Surface-modified ZrO2 nanoparticles with caffeic acid: Characterization and in vitro evaluation of biosafety for placental cells
Introduction
Zirconia ceramics has emerged as an attractive alternative to titanium-based materials, which have been the gold standard in implantology for decades, owing to their unique physical, biological, and esthetic properties. Zirconium dioxide nanoparticles (ZrO2 NPs) display great perspective in biomedical applications, particularly in dentistry, due to their strong resistance to corrosion, pure white color, and good compatibility [1]. Additional applications of ZrO2 NPs are in metal joints, as drug carriers, and in other fields, since they are biocompatible and chemically stable [2].
In terms of beneficial biological activity, zirconia has demonstrated a low affinity to bacterial biofilm formation, reduced inflammatory infiltration, and good soft-tissue integration. The surface properties of the implant are a prerequisite for achieving good tissue integration. Zirconia substrates showed a better effect on osteoblast cell adhesion and proliferation in comparison to titanium elements [3]. The initial biological interaction between the cells and nanomaterial strongly depends on the physicochemical properties of the material. In particular, the size and morphology of NPs are known to impact biosafety and affect the level of toxicity to cells [[4], [5], [6]]. Many studies reported the lack of cytotoxic effect in zirconia surfaces in vitro and any systemic inflammation [3,[7], [8], [9], [10]]. However, Ye et al. reported the toxicity of coated and bare ZrO2 and TiO2 NPs [11,12]. This report indicated that reactive oxygen species (ROS) play a crucial role in the TiO2 and ZrO2 NP-induced cytotoxicity in a concentration-dependent manner. Also, traces of zirconia are found in the peri-implant tissues and can diffuse to the other parts of the body [13].
The small size of nanoparticles enables their easy uptake inside the body, infiltration into the bloodstream, and the possibility to overcome different biological barriers, such as the blood-brain barrier and placenta. These properties of NPs hold great promise to revolutionize the treatment of diseases, as they could enable the delivery of drugs to the places in the body that are difficult to reach using conventional methods. However, the increasing use of nanomaterials in everyday life raised concerns about nanomaterials' influence on health, especially for vulnerable groups like pregnant women, since in utero exposure may adversely affect fetuses and health in later life [14]. Consequently, the increased incidence of some disorders is the consequence of prenatal exposure to nanosized materials [15,16]. The potential effects of nanoparticles during pregnancy are still controversial [[17], [18], [19], [20], [21]]. Thus, a more comprehensive understanding of NP effects at the placenta is required and needs thorough investigation. So far, a few studies have addressed the impact of TiO2 nanoparticles on fetal or maternal toxicity during pregnancy [18,[22], [23], [24]]. To the best of our knowledge, only Wang et al. investigated the influence of ZrO2 sol on pregnant rats [21]. They showed that oral exposures to ZrO2 NPs during pregnancy are dangerous to fetal brain development, especially in early pregnancy.
It is well-known that oxides (TiO2 and CeO2) and biologically important material hydroxyapatite form the interfacial charge transfer (ICT) complexes with small organic ligand molecules whose appearance indicates a red absorption shift [[25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36]]. So far, the ICT complex formation is reported for metal-oxide nanoparticles modified with either catecholate- or salicylate-a type of ligands. These two ligand groups include many valuable biological molecules such as antioxidants (caffeic acid, dopamine, natural polyphenols), drugs (salicylic acid, 5-aminosalicylic acid), and vitamins (ascorbic acid, vitamin B6). So far, the formation of ICT complexes with ZrO2 NPs never been reported.
Caffeic acid (3,4-dihydroxycinnamic acid) is a natural antioxidant, widely distributed in many fruits, vegetables, beverages, herbs, and spices, and, as such, is a component of the human diet. Caffeic acid (CA) and its derivatives have been extensively studied in the past years, and the obtained results unveiled a broad spectrum of biological activities, including anti-cancer, anti-inflammatory, antioxidant, antibacterial, antifungal, and protective effects on multiple organs [[37], [38], [39]]. Previous research suggests that CA possesses protective effects against reproductive tissues damage induced by harmful molecules such as free oxygen radicals, pesticides, methotrexate, etc. [40]. CA exposure per se is considered to be safe, as demonstrated in animal studies where no significant effect on reproductive toxicity, fetal teratogenesis, or pup development was observed. Mid and high-dose (5 and 150 mg/kg/day) mainly affected embryo implantation [41]. Also, caffeic acid exerts an antitumor effect through the inhibition of pathways of cancer metabolism [42].
The focus of this study is to evaluate the toxicity of hybrid ZrO2/CA NPs, prepared by condensation reaction between hydroxyl groups from inorganic and organic components of the hybrid, in a broad concentration range using four different cell lines (HTR-8 SV/Neo, JEG-3, JAR, and HeLa). These cell lines are chosen for the following reasons. The HTR-8 SV/Neo cells faithfully represent the primary first trimester of pregnancy cytotrophoblast in vitro since they behave in the same way in various functional assays [[43], [44], [45]]. Consequently, HTR SV/Neo cells are a suitable in vitro model for the initial testing of toxicity of nanoparticles passing to the placenta from the maternal bloodstream. The choriocarcinoma cell lines (JEG-3 and JAR) possess biological characteristics of syncytiotrophoblasts and present a convenient experimental model of placental barrier integrity, providing information about nanoparticles’ transfer ratios similar to ex-vivo placenta perfusion models [46]. HeLa is a human cervical carcinoma cell line universally employed as a reference cellular model in toxicity screenings of nanomaterials [47]. Before toxicity evaluation, the ICT complex is characterized in detail, applying transmission electron microscopy, X-ray diffraction analysis, and zeta-potential measurements. Also, spectroscopic measurements were supported with the density functional theory (DFT) calculations to provide an in-depth understanding of the optical properties of hybrid.
Section snippets
Synthesis and surface modification of zirconium oxide sol
All chemicals used in this study were of analytical grade quality and used without further purification.
Zirconium oxide sol was prepared by forced hydrolysis of zirconium salt, as described previously [48,49]. Briefly, a necessary amount of ZrOCl2 (Sigma Aldrich) was dissolved in an acidified solution (0.1 M HCl) and added dropwise into the boiling HCl (0.1 M) solution, and the obtained mixture was overnight left at elevated temperature and vigorously stirred. Then, the sol was cooled down, the
Results and discussion
TEM analysis of the ZrO2 sol (Fig. 1.) revealed snowflake-like loose agglomerates, 30–60 nm in size, consisting of much smaller NPs that are 2–5 nm in size having polyhedral shapes. The selected area electron diffraction (SAED) pattern is consistent with the monoclinic crystal structure of ZrO2. High-resolution TEM images indicated that the smaller NPs within each agglomerate share a close crystallographic alignment. For example, (200)/(002) cross-fringes are visible throughout an NP when
Conclusion
Surface-modified ZrO2 nanoparticles with caffeic acid, a natural polyphenol abundantly present in the human diet, are prepared for cytotoxic evaluation. Before the cytotoxic examination, microstructural and optical characterization of unmodified and surface-modified ZrO2 NPs was performed. Hybrid ZrO2/CA NPs display a red absorption shift due to the formation of the ICT complex. The spectroscopic data were supported with the DFT calculations that provide deeper insight into the electronic
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgment
This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (agreement No. 451-03-9/2021-14/200019 and No. 451-03-9/2021-14/200017).
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