The Effect of Chitosan Capped Copper Nanoparticles on Angiogenesis in Cam Assay

Angiogenesis is the growth of blood vessels from the existing
vasculature [1] that occurs during embryonic development,
wound healing, and the menstruation cycle. Moreover, unregulated
angiogenesis...


Introduction
Angiogenesis is the growth of blood vessels from the existing vasculature [1] that occurs during embryonic development, wound healing, and the menstruation cycle. Moreover, unregulated angiogenesis (upregulation or downregulation) is seen in pathological conditions, such as psoriasis, diabetic retinopathy, and cancer [2]. Chitosan is a natural cationic polysaccharide and one of the natural polymers and obtained by deacetylation of α-chitin [3,4]. It has been included in studies that chitosan molecule is an effective transportation tool for reaching the related target with the pharmaceutical properties of various drugs [5]. Chitosan-based nanoparticles also have major effects such as anti-cancer, antibacterial, anti-angiogenesis. Moreover, it was also demonstrated that chitosan nanoparticles suppress lipopolysaccharide-induced inflammatory cascade, which is prominent in Crohn's disease and ulcerative colitis [6]. It is known in the literature that chitosan has a procoagulant effect. Nanotoxicity study of chitosan nanoparticles was done in animal models. As a result of these studies, chitosan nanoparticles showed good biosafety [7].
Copper enters the human body through water and food, is mainly carried by plasmic proteins such as ceruloplasmin albumin, and its metabolism is in the co-factor structure of many enzymes [8]. They possess a great application in different fields such as antimicrobial activity, antifungal activity, sensors, catalysts, cytotoxicity "anticancer" activity, antioxidant activity [9]. The mesodermal layers of the allantoic and chorion fuse to form the chorioallantoic membrane (CAM) throughout avian development.
CAM is an effective model to determine the behavior of normal and pathologic tissues (as cultured cells or malignant tumors).
Using of various drugs, antibodies or biomaterials which placed on its surface allows us to observe angiogenesis [10]. CAM assay is preferred model by virtue of some advantages including, high embryo survival rate, easy methodology, sterility is not required, low cost, reproducibility, reliability [11]. In this study, the effects of chitosan capped copper nanoparticles on angiogenesis in chick chorioallantoic membrane (CAM) model were investigated.

Synthesis of Dithiocarbamate Chitosan (DTCCS)
DTCCS were prepared with minor revision according to the method defined by Muzzarelli et al. [12], A mixture of 4g of chitosan and 0.025 mol of ammonia was stirred in ethanol 95% (v / v) at room temperature for half an hour. A solution of carbon disulfide CS 2 (2 mL) was then added slowly dropwise to the solution. After stirring about 2 hours, the resulting mixture was filtered under vacuum with a filter paper and the remnant was washed by 100% of ethanol. After washed the polymer dried at 60°C. Finally, DTCCS was obtained.

Synthesis of DTCCS Capped Copper Nanoparticles
The synthesis of copper nanoparticle was done by alteration of the procedure used by Dang et al. [13], Firstly, 0.1 gr of dithiocarbamate chitosan DTCCS was dissolved in 100 ml of %1 acetic acid, then the polymer solution was added dropwise to the solution of 0.105 gr copper nitrate (Cu (NO 3 ) 2 .3H 2 O) in 10 ml distilled water, while the color of copper solution changed from blue to light blue color. After stirring at 10,000 rpm by a high-speed homogenizer for 15 minutes the PH of the solution was changed from PH=3 to PH=6 by 1M of NaOH solution, after adding NaOH the solation color changed to be dark blue. After stirring for about 30 minutes by high-speed homogenizer 10 ml of NaBH 4 was added to give a rapid brown coloration which turned to dark red after about 10 minutes of mixing. The solution was stirred for an additional 30 minutes to complete the reaction. after boiling the solution the copper nanoparticles were precipitated by centrifugation at 5,000 rpm for 15 minutes. The particles were washed repeatedly to ensure purity. Finally, the Cu-NPs were dried at 40 °C.

Fertilized Chicken Egg Preparation
For the experiment, we put 36 fertilized chicken eggs in an incubator at 37°C and 85-90% relative humidity. We kept it in the incubator for 5 days. Then, on the 5 th day, we pierced the eggshell in the place corresponding to the CAM of the chicken eggs. We applied the CuNPs solution on the CAM and covered the hole of each egg with paraffin tape. Then we put the eggs back in the incubator.
We kept it in the incubator at suitable temperature and humidity for 48 hours after the application. At the end of the 48th hour, we evaluated the vascular development macroscopically on the CAM and photographed with a stereomicroscope attached to a digital camera.

Application of the Solution to Fertilized Chicken Eggs
On the 5 th day, we pierced the eggshell in the place corresponding to the CAM of the chicken eggs. Before the application, we photographed the CAMs of the eggs with a stereomicroscope connected to a digital camera. We applied 15 mM and 25 mM of CuNPs solution to each CAM surface. After the application, we closed the holes of each egg with paraffin tape. We grouped and numbered eggs with 15mM and 25mM CuNPs solution applied [14]. Then we kept it in the incubator for 48 hours at suitable temperature and humidity. On the 7th day, we removed the paraffin tape on the CAM. All eggs were observed macroscopically and photographed with a stereomicroscope connected to the digital camera. Using the Knighton et al. score, the vascularization increases in the photographs taken on the 5th and 7th days were scored by 3 observers [15].

Result
On the 5 th day we pierced the eggshell. We used 36 eggs for this study. We established 3 eggs died before the application of CuNPs  Table 1.

Discussion
Angiogenesis is the process of generating new blood vessels and have essential roles in many pathological conditions [16].
Because of high sensitivity, being cheap and easy CAM model of chick embrios is mostly used as an in-vivo working model for angiogenesis [17]. Copper is a essential microelement required by animals and humans. Although the content of Cu in the human body is very low but it plays an important and multifunctional role as a cofactor of many enzymes [18]. Cu has essential role in angiogenesis as a stimulator of angiogenesis, vasculogenesis and endothelial cell migration [19]. Cu has been shown to stimulate angiogenesis in chick embryo chorioallantoic membrane (CAM) models [20].
Chitosan is the product of the complete or partial deacetylation of chitin. Chitosan is a widely used excipient for topical or oral drug administration. It is also now considered as a useful material for drug and gene delivery systems for parenteral administration [21].
Chitosan has been shown to inhibit angiogenesis in chick embryo chorioallantoic membrane (CAM) models [22].
Up to now, no study has been observed about angiogenic  [22]. In another study, Ribeiro and colleagues have shown that insulin-containing chitosan nanoparticles stimulate angiogenesis [24]. We thought this effect could be due to insulin.
Similarly, in our study, chitosan coated copper nanoparticles stimulated angiogenesis. We assumed that this effect may be due to copper nanoparticle as a result of our literature review.
Consequently, Chitosan and copper nanoparticles are biomaterials in which experimental research are carried out in many areas such as cancer treatments, drug administrations, scaffolds. Therefore, it is essential to know the mechanism of action of these molecules. In our study, we also focused on the effects on angiogenesis which has an important role in many pathology processes. As a result of the study, we established that chitosan coated copper nanoparticles stimulate angiogenesis. Further studies are required.