Many nanoparticle systems have been used and studied as potential candidates in the release, drug delivery and bioimaging purposes. A very good example in this area was hydroxyapatite nanoparticle (nHA) which is a natural source of calcium for human body being as a dispersed colloidal system of calcium phosphate with a size correlated to the colloidal calcium phosphate in milk’s casein micelles . Construction and in vivo application of these kind controlled drug delivery systems is one of the most important scientific activities due to their direct relationships with human health care. In this work, we speciﬁcally tried to create a colloidal and water soluble hydroxyapatite (nHA) nanocomposite using biologically active casein polymers as a matrix. [2-4]. Caseins are amphiphilic self-assembled proteins stable in aqueous solutions known as casein micelle. Their main biological function is the transport and delivery of proteins in the body. Noting that synthesis of nHA nanoparticles in casein substrate will be associated with certain technical difficulties because of its high tendency to aggregate. For reducing the size of nanoparticles ultrasonic treatment of the synthesis batch or addition a modifier agent have been applied. [5-7] Different methods which have been reported for preparation of the synthetic nHA were micro emulsion technique , wet chemical precipitation method , biomimetic preparation , sol–gel method , etc. Among these method, the most widely used technique for synthesis of HA nanoparticle was chemical inverse micro emulsion method  which we have used it to prepare nHA colloidal system using different concentration of the main milk protein, casein.
All the starting chemical materials used in this work, Ca(NO3)2.4H2O٫ (NH4)2HPO4, NaOH and cyclohexane were supplied from Merckcompany and used without any further purification. Casein proteinswere prepared in our Laboratory using commercial skimmed milk.
Isolation of Casein from Milk
50 ml skimmed milk was poured into a flask placed in a water bath heated to reach a critical temperature (40 °C). By addition 20 drops of acetic acid to the prepared sample and stirring with a glass rod, a clot of casein was obtained in the flask. The clots was separated from the mixture by vacuum filtration and added 25 ml of ethanol and stirred slowly to obtain dispersed casein in ethanol. To remove any remained impurities in the sample, the slurry was washed by 50 ml of a mixture of 1 : 1 ethanol / diethyl ether and dried at room temperature.
Synthesis of Hydroxyapatite nanocomposite
10 mL of aqueous solution of (NH4)2HPO4 (0.6 M) was directly added to a reverse micro emulsion system prepared by dispersion of 0.1, 0.2 and 0.3g of our prepared casein powder in 30 ml cyclohexane under vigorous stirring batch. By injecting slowly 10 mL of appropriate aqueous solution of Ca(NO3)2.4H2O in the above prepared mixture and stirring again for about 15 minutes, a transparent solution was obtained. To adjust the pH in the range of 9-10, a small amount of NaOH solution (1M) was added to the system and vigorously stirred at room temperature for one day. The colloidal solutions (with and without biopolymer) were centrifuged and dried at 80 °C for 48 hrs.
Characterization of as prepared products was performed using XRD patterns using Philips expert pro. Diffractometer equipped with CuKa radiation (λ=0.154 nm). The typical groups of the synthesized powders was analyzed by Fourier transform infrared spectroscopy (FT-IR, Model Thermo Nicolet’s Nexus 870), in the wave number range of 400-4000 cm-1, with a resolution of 4 cm-1. Field Emission Scanning Electron Microscopy (FESEM) technique (MIRA3TESCAN-XMU) and Transmission Electron Microscopy (TEM) were used to examine and estimate the size, distribution and morphology of the samples (Zeiss-EM10C-100 KV). Particle size of HA/casein nanoparticles was measured using Dynamic Light Scattering (DLS) technique (ZS/ZEN3600 Zeta sizer; Malvern, Instruments Ltd, Malvern, UK). This system was equipped with a 4 mV helium/neon laser at 633 nm wavelength and measures the particle size with the noninvasive back scattering technology at a detection angle of 173° after an at least 100-fold dilution with purified water. All of the DLS measurements were performed at 25.0 °C ± 0.1 °C at 20-second intervals for three repeat measurements. For zeta potential measurement, each diluted nanoparticle suspension was put in a universal folded capillary cell equipped with platinum electrodes. The electrophoresis mobility was measured and the zeta potential (ζ) was calculated by the Dispersion Technology Software provided by Malvern.
RESULTS AND DISCUSSION
X-Ray Diffraction (XRD) study
Fig. 1 shows XRD diffraction patterns of as prepared sample, at the presence of: a) 0.1g, b) 0.2g and c) 0.3 g Casein. All the main diffraction peaks recorded at (002), (211), (310), (222), (213) and (304) plane, were characteristic of the monophasic low crystalline HA with ICDD PDF No: 00—001-1008, matching well with the hexagonal system (P63/m). Very interesting point in this figure was appearance of the broad peaks width for the samples with different concentration of casein and a sample’s broadening peak’s width is in general a good evidence for explaining formation of nanostructured materials at the presence of a substrate. Average HA crystalline size was calculated using Scherrer`s  equation as follows:
Where, D is the average crystal size, K is a constant (here chosen as 1); λ is the wavelength of X-ray radiation (1.54056 A), β1/2 is the half width of the diffraction peak and θ (°) is Bragg angle. The result of D values calculation for the samples prepared at the presence of 0.1, 0.2 and 0.3 g of casein were 13, 8 and 6 nm. This crystalline size reduction can be explained by the effect of casein concentration on the crystalline size. Because, for an emulsion polymerization, higher amount of the surfactant can stabilizes smaller emulsion droplets because of their larger specific surface area.
FTIR Spectroscopy Study
Functional groups associated with hydroxyapatite were identified by FTIR spectroscopy. Recorded FTIR spectrum for the sample synthesized in the presence of 0.3 g casein is given in Fig. 2. In this figure, the presence of hydroxyl group in the apatite lattice is well confirmed by absorption broad bands at 3571 cm−1 and 629 cm−1, attributed to the stretching and flexural modes of hydroxyl (OH−) group. The IR characteristic peaks of phosphate groups appeared between 1090-1030 and 600-560 cm-1originated from bending and stretching vibration mode of phosphate of HAp respectively [14-15]. The absorption bands at 1420 cm-1 and 875 cm-1 suggest the presence of CO3-2 in hydroxyapatite structure . Likewise, the other stretching vibrations for carbonyl and phosphate groups were also observed as reported. The peaks appearing at 2924 cm–1, 2854 cm–1 and 2956 cm–1 are assigned to the vibrating absorption of C-H asymmetric and symmetric stretching in CH2 and C-H asymmetric stretching in CH3 respectively . All peak’s assignment were shown in Table 1.
The FE-Scanning electron microscopy
Fig. 3 shows FE-SEM micrograph for the sample prepared on 0.3 g casein in micro emulsion system. In this image, formation of homogeneous distribution of uniform spherical nanoparticles aligned in a rod like system can be distinguished.
The Transmission electron microscopy
Fig. 4 shows TEM image of the as prepared sample at the presence of 0.3 g casein. This image reveals formation of needle like morphology of HA nanoparticles with a mean crystalline size of approximately 10 nm in diameter and 60-70 nm in length. This value is much more different than of the crystallite size obtained via Scherrer’s equation. This discrepancy between crystalline and grain size can be due to the effect of casein addition as scaffold in the batch preparation to provide the necessary condition for formation grain size nHA at room temperature. Interesting point in this image was arrangement of the needle like HA around spherical casein micelles (shown in circle at Fig. 4).
Particle size and zeta potential distributions
Dynamic light scattering (DLS) is a valuable tool for studying size and degree of agglomeration of the nanoparticles as a function of time in the suspension solution. In this technique, one can determine hydrodynamic size of the nanoparticles by measuring intensity of laser scattered light that passes through a colloidal solution. Larger particles will diffuse slower than smaller particles. So, particle size can be mathematically provided by measuring time dependence of the scattered light. The DLS experimental data obtained in this work which is given in Fig. 5 shows a zeta particle size distribution of 265.6 nm. In fact, this is the average particle size of the hydrodynamic colloidal nanoparticle in HA/casein suspension. Simulated image of this particle size distribution (6a) which is supported by TEM image of nHA prepared at the presence of 0.3 g casein is presented in Fig. 6b.
The zeta-potential of the particles which is a basic parameter in controlling stability of colloidal suspensions is obtained by measuring particles mobility under an applied electric field which is applied and can be calculated using Smoluchowski equation :
Where, and are dielectric constants of vacuum and solvent respectively, η is viscosity and U is the mobility of the particles in suspended solution. In general, when an electric field is applied to the charged particles in a colloidal sample, particles move toward an electrode opposite to its surface charge. Because the velocity is proportional to the amount of charge of the particles, zeta potential can be therefore estimated by measuring the mobility of these particles. To determine the speed of the particles movement, the particles was normally irradiated with a laser light, and the light emitted from the particles was detected from the shifted scattered frequency. Since, shifted scattered light from incident light is proportional to the speed of the particles movement, and the electrophoretic mobility of the particles can be thereby measured [19-21]. The most widely used theory for calculating zeta potential from experimental data was developed by Marian Smoluchowski in 1903 . Experimental zeta potential value obtained in this work for ionically cross linked HA/casein suspension is given in Fig. 7 This result reveals a bimodal zeta potential distribution may be due to the two different populations of particles within the prepared suspension sample. About 42% of the particles have a zeta potential of +23 mV, and 58% of the particles have a lower value of +2.94 mV. Considering that and a dividing line between stable and unstable aqueous dispersions reported at either +30 or -30mV, particles with zeta potentials more positive than +30mV have been considered stable and particles with low absolute values of the zeta potential will agglomerate and the dispersion becomes unstable [23-31], we can conclude that colloidal samples prepared in this work at the our experimental condition even after 4 hours ultrasonic treatment, were unstable because of casein- calcium high tendency to aggregate. To assure stabilized colloidal suspensions, addition of some anticoagulant agent to regulate stability of the colloidal suspensions seems to be indispensable.
HA nanoparticles were successfully prepared in an inverse micro emulsion template phase consisting of cyclohexane, water, anionic surfactant and co-surfactant. The X-ray diffraction patterns confirmed formation of hexagonal nHA crystalline at the presence of casein micelles. Arrangement of needle like HA crystallite at around casein micelles in its colloidal solution, was clearly evidenced by TEM image. Dynamic light scattering (DLS) techniques used to measure particle size and zeta potential distribution revealed successful preparation of a colloidal HA/casein suspension with a hydrodynamic size distribution of 256.6 nm.
Bimodal zeta potential distribution in divergence with the DLS average zeta size data reveals unstability of the colloidal HA/casein suspension because of casein- calcium nanocomposite’s high tendency to aggregation.
We would like to thank research vice presidency of Science and Research Branch of Islamic Azad University for providing financial support.
CONFLICT OF INTEREST
All authors declare no conflicts of interest in this paper.
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