Chitosan/ HAP Nanoparticles: Antibacterial Study

INTRODUCTION
The World Health Organization reports were Diagnosed that the major health problem is dental caries, especially among disadvantaged social groups worldwide. This disease affects both sexes, all races and age groups, and different social and economic classes. Dental caries causes pain and sadness, and it imposes a relatively high financial burden on families. There are about 700 different bacterial species in human oral microbiota. Antimicrobials such as ampicillin, chlorhexidine, quaternary ammonium compounds and metronidazole are used to prevent tooth decay, but they have several side effects such as tooth staining, diarrhea, increased calculus formation and changes in bowel flora. Therefore, new methods for prevention and probably better management of this important challenge are needed. In recent years, the use of natural nontoxic alternatives for the control and prevention of dental caries has emerged. Chitosan is a natural polymer with specific properties, including nontoxicity, antimicrobial activity and biodegradability, which has attracted great attention for some years. Prior studies have described the antimicrobial potential of chitin, chitosan and their derivatives [1-3].
However, application of micro and nano crystalline chitosan structure have a clear impedance to the dissolution in neutral pH as well biodegradation prolongation due to high crystallinity of the Chitosan bio-composites. Hydroxyapatite (HAP), (Ca10(PO4)3OH), is present in mammalian as major inorganic compound and highly recognized and used for its bio-compatibility. The calcium phosphates are perfect for bone replacement, promote rates of resorption/replacement so appropriate which have been proven by the Environmental Physiology and the osseoconductive. The Hydroxyapatite is very similar to natural bone than other calcium phosphate formula (like ß-TCP, (Ca3(PO4)2)) [4-5]. The performance of a polymer medical device depends on bulk and surface material properties [6].
Many researchers are work on development of nano HAP composite with biopolymer, because of their identification to bone matrix when coupled with biological and mechanical performances [7]. Biopolymers materials are very widely applications in the fields of biomedical and orthopedic, because of their biocompatibility and biodegradability properties [8]. Many composited containing HAP/polymer such as HAP/ collagen [9], HAP/chitosan [10], HAP/ collagen/poly(lacticacid) [11], HAP/ alginate/collagen [12], HAP/gelatin [13] were used for bone tissue engineering. Chitosan and curcumin with HAPNPs showed reproductive toxicity via its ameliorative effects on the fertility, testicular tissue functions, antioxidant system and hormonal status [14]. 
In this study low cost technique was used to produce HAPNPs and mixing with chitosan at different percentage. The morphology testing shows unique structure for CS/HA NPs and cluster for HAPNPs alone. It showed a good antibacterial activity against E-coli and and Staphylococcus aureus bacterial.

MATERIALS AND METHODS
Raw Materials and Characterization
The raw materials used in this work are: Chitosan (grade of deacethylation 0.85, MW 110 kDa, Calcium nitrate (Ca(NO3)2.4H2O) tetrahydrate, diammonium hydrogen phosphate (NH4)2HPO4 were purchasing from various suppliers. All solvents and reagents were used without any additional purification. The morphology and diameter of the HAPNPs and/or CS/ HAPNPs were examined by using TE FEI technical G2 Split Biotic transmission electron microscope at (120 kv) and Field Emission Scanning Electron Microscopy, model Quanta 200FEG, which is configured to operate at 120 Kv with various magnification level. The crystallinity and crystal phase for HAPNPs and/or CS/ HAPNPs were studied by,using [ Rigaku X-ray-diffracto-meter, 6000, Shimadzu, Japan). The physical properties and chemical structure were characterized by using FTIR [FTIR 8400S,Shimadzu, Japan] and UV- Vis spectroscopy [AA 6300, Shimadzu, Japan]. The antimicrobial tests were made in vitro.

Method
First, the procedure used for Hydroxyapatite (HA) nanoparticles preparation in this study have followed according to our previous study [15]. In briefly, Hydroxyapatite nanoparticles (HAPNPs) were obtained from diammonium hydrogen phosphate and calcium nitrate tetrahydrate precursors, Ammonia used in control of the acidic function at 11. The use of ultrasound to study the impact on the particle sizes. Chitosan were mixed with HA nanoparticles in Distilled water with different percentage [5%, 10%, and 15% respectively]. The morphology were examined by mean of TEM, FESEM, XRD, and FTIR. CS/HA nanoparticles solutions were fabricated by using the agar diffusion test in same way as describe from Ammar Hamza et el. [16]. E. coli and Staphylococcus aureus, which both of them are positive gram are used to study The antibacterial properties of CS/HA nanoparticles composite.

RESULT AND DISCUSSION
The nanoparticles which get from HAP by using ultrasonic technique and Fig. 1a shows the morphology of HAPNPs. The results of obtained hydroxyapatite powders showed almost spherical particles with narrow size distribution. Average size of HAP particles is approximately ∼ 90 nm as shown in Fig.1a. The FESEM images indicated Individual spheres with agglomerated and porosity. We think the formation like a sphere for HAPNPs are due to the high level of correlation between ultrasonic waves and HAP molecules [17]. Fig. 1b show the transmission electron microscope (TEM) for CS/HAPNPs 15wt%. It was showed a unique shape of the prepared material, which was in the form of nanoparticles for HAP composed with chitosan. It is clear that the original HAP are highly complicated particles with average diameters of ∼35 nm. We think that two reasons caused the diameters of HAPNPs are less, as it composited with CS; first, the cluster was formed for HAP molecules. Second, CS-HAPNPs composite prepared by gel-formation indicated that the HAPNPs are relatively dispersed over a wide area in the chitosan matrix. The TEM results of HAPNPs were suggested that can be homogeneously incorporated with Chitosan.
The XRD patterns of the CS, HAPNPS, and CS/HAPNPs 15wt% samples are summarized in Fig. 2a. The result of XRD were confirmed by the Power Diffraction File (HAP: Card No. 090432; CS: Card No. 391894). It indicated that, the broad diffraction characteristic peak that noticed around 20° was correspond to CS (20.305°, 21.290°), when Chitosan alone or mixing with HAPNPs. The sharp peaks that noticed at around 31.8° and 25.9° assigned to the HAPNPs (31.773°, 25.879°). XRD patterns indicated the presence of peaks for both chitosan and HAPNPS with approximately no change in the position of angle (2θ). FTIR spectra are indicated that absorption bands at 3470, 2923, 1624, and 1383 cm-1 for chitosan molecular, and at 3470 and 3570 cm-1 is corresponded to the stretching and vibration of the lattice OH- ions in chitosan molecular and Hydroxyapatite nanoparticles [18]. The bands at 631 cm-1 are for the ν1 mode of characteristic bands PO4-3, and absorption bands at 1093/1040 cm-1 for the ν3 symmetric P-O stretching vibration were observed. The absorption bands for HPO4-2 were appeared at 1393 cm-1and 1460 cm-1 as shown in Fig. 2b. These results pointed out the presence of HAPNPS in the Chitosan samples, in accord with the XRD results [19-20].
Fig. 3 (a and b) showed the weight percentage of HAPNPs have activity against E. coli and Staphylococcus aureus because the antimicrobial activity is concerned with compounds that make locally killed microbial or decreased slowly their growth. Nowadays, most of current antimicrobial agents are produced from chemically modified natural compounds [21-22]. A ruler was used to measure Zone of inhibition, which is defined as the average diameter of the area around the discs that stops the growth of bacteria. It was observed that the CS/HAPNPs synthesized have antibacterial activities against Staphylococcus aureus more than E. coli as shown in Table 1. As the weight percentage of HAPNPs increased, the inhibition zones are increased for both bacterial.

CONCLUSION
The synthesis of HAP with spherical and a roughly ∼35 nm particle size in aqueous solution has been achieved in the presence of the chitosan which are adopted by TEM and FESEM equipment. Also, they showed hierarchical ordering compositionally from inorganic (HAP) to organic (chitosan), which suggested very interesting and novel biomedical applications such as drug delivery devices, hybrid tissue engineering and scaffolds. The XRD and FTIR spectrum conformed the structure for composites. Antibacterial test was highly dependent on the concentration of a nano-hydroxyapatite as shown in the inhibited zone. These results show a strong biomaterials property for CS/HAP nanoparticles.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.