Preparation and characterization of insulin nanoparticles using chitosan and Arabic gum with ionic gelation method

Preparation and characterization of insulin nanoparticles using chitosan and Arabic gum with ionic gelation method

Available online at Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 58 – 63 Original Artic...

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Available online at

Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 58 – 63

Original Article

Preparation and characterization of insulin nanoparticles using chitosan and Arabic gum with ionic gelation method Mohammad Reza Avadi, PhDa,b,⁎, Assal Mir Mohammad Sadeghi, PhDb , Nasser Mohammadpour, PharmDc , Saideh Abedin, PharmDb , Fatemeh Atyabi, PhDd , Rassoul Dinarvand, PhDd , Morteza Rafiee-Tehrani, PhDd a

Faculty of Pharmacy, Azad University of Medical Sciences, Tehran, Iran b Hakim Pharmaceutical Company, Tehran, Iran c Razi Vaccine and Sera Research Institute, Karaj, Iran d Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran Received 9 February 2009; accepted 19 April 2009

Abstract In the past decade, many strategies have been developed to enhance oral protein delivery. The aim of the current work was to develop a nanoparticulate system based on ionic gelation between chitosan and Arabic gum for loading of insulin. Various formulations were prepared using 23 factorial designs. The optimum association efficiency was obtained for formulations F2, F5, and F8. The release profile of insulin in phosphate buffer solutions (pH 6.5 and pH 7.2) is completely different than that in acidic medium (pH 1.2). Increased solubility of chitosan in acidic medium and better swelling of Arabic gum chains at pH N6.5 resulted in lower insulin release of nanoparticles at pH 6.5 in comparison with that of the other pH mediums. The values of the exponent n were 0.49 and 0.82 for formulations F8 and F5, respectively, indicating a non-Fickian transport. This suggests that release is possibly controlled by diffusion or relaxation of the polymer chains. From the Clinical Editor: This paper summarizes the development of a nanoparticulate system based on ionic gelation between chitosan and gum Arabic for oral delivery of insulin. If preclinical studies in animal models will indicate reliable and quantifiable delivery of insulin, this method may pave the way to a novel and less invasive way of administering insulin to diabetes patients. © 2010 Elsevier Inc. All rights reserved. Key words: Arabic gum; Chitosan; Factorial design; Ionic gelation; Nanoparticle

Many attempts have been made to improve the gastrointestinal uptake of poorly absorbable drugs such as insulin.1 The obvious disadvantages of invasive injections compared with noninvasive oral delivery are the low patient compliance, the high costs of a sterile manufacturing process, and the need for qualified personnel to administer the injections. Furthermore, a potential advantage of oral insulin delivery would be a hepatic versus systemic ratio more closely resembling that of the natural state than that of subcutaneous injections.2 In fact, oral delivery may circumvent the reduced patient compliance associated with prenatal delivery.3 However, the bioavailability of orally administered biotechnological drugs, such as proteins and vaccines, are usually low due to No conflict of interest was reported by the authors of this article. ⁎Corresponding author: Faculty of Pharmacy, Azad University of Medical Sciences, Tehran, Iran. E-mail address: [email protected] (M.R. Avadi).

the harsh gastric and enzymatic intestinal environments and the poor gastrointestinal mucosal permeability.4 In the past decade, many strategies have been developed to enhance oral protein delivery.5,6 Among these approaches, nanoparticulate systems have attracted special interest for three reasons. First, nanoparticles are able to protect active agents from degradation.7 The second reason is related to improving drug transmucosal transport8 and transcytosis by M cells. Third, the particulate systems can provide controlled-release properties for encapsulated drugs.9 Moreover, mucoadhesive properties also play an important role in oral drug delivery systems by prolonging the residence time of drug carriers and also increasing the intimacy of contact between drug and mucous membrane at the absorption sites; therefore, enhancing the permeability and reducing the degradation of drugs.10 Chitosan, a (1,4)-2-amino-2-deoxy-β-D-glucan, is a deacetylated form of chitin, an abundant polysaccharide present in crustacean

1549-9634/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2009.04.007 Please cite this article as: M.R. Avadi, et al, Preparation and characterization of insulin nanoparticles using chitosan and Arabic gum with ionic gelation method. Nanomedicine: NBM 2010;6:58-63, doi:10.1016/j.nano.2009.04.007.

M.R. Avadi et al / Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 58–63 Table 1 Parameters used in the factorial design


Table 2 Types of insulin nanoparticles based on 23 factorial designa


Low level

High level

Formulation code




y%, mean ± SD (N = 3)

x1, chitosan concentration (mg/mL) x2, Arabic gum concentration (mg/mL) x3, insulin amount (mg)

1 1 5

10 5 10

F1 F2 F3 F4 F5 F6 F7 F8 F9b

1 10 1 1 10 10 1 10 1

1 1 5 1 5 1 5 5 1

5 5 5 10 5 10 10 10 5

15.5 ± 1.81 31.2 ± 2.83 22.6 ± 4.32 18.4 ± 3.65 35.8 ± 4.31 20.6 ± 3.43 17.7 ± 4.21 37.5 ± 2.75 10.43 ± 3.65

shells. Chitosan is a nontoxic polymer that has been used in biomedical fields in the forms of sutures, wound coverings, and as artificial skin.11 Chitosan is a biodegradable and biocompatible polymer that due to its cationic nature has good mucoadhesive and membrane permeability–enhancing properties.12 Hence, chitosan has been extensively investigated for its potential as an absorption enhancer across intestinal epithelium for peptides and proteins.12 Chitosan is insoluble under alkaline and neutral conditions but is able to react with inorganic and organic acids such as hydrochloric acids, acetic acid, and glutamic acid under acidic conditions. The mucoadhesive property of chitosan is mediated by its ability to spread over the mucous layer and additionally through its positive ionic interactions with the negative charges of the mucus or of the cell surface membranes.13 Arabic gum (acacia), a biocompatible and biodegradable polymer, is mainly used in oral and topical pharmaceutical formulations as a suspending and emulsifying agent.14 It is also used in the preparation of lozenges and as a tablet binder. Arabic gum has also been evaluated as a bioadhesive in novel tablet formulations and modified-release tablets.14 The first chitosan nanoparticles were prepared by Calvo et al using an ion gelation technique between chitosan with positive charge and tripolyphosphate (TPP) with negative charge.15 However, TPP is a chemical agent with limited sites of interaction for ionotropic gelation. On the other hand, Arabic gum is a biocompatible and biodegradable polymer with more interaction sites and negative charge for interaction with polycationic polymers such as chitosan. The aim of this study was to develop nanoparticulate systems based on ionic gelation between chitosan and Arabic gum for loading of insulin nanoparticles. The obtained insulin nanoparticles were characterized in terms of particle size determination, zeta potential, and insulin loading and release. Moreover, the effects of some parameters, such as the concentration of chitosan and Arabic gum and the amount of insulin, on association efficiency of insulin nanoparticles were investigated using a 23 factorial design.

a Association efficiency (run in triplicate) was selected as the dependent variable (y). b The three independent variables are the same with formulation F1, except the chitosan solution was added to the Arabic gum solution.

Preparation of insulin nanoparticles The insulin nanoparticles were prepared by ionic gelation between positively charged chitosan and negatively charged Arabic gum. Initially, known amounts of chitosan were dissolved in 1.0% acetic acid aqueous solution to obtain concentrations of 1 mg/mL and 5 mg/mL (Table 1) under stirring at room temperature. Subsequently, different amounts of insulin according to the factorial design were added to chitosan solution under magnetic stirring at room temperature. Consequently, Arabic gum was dissolved in water to obtain a known concentration according to Table 1 under magnetic stirring at room temperature. Nanoparticles were prepared by adding Arabic gum solution dropwise to chitosan solution containing insulin under gentle magnetic stirring at room temperature. The nanoparticle suspension was centrifuged (sigma 3K30, Osterode, Germany) for 15 min at 14,000 rpm at 4°C, and the supernatant was used for measurement of free insulin by high-performance liquid chromatography (HPLC; SDV 505, Anyang, South Korea). Factorial design experiment Insulin nanoparticles were obtained based on 23 factorial designs. Chitosan concentrations (x1), Arabic gum concentrations (x2), and insulin amounts (x3) were selected as independent variables (Table 1). The drug entrapment efficiency of the nanoparticles (y) was the response parameter or the dependent variable (Table 2). Characterization of insulin nanoparticles

Methods Materials Chitosan (95% deacetylated with viscosity of 1% wt/vol solution, 30 mPa·s) was purchased from Primex (Siglufjordur, Iceland). Crystalline recombinant human insulin (28.3 IU/mg) and Arabic gum were purchased from Eli Lilly (Suresnes, France) and Arthur Branwell (Braintree, Essex, England), respectively. The other materials were of pharmaceutical and analytical grades and were used as received.

The sizes of the insulin nanoparticles were measured with a Malvern zetasizer (Malvern Instruments, Worcestershire, United Kingdom). The particle size distribution is reported as a polydispersity index (PDI). The range for the PDI is from 0 to 1: Values close to zero indicate a homogeneous dispersion, and those greater than 0.5 indicate high heterogeneity. The samples were placed in the analyzer chamber and readings were performed at 25°C with a detected angle of 90 degrees. The zeta potential of nanoparticles was measured with a zetasizer (Malvern). The samples were diluted with double-


M.R. Avadi et al / Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 58–63

distilled water. Each sample was measured three times, and values are presented as the mean ± standard deviation (SD). Transmission electron microscopy (TEM) was used to observe the morphology of insulin nanoparticles (Philips 400, KW 80 Eindhoven, Holland). Insulin association efficiency The association efficiency was determined indirectly after separation of insulin nanoparticles from the aqueous medium containing nonassociated insulin. The amount of the free insulin was measured by HPLC (SDV 505, Anyang, South Korea). The liquid chromatograph was equipped with a 210-nm detector and a C8 column (Chrompack, Edinburg, United Kingdom 4.6 mm × 15 cm, 5 μm). The flow rate was about 1.2 mL/min, and the mobile phase was a mixture of acetonitrile (28%), 72% phosphate buffer solution (KH2PO4), and 1% triethylamine adjusted to pH 3.0 with phosphoric acid. The association efficiency (AE) of insulin nanoparticles was calculated according to the following formulation: AE = ½ðTotal amount of insulin  Free insulinÞ =ðTotal amount of insulinÞ  100: All samples were measured in triplicate. The loading efficiency (LE) was determined as follows: LE = Weight of the total insulin  Weight of free insulin =Weight of nanoparticles: Release studies Insulin release from nanoparticles was performed at three different pH values: 1.2 (0.1 N HCl), 6.5 (phosphate buffer solution; PBS), and 7.2 (PBS). Freeze-dried nanoparticles (containing 4 mg insulin) were placed in a 25-mL test tube and set into a Erweka apparatus (DT6; Heusentamn, Germany). The amount of nanoparticles in the release medium was adjusted for sink condition. The temperature and rotation were set at 37°C and 75 rpm, respectively.16 Results Preparation of insulin nanoparticles The aim of this study was to investigate the influence of variables in the preparation of insulin nanoparticles using the ionic gelation method. Various formulations (F1 to F8) were prepared using 23 factorial designs. As shown in Table 3, the optimum AE was obtained for three formulations: F2, F5, and F8. The amount of chitosan in formulation F2 was at a higher level (Table 1) and caused an increase in AE, possibly due to the higher ability of ionic gel formation than that of F1 (lower level of chitosan), which prevents insulin movement to the external phase. Although increasing the Arabic gum concentration in F3 caused an increase in AE compared with that of F1, this effect was less important than the effect of chitosan concentration. The effect of insulin variable in AE is the least important effect

Table 3 Characteristics of the nanoparticles containing insulin for different formulations F1 to F9 Formulation Mean diameter, Polydispersity Zeta AE, % code nm (N = 3) index potential, mV F1 F2 F3 F4 F5 F6 F7 F8 F9

188 191 185 198 172 193 194 177 245

± 14 ± 17 ± 16 ± 14 ± 10 ± 19 ± 12 ± 10 ± 18

0.39 0.48 0.44 0.38 0.26 0.49 0.31 0.25 0.45

35.7 ± 2.1 42.6 ± 3.4 39.7 ± 4.1 38.8 ± 3.8 41.7 ± 2.9 43.4 ± 3.1 37.5 ± 2.8 40.5 ± 3.3 38.6 ± 3.6

15.5 ± 1.81 31.2 ± 2.83 22.6 ± 4.32 18.4 ± 3.65 35.8 ± 4.31 20.6 ± 3.43 17.7 ± 4.21 37.5 ± 2.75 10.43 ± 3.65

among the three variables. According to the polymer concentration effect of chitosan and Arabic gum in AE (formulation F5), this is possibly due to interaction between these two variables (Table 2). In formulation F8, the variables are at high levels, which results in the highest AE response. As shown in Table 3, we have an additional formulation (F9) where the three variables are at a low level, the same as formulation F1, except the chitosan solution was added to Arabic gum solution. The AE in this formulation is lower than that of F1 (Table 2), which may be due to the diffusion of insulin to the external phase. Indeed, when the chitosan solution containing insulin is added to Arabic gum solution, the insulin tends to be retained on the surfaces of nanoparticles that are in direct contact with the external phase. However, when insulin is dissolved in chitosan solution and when the Arabic gum with negative charge is added to the positively charged chitosan solution, the nanoparticles are formed rapidly and the insulin is surrounded by a polymeric network due to interaction between two polymers. As formulations F2, F5, and F8 had the highest AE, they were selected as the optimum formulations. However, as F8 showed the highest AE, the release studies were carried out with this formulation. Characterization of the nanoparticles The drug-loaded nanoparticles exhibited relatively narrow particle size distribution, as indicated by relatively low PDI values (Table 3). The diameter of nanoparticles F5 and F8 are smaller than that of other formulations, which may be due to the effect of more interaction between the negative charge of Arabic gum and the positive charge of chitosan in high level according to the factorial design experimental data. The other parameter for characterization of nanoparticles is the surface charge of the nanoparticles indicated by zeta potential. Chitosan nanoparticles are all positively charged as shown in Table 3. This could be related to the type of particle formation mechanism in ionic gelation, where positively charged amine groups of chitosan are neutralized by their interaction with negatively charged Arabic gum polymer. Arabic gum constituents are principally calcium,

M.R. Avadi et al / Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 58–63


Figure 1. TEM micrographs of insulin nanoparticles for formulation (A) F5 and (B) F8.

magnesium, and potassium salts of the polysaccharide of Arabic acid, which on acid hydrolysis yields L-arabinose, L-rhamnose, D-galactose, and aldobionic acid containing D-glucuronic acid and D-galactose. The solution of Arabic gum in water dissociates the salts and reveals the negative charge of Arabic gum, which allows the interaction with the positive charge of chitosan. The residual amine groups would be responsible for the positive zeta potential. It seems that the higher zeta potential in the determined range leads to more stable nanoparticles. This effect may be related to the absorption of the anionic groups by the long amino groups of chitosan to keep the high value of the electrical double-layer thickness, which in turn prevents the aggregation.17 As shown in Table 3, all the nanoparticle formulations are positively charged, but the values of charges for F2, F5, and F8 are higher than those of the other formulations, which may be related to stronger positive charges of the amino group of chitosan at high level in the factorial design experiment. The PDI is another factor that represents the dispersion homogeneity. The PDI for all the formulations as shown in Table 3 is smaller than 0.5, which indicates a relative homogeneous dispersion. The LE for all formulations was calculated and was shown to be between 25% and 35% (data has not shown). TEM micrographs of two formulations (F5 and F8) are presented in Figure 1. The nanoparticles look round, spherical, or oval with relatively smooth surfaces. According to Figure 1 and the result of zeta potential observations (Table 3), the positive charges on the surfaces of nanoparticles could prevent the agglomeration process. Release studies The release profile for insulin nanoparticles (formulation F8) was observed at three different pH values: 1.2 (0.1 N HCl), 6.5 (PBS), and 7.2 (PBS). Each experiment was done three times. As

Figure 2. In vitro release profile of insulin from chitosan nanoparticles in 0.1 N HCl, pH 1.2.

shown in Figure 2, a burst effect of insulin release in the initial steps of the study (about 30% to 50% between 5 to 15 minutes) was observed that is related to the high solubility of both chitosan and insulin in acidic medium (0.1 N HCl). Moreover, the structure of nanoparticles was broken in this medium, and insulin freely dissolves in 0.1 N HCl. Also, the binding of insulin molecules that accumulate on the surfaces of nanoparticles and the polymer matrix is not too strong, and therefore the insulin molecules tend to move out from the surfaces of nanoparticles and diffuse to the external medium. The release profile of insulin in phosphate buffer solutions (pH 6.5 and pH 7.2) is completely different than that in the acidic medium (pH 1.2). This difference is related to the solubility of chitosan and insulin in different media (Figure 3). Indeed, both insulin and chitosan are soluble in acidic pH (0.1 N HCl) and therefore a burst insulin release was observed. Moreover, the solubility of chitosan and insulin in PBS at pH 6.5 and 7.2 is lower than that of the acidic medium, and hence a burst effect in the initial steps of the release study was not observed (Figure 3). Mir-


M.R. Avadi et al / Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 58–63 Table 4 Kinetic constant (k), diffusional (n) and determination coefficient (r2) determined by the linear regression of Ln(Mt/M∞) against Ln t Formulation

n, mean ± SD (N = 3)

k, mean ± SD (N = 3)


F5 F8

0.823 ± 0.0341 0.494 ± 0.0268

0.0008 ± 0.0001 0.0019 ± 0.0003

0.956 0.958

transport is possibly controlled by diffusion and/or relaxation of the polymer chains. Figure 3. In vitro release profiles of insulin from chitosan nanoparticles in phosphate buffer solution, pH 6.5 and pH 7.2.

Mirmohammad Sadeghi et al investigated the release of nanoparticles using ionotropic gelation between chitosan and TPP in PBS at pH 6.8.18 They observed a burst release of about 15%, and maximum insulin release was about 40% after 45 minutes. In this study, we used Arabic gum with negative charge, which has more sites of interaction with chitosan for ionotropic gelation. It seems that the interaction between two polymers with different charges could obtain a strong matrix polymer network that caused insulin trapping in this matrix network and finally decreased insulin release in PBS at pH 6.5 medium (Figure 3). The release profile of insulin nanoparticles in PBS at pH 7.2 is different than that at pH 6.5 (Figure 3). This difference could be related to Arabic gum properties. A 5% wt/vol aqueous solution of Arabic gum has a pH value of 4.5 to 5.0 and its acidic value is about 2.0.19 Consequently, it seems that the chains of Arabic gum polymer in media with pH values higher than 6.5 tend to swell and disturb the structure of nanoparticles, resulting in more porosity in the nanoparticle structure and more insulin release. Fickian and non-Fickian (anomalous) behaviors have been used for determining the mechanism of drug release from polymeric systems. However, we tried to analyze the drug release data in phosphate buffer solutions at pH 6.5 and 7.2 using the following general equation: Mt =Ml = ktn where Mt is the amount of insulin released in a given time, M∞ is the total amount of insulin within the nanoparticles, k and n are equation constants, and t is the time. k is a constant incorporating structural and geometric characteristics of the drug dosage form, and n is the release exponent indicating the drug-release mechanism.20 Different values of n for cylindrical, spherical, and slab geometries are available in the literature. For spheres, values of 0.43, 0.43 b n b 0.85, and 0.85 are related to Fickian diffusion (case I transport), anomalous, and case II transport, respectively.21 When the exponent n takes a value of 0.85, the drug release rate is independent of time, which corresponds with zero-order kinetics. Regarding the data presented in Table 4, the values of the exponent n for the formulations F5 and F8 were between 0.43 and 0.85 indicating a non-Fickian transport. This suggests that

Discussion Insulin is the most effective and durable drug in the treatment of advanced-stage diabetes. Despite significant advancement in the field of pharmaceutical research, development of a proper, noninvasive insulin-delivery system remains a major challenge.22 The oral route represents the most convenient method of drug administration possibly due to high patient compliance and comfort. One of the most attractive applications of nanoparticulate systems is in the oral administration of peptide or protein drugs. Protein drugs such as insulin are intrinsically poorly absorbable owing to their high molecular weight and hydrophilicity; they are also susceptible to enzymatic degradation in the gastrointestinal tract.23 It is well known that proteins have low bioavailability when administered orally due to their instability in the gastrointestinal tract.4 New delivery approaches depend on protecting insulin against enzymatic degradation and enhancing its transport across the intestinal mucosa into the systemic circulation. Various approaches have been proposed to overcome barriers and to attain better oral bioavailability, including the use of surfactants, permeation enhancers, enteric coatings, carrier systems, and chemical modifications of insulin.24 Hydrophilic nanoparticles based on chitosan are receiving increased interest as they could control the rate of drug release, prolong the duration of the therapeutic effect, and deliver the drug to specific sites in the body.15 Chitosan is able to form nanoparticles using, among other methods, ionotropic gelation. The method is based on the gelation of chitosan with positive charge when it comes in contact with specific polyanions due to the formation of intramolecular and intermolecular cross-linking mediated by these polyanions.15 Ionic gelation of chitosan with Arabic gum was used to prepare insulin-loaded nanoparticles. The technique, an extremely mild and simple process, yields nanoparticles with reproducible size (150 to 200 nm) and encapsulation capacity of 25% to 35%. In this study, we developed a new nanoparticle system with Arabic gum for oral delivery of insulin. The effect of different variables on nanoparticle preparation was investigated. Results have indicated a small size, positive charge, and median AE for nanoparticles. Moreover, the results show that the chitosan and Arabic gum concentrations are the most important parameters that effect the AE of nanoparticles. It was shown that the release of insulin from nanoparticles was obtained with more than one

M.R. Avadi et al / Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 58–63

mechanism (possibly diffusion, dissolution, and relaxation of the polymer chains). The obtained data showed that all variables should be taken into account during the formulation of such a system. Finally, this study showed that although Arabic gum has more negative sites for interaction with the positive charge of chitosan, the loading of insulin nanoparticles with this polymer is less than that for TPP. Acknowledgments We would like to extend our thanks to Dr. H. Mir-Mohammad Sadeghi (board member of Hakim Pharmaceutical Company) and Dr. M. Alimian (The Managing Director of Hakim Pharmaceutical Company) for their kind support through out this study.

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