Highly Monodisperse Chitosan Nanoparticles Prepared by a Combined Triple-Method for Potential Use as Drug Carriers

Citation: Saleh TA (2016) Surface Enhanced Raman Scattering Spectroscopy for Pharmaceutical Determination. Int J Nanomater Nanotechnol Nanomed 2(1): 029-06. DOI: 10.17352/2455-3492.000012 farzadeh M, Tahe i S, Mohamad Sadeghi GM (2017) Highly Mon dispe se Chitosan Nanoparticles Prepared by C bined Tripl -Method for Potential Use as Drug Carriers. Int J Nanomater Nanotechnol Nanomed 3(1): 001-006. DOI: http://doi.org/10.17352/2455-3492.000013 Abstract


Introduction
Chitosan (CS) is a high molecular weight polysaccharide composed of -(1,4)-2-acetamido-2-deoxy-D-glucose and -(1,4)-2-amino-2-deoxy-D-glucose units. It is a natural polymer, non-toxic, biodegradable, which has been found a fascinating candidate in a broad spectrum of applications such as targeted drug delivery, wound dressing, tissue and cell engineering, radiopharmaceuticals and cosmetic industries, along with unique biological properties including biocompatibility, physiological inertness, remarkable affi nity to proteins, antibacterial, haemostatic, fungistatic, antitumoral and anticholesteremic properties [1][2][3]. In the form of microparticles and nanoparticles, CS has a variety of important applications. Loaded with DNA plasmids, CS nanoparticles could give rise to protective immune responses in rodents [4].
Likewise, the use of CS nanoparticles as nanomedicine and drug carriers has been extensively reported before [5][6][7]. For example, insulin-loaded chitosan nanoparticles could enhance intestinal absorption of insulin and increase its relative pharmacological bioavailability [8]. In enhancing genetransfer effi ciency in cells, CS nanoparticles have been proved to be valuable assets [9].
However, due to its high molecular weight and viscosity, chitosan has a low solubility (above pH 6.5) in a variety of solvents, which limit its applications, especially when functionalities such as "lipid binding" is required (nanomedicine and targeted drug delivery) [10]. To overcome this issue, a popular approach is to reduce the molecular weight of chitosan. It has been reported that low molecular weight chitosan (LWCS) shows an outstanding water solubility and biological, chemical, and physical functionality, providing that its chemical structure is not changed [11]. A common synthetic rout to obtain LWCS is enzymatic degradation of CS with hydrolytic enzymes or acidic degradation by hydrochloric acid and sulfuric acid [12,13]. Recently, LWCS was also formed by oxidative degradation with some oxidative systems [14][15][16]. The degradation patterns of chitosan using O 3  have been reported [14,15]. Tanioka et al. [16], and Chang et al. [17], respectively reported that Cu (II)-UV-H 2 O 2 and H 2 O 2 -Fe 2+ systems could decrease the molecular weights of chitosan.
They postulated that metal ions induced the decomposition of H 2 O 2 , which caused the degradation. Nonetheless, the capacity of chitosan to form complexes with metallic ions has made ionic-metal-driven oxidative systems less desirable; especially when it comes to biomedical application [18]. On the other hand, when H 2 O 2 is used alone, oxidative degradation is an ineffi cient method to obtain a monodisperse nanoparticle system. To improve the effi ciency, we propose a triple-method treatment of oxidative degradation, ultrasonication and ionic gelation.
The use of ultrasonication in nanotechnology has been ubiquitous in recent years. Nevertheless, its effects on CS nanoparticles are not well understood, in spite of a plethora of research works that are devoted to ultrasound-assisted techniques in CS nanoparticle preparation. Also, the ionic gelation method has been separately used to prepare CS nanoparticles. Bodmeier et al. [19], was the fi rst to report the ionic gelation of chitosan using sodium tripolyphosphate (TPP), while Alonso et al. [20] developed chitosan nanoparticles by adding a solution containing TPP into an acidic phase (pH 4-6) containing CS. The study of Wu et al. [21], showed that the formation of nanoparticles is possible only within specifi c, moderate concentrations of chitosan and TPP. Furthermore, the chitosan/TPP weight ratio should be within the range of 4:1-6:1 in order to obtain a high yield of nanoparticles [22].
The study of Tsai et al. [23], showed that the particle size of chitosan nanoparticles prepared by the ionic gelation method can be infl uenced by using different mechanical energies such as ultrasonic radiation or mechanical shearing, different treatment times, different chitosan concentrations, and different solution temperatures.
Despite a relatively large work that has been conducted in this area, a series of defi ciencies still prevail. For example, the feasibility of producing LWCS by using hydrogen peroxide has been overlooked due to the additional procedures that it requires. Moreover, the use of parallel techniques to reach the optimum system is terms of parameters such as mean average size, Zeta potential, and uniformity has been minimal. Taking the antecedents into account, the aim of the present study is to prepare low molecular weight, water-soluble chitosan nanoparticles by means of a unique combined treatment of oxidative degradation, ultrasonication and ionic gelation.
Specifi cally, achieving an extremely narrow size distribution is one of the priorities of the current research, since a narrow distribution is vital in drug delivery to ensure identical biological response by each particle.

Materials
Middle-viscous crab shell chitosan (CS) was purchased from Sigma-Aldrich (United States, cat. no. 28191). Then, WSCS was vacuum fi ltered with the aid of appropriate fi lter paper, while the LWCS was submitted to ultrasonic irradiation with an amplitude of 100 Hz for 20 minutes in order to break the chains further.

Ionic gelation
Ionic gelation was carried out for both LWCS and WSCS.
The pH of the solution was adjusted to approximately 5 by the addition of acetic acid. Then, the TPP solution was added to the chitosan solution drop wise until the resulting mixture became opaque. For separating nanoparticles from microparticles, the solution was centrifuged at 6000 rpm for 30 minutes again.
The upper phase was fi ltered by means of syringe fi lters with two different mesh sizes (450 nm and 220 nm). The fi ltered solution was freeze-dried for 72 hours to be used for further uses. The prepared samples were named indicating the type of nanoparticles and the used mesh size, as WSCS-450 or LWCS-220 stand for water-soluble nanoparticles that were fi ltered by a 450 nm mesh size, and low molecular weight CS nanoparticles that were fi ltered by a 220 nm mesh size and were submitted to ultrasonic irradiation, respectively.

Characterization
Chitosan nanoparticles were characterized by a Bruker Where [ ] is the intrinsic viscosity and M v is viscosity molecular weight of polymer. In order to calculate the M v , k and  are assumed to be constant (1.81×10 -3 mL/g and 0.93, respectively) in a solvent system of 0.1 M CH 3 COOH/0.2 M NaCl at 25℃ [25]. An AIS2100C scanning electron microscope (SEM) was used to evaluate the surface morphology of nanoparticles.
One drop of each chitosan solutions was put to the glass lam, dried at room temperature, gold-coated and prepared for SEM.
Transmission electron microscopy (TEM) was performed to monitor the shape and structure of water-soluble nanoparticles more accurately. The samples were immobilized on copper grids and dried at room temperature before testing by a Philips    [11]. It was particularly important to detect the 1647 cm -1 band, which showed that the carbonyl group is formed during the degradation of chitosan.

Results and Discussion
Vibrations in the range of 1154-896 cm -1 are assigned to the characteristics of chitosan's polysaccharide structure [11].
In comparison with the spectrum of CS, those of LWCS and WSCS revealed almost all of the characteristic vibrations of the original chitosan, indicating that the chemical structure of chitosan was not changed. A few changes about the intensity and wavenumber of a given peak might be due to the destruction of the intramolecular and intermolecular hydrogen bonds and the decrease in the degree of crystallinity in the polymer [11].
Specifi cally, an additional weak peak in the spectrum at 1250 Nevertheless, the small volume fraction of the bigger particles (24%) and the narrow size distribution (PDI = 0.158) can lead to the conclusion that a relatively uniform system was achieved for the aforementioned sample. By increasing the mesh size, the average size distribution and PDI both increased expectedly. affecting the degree of deacetylation of chitosan samples [27].
From Table 1, it can be seen that the Zeta potential for both systems were between 30-36 mV. This suggests that the electrostatic repulsion between neighboring particles were intense enough to yield a stable solution. Achieving stable, positively-charged CS nanoparticles is particularly important in drug delivery applications and cell-polymer interactions, where the cell membrane is often negatively charged [28].
The SEM micrographs of CS nanoparticles derived from corresponding thick solutions (LWCS, and WSCS) are shown in