Lecithin-based wet chemical precipitation of hydroxyapatite nanoparticles
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Hydroxyapatite Ca10(PO4)6(OH)2 nanoparticles have been successfully synthesized by the wet chemical precipitation method at 60 °C in the presence of biocompatible natural surfactant—lecithin. The composition and morphology of nanoparticles of hydroxyapatite synthesized with lecithin (nHAp-PC) was studied by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). Size distribution for nanoparticles was measured by nanoparticle tracking analysis in NanoSight system. We discuss in details influence of lecithin concentration in reaction system on nHAp-PC morphology, as well as on size distributions and suspendability of nanoparticles. Product exhibits crystalline structure and chemical composition of hydroxyapatite, with visible traces of lecithin. Difference in surfactant amounts results in changes in particles morphology and their average size.
KeywordsHydroxyapatite Nanoparticles Synthesis Lecithin Wet chemical precipitation
In current research, the calcium phosphate materials, including hydroxyapatite (HAp), gather a lot of attention. Biocompatibility, osteoinductivity, osteoconductivity, and non-toxic properties of HAp create an opportunity to apply this material in wide range of industries . Beginning with biomedical applications, HAp in different physical forms finds application in tissue engineering , drug delivery , implants , and bone cements . High stability of HAp in a wide range of physical conditions makes it a desirable material for other industries. Apart from biomedical application, one can utilize HAp in catalysis , in adsorption processes , or in chromatography . For the most cases of its applications, hydroxyapatite has a form of nanoparticles (nHAp), sometimes with different sizes and morphologies: from plates, through rod-shape nanoparticles, to spherical nanoparticles.
In order to apply any kind of material for practical usage, there is a great need to produce it quickly, without high-cost manufacturing equipment and in stable, controllable conditions. In the case of nHAp synthesis, the methods might be divided into three categories: (1) simple chemical processes, (2) advanced processes, and (3) processes with application of novel technologies. Such a division depends on the complication of the synthesis system or difficulties on maintaining the synthesis conditions. Looking at the simplest approaches for nHAp synthesis—mostly one-step processes—one can extract as the most common wet chemical precipitation technique [9, 10, 11], hydrothermal method [12, 13], sol-gel method , and microemulsion method . In all of these methods, addition of templates, surfactants, or crystallization mediation agents is possible for improvement of product physical properties control. However, for applications in field of biomedical engineering, nHAp need to be synthesized without addition of any toxic additives. Biomimicking process , aerosol pyrolysis , and combustion preparation  are the examples of the advanced processes. Effort made for those advances aimed on higher nHAp quality in terms of morphology, connected with its application. Nevertheless, the cost of nHAp synthesis in this manner has risen. The last group of nHAp synthesis processes contains methods using novel energy sources for nanoparticle formation: ultrasounds , microwaves , and laser ablation . Those methods yield with the highest quality of the nHAp (narrow size distribution, uniform particles morphology, etc.), on the other hand, rise the production cost.
Product properties, chemical as well as physical, play a key role in its application. Synthesis process of the product should give a possibility to control product properties in a reproducible and dependable manner, without cost rising. Among simple chemical processes, wet chemical precipitation method for synthesis of hydroxyapatite nanoparticles has raised up much interest owing to its good repeatability, low reaction temperature, and reaction system simplicity . In wet chemical precipitation of nHAp, chemical and morphological properties of the product can be tailored by variation of synthesis conditions: temperature; pressure; and pH , as well as by additives, polymers, surfactants, and small molecules. Research groups tested various polymers in order to control morphology of the synthesized hydroxyapatite nanoparticles or to provide a template for synthesis, including the following: polyvinyl alcohol (PVA) , polyethylene glycol (PEG) , polystyrene-acetoacetoxyethyl methacrylate (PS-AAEM) , etc. Addition of surfactants into synthesis system is another way of product properties control and includes substances like the following: cetyltrimethylammonium bromide (CTAB) , sodium dodecyl sulfate (SDS) , and ethanolamine . In some cases of wet chemical precipitation process and hydrothermal method, one can add small molecule substances like sodium citrate to affect physical characteristics of nHAp .
In light of currently used technologies described above, we propose a novel approach to the wet chemical precipitation method for synthesis of hydroxyapatite nanoparticles. The goal of this paper is to describe the wet chemical precipitation process based on the application of solution of lecithin—zwitterionic surfactant, mixture of phosphatidylcholines (PC) ubiquitous in cellular membranes—as a controlling agent of product properties. Proposed process results with hydroxyapatite nanoparticles chemically modified with lecithin—a product suitable for various applications. Furthermore, phosphatidylcholines bonded to the nanoparticle surface increase biocompatibility and suspendability of the synthesis product. Additionally, the paper presents results of investigation of the product chemical and crystalline structure (by Fourier transform infrared spectroscopy and X-ray diffraction) and morphology (by scanning electron microscopy) as a function of the amount of lecithin added to the precipitation system. We present size distributions and results of the estimation of suspendability property of the resulting nanoparticle powders in water (estimated by NanoSight measurements). Method for production of nanoparticles of hydroxyapatite bonded with lecithin (nHAp-PC) presented in this paper yields with product for many kinds of applications: from biomedical products to chemicals from and for various industries.
Materials and methods
Diammonium phosphate (NH4)2HPO4, calcium nitrate tetrahydrate Ca(NO3)2·4H2O, ammonium water NH3∙H2O (Chempur, Poland), lecithin (Serva, Germany), and commercial hydroxyapatite nanopowder (particle size <200 nm, Sigma-Aldrich, Germany) were used as received without any further purification.
Synthesis of HAp-PC nanoparticles
Reaction procedure was as follows: 0.0075 moles of Ca(NO3)2·4H2O was dissolved in 25 ml of demineralized water. Suspensions of lecithin in 37.5 ml of demineralized water were prepared, with concentrations 0.30, 0.75, 1.50, 3.00, and 9.00 % w/w. Ca(NO3)2·4H2O solution was added into reactor, and lecithin suspension was slowly and gently added to the reaction system. The initial value of pH of the resulting solution was adjusted to 10.0 with ammonia water. The temperature was controlled and set at 60 °C and mixture was stirred to maintain stable conditions. Then, 0.0046 moles of (NH4)2HPO4 was dissolved in 15 ml of demineralized water and the initial pH of the solution was adjusted to 10.0. Diammonium phosphate solution was added drop-wise into the mixture of calcium nitrate and lecithin at a feeding rate of 2 ml/min during the stirring. Then, mixture was stirred continually for 1.5 h in 60 °C, and then naturally cooled to room temperature and left under stirring overnight for aging the product. The resulting suspension was centrifuged (Centrifuge MPW-251, MPW Med.Instruments, Poland), decantated, and washed with hexane (washing process was repeated five times) to remove unbounded residues of lecithin. The final product (yellowish powder) was dried at 50 °C for 24 h.
The values of initial pH were measured with SevenMulti (Mettler Toledo, Switzerland) with measuring range of pH 0.000–14.000, resolution 0.001, and accuracy ±0.002. The degree of crystallinity was characterized by X-ray diffraction (XRD, D8 Advanced, Bruker-ASX) using Cu Kα radiation (λ = 0.15406 nm) at the X-ray tube voltage 40 kV and tube current 40 mA. The XRD data were collected at room temperature over the 2θ range of 3–60° at a step size of 0.02°/s. The characteristic groups of HA and lecithin were analyzed by Fourier transform infrared spectroscopy (FTIR, NicoletTM 6700 spectroscope, Thermo Scientific, USA). Morphology of final dry product was studied by scanning electron microscope (SEM, Zeiss Ultra Plus, Germany). Average size, size distribution, and assessment of suspendability of synthesized nHAp-PC powders were carried out based on nanoparticle tracking analysis (NTA). For the analysis, NTA 2.3 software on NanoSight system (LM10, Malvern Instruments Ltd., UK) was applied. The system measures diffusion coefficient for particles and then calculates, from Stokes-Einstein equation, their sphere-equivalent hydrodynamic diameter. In this paper, calculated hydrodynamic diameter is considered as a size of the analyzed nanoparticles. Powder samples were suspended, by sonication for 10 min, in ultra pure water (Direct-Q System, Merck Millipore, Germany) to obtain 10 ml of highly diluted suspension prior NTA measurements.
Results and discussion
Selected methods of morphology and size control in nHAp synthesis reported in literature
Synthesis of nHAp
Morphology and size control method
Particles size [nm]
Wet chemical precipitation
Dominant rod; sphere
Wet chemical precipitation
Addition of Schiff bases
Dominant sphere; rod
Wet chemical precipitation
Reactant addition rates (titration speed)
Shift from rod to sphere
Application of microwaves
up to 40 nm
Gas-phase laser ablation
up to 100
In this paper, we proposed a novel approach to wet chemical precipitation of hydroxyapatite nanoparticles in the presence of lecithin in the reaction system. We showed resulting nanoparticles crystallinity and chemistry analysis results, proving that lecithin-based approach allows formation of good quality hydroxyapatite nanoparticles, slightly modified by traces of lecithin (nHAp-PC). Based on SEM image investigation and additional particle tracking analysis experiments in NanoSight system, we described possibilities to control the product morphology and the average particle size by varying lecithin concentration in the reaction system only. We postulate the increased biocompatibility of the resulting nHAp-PC material by modification of nanoparticles by bonding with lecithin. This modification also increases suspendability property of nanoparticles in various media.
The patent of the presented method is pending.
The authors are thankful to Iga Wasiak, MSc. Eng., from Faculty of Chemical and Process Engineering, Warsaw University of Technology for the NanoSight measurements, and Andrzej Ostrowski, PhD Eng., from Faculty of Chemistry, Warsaw University of Technology for the XRD measurements.
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