Preparation of chitosan nanoparticles by spray drying ...

Preparation of chitosan nanoparticles by spray drying, and their antibacterial activity La Thi Kim Ngan, San-Lang Wang, Đinh Minh Hiep, Phung Minh Luong, Nguyen Tan Vui, Tran Minh Đinh & Nguyen Anh Dzung Research on Chemical Intermediates ISSN 0922-6168 Volume 40 Number 6 Res Chem Intermed (2014) 40:2165-2175 DOI 10.1007/s11164-014-1594-9

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Author's personal copy Res Chem Intermed (2014) 40:2165–2175 DOI 10.1007/s11164-014-1594-9

Preparation of chitosan nanoparticles by spray drying, and their antibacterial activity La Thi Kim Ngan • San-Lang Wang • Ðinh Minh Hiep • Phung Minh Luong Nguyen Tan Vui • Tran Minh Ðinh • Nguyen Anh Dzung

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Received: 25 December 2013 / Accepted: 25 January 2014 / Published online: 1 April 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Chitosan nanoparticles were prepared from chitosan of different molecular weight by spray drying. The morphology of the particles was characterized by SEM, and size distribution and zeta potential were determined. The effects of chitosan solution concentration, molecular weight of chitosan, and size of the spray dryer nozzles on average size, size distribution and zeta potential of chitosan nanoparticles were investigated. The effects of chitosan nanoparticles and chitosan nanoparticles–amoxicillin complex on Staphylococcus aureus were also tested. The results showed that the average size of chitosan nanoparticles were in the range 95.5–395 nm and zeta potentials were 39.3–45.7 mV, depending on the concentration and molecular weight of the chitosan. The lower the concentration and molecular weight of the chitosan, the smaller the chitosan nanoparticles and the higher the zeta potential. Testing for antibacterial activity against S. aureus indicated that chitosan nanoparticles strongly inhibited growth of the bacteria; the minimum inhibitory concentration, 20 lg/mL, was lower than those of chitosan solution or amoxicillin. The antibacterial capacity of chitosan nanoparticles also L. T. K. Ngan N. T. Vui T. M. Ðinh N. A. Dzung (&) Institute of Biotechnology and Environment, Tay Nguyen University, 567 Le Duan Str., Buon Ma Thuot, Vietnam e-mail: [email protected] S.-L. Wang (&) Department of Chemistry, Tamkang University, Taipei 251, Taiwan e-mail: [email protected] Ð. M. Hiep Department of Science and Technology, 244, Dien Bien Phu Str., District 3, Ho Chi Minh City, Vietnam P. M. Luong Faculty of Medicine and Pharmacy, Tay Nguyen University, 567 Le Duan Str., Buon Ma Thuot, Vietnam

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depended on the size, zeta potential, and molecular weight of the chitosan. Complexation of chitosan nanoparticles with amoxicillin improved the antibacterial activity of amoxicillin. Keywords Chitosan nanoparticles Antibacterial activity Amoxicillin Staphylococcus aureus

Introduction Chitosan, a biopolymer of glucosamine and N-acetylglucosamine, is produced from seafood by-products, for example shrimp and crab shells, and from the cell walls of fungi [1]. Chitosan and its derivatives are natural, non-toxic, biocompatible, and biodegradable polymers, and natural polycationic agents [1–3]. Therefore, chitosan and its derivatives have been used in biomedicine, for example as antibacterial agents [4–8]. The mechanism of antibacterial action of chitosan is: 1 The polycationic charge on the chitosan molecule enables aggressive binding to the microbial cell surface, leading to gradual shrinkage of the cell membrane and, finally, death of the cell; 2 The polycationic chitosan molecule can interact with predominantly anionic cell wall components (lipopolysaccharides and proteins) of the microorganism, resulting in leakage of intracellular components because of changes in the permeability barrier, preventing nutrient transport through the cell membrane; 3 Chitosan (especially low-molecular weight or nanoparticles) can enter the cell, binding to DNA (polyanion) and thus inhibiting RNA and protein synthesis; and 4 Polycationic chitosan can cause cells to coagulate, leading to their precipitation [9–11]. The antibacterial activity of chitosan nanoparticles has recently been reported. Qi et al. [5] reported that chitosan nanoparticles prepared by ionic gelation with tripolyphosphate (TPP) had much greater antibacterial activity than chitosan solution or doxycycline antibiotics [5]. Du et al. studied the antibacterial activity of chitosan nanoparticles and chitosan nanoparticle-loaded metal ions against Escherichia coli and Staphylococcus aureus [12]. The antibacterial activity of oleoylchitosan nanoparticles have been reported [8]. Preparation of chitosan nanoparticles by ionic gelation with TPP has been studied. The disadvantage of this method is the difficulty of obtaining sufficient chitosan nanoparticles for large-scale pharmaceutical application. Chitosan micro and nanoparticles can be prepared by such methods as emulsion cross linking, coacervation in NaOH–methanol, spray drying, ionic gelation in TPP, combined spray drying and ionic gelation, and by use of a reverse micellar and sieving method [13]. Spray drying has been widely used for preparation of micro and nanoparticles for use by the food and pharmaceutical industries, because, in comparison with other techniques, it is relatively low-cost technology, rapid, and easy to scale up. The process is flexible, adaptable to commonly used processing equipment, and produces particles with good properties. Microencapsulation of such

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Author's personal copy Preparation of chitosan nanoparticles

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antibiotics as ampicillin, amoxicillin, vancomycin, etc., by chitosan, by use of a spray drying method, to enhance the efficiency of their antibacterial activity has been widely used in industrial applications [14–16]. The spray-drying process produces relatively fine chitosan particles (10–50 lm) leading to an increase in their direct antibacterial activity [15, 17–20]. The objectives of this work were to determine the optimum spray-drying conditions, for example chitosan solution concentration, molecular weight of chitosan, and type of spray drying nozzle, for preparation of chitosan nanoparticles and the effect of these condition on size distribution, zeta potential, and antibacterial activity of the chitosan nanoparticles and of chitosan nanoparticles–amoxicillin complex.

Experimental Materials Chitosan was purchased from Taehoon Bio, Seoul, Korea. The degree of deacetylation of chitosan was approximately 80–90 %, as determined by IR [21]. The molecular weight of the chitosan was determined by viscosity measurement (Brookfield viscometer) and use of the conversion factors: very high molecular weight (VHMW) 1,200 cps (centipoise); high molecular weight (HMW) 760 cps, and medium molecular weight (MMW) 276 cps. Staphylococcus aureus ATCC 25923 (MRSA) was from The Pasteur Institute, Ho Chi Minh City, Vietnam. Acetic acid (Merck, Germany), amoxicillin trihydrate (Purimox), and nutrient broth were products of Himedia, India. Preparation of chitosan nanoparticles Chitosan nanoparticles were prepared by spray drying. In brief, chitosan solutions of different concentration (0.1; 0.075; 0.05 and 0.025 % w/v) and different molecular weight (equivalent to 276, 760, and 1,200 cps) were prepared by dissolving purified chitosan powder in 0.5 % acetic acid solution and storing the solutions overnight. Chitosan nanoparticles were produced by use of a nano spray dryer (Buchi Nano B90, Switzerland) with three types of spray drying nozzle (4.0, 5.5 and 7.0 lm). The flow rate was 2 mL/min; the drying gas flow 1.3 m3/min, the inlet temperature 120 °C, and the outlet temperature 80 °C. Chitosan nanoparticles were placed in glass tubes and stored at 5 °C [22]. Particle size, zeta potential, and morphology of the chitosan nanoparticles The morphology of the chitosan nanoparticles was investigated by scanning electron microscopy (SEM; FE SEM, S 4800; Hitachi, Japan) at an acceleration voltage of 15 kV. The zeta potential and size distribution of the chitosan nanoparticles were determined by use of a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK).

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Evaluation of the antibacterial activity of chitosan nanoparticles and of chitosan nanoparticles–amoxicillin complex Staphylococcus aureus was cultured in nutrient broth supplemented with chitosan nanoparticles (20 lg/mL) loaded with amoxicillin at a concentration of 10, 20, 30, 40 and 50 lg/mL. The control contained nutrient broth only. After adjusting the pH to 6.0 with 10 % NaOH, all samples were inoculated at 37 °C. Twenty-four hours after inoculation, growth of the bacteria was determined by measurement of turbidity at 610 nm (UV–Vis; Jasco, Japan). All measurements were performed on samples prepared in triplicate.

Results and discussion Effect of chitosan solution concentration on the characteristics of chitosan nanoparticles and their antibacterial activity Chitosan is a polysaccharide; its solutions are of high viscosity, which makes them difficult to control during spray drying and particle formation. For efficient production of particles, therefore, the drying conditions, i.e. initial chitosan solution concentration, molecular weight of chitosan, and the size of spray drying nozzle, should be optimized. In these experiments, the concentration of the chitosan solution with MMW (276 cps) was from 0.025 to 0.100 % (w/v) and the size of the nozzle was 4.0 lm. The results listed in Table 1 indicate that the average size of the chitosan nanoparticles produced by spray drying were from 95.4 to 358.3 nm, and increased with increasing concentration of the chitosan solution (Fig. 1). In contrast, the zeta potential of the particles increased with decreasing chitosan solution concentration and chitosan particle size, as shown in Fig. 2.

Table 1 Effect of chitosan concentration on the characteristics of chitosan nanoparticles and their antibacterial activity Conc. of chitosan (%)

Nozzle (lm)

Average size (nm)

Zeta potential (mV)

Recovery (%)

OD 610 nma

Controlb

–

–

–

–

2.1312

0.100

4.0

358.3

39.3

60.5

0.0415

0.075

4.0

235.7

41.3

60.9

0.0401

0.050

4.0

180.0

44.5

62.8

0.0322

0.025

4.0

95.4

45.7

62.6

0.0138 3

Operating conditions: nozzle size 4.0 lm, flow rate 2 mL/min, drying gas flow 1.3 m /min, inlet temperature 120 °C, and outlet temperature 80 °C a

The concentration of chitosan nanoparticles in the nutrient broth tested was 20 lg/mL

b

Control: S. aureus was cultivated in nutrient broth under the same conditions (37 °C, pH 6.0, 24 h, shaking at 150 rpm) without chitosan nanoparticles

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Fig. 1 Size distribution of chitosan nanoparticles (276 cps) obtained from chitosan solutions of different concentration: 0.1 % (a); 0.05 % (b), and 0.025 % (c). Nozzle size was 4 lm, the flow rate 2 mL/min, the drying gas flow 1.3 m3/min, the inlet temperature 120 °C, the and outlet temperature 80 °C

Fig. 2 Zeta potential of chitosan nanoparticles obtained from chitosan solutions of different concentration: from left to right: 0.1, 0.075, 0.050, and 0.025 % (w/v). The molecular weight of the chitosan nanoparticles was equivalent to 276 cps and the nozzle size was 4 lm

The morphology of the chitosan nanoparticles, observed by SEM (Fig. 3), indicated their shape was fine and of homogenous size. The size distribution was narrow, as shown in Fig. 1.

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Fig. 3 SEM of chitosan nanoparticles (276 cps) obtained from chitosan solutions of concentration: 0.1 % (a), 0.075 % (b), 0.050 % (c), and 0.025 % (w/v) (d). Nozzle size was 4 lm, the flow rate 2 mL/min, the drying gas flow 1.3 m3/min, the inlet temperature 120 °C, the and outlet temperature 80 °C

The size of particles produced by spray drying depend on the initial feed and on operating conditions. Most reports indicate that the size of chitosan particles produced by spray drying is from 3 to 50 lm, much larger than the size of the nanoparticles in our work [15–17, 19–21]. The different particle size in our work compared with previous work is because we used a lower initial concentration of chitosan and a smaller spray drying nozzle than almost all previous work [16]. Production yield in our work was 60.5–62.8 %, similar to that in most previous work [17, 18] and higher than that of Cevher [15] (production yield from 47.01 to 50.16 %) [15]. The antibacterial activity of chitosan depends on its positive charge, as expressed by the zeta potential. The results listed in Table 1 indicate that the zeta potential of the chitosan nanoparticles obtained in our work was from 39.3 to 45.7 mV, much higher than that in other work. Dudhani et al. [18] reported that chitosan nanoparticles prepared by dropping sodium TPP solution into chitosan solution had zeta potentials in the range 25–29 mV [18]. The zeta potential of chitosan microparticles prepared by cross linking and spray drying was from 15 to 32 mV [23] and from 11.5 to 18.9 mV [20]. The results in Table 1 indicate that chitosan nanoparticles at a concentration of 20 lg/L strongly inhibited growth of S. aureus; OD 610 nm values were 2.1312 for the control and 0.0415 and 0.0138 for samples to which chitosan nanoparticles had

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been added. These results showed that much smaller size and much higher zeta potential resulted in much greater antibacterial activity. The minimum inhibitory concentration (MIC) of the chitosan nanoparticles in this work was approximately 20 mg/L, which was smaller than that in previous work. Xing et al. [8] reported that the MIC of oleoyl chitosan nanoparticles against S. aureus was 125 lg/mL [8]. Ngo et al. reported that the MIC of chitosan nanoparticles against S. aureus was 234 lg/mL and that the MIC of chitosan solution against S. aureus was from 250 to 375 lg/mL [11]. Effect of nozzle size and molecular weight of chitosan on the characteristics and antibacterial activity of chitosan nanoparticles In the spray-drying process, the properties of the initial feed material and the operating conditions, for example nozzle size, flow rate, and inlet and outlet temperature, strongly affect the characteristics and production yield of chitosan nanoparticles. In this work we investigated the effect of nozzles of different size and of the molecular weight of the chitosan on the characteristics and antimicrobial activity of chitosan nanoparticles. The results, listed in Table 2, show that nozzles of different size furnished chitosan nanoparticles of different size and zeta potential. Particle size increased from 95.4 to 263.6 nm and zeta potential decreased from 45.7 to 35.8 mV when size of the nozzle was increased from 4.0 to 7.0 lm. When the 7.0 lm nozzle was used, however, production yield was nearly three times higher than for use of the 4.0 lm nozzle. Study of the morphology of the chitosan particles by SEM (Fig. 4) revealed that their shape and size were relatively homogenous and their size was relatively fine; this was much better than results reported after other work [17, 20, 23]. The effects of the molecular weight of chitosan (initial feed material) on the characteristics and antibacterial activity of the particles obtained are shown in Table 3 and Fig. 5. The results show that the size of chitosan nanoparticles prepared by spray drying depended on molecular weight. Increasing the molecular weight of the chitosan led to an increase in the average size of the chitosan nanoparticles and a reduction in the zeta potential from 45.7 to 29.6 mV, results similar to those in previous work [17]. The results also showed that the

Table 2 Effect of nozzle size on the characteristic of chitosan nanoparticles Nozzle size (lm)

Average particle size (nm)

Zeta potential (mV)

Recovery (%)

Production yield (mg/h-1)

4.0

95.4

45.7

62.6

7.72

5.5

215.1

40.2

60.8

10.13

7.0

263.6

35.8

60.0

20.05

Starting feed material: chitosan solution (0.025 %); molecular weight equivalent to 276 cps. Operating conditions: flow rate 2 mL/min, drying gas flow 1.3 m3/min, inlet temperature 120 °C, outlet temperature 80 °C

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Fig. 4 SEM of nanoparticles (276 cps) obtained from chitosan at a concentration of 0.025 % (w/v) by use of 4.0 lm (a); 5.5 lm (b), and 7.0 lm (c) nozzles. Operating condition: flow rate 2 mL/min; drying gas flow 1.3 m3/min, inlet temperature 120 °C, outlet temperature 80 °C

Table 3 Effect of molecular weight of chitosan on the characteristic of chitosan nanoparticles MW of CS (cps)

Average size (nm)

Zeta potential (mV)

OD 610 nm

Control

–

–

1.9737

267

95.4

45.7

0.0140

760

271.5

35.1

0.0439

1,200

335.9

29.6

0.0652

Starting feed materials: chitosan solution (0.025 %), molecular weight 276, 760, or 1,200 cps. Operating conditions: nozzle size 4.0 lm, flow rate 2 mL/min, drying gas flow 1.3 m3/min, inlet temperature 120 °C, outlet temperature 80 °C

antibacterial activity of the chitosan nanoparticles increased with decreasing molecular weight. Other workers have also reported reduced antibacterial activity of chitosan of higher molecular weight [15–17]. These results can be explained on the basis of that use of chitosan of lower molecular weight produced particles of smaller size and higher zeta potential, which led to stronger inhibition of the growth of the bacteria. Higher zeta potential helps the particles to bind and interact easily with the membrane of the bacteria. In addition, nanoparticles of smaller size can more easily penetrate cell walls then bind to DNA (polyanion), thus inhibiting RNA and protein synthesis [9, 10].

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Fig. 5 SEM of chitosan nanoparticles obtained from chitosan of molecular weight 276 cps (a), 760 cps (b), and 1,200 cps (c). The concentration was 0.025 %, nozzle size 4.0 lm, flow rate 2 mL/min, drying gas flow 1.3 m3/min, inlet temperature 120 °C, and outlet temperature 80 °C

Antibacterial activity of chitosan nanoparticles–amoxicillin complex Amoxicillin at doses of 10, 20, 30, 40, and 50 lg/mL was added to 20 lg/mL chitosan nanoparticles solution. Loading efficiency of amoxicillin on to chitosan nanoparticles prepared by spray drying was over 82.2 %, similar to results reported by Anal et al. [14], who loaded ampicillin on to ionotropic cross-linked chitosan microspheres [14]. In this experiment, amoxicillin (10, 20, 30, 40, or 50 lg/mL) and chitosan solution (10, 20, 30, 40, 50, or 60 lg/mL) were used as controls. The results shown in Fig. 6 indicate that chitosan nanoparticles completely inhibited growth of the bacteria at a concentration of 20 lg/mL only, a factor of three lower than the amoxicillin concentration with the same effect. The results in Fig. 6 also show that the activity was strongly enhanced when amoxicillin was loaded on chitosan nanoparticles. The MIC of chitosan nanoparticles–amoxicillin complex decreased into 10 lg/mL compared with 20 lg/mL for the particles and 60 lg/mL for amoxicillin. Chitosan nanoparticles–amoxicillin complex can enter cells and release amoxicillin, inhibiting the bacteria. These results are similar to those reported by Cevher et al., who encapsulated in chitosan microspheres [15].

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2.5

OD 610nm

2 Amoxicillin

1.5

Chitosan nanoparticles/amoxicillin Chitosan nanoparticles

1 0.5

Chitosan

0 0

10

20

30

40

50

60

Concentration, µg/mL Fig. 6 Antibacterial activity against S. aureus of chitosan nanoparticles and chitosan nanoparticles– amoxicillin complex. All samples were inoculated at 37 °C. Twenty-four hours after inoculation, growth of the bacteria was determined by measurement of turbidity at 610 nm

Conclusions Chitosan nanoparticles prepared by spray drying have sizes ranging from 95 to 358 nm, high zeta potential, and are homogeneous. It is easy to control their properties by adjusting the concentration of the chitosan solution used as the initial feed, the size of the nozzle, and the molecular weight of the chitosan. Chitosan nanoparticles have strong antibacterial activity, and efficiency of loading of amoxicillin is high. It is concluded that chitosan nanoparticles prepared by spray drying have much potential as an antibacterial agent and as a novel means of delivery for amoxicillin. Acknowledgments The authors would like to thank the Department of Science and Technology, Ho Chi Minh City, Viet Nam, for supporting this work (217/2013/HÐ-SKHCN). We also express our thanks to Professor Ro-Dong Park, Chonnam National University, South Korea for your gift of chitosan. This work was supported in part by a grant from the National Science Council, Taiwan (NSC 102-2313-B-032001-MY3 and NSC 102-2621-M-032-005).

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