Synthesis of Nanocomposition of Poly Acrylic Acid/Chitosan Coated-Magnetite Nanoparticles to Investigation of Interaction with BSA and IGG Proteins

Among the nanomaterial being applied for treatment and diagnosis fi eld, magnetic NPs especially magnetite phase of iron oxide have been signifi cantly interested due to their natural magnetic properties. In this study, a nanocomposition of poly (acrylic acid) and chitosan with Fe3O4 NPs were prepared in order to investigation for drug delivery systems and plasma protein adsorption. Synthesized NPs were studied by various technics including XRD, TEM, SEM, FT-IR, VSM, DLS, zeta potential and AAS. These NPs have superparamagnetic property that is essential for magnetic NPs to applying in body and drug delivery. Plasma protein adsorption is one of the most important issues for failure of nanocarriers function. In this work, BSA and IgG were used as models of proteins to study adsorption of protein onto NPs by UVVis spectroscopy method. Adsorption behavior of NPs for each protein was different. PAA-Fe3O4 NPs represent the lowest albumin adsorption and can be expected that have lower adsorption of protein in the body. The release profi le of ascorbic acid from PAA-Fe3O4 NPs represents no sensitivity to pH, and NPs showed approximately same release behavior in pH 7.4 and 5.8. Research Article


Introduction
Nanomaterials with special structural and physical properties can be used to improvement of treatment and diagnosis of disease [1]. Among the nanomaterials being applied in this fi eld, magnetic nanoparticles (NPs) especially magnetite phase of iron oxide (Fe 3 O 4 ) has been signifi cantly interested because of their natural magnetic properties [2]. Biocompatible polymers and surfactants have been applied with NPs to achieving these purposes [3].
There are barriers that can limit the role of NPs in the body. This may be happened by obstructing their movement, changing in physical properties of NPs or by produce a negative response from the body to NPs [4]. Interaction between NPs and proteins in biosystems has an important effect on the behavior of NPs [5]. Upon administration, proteins in plasma can attach to NPs leading to recognition by the reticuloendothelial system (RES) or mononuclear phagocyte system (MPS) tissues. This causes to collection of NPs in liver, spleen, and bone [6]. In addition, activation of immune system and non-targeted interaction are problems that can be happened because of interaction with plasma protein [4]. This attaching of protein onto NPs forms a shell around NPs which is named "protein corona". The physicochemical properties of this shell can infl uence on biological responses [7]. Therefore, nonspecifi c interaction with proteins in blood circulation is one of the most important that can limit NPs function in body and fi nally remove them from blood stream [4]. Grafting poly (ethylene glycol) to nanomaterials is the most applied approach to reduce nonspecifi c protein adsorption and phagocyte uptake.
Infl uences of nanomaterials properties like size and surface chemistry to adsorption of protein are poorly studied [1].
However, physicochemical features of nanomaterials and how long being in biological mediums can affect the forming of protein corona. Therefore, these infl uence on biodistribution of nanomaterials and their functions [8].
In this work, Fe 3 O 4 NPs were synthesized and then coated by chitosan. Then, chitosan coated NPs reacted with poly (acrylic acid) (PAA). These NPs have positive and negative surface charge respectively. Coating of NPs prevents them from agglomeration or non-specifi c interaction with cells.
PAA is poly anionic with carbocyclic acid groups which have good interaction with amine groups on the chitosan [9].
Here we studied NPs interaction with bovine serum albumin (BSA) and Immunoglobulin G (IgG). Albumin and IgG are the most plentiful proteins in human serum [10], but have a different isoelectric point which can infl uence on protein adsorption behavior [11]. Thus, these proteins are good choices to plasma protein adsorption. Moreover, other important properties of NPs for applying in drug delivery systems such as magnetic properties, size, shape and surface charge were studied. This nanocomposition showed relatively good characteristic for applying in biological environment.

Methods
Ch itosan modifi ed of Fe 3 O 4 NPs were synthesized by our pervious published method with some modifi cation [12].  Brin e shrimp mortality procedure was applied to assessment of NPs toxicity [13,14] h, the live brine shrimps were counted for each tube along with control (without NPs).

Results and Discussion
Fer r ic and ferrous salts were used for synthesized Fe 3  Cha r ged NPs can also infl uence on adsorbed protein structure. For example, BSA exposed to positive charged NPs has more changes in comparison of negative charged ones [22].
FT-I R spectroscopy was used to confi rm the reaction and characterization of the functional group at the surface of NPs.
The FT-IR spectra of obtained products were presented in  In presence of magnetic fi elds, NPs can be controlled and use in magnetic targeting. Furthermore, by applying a magnetic fi eld with a high frequency, these NPs can be used in hyperthermia treatment. In this treatment cancer cells can be killed at around 42-45 ºC [24,25].

Adsorption of biomolecules by NPs in biological environment
can affect or prevent functions of NPs [26]. Moreover, it is believed that interaction between nanomaterials and proteins is the primary step to toxicity of nanomaterials [27]. Plasma protein adsorption onto surface NPs and recognition by MPS phagocytes is called opsonization. This causes that RES recognize NPs in body and remove them from blood circulation [1,16]. Moreover, thrombosis or anaphylaxis can be happened by modifying the activation of enzymatic cascades through protein adsorption [1]. For achieving longer circulation in body, NPs must have low electrostatic interaction with proteins that can be obtained via net neutral surface [18]. Furthermore, cell internalization of NPs, can be affected by protein adsorbed NPs. In the absence of protein, NPs have stronger interaction with membrane of cells and internalization can be increased.
But, forming protein corona in presence of serum protein internalization reduces [21]. For achieving successful targeting, the targeting ligand on the NPs must have a more compatibility for its receptor than serum proteins. Adsorbing serum proteins onto targeting ligands on the NPs is responsible for the challenges related to in vivo NPs targeting [22]. NPs properties such as shape, size and surface chemistry infl uence on protein adsorption.
Protein adsorption of NPs was studied by UV-Vis spectroscopy method ( Figure 5). Albumin and IgG were selected as models of protein that are two most abundant proteins in human blood plasma, respectively [10]. Coulomb interactions are very important in forming protein corona. But, these interactions are actually effective only between charges placed at the surface of NPs and proteins that are in close contact [7]. Selected proteins have different isoelectronic points and charged NPs can interact with them. Isoelectric point of IgG is greater than 5.5 and can adsorb at the surface of negatively charged NPs. But, isoelectric point of albumin is less than 5.5 and can adsorb onto positively charged NPs [11]. As have showed in Figure 5,    [25].
Since the MRI contrast agents would eventually be used in living body, it was necessary to evaluate their toxicity.
Evaluation of toxicity of NPs is necessary to confi rm that NPs are suitable for applying in drug delivery systems. Toxicity of NPs was studied on brine shrimps. 10 brine shrimps were incubated in 10 mL dispersion of NPs (1 μg/mL) and after 24 h survival brine shrimp counted. The result was showed in Figure   7 and NPs were showed good biocompatibility and represented no signifi cant toxicity. FT-IR, DLS, zeta potential and AAS showed that reactions have successfully curried out. Our study showed that both