, and particularly when using the Tb3+ -doped particles. Diclofenac-release efficiencies were higher at pH 5.2, a condition simulating a local inflammation. The luminescence properties of diclofenac-loaded Tb3+ -doped particles were affected by pH, being the relative luminescence intensity higher at pH 5.2 and the luminescence lifetime higher at pH 7.4, but not influenced either by the temperature or by the diclofenac-loaded amount. Both undoped and Tb3+-doped nanoparticles were cytocompatible. In addition, diclofenac release increased COX-2 mRNA expression and decreased PGE2 production in an in vitro inflammation model. These findings evidence the potential of these nanoparticles for osteo-localized delivery of anti-inflammatory drugs and the possibility to localize the inflammation, characterized by a decrease in pH, by changes in luminescence.

Development of ATP13A2-deficient In vitro Model for PARK9 Parkinson’s Disease

Background: PARK9 familial Parkinson’s disease (PD) is caused by a loss-of-function mutation in the ATP13A2 gene in which the mutation impairs the autophagic-lysosomal degradation pathway and induces intraneuronal accumulation of alpha-synuclein. RNA interference has been a useful tool in generating in vitro knockdown model to study the physiological role of the gene. However, the availability of a validated ATP13A2-deficient in vitro model is limited. Objective: This study aimed to develop the ATP13A2-deficient PD model by delivering ATP13A2 siRNA into neuroblastoma cells using carbonate apatite nanoparticles (CA NPs). Methods: CA NPs were fabricated using different concentrations of calcium chloride and characterised in the presence or absence of ATP13A2 siRNA. Time-dependent stabilities of CA NPs and CA NPs-associated siRNA (CA-siRNA) complex were evaluated by pH, turbidity, size, and zeta potential measurements. The dissolution abilities at acidic conditions of both complexes were investigated. Following that, green fluorescence protein (GFP) and four different siRNAs targeting ATP13A2 (siRNA_5, 6, 7, and 8) were transfected to cells with the fabricated CA NPs. Western blot was performed to determine the knockdown effect of the four siRNAs. Results: It was found that 4 mM calcium chloride was ideal for CA NP formation, while an incubation time of 48 hours was required to maintain the stability of nanoparticles. Successful transfection was confirmed by detection of fluorescence signal from the GFP plasmid and the subsequent silencing of this signal by transfecting GFP siRNA. Western blot analysis revealed that ATP13A2 protein expression was significantly reduced to 20% upon transfection with 20 nM of siRNA_5. Conclusion: ATP13A2-deficient PD model was successfully developed.

Inhibition of Breast Tumour Growth with Intravenously Administered PRKCA siRNA- and PTEN Tumour Suppressor Gene-Loaded Carbonate Apatite Nanoparticles

Gene therapy aims to silence an oncogene through RNA interference, or replace an abnormal tumour suppressor via gene augmentation. In this study, we intended RNA interference for PRKCA oncogene and gene augmentation for PTEN tumour suppressor with a view to reduce tumour growth in a mouse model of breast cancer. Inorganic carbonate apatite nanoparticles (CA NPs) were utilized to deliver the synthetic siRNA and the purified gene-carrying plasmid DNA both in vitro and in vivo. Effects of PRKCA siRNA- and PTEN plasmid-loaded NPs on viability of MCF-7, MDA-MB-231 and 4T1 breast cancer cells were assessed by MTT assay. The cell viability data in MCF-7 cell line demonstrated that combined delivery of PRKCA specific siRNA and PTEN plasmid with CA NPs had an additive effect to significantly decrease cellular growth compared to individual treatments. In addition, we observed a similar pattern of cumulative influence for combined treatment in triple negative MDA-MB-231 breast cancer cell line. Upon treatment with PRKCA siRNA+PTEN plasmid-loaded NPs, a remarkable decrease in the phosphorylated form of AKT protein of PI3K/AKT pathway was observed in Western blot, indicative of diminished proliferative signal. Moreover, in vivo study in MCF-7 xenograft breast cancer mouse model demonstrated that the rate of growth and final tumour volume were reduced significantly in the mouse group that received intravenous treatment of PRKCA siRNA+NPs, and PTEN plasmid+NPs. Our findings demonstrated that PRKCA siRNA and PTEN plasmid loaded into CA NPs attenuated breast tumour growth, suggesting their therapeutic potential in the treatment of breast cancer.