Chitosan (CS) has shown high potential for a wide range of pharmaceutical purposes [1, 2]. Many studies have been conducted to evaluate the use of CS in the design of sustained release dosage forms, such as matrix tablets, and have shown that CS generally provides magnificent sustained release properties in vitro, especially at low pHs where a gel matrix is developed [3, 4]. A large number of studies have been recently performed on polymer nanoparticles as carriers for delivery of drugs and antigens [5, 6]. CS-NPs were investigated as an adjuvant and vaccine delivery system[7-9]. The absorption enhancing effect of CS-NPs has been proven to improve the mucosal immune response. The mechanism of CS-NP to improve the delivery of antigens across mucosal membranes can be explained using the same theory discussed earlier in the section about peroral administration [10, 11]. Researchers successfully developed CS and CS-NPs vaccines containing antigens of influenza, pertussis, and diphtheria for nasal delivery. They revealed that the vaccines produced a significant amount of antibody in mice, which included serum and secretory Immunoglobulin A (IgA) [12-15]. Besides the potential carrier for mucosal delivery vaccines, it has been reported that CS-NPs could act as an adjuvant for systemic vaccine delivery, such as making the macrophages and poly morphonuclear cells further proliferate and active. Macrophages are activated once the CS-NPs have been absorbed [16-18]. Today, to produce the antivenom, usually antigen are delivered to the horse mostly via intramuscular route . These delivery routes have several weaknesses: high initial concentrations of the antigen that cause side effects. Another reason is related to requirement of multiple injections, because only a very small percentage of the injected antigen reaches the affected area to induce enough immune system. It is therefore important to reduce the number of repeated venom administrations required for immunization over a long period of time and improvement of the production yield [7, 20, 21].
This study was thus conducted to examine the influence of a number of factors on the encapsulation of Vipera albicornuta and evaluate physicochemical properties of CS-NPs supposed to be used as a new antigen delivery system, using FTIR, DLS, and SEM.
MATERIALS AND METHODS
The medium molecular weight (MMW) CS derived from shrimp shells (Pandalus borealis) was purchased from Primex Co (Iceland). The degree of deacetylation was a minimum of 95%. Sodium tripolyphosphate (TPP) and coomassie blue G250 were supplied from Sigma (USA). Phosphoric acid (85%), acetic acid, and absolute ethanol were purchased from Merck (Darmstadt, Germany). Vipera albicornuta venom, prepared as a freeze-dried powder, was provided from Razi Vaccine and Serum Research Institute (Karaj, Iran). Other materials were of pharmaceutical and analytical grades and were used as they were received.
Nanoparticle preparation and venom-loaded
The CS-NPs were prepared using ionic gelation technique of CS with TPP anions [22, 23]. Different concentrations of CS (1.5, 2.0, 2.5, 3.0 mg/mL) were dissolved in acetic acid aqueous solution. Solutions of TPP with different concentrations were prepared afterwards. Eventually, 5 mL of the CS solution was added to 2 ml of the TPP solution dropwise under constant magnetic stirring (1100 rpm, 1 hour) at room temperature. An opalescent suspension formed spontaneously under the above-mentioned conditions. The nanoparticles were separated through centrifuging the suspension at 11,200 g, 14°C for 30 minutes. The freeze-dried nanoparticles were then weighed and stored at 4°C–8°C. In this study, the influence of different parameters, including venom concentrations (20, 30, 40, 50, 100, 200, 300, 400, 500, 750, and 1000 μg/mL), TPP concentrations (0.75, 1 and 1.25 mg/mL) and CS concentrations (1.5, 2, 2.5, and 3 mg/mL) on EE and LC was assessed. Once one of the above-mentioned parameters was under evaluation, the other parameters were kept constant.
Characterization of morphology and structure of nanoparticles
The characterization of morphology and particle size was carried out using a scanning electron microscope (SEM) (INCA-500, United Kingdom) as an accelerating voltage of 15.0 kv after sputtering with gold. The CS-NPs separated from suspension were dried with a freeze dryer, and the FTIR was performed using KBr pellets on Bomem Spectrum. The dynamic light scattering technique was performed to measure the particles’ size, size distribution [polydispersity index (PDI)], and zeta potential, using Zetasizer (Malvern Instruments, UK) [24, 25].
Venom loading percentage
The venom-loaded nanoparticles were removed from the aqueous suspension through centrifugation of the suspension at 11,200 g and 14°C for 30 minutes. The supernatant was removed, and the protein content (free venom) of the supernatant was determined through the Bradford protein assay spectrophotometric method at 585 nm. Equations 1 and 2 were used to respectively calculate the venom LC of nanoparticles and the venom EE of the process. Equation as follows [26, 27]:
LC = (A - B) / C × 100 (1)
EE = (A - B) / A × 100 (2)
A: Total amount of venom; B: Amount of free venom; C: Nanoparticle weight.
Studies on in vitro release of venom from CS-NPs
A certain amount of venom-loaded CS-NPs was suspended in separate test tubes containing equal volumes of 0.2 mol/L PBS solution (pH 7.4) and incubated at 37°C and 600 rpm. At appropriate time intervals (1, 2, 4, 6, 10, 22, 34, 48, 72 hours), the content of one tube was removed and centrifuged at 19,000 g and 10°C for 35 minutes. The Bradford assay was performed to measure the amount of venom released .
RESULTS AND DISCUSSIONS
Characterization of nanoparticles in terms of physiochemical properties
Fig. 1 shows morphological characteristics of nanoparticles. As shown in Fig. 1A as well as Fig. 1B, the shapes of particles are almost similar to each other with smooth semi-spherical or nearly oval appearance. According to DLS results, the mean size of the particles was 155 and 187 nm for CS and loaded CS-NPs, respectively. The mean size of empty and loaded nanoparticles was found to be bigger than that determined by SEM. In comparison with the SEM, the DLS technique measures the hydrodynamic diameter of the particles, including hydration layer and polymer shells, and generally shows a larger particle size . The aggregates seen in SEM images could be attributed to the drying process during sample preparation . The PDI value of CS was approximately the same as venom loaded nanoparticles (0.500 and 0.577), it means that no significant change was observed in PDI (Table 1).
The stability of venom loaded CS-NPs is related to Zeta potential . The particles’ surface charge was somewhat positive, with CS zeta potentials of 46.7 mV and venom loaded nanoparticles of 33.2 mV. The result of this study indicated that venome loading can cause minor reduction in Zeta potential. The venome involves with long chains of CS molcules, and it is not uniform. Due to electrostatic repulsive forces existing between amine groups and the molecular chain, CS molecules probably adopt a diffuse conformation in the solution. On certain sites along the CS chain, hydrogen-amine group bonds might form by carboxyl groups on a large proten molecule’s surface. The CS chain, however, still maintains its own compact three-dimensional structure without diffusing in the relatively acidic solution in order to keep its hydrophobic core . The positive surface charge of CS molecules is thus not suppressed adequately by the protein molecule attachment. There are still possibly a large number of unoccupied amine groups on the CS chain .
The potential of the ionic gelation process for formation of venom-loaded CS-NPs was evaluated through employing FTIR to detect venom-CS interactions. Fig. 2 shows the FTIR spectra of CS-NPs, venom loaded CS-NPs, and venom. In CS-TPP nanoparticles, the wide peak in the 3000 cm-1–3500 cm−1 range, related to the combined peaks of O-H stretching and intermolecular hydrogen bonding, led to decrease bending vibration of amine group. Similar to the primary amines, the N-H stretching is overlapped in the same area. The bond at 3420 cm-1 is related to to NH2 and OH group stretching vibration in CS. The peak around 1075 cm-1 is related to the symmetric stretch of C-O-C that usually appears between 1000 to 1300 cm-1 and 1320 cm-1 representing the C-N stretching vibration. Other peaks under 1000 cm-1 are related to the CS polysaccharide structure. Furthermore, the crosslinked CS indicates a P=O peak at 1157 cm–1 . These results might arise from the link between phosphoric and ammonium ions. It was therefore supposed that the tripolyphosphoric groups of TPP were linked with the ammonium group of CS, and the inter- and intramolecular actions are improved in CS-NPs. The spectrum of Vipera albicornuta venom-loaded CS-TPP nanoparticles and venom were compared, it showed the same peak at 1455 cm–1 and 1541 cm–1, so we conclude that the structure of venom remained intact in loading duration [27, 28].
Effects of CS concentrationss
A particle size analyzer was applied to evaluate the effect of different CS concentrations (2, 2.5, & 3 mg/mL) on particle size. The results showed that an increase in the CS concentration from 2 mg/ml to 3 mg/mL while the TPP concentration (1 mg/mL) was constant, led to an increase in the size of nanoparticles (Table 1). The nanoparticles with CS concentrations of 2 mg/ml and 3 mg/mL did not show a PDI value within the acceptable range due to the formation of large-diameter aggregates. The PDI value of nanoparticles was desirable at the CS concentration of 2.5 mg/mL. The results also revealed the effect of CS/TPP mass ratio on EE and protein release profiles. It can be therefore concluded that lower CS/TPP ratios lead to higher encapsulation of proteins . Resistance of liquid phase decreases against dispersion with a lower concentration of CS leading to formation of smaller size of nanoparticles and increase venom encapsulation [33, 34]. The molecular weight of CS is generally a significant factor for specifiying the optimum CS/TPP mass ratio [32, 35]. Considering the results of this study and results of previous studies about the optimum encapsulation and overall release, it may be argued that the venom loading is well supported using the CS concentration of 2.5 mg/mL and TPP concentration of 1 mg/mL.
Effects of venom concentrations on LC and EE
The effect of venom concentrations (20, 50, 100, 200, 300, 400, 500, 750 and 1000 μg/mL) on EE and LC was examined in this study. The results indicated that an increase in the venom concentration from 20 μg/mL to 750 μg/mL led to an increase in EE though it remained unchanged atvenom concentrations higher than 750 μg/mL, (Fig. 3). The results also showed that the concentrations higher than 50 μg/mL of venom highly contributed to the venom loading . As shown in Fig. 4, the nanoparticles’ loading capacity increased even more than 100% at higher venom concentrations (400 μg/mL to 1000 μg/mL). Previous reports on the effect of protein concentration on encapsulation are incomprehensive and sometimes contradictory. However, this study conforms to those of some researchers. The results of this study do not agree with those obtained by Xu and Du , who reported reverse results about encapsulation on bovine serum albumin (BSA) at pH 6.0. Jarudilokkul S et al. also reported the same result for α-lactalbumin, cytochrome C, and ribonuclease A . As a cross linking agent, TPP may react with free amine groups on both protein and CS molecules, developing more compact protein CS-NPs. The amount and size of protein on the surface of the nanoparticles increase by the excessive adsorption of protein on the surface of the nanoparticles . In this respect, the suggestion is that venom concentrations higher than 400 μg/mL are not suitable for this application, because they may lead to additional adsorption of venom on the surface of nanoparticles, which in turn results in a decrease in zeta potential and aggregation of particles .
The antivenom product is derived from equine hyperimmune serum and capable of neutralizing the poison of the six greatest common snakes in Iran, including 5 viper species (Echis carinatus sochureki, Vipera lebetina obtusa, Vipera albicornuta, Agkistrodon halys, and Pseudocerastes persicus) and an elapid species (Naja naja oxiana) . This product has been professionally used to treat snakebites.
Our group previously reported the CS-NPs loaded with various antigens such as Naja-Naja Oxiana , Mesobuthus eupeus , Hemiscorpius lepturus , Echis Carinatus  and Tetanus toxoid (under publication). For Naja-Naja Oxiana, the optimum LC and EE of venom at a concentration of 400 μg/mL and TPP concentration of 1 mg/mL were obtained for LMW of CS at a concentration of 2 mg/mL. In respect of the Mesobuthus eupeus’ optimum encapsulation and loading capacity, it may be suggested that 2 mg/mL of CS and 1 mg/mL of TPP are suitable concentrations for 400 µg/mL venom loading (LMW CS). Moreover, optimum EE and LC were observed when the CS concentration (with low-MW CS) and Hemiscorpius lepturus venom were 2 mg/mL and 400 μg/mL, respectively. But in our study we used CS with MMW. In comparison, report of Mohammadpour, et al for Naja-Naja Oxiana, Mesobuthus eupeus and Hemiscorpius lepturus venom, with LMW of CS loading capacity can be increased about Vipera albicornuta venom with MMW of CS, however we could reduce concentration of venom (Table 2). Also Table 2 shows greater EE for MMW-CS-NPs than that for LMW-CS. This is probably caused by the longer chains of MMW CS, which can generate higher amount of venom when being gelated with TPP.
In vitro release
The result of this study revealed that about 40% of the loaded venom was released during the first 36 hours of incubation in PBS. The release of venom from the NPs showed an initial burst release within initial hours of incubation followed by slow release for 40 hours (Fig. 5). The observed burst was caused by the breakdown of protein molecules that were loosely bound to the surface of CS-NPs and released from the surface of nanoparticles. [11, 41]. Regarding the second part of the release profile, the slow release of entrapped protein molecules at an approximately constant rate arouse from the slow decomposition of nanoparticles. After 36 hours, the protein degradation rate preceded the release rate .
In this study, CS-NPs loaded with Vipera albicornuta venom were prepared through recently recently optimized ionotropic gelation method, using TPP as the crosslinker to examine the physicochemical properties of nanoparticles. The MMW chitosan was used in this study. The optimum concentrations of CS, venom, and TPP were respectively 2.5 mg/mL, 400 μg/mL, and 1 mg/mL; the optimum CS/TPP ratio was 5:2. Venom-loaded nanoparticles with size of 187 nm, LC of 86%, EE of 94% and acceptable PDI were prepared under the above-mentioned conditions. Moreover, it was found that the controlled release properties may be affected by different factors, such as CS and venom concentration. Therefore, the CS-NPs prepared in this study appears to be an alternative for conventional delivery systems.
The researchers hereby appreciate the laboratory staff and library staff in Razi Vaccine and Serum Research Institute (RVSRI) for their assistance.
CONFLICT OF INTEREST
The researchers report no conflicts of interest and are responsible for the content and writing of the article.