Investigation of the Antimicrobial Activity and Hematological Pattern of Nano-Chitosan and its Developed Nano-Copper Composite

Novel synthesized Chitosan-Copper oxide nanocomposite (Cs-CuO) was prepared using pomegranate peels extract as green precipitating agents to improve the biological activity of Cs-NP's which was synthesized through ionic gelation method. The characterization of biogenic nanoparticles Cs-NP's and Cs-CuO-NP's were investigated structurally, morphologically to determine the full descriptive features of those nanoparticles. Antimicrobial activity was tested for both Cs-NP's and Cs-CuO-NP's via Minimum inhibition concentration and zone analysis against fungus, gram positive and gram negative. The results of the antimicrobial test showed high sensitivity of Cs-CuO-NP's to all microorganisms that are tested in concentration less than 20 mg/ml while the sensitivity of Cs-NP's against all microorganisms under test started from a concentration of 20 mg/ml to 40 mg/ml except for the C.albicans species. Hematological activity was also tested in via measuring the RBCs, platelets count and clotting time against healthy, diabetic and hypercholesterolic blood samples. Measurement showed a decrease in RBCs and platelets count by adding Cs-NP’s or Cs-CuO-NP's to the three blood samples. Cs-NP’s success to decrease the clotting time for healthy and diabetic blood acting as a procoagulant agent, while adding biogenic CuO-NP’s to Cs-NP’s increased clotting time considering as an anticoagulant agent for hyperchloesterolic blood samples.


Introduction
Throughout the history, wound healing has been a crucial challenge facing all wound care researchers in the medical eld. Wound healing is a process of repairing tissue integrity through a series of phases including hemostasis, in ammation, proliferation, and remodeling [1]. The initial stage which is the initiation of coagulation cascade to prevent excess blood loss leading to platelet accumulation and brin clot formation is known as Haemostasis. Halting in any healing stage leads to chronic wound susceptible to anincrease in microbial infections, large exudates, and necrosis in tissues due to an upsurge in pus cells number [2] [3]. Therefore, recent research has focused on improving wound dressing materials speci cally those that originated from natural polymers to becoming interactive and bioactive materials.
Chitosan is the most naturally abundant biopolymer and the second most abundant polymer coming after cellulose [4]- [6]. Chitosan being polycationic at acidic media (pH < 6) allows it to interact easily with negative charged molecules, such as phospholipids, anionic polysaccharides, proteins and fatty acids. Nonetheless, chitosan may also chelates metal ions selectively such as copper, iron, cadmium and magnesium [7]. Chitosan plays an important role in the regeneration of the wounded area via broblast proliferation and precence of glucosamine which enhances earlier synthesis of hyaluronic acid to acclerattethe healing process with minimal scarring [8], it also helps in revascularization, and plays a role in protecting against atherosclerosis [9]. Additionally, chitosan is able to control in ammatory mediators to accelerate the healing process [9].
Chitosan based nanoparticles, being versatile, nontoxic, biocompatible and biodegradable snatched the attention of researchers in the biomedical eld [10] [11]. Interests in improving nano chitosan properties via chemical modi cation have been growing rapidly [11]. Chitosan chemical modi cation is of great interest because it can retain its basic skeleton, which in turn keeps its physicochemical and biological properties [10].
Chitosan with various modi cations [8] [9] [12] and several reactive functional sites has shown high activity as an innate antibacterial agent specially when mixed with metallic nanoparticles. Copper is one of the metallic nanoparticles, which is a vital element, in trace amounts, that facilitates the activity of a variety of enzymes, and it also helps in skin regeneration, wound healing process, and angiogenesis [12].
Although, some previous studies showed restraints concerning copper due to its toxicity which is known to emerge from the production of oxy-radicals which initiates the formation of ROS resulting in oxidative stress [12] [13]. However, the literature revealed that the hybridization of Copper with chitosan reduces the toxicity level [14]. Also it was repoted that nano Cu and CuO are considered as effective antibacterial agents [14][15] [16], According to the literature, chemical reduction method is a facile process for the synthesis of NP's using biopolymeric materials [17] in achieving a better substantial bacteriostatic/bactericidal property.
Copper/Copper oxide nanoparticles (Cu/CuO-NP's) were biologically synthesized using different plant extracts as reducing agent as well as capping agent. These plant extracts have promising advantages towards enhancing biological activity of the CuO-NP's. Pomegranate peel is rich with signi cant amounts of polyphenols, that is, phenolic acids, such as ellagic and gallic acid, avonoids and Tannis [18], [19] which are effective as antimicrobials, antianxiety, antidepressant, antiproliferative, antitumor, antioxidant agents [20] [21] anticoagulants, antiplatelets, and antianemic agents [22] [23] and play a preventive role in cardiovascular diseases by inhibiting coagulation and thrombus [24]. Also it was proved that it had vital role in treating the blood vessels and heart, such as heart attack, atherosclerosis, and high cholesterol.
It is also used for conditions of the some digestive tract diseases, including diarrhoea and intestinal parasites [25]. Anticoagulant, antiplatelet, and hypo brinogenemic effects of P. granatum may be due to impaired activity of thrombin predominantly by TAT complex and PC [26].
The aim of this work is to synthesize a hybrid bioactive nanocomposite from marine based polymer, which provides antibacterial e ciency, playing a pivotal role in the healing process.  The indication of bands

3550-2902
Overlapping between N -H, -OH starching from chitosan [32][33]with -OH of carboxylic acid, phenol or alcohol and C -H starching vibration of aliphatic compound of pomegranate peel [34] 1626 Carbonyl group of amides [33] 1521 N -H bending vibration of amide [32] 1371 -CH 3 Symmetricaldeformation mode of amide group [32] 1327 Skeletal vibration of aromatic ring of pomegranatepeel [34] 1094 Interaction between chitosan and Cu-NP's [33] The data listed in table (1) illustrates that the difference in the FT-IR pattern between Cs-NP's and Cs-CuO-NP's is due to the existence of a few peaks belonging to Cs-CuO-NP's at 1327 cm -1 which indicate the existence of skeletal vibration of aromatic ring of pomegranate peel; this is besides peaks at 3550-2902 and 1094 cm -1 , which overlap with the peaks of chitosan. On the other hand, the appearance of a peak at 616 cm -1 and the shifting in many of the peaks' location clearly refers to the interaction between nano chitosan and CuO [32][33][34] [35]. Finally, the data from the above table reveals the formation of hybrid nano-composite between nano-chitosan and copper oxide, capped by pomegranate peel extract residual.

XRD Analysis
XRD analysis describes crystalline structure and assesses the compatibility of each component present in the synthesized composite. Figure 2 shows the XRD patterns of Cs-NP's and Cs-CuO-NP's. The XRD of chitosan nanoparticles (Cs-NP's) had a broad peak at 2θ=25° due to the deformation of the crystalline regions which lead to ionic crosslinking with tripolyphosphate, increasing the packing of chitosan chains resulting in the formation of amorphous chitosan nanoparticles [36]. This could be ascribed as a result of the substitution of hydroxyl and amino groups due to the deformation of the hydrogen bond in the original chitosan chain [37], which e ciently breakdown the regularity of the of the main chitosan chains packing. It is well established in the literature that chitosan derivatives have been signi cantly inhibiting the grampositive bacteria [42], while copper oxide NP showed greater activity against gram negative microorganism which is consistent with the ndings in this study.

Cs
Pomegranate peel extract which acts as the capping agent (con rmed by XRD) was not randomly selected, but, its high tannins and polyphenolic content has been reported as the key factors for the peel antimicrobial activity. The pomegranate peel extract showed a potent sensitivity towards Gram-positive bacteria [43], which is similar to our results; B. subtilis was more sensitive than S. aureus, followed by E.
coli [44] as it could affect the transport of substrates into the cell [45]. Additionally, pomegranate peel extract has signi cant fungal inhibitory activity. Thus, Cs-CuO-NP's were successfully tailored to merge together the activity of chitosan nanoparticles,CuO, and pomegranate peel capping extract to obtain a broad spectrum antimicrobial novel composite. The activity of the nanoparticles is usually ascribed to their small size enabling them to permeate through the bacterial cell membrane [46]. Besides, the positively charged hybrid Cs-CuO-NP's could block the nutrient intake of the cells due to their interaction with negatively charged lipidic bacterial membrane, and thus reducing both cell growth and viability [47]. It is also worth noting that the e cient antibacterial activity of hybrid Cs-CuO-NP's could be due to reactive oxygen species generation by the nanoparticles attached to the bacterial cells, which in turn provoked an enhancement of intracellular oxidative stress [48].

Haematological Test
Undoubtedly that RBC's and platlets play an important role in both thrombosis and hemostasis. RBC's affect the Rheological blood viscosity and platelet aggregation which enable them to act as a procoagulant and prothrombotic blood component. RBC's interact with platelets, endothelial cells, and brinogen, which inturn leads to their incorporation into the thrombin.
In comparison with control blood samples, a noticeable decrease in mean RBC's and platelets counts was observed by adding Cs-NP's to the blood rather than adding Cs-CuO-NP's as shown in gure 5 a & 5 b. Figure 5a shows that adding Cs-NP's decrease the mean RBC's count by 2.8%, 11.1%, and 6.7% in healthy, diabetic, and hyperchlosterolic blood samples respectively. The same decreasing pattern was observed which is determined to be 1.44%, 3.8%, and 5.0% when adding Cs-CuO-NP's into healthy, diabetic and hyperchlosterolic blood samples respectively. However, adding Cs-NP's leads to a decrease in mean platlets count to 22.4%, 24.4%, and 3.0 % in healthy, diabetic and hyperchlosterolic blood samples respectively, in comparison to 11.0% and 2.7% lesser in platlets count upon adding Cs-CuO-NP's into healthy and diabetic blood samples respectively while it increases the platelets count in hyperchlosterolic blood sample (Figure 5b).
The effects of Cs-NP's and Cs-CuO-NP's on the coagulation time of healhy, diabetic and hyperchloesterolic blood samples in vitro were also investigated ( Figure 5c). It was shown in Figure 5c that Cs-NP's are able to decrease clotting time for healthy and diabetic blood samples. An opposite effect was observed in hyperchlosterolic blood sample, while adding CuO capped with P. granaum extract to Cs-NP's (synthesized nanocomposite) act as anticoagulant by increasing clotting time.
The chitosan nanoparticle (Cs-NP's) gains hemostatic properties from its net positive charge which depend on the DD and number of pronated amine groups [50]. These amine groups initiate attraction with negatively charged red blood cells and platlets (Figure 5a and 5b) enabling chitosan to build a mesh-like spatial structure, which promoted interaction between chitosan and blood components facilitating formation of blood clotting. Also Cs-NP's is able to gradually depolymerized to release N-acetyl-Dglucosamine, which is transported to cells via glucose receptors and has arole in protecting against atherosclerosis. N-acetyl-D-glucosamine which initiates broblast poliferation, aids in providing collagen deposition orders and stimulates increased synthesis of natural hyaluronic acid levels at wound sites. It was proved in a previous study that chitosan with moderate DD nearly 68.36% had the most signi cant procoagulant effect [51] [52]. This is attributed to higher degree of DD had more amino groups and hydroxyl groups in the molecules, which form a stronger hydrogen bonds inside the molecules, leading to a crystalline structure of chitosan that could hardly interact with blood components to promote coagulation [51].
Adding Copper oxide nanoparticles (nCuO) to Cs-NP's play a vital role in masking and inhibiting the in ammatory activity of chitosan in addition to enhancing wound healing properties of chitosan [53]. It was proved histologically that nCu are able to stimulate proliferation and migration of broblasts. Some copper dependent enzymes help in the synthesis of collagen to facilitate wound healing. It was clearly known that chitosan is polycationic at acidic media so it chelate metallic ions such as Fe, Cu or Mg [54]. This prove that Cu ions chelate chitosan nanoparticles suppressing sites of interaction with RBCs and platlets. This could account for the increasing RBCs and platlets count in Figure 5 a and b.
On the other side, when comparing the results of Cs-NP's with Cs-CuO-NP's, it was observed that adding Cs-CuO-NP's lead to more RBC's and platlets and clotting time ( Figure. 5) , this is due to presence of P.
granatum extract as capping agent for synthesized composite. It was suggested in previous work that presence of P. granatum inhibit platlets aggregation due to the presence of anthocyanidins in P. granatum that are responsible to supress cyclooxygenase [55] or may be due to the decrease in brinogen level [56]. Increasing clotting time is due to the anti-coagulant effect of P. granatum which inhibit thrombin and intrinsic coagulation factor [57].
This proves that Cs-NP's particles are hemostat, can act as a protrombin or procoagulant while Cs-CuO-NP's are recommended as anti-coagulant.

Preparation of the Green Extract:
A mass of 40 g of pomegranate peel powder is added into 1L of bidistilled water. The mixture is boiled for 30 minutes, followed by ltration to obtain a clear ltrate. This clear ltrate is kept in the fridge at 4 C and is considered as the plant's extract.

Chitosan Nanoparticles Synthesis:
The nano-chitosan has been prepared by the ionic gelation method [27], where 0.5g chitosan was dissolved in 50 ml of 1.0% (v/v) acetic acid. Afterwards, 1.0% (w/v) of the trisodium polyphosphate (TPP) was added to the former solution with constant stirring for 1 hour. The produced white precipitate (nano chitosan) was isolated and washed several times with deionized water. Finally the product was dried in an oven overnight at 60 °C.
3.3 Bio-Synthesis of Chitosan-Copper Oxide Nanoparticles: 0.5 gram of chitosan was dissolved in 50 ml of 1.0% acetic acid. By drop wise, 1.0% of TPP was adding to the former solution. Then 50 ml of 1.0M of copper sulphate was added to the mixture of chitosan and TPP, followed by adding 50 ml of plant extract drop wise with constant stirring and heating at 80°C for 1 hour. The resulting nanoparticles were isolated and washed several times with deionized water, and then dried in an oven at 80 °C.

Structural Assessment
Synthesized nanoparticles were examined via Fourier Transform Infrared (FTIR) spectra to investigate the the presence of functional and characteristic groups using a Shimadzu FTIR spectrophotometer. The spectra were carried out at a resolution of 4.0 cm -1 . To obtain a reasonable signal to noise ratio, 64 scans were completed. The dried nanoparticles were pressed with KBr and tested [28]. To identify the speci c peaks of the Cs-NP's and CuO-NP's, x-ray powder diffractometer using Shimadzu XRD with Cu Kα radiation (λ = 1.5418 Å) at a scanning speed of 0.2 S, was used.

Morphological Characterizations
The morphology and the elemental analysis of the synthesized nanoparticles were performed using SEM (FEI, ISPECT S50, and Czech Republic). SEM was operated at 20 kV with a working distance around 10 mm. The samples were xed on a metallic stub with double-sided adhesive tape. Images were taken at different magni cations to obtain a better visual inspection, and noting important features of the specimens. For TEM, the synthesized nanoparticles were dispersed in ethanol under sonication for 5 minutes, and deposited onto TEM grids with carbon support lm. TEM grids were mounted into the TEM upon evaporation of water in the air at room temperature. The images of the specimens were recorded using TEM, FEI, Morgagni 268, and Czech Republic at 80 kV. Finally, the EDAX analyses were performed using EDX-8000 and , Shemadzu [29][30].

Antimicrobial Test
Antimicrobial activities of Cs-NP's and Cs-CuO nanocomposites were carried out according to NCCLS recommendations (National Committee for Clinical Laboratory Standards,1993). Inhibition zone primary screening tests were performed by the well diffusion method [31]. Inoculums suspension was prepared from using the tested organisms colonies grown overnight on an agar plate. Chitosan nanoparticles and synthesized nanocomposites were dissolved in DMSO with different concentrations (50, 10, 5, and 2.5 mg/ml). The diameter of the inhibition zone indicating antimicrobial activities were measured after a 24 hours incubation at 37 o C. This study investigated Cs-NP's and Cs-CuO-nanocomposites against fungi (C. neoformans and C. albicans), gram-positive (S. aureus and B. subtilis) and gram-negative bacteria (E. coli and P. aeruginosa). BCT was measured by adding a solution of 0.5 ml of Cs-NP's and Cs-CuO nanocomposite into 1.5 ml from each blood sample. The blood was incubated in a water bath at 37ºC for 5 minutes, and then the blood coagulation was observed by inclining the tube at 30 second intervals until the blood is clotted.

Haematological
When the blood ow was not observed up on inclination of the tube at a 90º angle, which indicated blood became coagulant. BCT was measured from the immediately after blood collection until blood coagulation was observed.

Conclusion
Cs-CuO-NP's and Cs-NP's were greenly synthesized and characterized then biological applied to illustrate the following: rstly, the characterization of prepared Cs-CuO-NP's and Cs-NP's found that both biogenic nanoparticles are in spherical shape with particles size around 20-40 nm. The characterization also provide the formation of Cs-CuO-NP's as hybrid nano-composite from nanochitosan and copper oxide capping with pomegranate peel residual. Secondly, the anti microbial activity inhibition zone test for both Cs-NP's and Cs-CuO-NP's show the superiority of Cs-CuO-NP's as antimicrobial agent over Cs-NP's. The results obtained that Cs-CuO-NP's is highly sensitive to C. neoformas, B. subtilis and E. coli at low concentration 10 mg/ml, in opposite at 10 mg/ml concentration of Cs-NP's aganist all microorganisms, under examination, was effected only on C. neoformas. on other hand increasing the concentration of both Cs-NP's and Cs-CuO-NP's to 50 mg/ml increase the sensitivity of Cs-NP's as an antimicrobial agent and also rises by high migraine the ability Cs-CuO-NP's to be lethal for all microorganisms under investigation. While the MIC test clears the role of hybrid composite Cs-CuO-NP's as antimicrobial which found lethal for all microorganisms under test in range of concentration below 20 mg/ml Cs-CuO-NP's, and Cs-NP's found affected only after 20 mg/ml at least for all microorganism except for C.albicans species, it was found lethal at 5mg/ml. This comparison between both biogenic nanoparticles proves the importance of hybrid composite of copper and capping agent with nano chitosan in enhances the antimicrobial properties of any biogenic nanoparticals.
Finally, the hematological activity of both Cs-NP's and Cs-CuO-NP's were examined to prove that Cs-NP's particles are hemostat, acts as a protrombin or procoagulant activator used to accelerate blood clotting process for healthy and diabetic patient to prevent Scar. While Cs-CuO-NP's act as anti-coagulant could be used as a coating for coronary stent or drug delivery to prevent arteriosclerosis.