Preparation of alginate and chitosan nanoparticles ...

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Preparation of alginate and chitosan nanoparticles using a new reverse micellar system Morteza Hasanzadeh Kafshgari, Mohammad Khorram, Mohsen Mansouri, Abdolreza Samimi & Shahriar Osfouri Iranian Polymer Journal ISSN 1026-1265 Iran Polym J DOI 10.1007/s13726-011-0010-1

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Author's personal copy Iran Polym J DOI 10.1007/s13726-011-0010-1

ORIGINAL PAPER

Preparation of alginate and chitosan nanoparticles using a new reverse micellar system Morteza Hasanzadeh Kafshgari • Mohammad Khorram Mohsen Mansouri • Abdolreza Samimi • Shahriar Osfouri



Received: 22 April 2011 / Accepted: 2 November 2011 Ó Iran Polymer and Petrochemical Institute 2011

Abstract Alginate and chitosan nanoparticles were prepared using a new reverse micelle system, composed of cetyltrimethylammonium bromide (CTAB) as a surfactant, isooctane as a solvent, and 1-hexananol as a co-solvent. The obtained nanoparticles were characterized by FTIR, DLS and TEM techniques. The main objective of this study was to investigate the effects of polymer concentration, water content, and volumetric ratio of co-solvent to solvent on the physical and morphological properties of the prepared nanoparticles. To evaluate the results, the design of experimental was initially carried out and then the obtained data were statistically analyzed using the Qualitek-4Ò software. Results revealed that the size of the prepared alginate and chitosan nanoparticles varied in the range 220–490 and 210–1,050 nm, respectively. Furthermore, increasing either alginate or chitosan concentration increased the size of their nanoparticles. The results also showed that the size of nanoparticles was decreased with increasing the volumetric ratio of co-solvent/solvent. Finally, the size of alginate nanoparticles was increased by increasing the water content while it decreased the size of chitosan nanoparticles. Considering the statistical analysis of experiments, the polymer concentration is the major parameter affecting nanoparticles’ size. In contrast, water content has the smallest effect on the size of nanoparticles. However, the difference between the particle sizes of

M. H. Kafshgari  M. Khorram (&)  M. Mansouri  A. Samimi Department of Chemical Engineering, University of Sistan and Baluchestan, P.O. Box 98164-161, Zahedan, Iran e-mail: [email protected] S. Osfouri Department of Chemical Engineering, Persian Gulf University, 7516913817 Bushehr, Iran

chitosan and alginate nanoparticles cab be attributed to the electrostatic repulsion between chitosan and CTAB. Keywords Reverse Micelle  CTAB  Nanoparticles  Chitosan  Alginate

Introduction Alginate and chitosan are both natural polymers that are mucoadhesive, biodegradable and biocompatible polymers that enable numerous pharmaceutical and biomedical applications such as drug delivery and cell encapsulation [1, 2]. Sodium alginate which is widely used in food, drinks, bioengineering and pharmaceutical industries is the water soluble salt of alginic acid, a naturally occurring nontoxic polysaccharide found in all species of the brown algae [3]. Soluble sodium alginate can be cross-linked by divalent cations, e.g., Ca2? and Mg2?, forming the ‘egg box junctions’ and insoluble calcium alginate [4]. Meanwhile, biopolymer chitosan is a polysaccharide derived from chitin by deacetylation, although this Ndeacetylation is almost never complete. Chitosan has a pKa value of approximately 6.5 and its free amino groups can be protonated in mild acidic solutions, provides positive charges to the glucosamine residue. Positive charges on chitosan import very different physical and chemical properties contrasted to chitin. Chitosan is inexpensive and exhibits an excellent film-forming ability, biocompatibility, nontoxicity, physiological inertness, antibacterial properties, high mechanical strength and a susceptibility to chemical modifications [5–7]. Because of the positive charges on chitosan, it has found a number of applications such as bandage materials for wounds [8], absorption enhancers across intestinal epithelium, mucosal sites for

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drugs, peptides and proteins [9], plant growth enhancers, aids in the defense of plants against fungal infections, in sand filtration systems [10] and metal complexation agents in water purification [11]. Chitosan has also shown usefulness for the immobilization of enzymes and cells [12, 13]. Alginate and chitosan in nanoparticle forms are preferred in many applications. Nanoparticles exhibit superior activity that can be attributed to their small size and quantum size effect [14]. Alginate nanoparticles are prepared by pregelation method. In this method, the interaction between alginate and Ca2? ions occurs at a certain ion concentration in dilute solution. A pre-gel state results by stirring, avoiding the gel point, and forming a continuous system. Subsequent addition of an aqueous polycationic solution (i.e., chitosan) results in a polyelectrolyte complex, stabilizing the alginate pre-gel nucleus into individual spongelike nanoparticles [15]. While, common methods to prepare chitosan nanoparticles are ionic gelation, coacervation or precipitation emulsion-droplet coalescence, reverse micelles and self-assembly chemical modification [16]. Besides the economical considerations in many fields of applications, the best method for preparation of alginate and chitosan nanoparticles has to provide a safe medium for biomolecules and a narrow particle size distribution. Reverse micelles are monodisperse spherical aggregates of surfactant molecules containing microscopic polar cores of solubilized water, the so-called water pools. The hydrophilic compounds, such as biomolecules, are solubilized inside the polar core of the reversed micelles and are stabilized in the organic solvent by a surfactant shell layer that protects them from denaturation against the organic phase [17, 18]. Many studies have been made on preparation of chitosan nanoparticles by reverse micellar method [19–24]. Parameters such as molecular weight and concentration of chitosan have already been studied to some extent [25]. However, most of these studies have not investigated the effects of water content (water to surfactant molar ratio, w0) and volumetric ratio of co-solvent to solvent on the size and size distribution of the prepared nanoparticles. Furthermore, few attempts have been focused on the effect of electrostatic interaction between polymer (chitosan or alginate) and surfactant. In this work, alginate and chitosan nanoparticles were prepared using a new reverse micellar system composed of cetyltrimethylammonium bromide (CTAB) as a surfactant, isooctane as a solvent, and 1-hexananol as a co-solvent. Alginate and chitosan are polyanion and polycation polyelectrolyte, respectively, whereas CTAB is a cationic surfactant. Therefore, the effects of electrostatic interaction between polymer (chitosan or alginate) and surfactant on the mean size and size distribution of nanoparticles were also compared.

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This reverse micellar system is more biocompatible as compared to those that have been used by other researchers. Thus, it is anticipated that the microemulsion used in this work can be employed in encapsulating bioactive agents into alginate or chitosan carriers which used in controlled release systems. Furthermore, according to our search, alginate nanoparticles have not been yet produced using reverse micelles method. The effects of polymer concentration, water content, and volumetric ratio of co-solvent to solvent were investigated on the physical and morphological properties of the prepared nanoparticles. To study the effects of these parameters on the size of the prepared nanoparticles, experiments were set based on the statistical design of experiments. The data obtained from the experiments were then statistically analyzed using the Qualitek-4Ò software. As stated in the previous paragraph the effects of these parameters, especially water content and co-solvent to solvent ratio, have not been investigated systematically by the other researchers.

Experimental Materials Alginic acid sodium alginate (medium viscosity, 3,500 cP, 2% w/v aqueous solution at 25 °C, Sigma-Aldrich, USA) and high molecular weight chitosan (Mw = 6 9 106 Da, degree of deacetylation = 76%, Fluka, USA) were employed as polyanionic and polycationic polymers, respectively. Cetyltrimethylammonium bromide (CTAB) was obtained as cationic surfactant from Merck (Germany) with 99% purity, and was used without any further purification. Isooctane as solvent (oil phase) and 1-hexanol as co-solvent were purchased from Merck (Germany) and Sigma-Aldrich (USA), respectively. The other chemicals such as acidic and basic solutions were all commercially available reagents of analytical grades. Preparation of nanoparticles Alginate and chitosan nanoparticles were prepared using reverse micellar method. The oil phase of the reverse micellar system was composed of CTAB dissolved in the mixture of isooctane as solvent and 1-hexanol as co-solvent. To prepare alginate nanoparticles, the aqueous phase was made of sodium alginate dissolved in the deionized distilled water. Based on the desired values of the water content, wo, the alginate-containing aqueous phase was added to the prepared oil phase in a glass test tube. The mixture was shaken for 3 min until the mixed emulsion became transparent or semi-transparent, indicating the formation of w/o microemulsion. To solidify the entrapped

Author's personal copy Iran Polym J Table 1 Formulations of the prepared alginate nanoparticles based on L9 arrays of Taguchi Sample code

C (w/v%)

V (v/v)

Water content (mol/mol)

ALG-1

0.5

1/3

ALG-2

0.5

1/3.5

11

ALG-3 ALG-4

0.5 1

1/4 1/3

14 11

ALG-5

1

1/3.5

14

ALG-6

1

1/4

7

ALG-7

2

1/3

14

ALG-8

2

1/3.5

ALG-9

2

1/4

7

Characterization of the prepared nanoparticles

7 11

FTIR analysis

Table 2 Formulations of the prepared chitosan nanoparticles based on the full factorial design of experiments Sample code

Water content (mol/mol)

required for preparing chitosan nanoparticles are shown in Table 2. It is worth noting that the concentration of CTAB was kept constant (50 mM) in all sample preparations. The nanoparticles were then precipitated with centrifugation (9,000 rpm for 5 min) at room temperature and were rinsed with de-ionized distilled water for thrice. The procedure employed in preparing nanoparticles is shown schematically in Fig. 1. Finally, the nanoparticles were dried in air at room temperature for 48 h.

V (v/v)

C (w/v%)

ChN-1

7

1/4

0.5

ChN-2

7

1/4

1

ChN-3

7

1/3

0.5

ChN-4

7

1/3

1

ChN-5

14

1/4

0.5

ChN-6 ChN-7

14 14

1/4 1/3

1 0.5

ChN-8

14

1/3

1

alginate in reverse micelles, 1.5 (w/v%) of CaCl2 aqueous solution (10 lL) was quickly added to the prepared reverse micelles and the mixture was then shaken. The sodium alginate was cross-linked with Ca2? and the calcium alginate nanoparticles were eventually formed. In order to investigate the effects of polymer concentration, water content, and volumetric ratio of co-solvent to solvent on the size of the prepared alginate nanoparticles, Taguchi statistical method was used for design of experiments in three levels and on the basis of L9 arrays. The formulations of all alginate samples are presented in Table 1. Chitosan nanoparticles were prepared by the same procedure, except that in this case aqueous phase was prepared by dissolving chitosan in 0.1 M acetic acid solution. The solidification of entrapped chitosan nanoparticles in reverse micelles was carried out by adding 2 M NaOH solution (10 lL) to the system, where chitosan was solidified with the increase of pH. In this case, to study the effects of above mentioned parameters on the size of the obtained chitosan nanoparticles, full factorial statistical experimental design, with 3 variables at 2 levels was performed. The concentration of chitosan and water content, wo, as well as, the volumetric ratio of co-solvent to solvent

To analyze the chemical structure of chitosan, alginate, and nanoparticles, FTIR absorption spectra for all samples were recorded in the range 400–4,000 cm-1 with a 460 Plus Jasco spectrophotometer (USA) using KBr pellets at room temperature. Each sample was recorded with 32 scans at an effective resolution of 4 cm-1. Size measurement The mean particle size and size distribution of nanoparticles were determined by dynamic light scattering (DLS), using a Malvern Zetasizer Nano-ZS-ZEN 1600 (Malvern Instruments, UK). The analysis was carried out at a scattering angle of 90o at a temperature of 25 °C, using solidified nanoparticles dispersed in the microemulsion system. Each sample measurement was repeated thrice to obtain representative results. TEM analysis The morphology of dried nanoparticles was observed using TEM. In this regard, the prepared chitosan nanoparticles were dispersed in about 3 mL ethanol. One droplet of the dispersed nanoparticles solution was then dripped onto copper grid where the sample was dried by natural airflow before TEM analysis.

Results and discussion FTIR analysis FTIR analysis was employed to study the effectiveness of the method for preparation of nanoparticles in terms of chemical structure. The FTIR spectra of sodium alginate, chitosan, and the prepared alginate and chitosan nanoparticles are presented in Fig. 2. The characteristic peaks of sodium alginate (Fig. 2a) represent O–H stretching at

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Author's personal copy Iran Polym J Fig. 1 Schematic representation of preparation steps

3,397 cm-1, COO–(asymmetric stretching) at 1,617 cm-1, COO–(symmetric stretching) at 1,429, and C–O–C stretching at 1,028 cm-1 [26]. An obvious shift to lower wavenumbers and a decrease in the intensity of COO– stretching peak are as a consequence of the cross-linking process of sodium alginate with Ca2?. Furthermore, as it is seen in Fig. 2b, the C–O–C stretching peak of sodium alginate has been shifted to the lower wavenumber, where the intensity of the peak decreases. This indicates the presence of the ionic bonding between the calcium ions and the carboxyl groups of sodium alginate and partial covalent bonding between the calcium and oxygen atoms of the ether groups [26]. As it is observed in Fig. 2c, three characteristic absorption bands at 3,413, 1,672, and 1,423 cm-1 are due to the N–H, amide I and amide III groups presented in chitosan, respectively [27]. As it can be seen in Fig. 2d, these characteristic absorption bands are also observed at the FTIR spectra of the prepared chitosan nanoparticles. Furthermore, FTIR spectra of the prepared chitosan nanoparticles do not show any additional absorption peak, representing that the chemical structure of the chitosan nanoparticles has not changed during the preparation of the nanoparticles.

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Effect of polymer concentration on size of nanoparticles Tables 3 and 4 present the average size and PDI of the samples represented in Tables 1 and 2, respectively. PDI is a measure of homogeneity in dispersed systems and ranges from 0 to 1. Homogeneous dispersion has PDI value close to zero while PDI values greater than 0.3 indicate high heterogeneity. As it is seen, PDI of all samples are too high. It was noted earlier in the introduction that the reverse micelles are very small aggregates in range smaller than 10 lm. Thus, it is anticipated that solidified particles in water pool of reverse micelles have similar size range. It seems that these very small nanoparticles would be aggregated forming different sizes rapidly after the solidification process. This phenomenon is the reason of high PDI in the methodology employed in this study. The statistical analysis of the data reported in Tables 3 and 4 were performed using the Qualitek-4Ò software. Figure 3 shows the variation of nanoparticles average size as a function of alginate or chitosan concentration obtained from this analysis. As it is seen, increasing the

Author's personal copy Iran Polym J Table 4 Mean particle size and PDI of the prepared chitosan nanoparticles Sample code

Polydispersity index

Mean size (nm)

ChN-1

0.681

531 ± 68.1

ChN-2

0.209

1,050 ± 227

ChN-3 ChN-4

0.492 0.586

459 ± 108 720 ± 142

ChN-5

0.404

250 ± 81.2

ChN-6

1

518 ± 75.3

ChN-7

1

212 ± 56.6

ChN-8

0.819

680 ± 158

Fig. 3 Effect of polymer concentration (w/v%) on average size of (open diamond) alginate nanoparticles and (open triangle) chitosan nanoparticles

Fig. 2 FTIR spectra of a alginate, b alginate nanoparticles, c chitosan, d chitosan nanoparticles Table 3 Mean particle size of the prepared alginate nanoparticles

a

All the samples has polydispersity index = 1

Sample code

Mean sizea (nm)

ALG-1

223 ± 81.5

ALG-2

300 ± 53.2

ALG-3

390 ± 80.2

ALG-4

343 ± 75.3

ALG-5

375 ± 82.7

ALG-6

368 ± 89.6

ALG-7

440 ± 151.4

ALG-8

390 ± 79.3

ALG-9

485 ± 105

alginate or chitosan concentration leads to the increase of the average nanoparticles size. According to the previous reports [28, 29] increasing the polymer concentration at

constant water content resulted in increasing the size of the reverse micelles. Although it cannot be generalized, however, several studies have already shown that the size of the reverse micelles controls the size of the nanoparticles [30–37]. By considering this fact, it is reasonable to consider that increase in the size of nanoparticles results from the increase of the polymer concentration. Effect of volumetric ratio of co-solvent to solvent on size of nanoparticles The effect of volumetric ratio of co-solvent to solvent on the average size of alginate and chitosan nanoparticles is shown in Fig. 4. These results were obtained according to statistical analysis performed on the presented data in Tables 3 and 4 using the Qualitek-4Ò software. The results represent that the samples prepared with the higher co-solvent to solvent ratios lead to smaller mean particle sizes as compared to those prepared with lower ratios. Previous studies on reverse micellar systems showed that using co-solvent such as alcohol could facilitate formation

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Fig. 4 Effect of volumetric ratio of co-solvent to solvent (v/v) on average size of (open square) alginate nanoparticles and (open triangle) chitosan nanoparticles

Fig. 5 Effect of water content (mol/mol) on average size of (open square) alginate nanoparticles and (open triangle) chitosan nanoparticles

of reverse micelle when cationic surfactant was employed [28]. This work revealed that the aggregates of surfactant molecules were compacted and the size of reverse micelles was reduced by increasing the alcohol concentration. The size of the reverse micelles controlled the size of the nanoparticles, thus, the size of alginate nanoparticles was decreased with increase of the volumetric ratio of co-solvent to solvent. Effect of water content on the size of nanoparticles Figure 5 demonstrates the effect of water content, wo, on the mean size of alginate and chitosan nanoparticles. The data were obtained according to statistical analysis of the data reported in Tables 3 and 4 using the Qualitek-4Ò software. The increase of the size of reverse micelle with the increase of water content was reported by Kuboi et al. [38] and Kiyoyama et al. [39] where the latter was under constant surfactant concentration. As noted earlier, the size of the reverse micelles controls the size of the nanoparticles. As it is seen in Fig. 5, the particle size of alginate nanoparticles increases with increasing the water content, wo. The effect of water content, wo, on the chitosan nanoparticles average size was also studied as shown in Fig. 5. It is worth noting that according to the work of Jiang and co-workers [40], the size of chitosan nanoparticles increased with increase of water content, wo. However, presented data in Fig. 5 reveal reverse pattern for chitosan. It seems that the reverse micelles which capsulated imperfectly are very weak and therefore, easily collapse. This is thought to be the main reason in our study, when the water content was increased from 7 to 14 (mol/mol), the phenomenon which was also observed by Kiyoyama and co-workers [39]. It seems that electrostatic repulsion between cationic chitosan and cationic surfactant (CTAB)

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Fig. 6 Column chart of individual contribution of the effective parameters on the size of the alginate and chitosan nanoparticles

used in this study, led to weak and imperfectly capsulated micelles. Analyzing the experimental results illustrated in Fig. 6 reveals the most effective parameters on the size of alginate and chitosan nanoparticles. The results show that polymer concentration affects the particle size more than any other studied parameters. The optimum conditions for preparation of alginate and chitosan nanoparticles with minimum size were also determined using the Qualitek-4Ò software. The results show that the optimum alginate nanoparticles size would be estimated as 230 nm using the alginate concentration 0.5 (w/v%), volumetric ratio 1/3 (v/v) and water content 7. This result is in agreement with the measured experimental value using DLS method for the sample ALG-1 in Table 3. In addition, the optimum chitosan nanoparticles size would be estimated as 260 nm using the chitosan concentration 0.5 (w/v%), volumetric ratio 1/4 (v/v) and water content

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Fig. 7 TEM micrographs and diameter distributions of a alginate nanoparticles (ALG-1 sample), and b chitosan nanoparticles (ChN-3 sample)

14. The size characterized with DLS for sample ChN-5 in Table 4 was found 250 ± 81.2 nm. This size is in rational agreement with the estimated size of optimum chitosan nanoparticles. TEM micrograph and the size distribution of ALG-1 and ChN-3 samples have been presented in Fig. 7a ,b, respectively. The figure clearly shows that the alginate nanoparticles are almost spherical. On the contrary, the shape of chitosan nanoparticles does not look precisely spherical. The reason of this difference can be referred to electrostatic repulsion between cationic chitosan and cationic CTAB in water pool that results in weak and imperfectly chitosan reverse micelles. In order to characterize the stability of the particles as a function of time, the DLS measurements were repeated for all samples in ten succeeding days. During the course of investigation no changes were observed in the particle size of almost all samples. Thus, we can conclude that the prepared nanoparticles were stable using the developed method of this study. As noted in the introduction section, some of researchers [19–24] have produced chitosan nanoparticles using W/O microemulsion which mostly composed of cyclehexane as solvent, n-hexanol as co-solvent and Triton X-100 as surfactant. The reported particle sizes in these works were smaller than those we reported in our study. The difference

could not be due to the new reverse micellar system employed in our research. We think that the method for particle size analysis is the main reason of this difference. The researchers mentioned above had used TEM for the particle size analysis. Furthermore, they had employed sonication for TEM sample preparation. Sonication may lead to particle breakage and therefore producing smaller particles. Whereas, in our study, DLS analysis was used for particle size analysis and sonication was not considered for the sample preparation.

Conclusion Alginate and chitosan nanoparticles were produced using a new reverse micellar system, composed of isooctane as a solvent, 1-hexanol as a co-solvent and CTAB as a surfactant. Such nanoparticles are suitable for encapsulation of bioactive agents for using in drug delivery systems. The size of prepared alginate and chitosan nanoparticles ranged between 220–240 and 210–1,050 nm, respectively. The results showed that by increasing polymer (alginate or chitosan) concentration, both alginate and chitosan nanoparticles size are increased. Considering the results presented in this study, the size of alginate and chitosan nanoparticles decreased with increase of the volumetric

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ratio of co-solvent to solvent. The results revealed that the size of alginate nanoparticles was increased with increasing water content while the size of chitosan nanoparticles was decreased with increasing the water content. Based on the statistical analysis of experiments, among studied parameters, polymer concentration was the most effective parameters on the size of nanoparticles. In contrast, water content had the lowest effect on the size of nanoparticles. In general, due to electrostatic repulsion between chitosan and CTAB, chitosan nanoparticles size is higher than that of alginate nanoparticles.

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